Notoginsenoside R1 Mitigates UVB-induced Skin Sunburn Injury through Modulation of N4-acetylcytidine and Macroautophagy

Notoginsenoside R1 Mitigates UVB-induced Skin Sunburn Injury through Modulation of N4-acetylcytidine and Macroautophagy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Notoginsenoside R1 Mitigates UVB-induced Skin Sunburn Injury through Modulation of N4-acetylcytidine and Macroautophagy Shuyun Liang, Xiaokang Liu, Yuting Yang, Fangyuan Zhang, Xiaobo Sun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6678920/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Chinese Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Background Panax Notoginseng Saponins (PNS) have exhibited therapeutic effects in the repair of skin photoaging induced by UVB radiation; however, the precise mechanism of action remains to be elucidated. Purpose This study is designed to utilize network pharmacology prediction methods to explore the mechanisms by which PNS repair UVB-induced skin photoaging. Furthermore, the chemical composition of PNS was characterized using UHPLC-Q-Orbitrap-MS/MS. An in-depth analysis of the pharmacodynamics of a specific component of PNS, Notoginsenoside R1 (NGR1), was also performed. Methods Qualitative and quantitative analyses were conducted utilizing UHPLC-Q-orbitrap-MS/MS and UHPLC-Q-Trap-MS/MS to investigate the chemical constituents of PNS. Furthermore, network pharmacology predictions were employed to explore the key targets and mechanisms by which PNS mitigates UVB-induced skin sunburn injury. Furthermore, a nude mouse model was employed to validate the therapeutic efficacy of PNS and its constituent NGR1, whereas HaCaT cells were utilized to elucidate their target mechanisms. Results This study was designed to thoroughly examine the fundamental mechanisms responsible for the efficacy of PNS in alleviating UVB-induced skin sunburn injury. Additionally, 16 primary saponin components within PNS were identified and subjected to quantitative analysis. Network pharmacology methodologies were utilized to identify 49 key targets of PNS in alleviating UVB-induced skin photoaging. Administration of PNS and NGR1 ameliorates UVB-induced photoaging symptoms through the reduction of inflammation, enhancement of antioxidant defense, inhibition of PI3K/AKT/mTOR signaling pathway activation, and regulation of cellular homeostasis proteins. Furthermore, it provides protection against apoptosis in HaCaT cells by upregulating essential cellular homeostasis proteins, such as p62, while concurrently downregulating autophagy-related proteins, including Beclin-1 and LC3-II. NAT10 expression is reduced by UVB radiation; however, this reduction can be reversed by the administration of drugs PNS and NGR1. The autophagy pathway, which is regulated by NBR1 and p62, is likely involved in the degradation of NAT10 under both physiological and UVB-induced conditions. Conclusion The potential of PNS and NGR1 in skin sunburn injury therapies is evidenced by their capability to mitigate UVB-induced skin damage via the inhibition of PI3K/AKT/mTOR signaling pathway activation, the reduction of cellular apoptosis and autophagy, and the enhancement of RNA acetyl transferase NAT10 regulation. The findings not only lay robust groundwork for subsequent clinical trials of PNS and NGR1 ointment but also furnish compelling evidence to elucidate the therapeutic mechanisms of PNS and NGR1 in the prevention and treatment of UVB-induced skin sunburn injury. Panax Notoginseng Saponins (PNS) Panax Notoginsenoside R1 (NGR1) N4-acetyltransferase 10 (NAT10) UVB Irradiation PI3K/AKT/mTOR Pathway Autophagy. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights 1. The combination of PNS and NGR1 effectively mitigates UVB-induced skin sunburn injury in a nude mice model. 2. PNS effectively inhibits the activation of the PI3K/Akt/mTOR signaling pathway in response to UVB irradiation. 3. The peripheral nervous system regulates the expression levels of apoptotic proteins and autophagy-related proteins in response to UVB irradiation. 4. The autophagic degradation of NAT10 under UVB stress was facilitated by NGR1 through the involvement of NBR1 and p62. Introduction UVB, the most biologically active component of solar radiation, is characterized by its high energy and strong penetrative capabilities. Exposure to UVB induces skin damage, which elicits a complex biological response marked by systemic changes in skin morphology and function. This damage mechanism encompasses structural remodeling of the skin, characterized by epidermal hyperplasia, degradation of the extracellular matrix resulting in reduced skin elasticity, and the onset of cellular damage along with inflammatory cascades. Chronic exposure to UVB not only induces direct damage to skin tissues but also expedites skin aging processes and markedly elevates the risk of cutaneous malignancies. The process of skin aging involves both endogenous and exogenous factors, manifesting as phenotypic features such as roughness, pigmentation, and reduced skin elasticity. Exogenous aging is attributed to environmental exposure[ 1 , 2 ], while endogenous aging occurs gradually over time. The term "(Ultraviolet B) UVB-induced skin sunburn injury of the skin" refers to the thickening of the skin and reduction in its elasticity caused by exposure to sunlight, resulting in significant cellular damage and inflammation factors. This condition is commonly associated with pronounced wrinkles, pigmentation issues, laxity, and sagging [ 3 ]. Excessive UVB exposure can lead to the development of skin lesions, accelerated sunburn injury, and even an increased risk of skin tumorigenesis [ 4 ].Therefore, the search for and development of drugs to repair skin photo-damage has received significant attention from researchers both domestically and internationally. In recent years, with an increasing number of reports on the beneficial effects of natural remedies on skin health, there has been a growing trend among individuals to develop natural drug treatments for skin UVB damage. Many effective ingredients found in natural remedies, such as flavonoids, isoflavones, and ginsenosides, possess properties that protect the skin[ 5 – 9 ]. Within the field of skincare, saponin compounds are widely recognized as safe and effective natural anti-aging agents. Many studies have demonstrated the significant impact of saponin compounds on skin care and repair. For instance, ginsenoside Rg1 has been found to attenuate UVB-induced glucocorticoid resistance in keratinocytes through the Nrf2/HDAC2 signaling pathway [ 10 ]. Treatment with ginsenoside Rc can effectively inhibit ROS production and prevent the elevation of pro-MMP-2 and − 9 levels in UVB-induced HaCaT keratinocytes [ 11 ]. The presence of ginsenoside Rc in keratinocytes plays a crucial role in protecting the skin from photooxidative stress caused by UV radiation, as it acts as an effective anti-sunburn injury agent and enhances barrier function [ 12 – 14 ]. Panax Notoginseng Saponins (PNS) extract is derived from Panax Notoginseng (Burk.) F.H.Chen , a member of the Araliaceous family. PNS, a complex bioactive compound derived from plants in the Araliaceous ginseng family, has attracted considerable attention in medical research. Its diverse pharmacological properties, including promotion of hematopoietic function, modulation of immune responses, alleviation of inflammation, and inhibition of aging processes, have positioned it as a key focus in clinical studies. This component was officially incorporated into the 2020 edition of the Chinese Pharmacopoeia and has exhibited substantial clinical value in the treatment of traumatic diseases. In the realm of skin injuries, PNS has demonstrated promising application potential, especially in mitigating UVB-induced skin damage. Nevertheless, further in-depth scientific investigation is warranted to clarify its precise cellular repair mechanisms. Notoginsenoside R1 (NGR1), a specific component of Panax Notoginseng, is a crucial constituent of Panax Notoginseng total saponins. It demonstrates a wide range of benefits, encompassing blood pressure reduction, heart rate decrease, analgesic effects provision, inflammation combatting, and aging processes delay. The isolated Ginsenoside C-Mx from Panax Notoginseng leaves' total saponins could inhibit intracellular ROS, MMP-1, and Interleukin-6 (IL-6) expression induced by UVB radiation. It effectively reverses the degradation of type I procollagen caused by UVB exposure through regulation of the TGF-β/Smad signaling pathway[ 15 ]. The application of Ginsenoside C-Y significantly decreases the levels of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-α) in the body after UVB exposure. Additionally, it restrains matrix metalloproteinase-1 (MMP-1) production, promotes the synthesis of type I collagen, inhibits melanin secretion and tyrosinase activity, and reduces the amount of melanin in the skin. These effects contribute to the inhibition of skin photodamage and prevention of excessive skin pigmentation [ 16 ]. Ginsenoside Rk3 significantly enhances the activities of hydroxyproline, Superoxide Dismutase (SOD), and GSH-PX enzymes in mouse skin tissue and blood, while reducing the expression levels of malondialdehyde, MMP-1, MMP-3, IL-6, Interleukin-1β (IL-1β), and TNF-α. This leads to suppression of inflammation and skin sunburn injury, thereby providing protection for skin health [ 17 ]. Research has demonstrated that saponin components derived from Panax Notoginseng are extensively utilized in diseases related to skin injuries. Although the potential efficacy of PNS against UVB-induced skin sunburn injury has been discovered, currently its repair mechanism remains unclear. The regulation of molecular functions is significantly influenced by RNA modifications, and accumulating evidence increasingly suggests that targeting the pathways governed by RNA modifications holds great promise in the field of cancer therapy [ 18 ]. The N 4 -acetylcytidine (ac 4 C) modification is a prevalent chemical alteration found in mRNA, playing a pivotal role in the regulation of mRNA stability and translation. However, the precise impact of ac 4 C modification on diseases remains elusive[ 19 ]. The N-acetyltransferase-like protein (NAT10) is the primary eukaryotic RNA enzyme responsible for catalyzing the production of ac 4 C, exhibiting both acetyltransferase activity and RNA-binding capability. Despite its crucial role as the sole recognized ac 4 C "writer" protein, the impact of NAT10 on disease progression remains to be elucidated. Drawing on a review of the literature and preliminary laboratory findings, it has been demonstrated that the NAT10 protein undergoes degradation under UVB-induced conditions. This process not only influences the formation of UVB-induced photoproducts, such as cyclobutane pyrimidine dimers (CPDs), but also contributes to the regulation of UVB-induced DNA damage and carcinogen-related repair mechanisms[ 20 ]. In this study, we unveil the mechanism through which NAT10-mediated mRNA ac 4 C modification regulates UVB-induced skin damage. Network pharmacology is an interdisciplinary field that merges principles from systems biology and network informatics. In recent years, this approach has gained widespread application in the development of novel therapeutics, with a particular focus on elucidating the synergistic interactions among multiple components, pathways, and targets[ 21 – 23 ]. The integration of vast amounts of information enables the discovery of novel drug targets and molecular mechanisms[ 24 – 26 ]. Network pharmacology facilitates the comprehensive analysis of multi-component drugs' effects on human physiology, enabling the identification of therapeutic targets for effective drug components, thereby augmenting drug efficacy while minimizing adverse reactions[ 27 , 28 ]. Presently, network pharmacology is increasingly employed to investigate the treatment potential of traditional Chinese medicine across diverse diseases. The aim of this study was to investigate the therapeutic efficacy of PNS and NGR1 in mitigating skin UVB-induced skin sunburn injury and elucidate its underlying mechanism. Initially, liquid chromatography-mass spectrometry was employed to analyze the chemical composition of PNS. The subsequent step involved the utilization of network pharmacology to predict potential targets and molecular mechanisms associated with PNS in mitigating skin aging caused by UVB light, followed by preliminary experimental validation. The aim of this article is to investigate the therapeutic mechanisms of PNS and NGR1 in treating UVB-induced skin sunburn injury and provide supportive data for the development of products targeting such treatment. Materials and Methods Chemical Reagent The specific preparation method for PNS is primarily outlined in the dedicated chapter on PNS in the 2020 edition of the Chinese Pharmacopoeia. The procedure involves crushing Panax Notoginseng into coarse powder, extracting it with 70% ethanol, filtering the mixture, concentrating the filtration under reduced pressure, passing it through a column of non-polar or weakly polar styrene-type macro-porous adsorption resin, washing with water, and discarding the washings. The resin is subsequently subjected to elution with 80% ethanol, followed by concentration under reduced pressure, decolorization, further concentration, and refinement to obtain the extract. Finally, the extract is dried to yield the ultimate product. Notoginsenoside R1 is one of the active components isolated from the total saponins of PNS. As a unique active component of Panax Notoginseng, it holds an important position within the total saponins. PNS with a purity of over 75% was procured from the China Food and Drug Research Institute, batch number: 110870-202105. The purities of the substances Notoginsenoside R1 (NGR1) (C 47 H 80 O 18 ) (110745-202322). Rg1 (C 42 H 72 O 14 ) (110703-202436), Rb1 (C 54 H 92 O 23 ) (110704-202331), Rd (C 48 H 82 O 18 ) (111818-202305), and Re (C 48 H 82 O 18 ) (110754-202330) are all greater than 95% and were obtained from the same institute. All-Trans Retinoic Acid (ATRA) was sourced from Sigma-Aldrich, located in St. Louis, Missouri, USA. The formic acid used, which was of mass spectrometry grade, was also provided by Sigma-Aldrich, St. Louis, MO, USA. Chromatic-grade acetonitrile and methanol were purchased from Fisher Scientific, based in Pittsburgh, Pennsylvania, USA. UHPLC-Q-Orbitrap-MS/MS Preparation of PNS Sample Solution: Accurately weigh 10 mg of PNS and subject it to ultrasonic extraction using 10 mL of 70% (v/v) methanol in water for 15 minutes at ambient temperature. Filter the resulting mixture through a 0.22 μm membrane filter. Place the filtered solution into a vial for subsequent analysis by Ultra-High-Performance Liquid Chromatography coupled with Quadrupole Orbitrap Mass Spectrometry (UHPLC-Q-Orbitrap-MS/MS). Chromatographic separation was carried out using an Ultimate 3000 ultra-high performance liquid chromatography system (Thermo, San Jose, CA, USA) equipped with a Supelco C18 column (dimensions: 3.0 × 50 mm, particle size: 2.7 μm; supplied by Sigma-Aldrich). The column temperature was set at 35°C. The mobile phases consisted of acetonitrile (phase A) and water (phase B). The separation of the experimental samples was achieved using a gradient elution program as follows: 80% solvent B from 0 to 20 minutes, decreasing to 54% B from 20 to 45 minutes, further reduced to 45% B from 45 to 55 minutes, held at 45% B from 55 to 60 minutes, then increased back to 80% B from 60 to 65 minutes, and maintained at this concentration for an additional 5 minutes. The sample injection volume was set at 5 μL, with a flow rate of 0.4 mL per minute. Mass spectrometric analysis was performed using a Q-Orbitrap-MS/MS instrument (Thermo, San Jose, CA, USA) with an electrospray ionization source operated in negative ion mode. The ion source parameters were configured as follows: sheath gas flow at 40 arbitrary units, auxiliary gas flow at 10 arbitrary units, and sweep gas flow at 1 arbitrary unit. The S-Lens RF level was set to 55%. The capillary voltage was configured at -3.5 kV, and the capillary temperature was maintained at 350°C. Full MS data were collected in centroid mode over an m/z range of 150 to 1,500 Da, employing a resolution of 70,000. The automatic gain control (AGC) target was set to 1×10 6 , and the maximum injection time was 100 ms. For tandem mass spectrometry, data were acquired in Full-MS/ddMS 2 mode with the following parameters: a resolution of 17,000, an AGC target of 1×10 5 , a maximum injection time of 50 ms, a loop count of 5, a Top N value of 5, an isolation window of 4.0 m/z, and stepped collision energies of 35, 45, and 55. UHPLC-Q-Trap-MS/MS Preparation of the PNS sample solution: Accurately weigh 1 mg of PNS at room temperature, followed by subjecting it to ultrasonic extraction using 10 mL of a 70% (v/v) methanol/water solution for a duration of 15 minutes. Subsequently, filter the resulting mixture through a 0.22 μm membrane filter to obtain a purified PNS sample solution. To prepare the standard solution, accurately weigh appropriate amounts of NGR1, Rg1, Rb1, Re, and Rd. Under controlled room temperature conditions, subject them to ultrasonic extraction using 1 mL of a 70% (v/v) methanol/water solution for a duration of 15 minutes. This process will result in the creation of a mixed solution with a concentration of 100 ng/mL, Similarly, this mixture is also passed through a 0.22 μm membrane filter for filtration. The filtered solutions will then be transferred to dedicated sample vials for subsequent analysis using a Triple Quadrupole Mass Spectrometer (UHPLC-Q-Trap-MS/MS). Using a quadrupole mass spectrometer (QTRAP6500; ABSciex, Framingham, MA), chromatographic separation was performed on an ultra-high-performance liquid chromatography (UPLC) system in the reversed-phase mode using a Waters ACQUITY BEHC 18 column (length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 μm, manufactured by Waters Corporation in Milford, MA, USA). Mobile phases A and B consist of water with 0.1 mmol/L ammonium acetate and acetonitrile respectively at a flow rate of 0.35 mL/min and maintained at a temperature of 35°C. The multi-component quantification of PNS was achieved using a gradient separation scheme, which involved the following steps: mobile phase B concentration started at 30% from 0 to 2.0 minutes, increased to 37% up to 5.0 minutes, further rose to 42% at 9.0 minutes, adjusted to 47% at 14.0 minutes, reached a concentration of mobile phase B of 51.0% at 17.0 minutes and swiftly increased between 20.0 and 21.0minutes until it reached 95%, before returning back to the initial concentration of 30% during 22-23 minutes. The operational parameters of the mass spectrometer were optimized with the following specific settings: ion spray voltage set to -4.5 kV in negative ion mode; ion source temperature maintained at 550 °C; gas 1 pressure set to 20 psi; gas 2 pressure adjusted to 30 psi; and curtain gas pressure regulated at 10 psi. During the analysis, the multiple reaction monitoring (MRM) mode was employed for precise quantification of each analyte of PNS. Cell Culture The human keratinocytes (HaCaT) (Catalog number: ATCC CRL-2404) and embryonal kidney 293T cells (293T) (Catalog number: ATCC CRL-3216) were obtained from Proteintech (Proteintech, Wuhan, China). The cell lines were maintained in DMEM (Gibico) enriched with 10% fetal bovine serum (FBS, Gibico), along with penicillin (at a concentration of 100 units per milliliter) and streptomycin (at a concentration of 100 micrograms per milliliter), all sourced from Gibico, USA. Animal Experiments Female BALB/c-nude mice, aged 6 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). The animals were housed under specific pathogen-free conditions for one week to acclimate. The environmental conditions included a temperature range of 15-25°C and relative humidity between 40% and 65%. A 12-hour light/dark cycle was maintained, and the mice had unrestricted access to both food and water. Forty nude mice were divided into five groups: the control group without treatment (Sham), the UVB-induced model group treated with cream base only (UVB, cream base), The positive control group is treated with a 0.1% ATRA cream and exposed to UVB radiation (UVB, 0.1% ATRA), the low-dose PNS-treated group using 1.5% PNS cream along with UVB exposure (UVB, PNS-Low), and the high-dose PNS-treated group using 3.0% PNS cream along with UVB exposure (UVB, PNS-High). The low-dose NBR1-treated group received 1.5% NBR1 cream in conjunction with UVB exposure (UVB, NBR1-Low), while the high-dose NBR1-treated group received 3.0% NBR1 cream along with UVB exposure (UVB, NBR1-High). After receiving UVB irradiation at a dose of 80 mJ/cm², administered 5-6 times per week for one week, the nude mice underwent a fasting period of 12 hours and were subsequently anesthetized following the final drug administration. Blood samples and skin tissues were collected for analysis. The animal experiment was conducted in accordance with the approved protocol of the Institutional Animal Care and Use Committee, which was authorized by the Animal Ethics Committee of Changchun University of Chinese Medicine. The registration number is 2024299, and the specific date is June 13, 2024. Hematoxylin and Eosin (H&E) Staining Skin samples were collected from each group of nude mice for histological examination, following established protocols. Briefly, the skin samples were fixed in 4% neutral formaldehyde for 24 hours, embedded in paraffin under vacuum, and sectioned into 10μm-thick slices. These sections were deparaffinized using xylene and then subjected to a series of ethanol rehydration and dehydration steps before being stained with hematoxylin and eosin. Identical cross-sections from each sample on three slides were selected, and four different microscopic fields (at×200 magnification) per slide were photographed. Stained images were acquired using an optical microscope. Enzyme-linked Immunosorbent Assay (ELISA) To investigate the effects of PNS and NBR1 on UVB-induced inflammatory and antioxidant factors, serum samples were collected from nude mice in the control group, model group, positive control group, PNS treatment group, and NBR1 treatment group. The expression levels of inflammatory factors including TNF-α, IL-1β, IL-6 and Interleukin-10 (IL-10)(Jiangsu Enzyme-linked Immunosorbent Assay Industry Co., Ltd., Batch Numbers MM-013M1, MM0040M1, MM0163M1, MM-0176M1) and antioxidant factors MOD, SOD, Total Antioxidant Capacity (T-AOC), Catalase Micrococcus lysodeikticus (CAT) (Nanjing Jiancheng Bioengineering Institute, Batch Numbers A003-1, A001-1, A015, A007-1) were analyzed following the manufacturer's instructions. UVB Irradiation The cell UVB radiation was performed in accordance with our previously established protocols [29]. Following two rinses with phosphate-buffered saline (1×PBS buffer, Invitrogen), the cells were subjected to UVB irradiation (20 mJ/cm², unless otherwise indicated) using a UV Stratalinker 2400 device fitted with UVB lamps (Stratagene). For control groups, mock irradiation was performed. The UVB dosage was consistently measured using a Goldilux UV meter equipped with a UVB sensor (Oriel Instruments). Cell Viability To evaluate the cytotoxic impact of UVB exposure on HaCaT keratinocytes and examine the possible protective roles of PNS and NBR1, we employed the CCK-8 assay to determine cell viability. Cells were plated in a 96-well plate at a concentration of 1×10^4 cells per well and incubated beforehand for 24 hours. Following this, the cells were exposed to different concentrations of PNS and NBR1 (0, 250, 500, 1000 μM) for a period of 24 hours. Subsequently, the cells were subjected to UVB irradiation and then incubated with various concentrations of PNS and NBR1 (0, 250, 500, 1000 μM) for a period of 24 hours. Additionally, PNS and NBR1 were administered at a fixed concentration of 250 μM but across different time points (0, 24, 48, 72, and 96 hours). Following these treatments, 10 μL of CCK-8 solution (Beyotime, Shanghai, China) was added to each well, and the plates were incubated for 4 hours under conditions of 37°C with 5% CO2 in a humidified environment. Finally, the absorbance was recorded at 450 nm using a Tecan Infinite1000 microplate reader (Tecan, Switzerland). Immunofluorescent Staining After UVB irradiation, separate treatments of PNS and NBR1 were administered, HaCaT cells (2×10 5 cells/well) were seeded onto cell climbing membranes in 6-well plates for a duration of 24 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized using PBS containing 0.2% Triton X-100 for a period of 20 minutes. Subsequently, the cells were incubated with a solution of 3% bovine serum albumin for 30 minutes to block non-specific binding sites. Following this, the cells were subjected to overnight incubation at a temperature of 4°C with Microtubule-associated proteins 1A/1B light chain 3B (LC3-II) antibody (diluted at a ratio of 1:75), followed by co-incubation with the corresponding fluorescent secondary antibody. Finally, DAPI staining was performed using a concentration of 500 ng/mL for a duration of five minutes, and cell staining was observed utilizing an Olympus fluorescence inverted microscope. Western Blot To obtain protein extracts, cells were first washed with cold PBS and then lysed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The resulting protein solutions were subjected to sonication and subsequently centrifuged at 13,000 RPM for 20 minutes at 4°C. Protein concentrations were determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Following quantification, the samples underwent heat treatment at 70°C for a duration of 10 minutes. The protein levels were then evaluated by performing SDS-polyacrylamide gel electrophoresis, which was subsequently followed by immunoblot analysis. The antibodies utilized in this study are listed as follows: Anti-GAPDH (Proteintech, Cat#10494, 1:2000), Anti-β-actin (Bioss, bs-0061R, 1:2000), Anti-AKT1 (Servicebio, GB13011, 1:500), Anti-PI3K (Proteintech, Cat#27921, 1:500), Anti-mTOR (Proteintech, Cat#66888, 1:500), Anti-Bax (Proteintech, Cat#68111, 1:500), Anti-Bcl-2(Proteintech, Cat #68103, 1:500), Anti -Caspase 3 (Proteintech, Cat #19677, 1:500) Anti-P62 (Abcam, ab207305, 1:1000) Anti-Beclin-1 (Abcam, ab207612, 1:1000) Anti-NAT10 (Abcam, ab194297, 1:2000) Anti-NBR1 (Abcam, ab55474, 1:1000). Flow Cytometry Using flow cytometry (FACSCalibur, Becton Dickinson, USA) for the flow cytometric analysis of cells. After knocking down NAT10 using siRNA, 293T cells were pre-treated with a UVB dose of 20mJ/cm². The cells were then collected 24 hours later for further analysis. HaCaT cells were first pretreated with a UVB dose of 20 mJ/cm². Following this, the cells were incubated for 24 hours in the presence or absence of PNS and NGR1. After incubation, the cells were collected, washed with PBS, and resuspended in 0.5 mL of binding buffer. To differentiate between early apoptotic cells (which stain positive for annexin V (AV) but negative for propidium iodide (PI); AV + /PI - ) and late apoptotic or necrotic cells (which stain positive for both AV and PI; AV + /PI + ), dual-color flow cytometry analysis was conducted. The Evaluation of The Degradation Pathway of NAT10 After 24 hours of UVB irradiation, HaCaT cells were subjected to treatment with either MG132 (MG, 10 μM) or bafilomycin A1 (BafA1, 50 ng/mL) for a duration of 6 or 24 hours. Another group of cells was left untreated. Subsequently, immunoblot analysis was performed on both the treated and untreated cells to detect NAT10 and GAPDH proteins. siRNA Transfection The siRNAs utilized were as follows: siRNA-ATG7 (sc-41447), siRNA-ATG5 (sc-41445), and siRNA-control (sc-37007) obtained from Santa Cruz Biotechnology. Each of these was transfected into distinct cells using a specialized medium for siRNA transfection (sc-36868) in conjunction with a specific transfection reagent for siRNAs (sc-29528), in accordance with the guidelines supplied by the manufacturer. The Application of NAT10 Pulldown Combined with Proteomic Analysis In NAT10 pull-down experiments, the Universal Magnetic Beads Co-Immunoprecipitation (Co-IP) Kit (Proteintech, Cat#54002) was utilized in accordance with the provided instructions. The sample was treated with a reaction solution containing 1% SDC, 100 mM Tris-HCl (pH 8.5), 10 mM TCEP, and 40 mM CAA. Incubation was performed at 95°C for 10 minutes to achieve thorough denaturation, reduction, and alkylation of the proteins. The mixture obtained was then processed through centrifugation, after which the supernatant was diluted with an equivalent volume of ddH 2 O. For the overnight digestion at 37°C, trypsin was introduced in a proportion of 1:50 (enzyme to protein, by weight). The next day, the pH was adjusted to 6.0 using TFA to halt the digestion. Following another centrifugation step (12,000 g for 15 minutes), peptide purification was carried out on the supernatant using a custom-made SDB-RPS desalting column. The peptides that were eluted were then dried under vacuum and stored at -20°C for subsequent use. The analysis of the samples was conducted using a timsTOF Pro instrument (Bruker Daltonics), an integrated device that combines trapped ion mobility spectrometry (TIMS) with quadrupole time-of-flight mass spectrometry. This timsTOF Pro system was connected to an UltiMate 3000 RSLC nano liquid chromatography system (Thermo) equipped with a Captive Spray nano ion source, provided by Bruker Daltonics. Peptide samples were introduced into a C18 Trap column (75 µm × 2 cm, 3 µm bead size, 100 Å pore diameter, Thermo) and subsequently separated using a reversed-phase C18 analytical column (75 µm × 15 cm, 1.7 µm bead size, 100 Å pore diameter, IonOpticks). The separation gradient was created using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nL per minute. Mass spectrometry analysis was conducted in diaPASEF mode with the capillary voltage configured at 1500 volts. Both MS and MS/MS spectra were recorded within an m/z range of 100 to 1,700, while ion mobility scans were performed over a range of 0.6 to 1.6 Vs/cm². The accumulation time and ramp time were each set to 50 milliseconds. The diaPASEF acquisition method was configured on the m/z-ion mobility plane utilizing the timsControl software from Bruker Daltonics. Collision energy varied linearly with ion mobility, starting at 59 eV when 1/K0 equaled 1.6 Vs/cm² and decreasing to 20 eV when 1/K0 reached 0.6 Vs/cm². The DIA raw data were processed using DIA-NN (version 1.8.2 beta 11) in a library-independent manner. The spectral files were then matched against the Human protein sequence database (obtained from Uniprot on August 7, 2024, which includes 20,654 entries). The search parameters largely adhered to the default configurations, with several adjustments: options for precursor ion generation were activated to create an in silico-predicted spectral library; the enzyme Trypsin/P was utilized, permitting up to 2 missed cleavages. Carbamidomethyl modification on cysteine residues was defined as a fixed alteration, whereas oxidation on methionine residues and N-terminal acetylation of proteins was treated as variable modifications. Both the mass tolerance and MS1 accuracy were configured to 15 ppm, with match-between-runs (MBR) and heuristic protein inference enabled. To ensure reliable identifications, the precursor false discovery rate (FDR) was controlled at 1%. Protein intensities were normalized utilizing the MaxLFQ algorithm. The protein-level quantification analysis was conducted using the 'pg_matrix.tsv' file derived from the DIA-NN search outcomes. Further bioinformatics analyses were carried out in the R statistical programming environment. To address the missing values, we employed a random Gaussian distribution centered at the mass spectrometer's detection limit. This distribution was adjusted with a downshift of 1.8 times the standard deviation and a width of 0.25 times the standard deviation. For valid value filtering, we required that at least 50% of the values within each group be non-missing. Significantly differentially expressed proteins (DEPs) were identified using P-values from Student’s T-tests and specified fold change thresholds. Functional annotations were performed using several databases, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), EggNOG, Pfam, and UniProt for subcellular localization. To determine significantly enriched terms and pathways in the regulated set, a Fisher's Exact Test was applied. Additionally, protein-protein interaction networks were built and evaluated utilizing the STRING database. Co-IP The cells underwent two washes with chilled PBS, were collected on ice using a cell scraper, and then centrifuged at 4°C for 5 minutes at 1000 rpm. Following this, the supernatant was removed. Next, cold Co-IP lysis buffer (Proteintech, Cat#PR20037) was introduced, and the cell pellet was carefully resuspended through repeated pipetting until no aggregates remained. The sample was first incubated at 4°C for a period of 30 minutes, then subjected to centrifugation at 13,000 revolutions per minute (rpm) for 15 minutes at the same temperature. Following this step, the resulting supernatant was carefully transferred into a fresh tube. The reaction tubes were each supplemented with 20 μL of magnetic beads and an optimized amount of immune precipitation antibody (the appropriate dilution of the antibody was determined). The mixture was incubated at 4°C on a rotator for 4 hours. After incubation, the supernatant was removed using a magnetic stand. The beads were incubated with pre-prepared cell lysis buffer at 4°C overnight. Following the overnight incubation, the supernatant was discarded to eliminate non-binding proteins, and the beads were washed with PBS 2-3 times. Subsequently, the magnetic bead-bound immunocomplexes were resuspended in 25 μL of 5×Laemmli buffer, subjected to boiling for 5-10 minutes, and subsequently analyzed through immunoblotting. Network Pharmacology Analysis The saponin composition of PNS was determined by UHPLC-Q-Orbitrap-MS/MS. After being identified, the SMILES codes corresponding to each saponin were retrieved from the PubChem database. Subsequently, these SMILES were inputted into the Swiss Target Prediction platform (http://www.swisstargetprediction.ch/) to identify potential targets of these saponins, selecting proteins with a probability ＞0. By integrating reported targets from published studies, the final targets of the saponins were identified. Using the search terms "skin sunburn injury" "skin inflammation" and "skin cancer" as keywords, we conducted searches in three databases: GeneCards database (https://www.genecards.org/), OMIM database (https://www.omim.org/), and DrugBank database (https://www.drugbank.ca/) to retrieve targets associated with skin sunburn injury. Subsequently, we employed Draw Venn (http://bioinformatics.psb.ugent.be/webtools/Venn/) to generate a Venn diagram for integrating the disease targets, aiming to identify potential targets for skin photodamage. The construction of protein-protein interaction (PPI) networks was performed, and the gene targets that overlapped in both datasets were imported into the STRING database (http://string-db.org). The species selection in the operation interface was restricted to "Homo sapiens", with a confidence score threshold set at ≥0.9. Utilizing Cytoscape 3.7.2 (https://www.cytoscape.org/), we established a potential key target network and systematically analyzed its network parameters. To investigate the impact of the peripheral nervous system (PNS) on signaling pathways and gene functions in skin UVB light damage, we utilized the DAVID database (https://david.ncifcrf.gov/) for GO functional analysis and KEGG pathway enrichment analysis. The obtained results were visualized using R version 3.4.1, where we focused on the top 10 outcomes from GO analysis and the top 30 outcomes from KEGG analysis, with significance levels set at FDR < 0.05 and P < 0.05. We then developed ginsenoside-target-pathway (G-T-P) networks to graphically illustrate the interactions between compounds, their targets, and associated pathways. Molecular Docking Analysis Obtain the high-resolution crystal structure of the target protein from the Protein Data Bank (RCSB, https://www.rcsb.org). Use PyMOL 2.5.2 software to remove any non-protein atoms and solvent molecules, then save the cleaned structure in "pdb" format. Extract the three-dimensional structure of the compound from the PubChem database and leverage Open Babel 3.1.1 software to transform the file into either "mol2" or "pdb" format. Combine the pre-processed protein and compound structures within AutoDock 4.2.6 for molecular docking analysis, selecting the conformation with the lowest binding energy as the result. A docking energy less than 0 kcal/mol suggests potential molecular interaction, while a value below -5 kcal/mol indicates a strong binding affinity. Finally, use PyMOL software to visually analyze the docking results. Statistical Analysis The data were presented as mean±standard deviation (S.D.). Statistical significance of inter-group differences was assessed using one-way analysis of variance, followed by Dunnett's test or Tukey-Kramer multiple comparison test to analyze differences between the groups of the Means. SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA) was employed for statistical analysis. A P-value ＜ 0.05 was considered statistically significant. Results Saponin Composition Analysis of PNS The composition of PNS was comprehensively analyzed utilizing UHPLC-Q-Orbitrap-MS/MS, with the total ion chromatogram depicted (Figure 1A and Supplementary Figure 1) . Through meticulous high-resolution mass spectrometry analysis of individual ions, subsequent fragmentation data, and comparative studies with entries in the PNS database, in conjunction with pertinent literature references, a definitive identification was made for a total of 16 saponin compounds. Extensive details regarding these constituents are provided (Table 1 and 2) . The total content of five components, namely NGR1, ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rd, and ginsenoside Re, was determined in accordance with the methods specified in the 2020 edition of the Chinese Pharmacopoeia, the chromatogram of the reference was shown in (Figure 1B) .Quantitative analysis of NGR1, Rg1, Rb1, Re, and Rd was performed using UHPLC-Q-Trap-MS/MS (Figure 1C and 1D) , the analysis revealed a combined content of 84.13%, establishing them as crucial constituents within PNS, therefore, we selected these 5 saponins for follow-up network pharmacological analysis. The UHPLC-Q-Orbitrap-MS/MS spectra were summarized, and potential fragmentation pathways were analyzed to identify four saponins: NGR1, ginsenoside Rd, ginsenoside Re, and ginsenoside Rg1 (Figure 1J, 1K, 1M, 1N, 1O, 1P and 1Q) . while the specific contents of 16 ginsenosides were determined by HPLC (Table 7) . Protein-Protein Interaction (PPI) Analysis We conducted a search in the PubChem database to retrieve the SMILES identifiers of the 5 crucial saponin compounds found in PNS, namely NGR1, ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rd, and ginsenoside Re. Subsequently, these identifiers were imported into the Swiss Target Prediction platform for predictive analysis, resulting in a total of 198 target predictions (Figure 1E) . Through GeneCards, OMIM, and DrugBank databases, we retrieved a total of 31,465 target genes associated with UVB-induced skin sunburn injury (Figure 1F) . After thorough screening and standardization using Uniprot to eliminate duplicates, we ultimately identified 19,689 unique targets. By intersecting the 198 drug-derived targets with the 19,689 UVB-induced skin sunburn injury targets, we obtained a final set of 94 overlapping targets (Figure 1G) . We imported the 94 common targets into the STRING database, resulting in the construction of a Protein-Protein Interaction (PPI) network consisting of 82 nodes and 1,015 edges (Supplementary Figure 2) . The initial network was obtained by applying a condition where the parameter degree was set to be greater than or equal to the median value (≥23). The network is characterized by highly interconnected nodes, including AKT serine/threonine kinase 1(AKT1), IL6, Bcl-2-Associated X protein (Bax), B-Cell Lymphoma 2 (Bcl-2), CXCL8, FN1, MAPK8, IL1B, HSP90AA1 and GSK3B, etc. These proteins have been identified as the top 11 hubs in the network based on their Degree, BC and DC values (Table 3) . Consequently, these proteins represent potential targets for mitigating skin UVB-induced sunburn injury pathways in the PNS. Conducting Gene Ontology (GO) and KEGG Pathway Enrichment Analysis The DAVID 6.8 platform was utilized to conduct GO functional enrichment and KEGG pathway enrichment analyses on 94 targets associated with ginsenoside and skin sunburn injury. The bar charts illustrate the top 10 results for each of the three GO enrichment categories (Figure 1H) . Additionally, the bubble plot illustrates the enrichment of the top 20 KEGG pathways, providing a schematic diagram of how PNS mediates prevention of UVB-induced skin sunburn injury through multiple signaling pathways (Figure 1I) . Through KEGG analysis, we have identified that total saponins derived from Panax Notoginseng may potentially participate in 30 signaling pathways associated with the treatment of UVB-induced skin damage. The major signaling pathways encompass the PI3K-AKT, Notch, Rap1, neurotrophins, thyroid hormone, Ras, JAK-STAT, and MAPK signaling cascades. The PI3K-AKT signaling pathway, Notch signaling pathway, and Rap1 signaling pathway exhibit greater significance in comparison to the remaining pathways. These pathways encompass 16 crucial PPI network target points such as GF, Cytokine, ECM, ITGA, ITGB, AKT, HSP90, Bcl-2, and NF-kB. Moreover, considering the number and proportion of genes involved in each pathway, the PI3K-AKT pathway emerges as the most plausible candidate for repairing UVB-induced skin sunburn injury (Table 4) . These findings suggest that the potential mechanism underlying PNS-mediated repair of UVB-induced skin sunburn injury may be associated with the PI3K-AKT signaling pathway. Based on the enrichment analysis from the GO and KEGG databases, it is suggested that PNS may implement its therapeutic benefits in treating UVB-induced skin sunburn injuries via the activation of the PI3K-AKT and mTOR signaling pathways. We employed the "Path-view" package in R to visualize the PI3K-AKT and mTOR signaling pathways (Supplementary Figure 3 and 4) . These findings suggest that the potential mechanism underlying PNS-mediated repair of UVB-induced skin sunburn injury may be associated with modulation of the PI3K-AKT-mTOR signaling pathway. Ginsenoside-Target-Pathway (G-T-P) Analysis To gain a deeper comprehension of the interconnections among ginsenosides, targets, and biological pathways in PNS, we constructed a comprehensive ginsenoside-target-pathway (G-T-P) network (Supplementary Figure 5) . Additionally, a summary was provided for the analysis of the 16 saponins in PNS and their associated pathways in relation to UVB-induced protein damage (Table 5 and Supplementary Figure 6) . This intricate network comprises 65 nodes consisting of 5 compounds, 50 targets, and 10 pathways. The green nodes represent potential targets, the purple nodes symbolize ginsenosides, the yellow nodes depict signaling pathways, while the connecting lines signify their interactions. The larger and opaquer the nodes are, the higher their degree of association. The illustrated G-T-P network demonstrates that each ginsenoside interacts with numerous targets implicated in diverse pathways (Supplementary Figure 5) , suggesting a multi-target and multi-pathway mechanism in PNS for mitigating UVB-induced skin sunburn injury. The degrees of Rela, Jun, Stat3, IL2, Fos and Bcl-2 are 11, 11, 11, 10, 10 and 10 respectively. This indicates a close association between these proteins and the therapeutic efficacy of PNS in repairing UVB-induced skin sunburn injury. It highlights the significant role played by the PI3K-AKT pathway. The Administration of PNS Effectively Alleviates UVB-induced Skin Sunburn Injury in A Nude Mice Model The H&E staining results revealed pronounced skin sunburn injury nude mice following UVB irradiation, characterized by an increase in epidermal thickness and evident signs of sunburn injury on the dorsal skin. However, treatment with PNS significantly alleviated epidermal hypertrophy and effectively restored UVB-induced skin sunburn injury (Figure 2A and 2B) . The treatment with PNS significantly reduced the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in UVB-exposed nude mice, while also increasing the expression of the anti-inflammatory cytokine IL-10 (Figure 2C) . Additionally, PNS significantly augmented the expression of antioxidant enzymes SOD, T-AOC, and CAT in nude mice subjected to UVB irradiation, while concurrently suppressing MOD expression (Figure 2D) . The findings suggest that PNS possess the ability to effectively counteract UVB-induced skin sunburn injury in nude mice. The Application of PNS Enhances the Survival Rate of HaCaT Cells under UVB Radiation After treatment with various concentrations of PNS (0, 250, 500, and 1000 μM), HaCaT cells demonstrated no significant cytotoxicity as indicated by cell viability exceeding 95% (Figure 2E) . Compared to the control group, HaCaT cells exhibited cell death and a decrease in cell viability to 50% following irradiation with 20 mJ/cm 2 of UVB. Subsequent treatment with different concentrations of PNS (0, 250, 500, and 1000 μM) significantly enhanced cell viability, demonstrating a pronounced dose-dependent effect (Figure 2E) . Following exposure to PNS for various time periods (0, 24, 48, 72 and 96 h), HaCaT cells exhibited minimal cytotoxic effects, as indicated by cell viability exceeding 95% (Figure 2F) . The findings suggest that the presence of PNS enhances the viability of HaCaT cells under UVB radiation exposure. The Application of PNS Mitigates UVB-induced Apoptosis in HaCaT Cells The impact of PNS on the apoptosis of HaCaT cells under UVB irradiation was assessed through flow cytometry. Flow cytometry analysis revealed that the apoptotic rate of HaCaT cells under UVB irradiation was 4.62%. After treatment with 250μM and 500μM PNS, the percentages of apoptotic cells were merely 3.63% and 1.86%, respectively, indicating a significant inhibition of cellular apoptosis (Figure 2G and 2H) . These findings suggest that PNS effectively inhibit apoptosis in HaCaT cells under UVB irradiation. The Activation of the PI3K/AKT/mTOR Signaling Pathway under UVB Irradiation is Inhibited by PNS The network pharmacology analysis reveals that PNS exhibits therapeutic potential in repairing UVB-induced skin sunburn injury, possibly through the modulation of multiple signaling pathways including PI3K-AKT, Proteoglycans in cancer, Pathways in cancer, Notch and Rap1 signaling pathways. The PI3K/AKT/mTOR signaling pathway exhibits significant enrichment in KEGG analysis and may serve as a pivotal pathway. We observed a substantial upregulation of Phosphatidylinositol-3-kinase (PI3K), AKT1, and mTOR proteins in HaCaT cells upon UVB irradiation. The expression of PI3K, AKT1, and mTOR proteins were significantly downregulated after irradiation when treated with 250 and 500 μM PNS (Figure 3A and 3B) . This suggests that PNS effectively inhibits the activation of the PI3K/AKT/mTOR signaling pathway under UVB irradiation. The Expressions of Apoptotic Proteins are Regulated by PNS in Response to UVB Irradiation The network pharmacology findings suggest that PNS may play a role in regulating cellular apoptosis for the repair of UVB-induced skin sunburn injury. Our experimental results further validate the inhibitory effect of PNS on apoptosis in HaCaT cells exposed to UVB irradiation. Further verification revealed that PNS (250, 500μM) upregulates the expression of anti-apoptotic factor Bcl-2 protein and downregulates pro-apoptotic factors Bax and Cysteine-aspartic proteases 3 (Caspase 3) proteins in HaCaT cells on UVB irradiation (Figure 3C and 3D) . This demonstrates that PNS regulates apoptotic protein expression on UVB irradiation, thereby inhibiting cell apoptosis and playing a role in repairing UVB-induced skin sunburn injury. The Expressions of Autophagy-related Proteins are Regulated by PNS in Response to UVB Irradiation The network pharmacology findings suggest that PNS may be involved in the regulation of autophagy for repairing UVB-induced skin sunburn injury. This study aims to investigate the impact of PNS on autophagy in HaCaT cells exposed to UVB radiation. The protein expression of autophagy markers LC3-II, Beclin 1 coiled-coil domain autophagy regulator (Beclin-1), and Sequestosome 1 (P62) were assessed using Western blot and immunofluorescence techniques. Our findings revealed that high doses of PNS significantly upregulated the expression of P62 protein in UVB-irradiated HaCaT cells while downregulating the expression of Beclin-1 protein (Figure 3E and 3F) . The immunofluorescence results demonstrated that high doses of PNS significantly downregulated the expression of LC3-II in UVB-irradiated HaCaT cells (Figure 3I and 3J) . PNS modulates the expression of autophagy-related proteins on UVB irradiation, thereby inhibiting cellular autophagy and exerting a reparative effect on UVB-induced skin sunburn injury. The Downregulation of RNA Acetylase NAT10 Induced by UVB can be Effectively Reversed in a Dose-dependent Manner by PNS We observed a significant reduction in NAT10 protein expression in HaCaT cells following exposure to UVB irradiation. Treatment of irradiated cells with 250 and 500 μM PNS led to a marked restoration of NAT10 protein expression (Figure 3G and 3H) . These findings indicate that PNS effectively mitigates the UVB-induced downregulation of RNA acetyltransferase NAT10. NGR1 has the Most Optimal Interaction Mode for Treating UVB-Induced Skin Sunburn Injury To elucidate the binding activity between targets and components, this study selected the top five core targets with the highest degree values in the protein-protein interaction network and their corresponding chemical components for molecular docking analysis (Table 6). The results demonstrated that all docking modes exhibited binding energies lower than -5 kJ/mol, with hydrogen bonds formed between the ligands and receptors. These findings suggest that the corresponding protein targets and chemical components possess strong binding affinity. The docking results were visually analyzed using PyMOL software. It was observed that NGR1 exhibited a significant docking effect on the core targets (top 5 in degree value ranking) within the protein-protein interaction network, with its docking RMSD values ranking among the highest. The binding energies of these core targets were all lower than -5.0 kJ/mol, and clear hydrogen-bond interactions were identified (Figures 4A) . Through in-depth analysis, it was determined that NGR1 demonstrated the most optimal binding affinity during the process of PNS treating UVB-induced skin damage. Consequently, it can be reasonably inferred that NGR1 plays a pivotal role in the therapeutic efficacy of PNS in mitigating UVB-induced damage. In the visualization results, green dotted lines represent hydrogen bonds, the orange-yellow color indicates the chemical component structure, and blue highlights the binding sites of target proteins. The Administration of NGR1 Effectively Alleviates UVB-induced Sunburn Injury in A Nude Mice Model The H&E staining results demonstrated significant skin sunburn injury in nude mice following UVB irradiation, as evidenced by increased epidermal thickness and prominent signs of sunburn on dorsal skin. Notably, treatment with NGR1 markedly attenuated epidermal hypertrophy and efficiently mitigated UVB-induced skin sunburn injury (Figure 4B and 4C) . The treatment with NBR1 significantly decreased the levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in UVB-exposed nude mice, while concurrently enhancing the expression of the anti-inflammatory cytokine IL-10 (Figure 4D) . Additionally, NBR1 significantly enhanced the expression of antioxidant enzymes, including SOD, T-AOC, and CAT, in nude mice exposed to UVB irradiation, while simultaneously inhibiting MOD expression (Figure 4E) . These findings indicate that NBR1 has the potential to effectively alleviate UVB-induced skin sunburn injury in nude mice. The Application of NGR1 Significantly Enhances the Survival Rate of HaCaT Cells Exposed to UVB Radiation After treatment with various concentrations of NGR1 (0, 250, 500, and 1000 μM), HaCaT cells showed no significant cytotoxicity, as evidenced by cell viability remaining above 95% (Figure 5A) . In contrast, exposure to 20 mJ/cm² UVB irradiation resulted in a marked decrease in cell viability to approximately 50% compared to the control group. Subsequent treatment with various concentrations of NGR1 (0, 250, 500, and 1000 μM) significantly increased cell viability in a dose-dependent manner (Figure 5A) . Upon exposure to NGR1 for different time intervals (0, 24, 48, 72, and 96 h), HaCaT cells demonstrated minimal cytotoxicity, as evidenced by cell viability remaining above 95% (Figure 5B) . These results indicate that NGR1 treatment enhances the viability of HaCaT cells under UVB radiation exposure. The Application of NGR1 Inhibits UVB-induced HaCaT Cells Apoptosis and Regulates the RNA Acetylase NAT10. The impact of NGR1 on the apoptosis of HaCaT cells under UVB irradiation was evaluated using flow cytometry. Flow cytometry analysis demonstrated that the apoptotic rate of HaCaT cells exposed to UVB irradiation was 4.62%. Following treatment with 250 μM and 500 μM NGR1, the percentages of apoptotic cells decreased to 3.09% and 1.30%, respectively, indicating a significant reduction in cellular apoptosis (Figure 5C and 5D) . The immunofluorescence results demonstrated that high doses of NGR1 significantly downregulated the expression of NAT10 in UVB-irradiated HaCaT cells (Figure 5E and 5F) . These results indicate that NGR1 can effectively inhibit the apoptosis of HaCaT cells under UVB irradiation and regulate the down - regulation of NAT10 induced by UVB. Quantitative Proteomic Analysis of Distinct Cohorts following NAT10 Affinity Purification To compare the proteomes of different groups, we conducted NAT10 pull-down treatments on the KB group, UVB group, and UVB-induced group separately with NGR1 (250μM) drug administration, followed by comprehensive proteomic analysis. We performed a combined analysis of three replicates from all groups and successfully identified a total of 44,618 peptides corresponding to 6,398 distinct proteins (Figure 6A) . By calculating the Pearson correlation coefficient (R) between each pair of samples, we evaluated the quantitative reproducibility among replicate samples. Subsequently, a heatmap was generated based on the quantitative correlation coefficients to visually depict the consistency in measurements across samples (Figure 6B) . Differential Proteomic Analysis of Distinct Cohorts Following NAT10 Pull-down The detection signal intensities of each peptide were obtained through a database search of mass spectrometry raw data, and the quantitative information of the corresponding proteins was calculated. Following normalization of the results, we compared the quantities of the same proteins among different samples. By comparing the proteomic data of the UVB group and the NGR1 group, we constructed volcano plots to visually depict differential protein expression (Figure 6C) , performed principal component analysis (PCA) to analyze sample clustering patterns (Figure 6D) , and generated quantitative heatmaps to illustrate protein expression differences between the two groups (Figure 6E) . Through our comparative analysis, we have identified several autophagy-related proteins, namely p62, NBR1, ATG5, MTOR, and MAP1LC3B2 among the differential proteins. This discovery suggests that NGR1 may counteract the downregulation of RNA acetyltransferase NAT10 induced by UVB through leveraging these autophagy proteins. The Proteome was Subjected to Annotation and Enrichment Analysis Across Distinct Groups after Performing NAT10 Pull-down We performed Gene Ontology (GO) annotation analysis on all identified proteins, elucidating their molecular functions (MF) (Figure 6H) , cellular locations (CC) (Figure 6G) , and biological processes (BP) (Figure 6F) . Additionally, we conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the identified proteins (Figure 6I) . The results clearly demonstrate a close association between these proteins and the mTOR pathway, a well-established pathway involved in the regulation of autophagy. This further confirms that NGR1 mediates the downregulation of acetyltransferase NAT10 under UVB through mechanisms related to autophagy regulation. COG (Clusters of Orthologous Groups of proteins, orthologous protein clusters) is constructed based on classifying coding proteins from complete genomes of bacteria, algae, and eukaryotes according to their evolutionary relationships. We conducted COG annotation analysis on all identified proteins from both the UVB and NGR1 groups (Figure 6J) . The findings suggest a close association between these proteins and the "Inorganic ion transport and metabolism" pathway. Additionally, we performed Pfam protein domain annotation analysis on all identified proteins from the UVB and NGR1 groups, with the results depicted in the figure (Figure 6K) . The Analysis of PPI Networks after NAT10 Pull-down was Conducted Across Different Groups We utilized the STRING database to analyze the differential proteins between the UVB and NGR1 groups, subsequently constructing protein interaction network diagrams. These two diagrams were generated based on the log2 FC value and Degree value, respectively (Figure 6L and 6M) . In the diagrams, each node represents a differential protein, and the lines connecting the nodes indicate established or potentially predicted interactions between the proteins. The color of the nodes corresponds to the differential fold change or other scores of the proteins, while the size of the nodes reflects their connectivity within the interaction network. The associations we have discovered involve proteins such as PLG, MRPS35, GAK, MRPS5, EIF3I, and F13A1. The Deficiency of NAT10 Increases UVB-Induced Cell Apoptosis Efficiency The impact of NAT10 on UVB radiation-triggered apoptosis in 293T cells was evaluated using flow cytometry. The results demonstrated that the apoptosis rate increased to 6.00% in cells treated with siRNA-mediated NAT10 knockdown, compared to 3.64% in the control group (Figure 7A) . This finding indicates that depletion of the RNA acetyltransferase NAT10 enhances UVB-induced apoptosis in 293T cells, highlighting its critical role in modulating UVB-induced skin damage. The Downregulation of NAT10 by UVB Occurs through the Process of Autophagy In our initial investigations, we assessed the impact of UVB irradiation on the abundance of NAT10 in keratinocytes. Our findings revealed that exposure to UVB radiation leads to a downregulation of NAT10 expression in HaCaT cells. To further elucidate the underlying mechanism behind this downregulation, we examined the roles of two crucial protein degradation pathways: the proteasome and the autophagy-lysosome system. The proteasome inhibitor MG132 did not exert any influence on the UVB-induced downregulation of NAT10. In contrast, bafilomycin A1 (BafA1), a lysosome function inhibitor, effectively blocked the UVB-induced downregulation of NAT10 (Figure 7F and 7G) . This implies that UVB-induced autophagy may play a role in mediating the downregulation of NAT10. To test this hypothesis, we genetically suppressed autophagy by deleting the essential autophagy genes ATG5 or ATG7 in HaCaT cells, resulting in effective inhibition of UVB-induced NAT10 downregulation (Figure 7H and 7I) . Collectively, these findings suggest that autophagy mediates the UVB-induced downregulation of NAT10. The Downregulation of RNA Acetylase NAT10 Mediated by UVB can be Effectively Reversed in A Dose-dependent Manner by NGR1 We observed a significant decrease in NAT10 protein expression in HaCaT cells upon exposure to UVB irradiation. The treatment of irradiated cells with 250 and 500 μM NGR1 resulted in a significant increase in NAT10 protein expression (Figure 7B and 7C) . After administering various concentrations of NGR1 (0, 250, 500, and 1000 μM), we observed a dose-dependent regulation of NAT10 protein expression in HaCaT cells by NBR1 (Figure 7D and 7E) . The results suggest that NGR1 effectively counteracts the UVB-induced downregulation of RNA acetyltransferase NAT10. The Autophagic Degradation of NAT10 under UVB Stress was Mediated by NGR1 through The Involvement of NBR1 and p62 To identify the autophagic receptor responsible for UVB-induced downregulation of NAT10, based on proteomics results, our specific focus was directed towards elucidating the roles played by autophagic receptors P62 (also known as SQSTM1) and NBR1. After exposing HaCaT cells to UVB irradiation, we administered NGR1 at concentrations of 250 μM and 1000 μM, respectively. Co-immunoprecipitation analysis revealed that NAT10 interacted with both p62 and NBR1, indicating that NGR1 facilitated the decrease in NAT10 abundance through its association with NBR1 and p62 (Figure 7J) . Discussion Due to current environmental factors, the process of ozone depletion has commenced, leading to the formation of ozone holes that can result in a diminished protective effect against UVB radiation[ 30 ]. Consequently, the augmented influx of UVB radiation reaching the Earth's surface may induce various dermatological conditions and suppress immune response[ 31 ]. The investigation into the molecular mechanisms underlying UVB-induced damage to organisms and its protective effects hold both theoretical and practical significance. Exposure to ultraviolet radiation can result in damage to the dermal extracellular matrix, including collagen and elastin proteins, within the skin. This is histologically characterized by excessive epidermal damage, abnormal proliferation of keratinocytes, and focal melanocyte hyperplasia, among other effects [ 32 – 34 ]. Exposure to UVB radiation can lead to detrimental alterations in skin tissue. These changes compromised integrity of the skin barrier, augmented cutaneous thickness and wrinkling, as well as diminished elasticity [ 33 , 35 ]. Notable transformations involve irregular thickening of the epidermal layer, excessive hyperkeratosis within the stratum corneum, flattening at the junction between epidermis and dermis, along with vanishing dermal papillae. In the dermis, there is often infiltration of inflammatory cells, accompanied by irregular arrangement of elastic fibers characterized by fragmentation or aggregation into clusters, alterations in the structure and quantity of collagen fibers, as well as abnormal accumulation of elastin[ 36 ]. Furthermore, significant degenerative changes are observed in skin collagen. The collagen fibers exhibit fragmentation and disordered arrangement, while the micro vessels display twisted and dilated characteristics. Additionally, there is evidence of surrounding infiltration by inflammatory cells, along with focal proliferation of melanocytes[ 36 ]. Skin sunburn injury is a multifaceted process that involves various intricate mechanisms, including but not limited to cell apoptosis, oxidative stress-induced damage, matrix metalloproteinases activation, and cellular autophagy impairment [ 37 – 39 ] The most potent active components extracted from Panax Notoginseng are PNS, which have been widely used in clinical applications [ 40 , 41 ]. The primary individual components in PNS are predominantly NGR1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, and ginsenoside Re. Previous studies have demonstrated the potent anti-inflammatory properties of ginsenoside Rb1, ginsenoside Rg1, and NGR1[ 42 – 44 ].The ginsenosides Rg1, Re, Rb1, and Rd derived from Panax Notoginseng have been extensively utilized for their anti-aging, anti-cancer, and immune-modulating properties. However, the current development and utilization of PNS and specific ingredients NGR1 remain relatively limited. NGR1, a representative monomeric component of PNS, is distinguished by its well-defined chemical structure and properties. Unlike the complex mixture of PNS components, utilizing NGR1 as a single ingredient provides significant advantages in quality control. The high purity of NGR1 ensures consistent drug dosages, which enhances both the safety and efficacy of clinical treatments. Unlike the potential allergic reactions associated with PNS, which are often unpredictable, the adverse effects of NGR1 are more predictable and manageable. Existing studies have demonstrated no significant toxic side effects at standard doses. The literature confirms that NGR1 exerts significant protective and reparative effects on UVB-induced skin damage through multiple mechanisms, such as anti-inflammatory activity, antioxidant properties, promotion of DNA damage repair, and protection of dermal fibroblasts. The PI3K/AKT/mTOR signaling pathway is a crucial regulatory pathway that governs cellular apoptosis and autophagy, intricately linked to the progression of diverse malignancies [ 45 ]. The PI3K enzyme functions as an intracellular kinase, whereas AKT operates as a serine/threonine kinase. PI3K interacts with growth factor receptors to regulate the conformation of the AKT protein, thereby playing a pivotal role in cellular signaling pathways. The subsequent activation of apoptotic proteins, including phosphorylated Bad and Caspase 9, is triggered by this initial activation, thereby regulating the process of apoptosis. [ 46 , 47 ]. The UVB-induced PI3K/AKT signaling pathway can suppress autophagy through mTOR[ 48 ], with UVRAG typically serving as an initiator of autophagy. Suppression of UVRAG levels leads to activation inhibition of autophagy[ 49 ]. The transcriptional activity of AMPK, Sesn2, TSC2, and UVRAG is influenced by UVB, thereby interfering with the process of autophagy in the body. In summary, UVB exposure can induce autophagy in organisms, which is regulated by various UVB-mediated signaling pathways. The dynamic process of autophagy is typically tightly controlled. Upon initiation of autophagy, multiple signaling pathways converge on the same target, specifically the complex comprising mammalian target of rapamycin complex 1 (mTORC1) [ 46 , 50 ], consisting of TOR, Raptor, GβL/mLST8, PRAS40, and DEPTOR. Among these components, mTOR plays a particularly significant role. Therefore, the upregulation of LC3-II protein expression induces the formation of autophagosomes that merge with lysosomes, thereby facilitating the elimination of damaged organelles. In our study, we observed a reduction in PI3K, AKT, and mTOR protein expression upon treatment with PNS, suggesting that PNS exerts inhibitory effects on the activation of the PI3K/AKT/mTOR signaling pathway. We constructed a PPI network of the primary active ingredients in PNS and identified shared targets associated with UVB-induced skin sunburn injury. Subsequently, we conducted GO enrichment analysis and KEGG pathway analysis to elucidate the functions of their key genes. Later, we conducted a series of in vitro and in vivo experiments to further validate that the total saponins from Panax Notoginseng exert their effects by effectively inhibiting the PI3K/AKT/mTOR pathway. Our study revealed that PNS significantly downregulates the expression of PI3K, AKT, and mTOR proteins, thereby indicating its potent ability to inhibit the activation of the PI3K/AKT/mTOR signaling pathway. Furthermore, we investigated the pharmacological effects of PNS to identify a safe and efficacious therapeutic mechanism and pathway for the treatment of UVB-induced skin sunburn injury. Following treatment with PNS, no significant cytotoxicity was observed in keratinocytes. In this study, protein expression levels were utilized as primary indicators of UVB-induced damage. Our findings suggest that PNS not only significantly attenuates UVB-induced apoptosis, but also modulates the levels of P62, Beclin-1, and LC3-II. Thus, PNS exhibits a regulatory effect on UVB-induced autophagy. Considering its established effects on other pathologies, PNS holds promise as a potential candidate for preventing UVB-induced skin sunburn injury. RNA acetylation, specifically referring to the N 6 -acetyladenosine (m 6 A) modification of RNA, is a prevalent chemical modification in RNA molecules that holds significant biological implications. However, the mechanism and regulatory factors of ac 4 C acetylation, a recently discovered RNA chemical modification, are still in the exploratory stage. Specifically, the modulating factors of this modification remain unclear, with only one key enzyme identified so far - NAT10 [ 51 ]. The N-acetyltransferase 10 (NAT10) enzyme plays a crucial role in catalyzing RNA acetylation modifications, and its aberrant expression or dysfunction has been closely linked to the pathogenesis and progression of various diseases, including cancer and [ 52 , 53 ]. Manipulating the activity or expression level of NAT10 may offer novel strategies and tools for the prevention and treatment of these conditions. We have demonstrated several innovations in the treatment of UVB-induced skin sunburn injury using PNS and NGR1. Our research findings indicate that PNS effectively neutralizes free radicals generated by UVB radiation and suppresses the release of inflammatory factors, thereby ameliorating UVB-induced skin sunburn injury. The study proposes that modulation of the PI3K/AKT/mTOR signaling pathway and inhibition of apoptosis and autophagy through involvement of the PNS play a crucial role in attenuating UVB-induced skin sunburn injury. To ensure scientific validity and reliability, our research design encompasses animal and cell models along with multiple evaluation metrics, offering valuable insights for potential clinical applications of other natural drugs. Our study also investigates the degradation pathway of NAT10 induced by UVB radiation and confirms the interaction between NGR1 and autophagy receptors p62 and NBR1 in regulating NAT10's degradation. These findings not only offer a novel perspective on the response of skin cells to ultraviolet radiation but also present potential targets for the development of innovative strategies to prevent and treat UVB-induced skin damage. Through these investigations, our objective is to comprehensively elucidate the role of NAT10 in UVB-mediated skin injury, thus establishing a robust foundation for future clinical applications. Due to their natural origin, PNS and NGR1 demonstrate exceptional tolerance and market potential, positioning them as a significant innovation in the advancement of contemporary skincare products. In summary, both PNS and NGR1 effectively mitigate UVB-induced apoptosis and autophagy in cells. Importantly, the protective effects of PNS were partially mediated through the PI3K/AKT/mTOR signaling pathway, while NGR1 primarily exerted its effects by modulating the RNA acetyltransferase NAT10. However, it remains a subject for further investigation whether PNS and NGR1 are involved in other mechanisms during the process of UVB-induced skin photodegradation. Moreover, PNS and NGR1 possess significant research value and application potential as potential agents for mitigating chronic skin inflammation or preventing UVB-induced skin carcinogenesis. Conclusion We have demonstrated several innovations in the treatment of UVB-induced skin sunburn injury using PNS. Our research findings indicate that PNS effectively neutralizes free radicals generated by UVB radiation and suppresses the release of inflammatory factors, thereby ameliorating UVB-induced skin sunburn injury. The study proposes that modulation of the PI3K/AKT/mTOR signaling pathway and inhibition of apoptosis and autophagy through involvement of the peripheral nervous system play a crucial role in attenuating UVB-induced skin sunburn injury. To ensure scientific validity and reliability, our research design encompasses animal and cell models along with multiple evaluation metrics, offering valuable insights for potential clinical applications of other natural drugs. For the specific component NGR1, our primary focus was on its effects on the RNA acetyltransferase NAT10 (Fig. 7K) . Through experimental validation, we not only discovered that NGR1 can regulate the downregulation of NAT10 under UVB-mediated conditions but also revealed that NGR1 controls the degradation of NAT10 under UVB stress through autophagic receptors p62 and NBR1. The naturally derived ingredients PNS and NGR1 demonstrate exceptional tolerance and market potential, positioning them as significant innovations in the advancement of contemporary skincare products. Abbreviations Panax Notoginseng Saponins (PNS) Ultra-high-performance Liquid Chromatography Coupled with Quadrupole Orbitrap Mass Spectrometry (UHPLC-Q-Orbitrap-MS/MS) Hematoxylin and Eosin (HE) Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Interleukin-1β (IL-1β) Interleukin-6 (IL-6) Interleukin-10 (IL-10) Catalase Micrococcus Lysodeikticus (CAT) Total Antioxidant Capacity (T-AOC) Superoxide Dismutase (SOD) Sequestosome 1 (P62) Beclin 1 Coiled-coil Domain Autophagy Regulator (Beclin-1) Microtubule-associated Protein Light Chain 3B (LC3-II) AKT Serine/Threonine Kinase 1(AKT1) Phosphatidylinositol-3-kinase (PI3K) Mammalian Target of Rapamycin (mTOR) Bcl-2-associated X Protein (Bax) B-cell Lymphoma 2 (Bcl-2) Cysteine-aspartic Proteases 3 (Caspase 3) Notoginsenoside R1(NGR1) All-trans Retinoic Acid (ATRA) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). N-acetyltransferase 10 (NAT10) Declarations Funding Declaration This study was endorsed by National Natural Science Foundation of China, 82104494; Shanghai Pujiang Program, China, 23PJ1412300; The Project of "Taking on Challenges and Accepting Responsibilities" of The Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine, QYCXZY250303. Credit Author Statement Shuyun Liang, Xiaokang Liu, Yuting Yang and Fangyuan Zhang Wrote the original draft preparation, collected the data and drew the figures; Zizhao Yang, Tong Zhang, Xiaobo Sun, and Dean Guo provided the editing and writing assistance and suggestions; Zizhao Yang and Jiyu Gong approved the final version of manuscript for publication. All the authors have made great contributions during the periods of the manuscript's initiation and submission. The data were exclusively generated internally, with no involvement of external paper mills. All authors willingly assume responsibility for all aspects of the work to ensure its integrity and accuracy. Declaration of Interest Statement The authors affirm that they possess no known conflicting financial interests or personal relationships that could have potentially influenced the findings presented in this paper. The animal experiment was conducted in accordance with the approved protocol of the Institutional Animal Care and Use Committee, which was authorized by the Animal Ethics Committee of Changchun University of Chinese Medicine. The registration number is 2024299, and the specific date is June 13, 2024. The procedures used in this study adhere to the tenets of the Declaration of Helsinki. References El-Domyati M, Attia S, Saleh F, Brown D, Birk DE, Gasparro F, Ahmad H, Uitto J. Intrinsic aging vs. photoaging: a comparative histopathological, immunohistochemical, and ultrastructural study of skin. Exp Dermatol. 2002;11(5):398–405. Gilchrest BA. 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Signal Transduct Target therapy. 2022;7(1):334. Balmus G, Larrieu D, Barros AC, Collins C, Abrudan M, Demir M, Geisler NJ, Lelliott CJ, White JK, Karp NA, et al. Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome. Nat Commun. 2018;9(1):1700. Liao L, He Y, Li SJ, Yu XM, Liu ZC, Liang YY, Yang H, Yang J, Zhang GG, Deng CM, et al. Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner. Cell Res. 2023;33(5):355–71. Tables Table 1 to 7 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supplementalmaterial.docx GraphicalAbstract.docx Table.docx Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Chinese Medicine → Version 1 posted Editorial decision: Revision requested 27 Jun, 2025 Reviews received at journal 26 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviewers agreed at journal 09 Jun, 2025 Reviewers invited by journal 06 Jun, 2025 Editor assigned by journal 19 May, 2025 Submission checks completed at journal 19 May, 2025 First submitted to journal 16 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6678920","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467446047,"identity":"ac864c84-fda3-47cc-ae27-0e017cabd246","order_by":0,"name":"Shuyun Liang","email":"","orcid":"","institution":"Changchun University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuyun","middleName":"","lastName":"Liang","suffix":""},{"id":467446048,"identity":"3999c56a-163b-4d4c-8149-824dcc0474b3","order_by":1,"name":"Xiaokang Liu","email":"","orcid":"","institution":"Shanghai Institute of Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"Xiaokang","middleName":"","lastName":"Liu","suffix":""},{"id":467446049,"identity":"89cd042b-2097-4059-9eb1-b9c251700070","order_by":2,"name":"Yuting Yang","email":"","orcid":"","institution":"Changchun University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Yang","suffix":""},{"id":467446050,"identity":"5dffc4fa-50ac-42a3-9ed9-4221bb8f6fcc","order_by":3,"name":"Fangyuan Zhang","email":"","orcid":"","institution":"Changchun University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fangyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":467446051,"identity":"c83c966a-cb7c-4ba2-aaa2-c299e6d51f14","order_by":4,"name":"Xiaobo Sun","email":"","orcid":"","institution":"Peking Union Medical College, Chinese Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaobo","middleName":"","lastName":"Sun","suffix":""},{"id":467446052,"identity":"0f775c5c-4f44-46b7-a60b-38f703b93fc8","order_by":5,"name":"Tong Zhang","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Zhang","suffix":""},{"id":467446053,"identity":"5ac02db7-68e6-48fb-9566-f3283005a6f0","order_by":6,"name":"Dean Guo","email":"","orcid":"","institution":"Shanghai Institute of Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"Dean","middleName":"","lastName":"Guo","suffix":""},{"id":467446054,"identity":"d454c53a-7060-44cc-9c1d-900983877c66","order_by":7,"name":"Jiyu Gong","email":"","orcid":"","institution":"Changchun University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiyu","middleName":"","lastName":"Gong","suffix":""},{"id":467446055,"identity":"f1489e9b-ecdf-4c5b-bf3d-129c849de9ae","order_by":8,"name":"Zizhao Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYPACGwYDKIuxgUgtaUAtzKRpOUyCFvn+M4afC36dtzeXyD/46QaDjeyGA8zPHuDTwthwxlh6Zt/txJ0zkpmlcxjSjDccYDM3wKeFmbHHQJq353aCwY1kNuYchsOJGw7wsEng08LGzGP8m7fnnD1Uy3/CWnjYeMykeX4cYNwA0XKAsBYJHrYya96G5MQNZx4bS+cYJBvPPMxmhleLfP/hzbd5/tjZGxxPfPg5p8JOtu948zO8WhgYOAwYGNtgHFBQMeNXDwTsDxgY/hBUNQpGwSgYBSMZAAD1AUTWYH/i2gAAAABJRU5ErkJggg==","orcid":"","institution":"The Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Zizhao","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-05-16 08:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6678920/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6678920/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13020-025-01270-3","type":"published","date":"2025-12-18T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84245710,"identity":"9d6f4e16-74fa-4ad4-a5e9-0b4632316c3d","added_by":"auto","created_at":"2025-06-09 16:40:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5150466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical Component Analysis and Network Pharmacology Analysis of PNS.\u003c/strong\u003e (A) The total ion chromatograms of the standard Panax Notoginseng's total saponins. (B) Test sample in negative ion mode. 1.NGR1；2.Rg1；3.Re；4.Rb1；5.Rd；(C, D) The sample quality mass spectrum was tested using UHPLC-Q-Trap\u003cstrong\u003e-\u003c/strong\u003eMS/MS. 1. NGR1；2.Rg1；3.Re；4.Rb1；5.Rd. Fragmentation pathways of NGR1 and its mass spectrum (MS) in negative ion mode. (E) The targets of peripheral nervous system (PNS) overlap with ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rd, NGR1, and ginsenoside Re. (F) The overlapping targets of UVB damage encompass skin cancer, cutaneous inflammation, and skin sunburn injury. (G)The Venn diagram demonstrates the convergence of PNS targets associated with UVB-induced skin sunburn injury. (H) The top 10 biological process (BP), cellular component (CC), and molecular function (MF) terms identified in the Gene Ontology (GO) analysis are visually represented by bars colored orange, green, and blue respectively. (I) The bar graph displays the top 30 pathways determined through KEGG enrichment analysis. (J, K) Fragmentation pathways of Notoginsenoside R1 and its MS spectrum in negative ion mode. (L, M) Fragmentation pathways of ginsenoside Rd and its MS spectrum in negative ion mode. (N, O) Fragmentation pathways of ginsenoside Re and its MS in negative ion mode. (P, Q) Fragmentation pathways of ginsenoside Rg1 and its MS in negative ion mode.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/7d69a27be4c8a8f7d0ba58ab.png"},{"id":84245713,"identity":"916460ab-b497-4f14-a5cd-eee39f2452a5","added_by":"auto","created_at":"2025-06-09 16:40:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12135058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe administration of PNS effectively alleviates UVB-induced sunburn injury in nude mice and enhances the viability of HaCaT cells when exposed to UVB irradiation, while also inhibiting apoptosis in these cells under UVB irradiation. \u003c/strong\u003e(A) The photographs depict mouse skin samples from various experimental groups following exposure to UVB radiation. (B) Microscopic images of skin sections stained with H\u0026amp;E were acquired from the control group, UVB irradiation group, ATRT positive drug-treated group, and PNS-treated group. (C) The levels of TNF-α, IL-1β, IL-6, and IL-10 in the serum of UVB-irradiated nude mice were determined through ELISA analysis following PNS treatment. (D) ELISA analysis showed that after PNS treatment, the levels of MDA, SOD, T-AOC, and CAT in the serum of UVB-irradiated nude mice were determined.(E) Viability diagram of HaCaT cells after treatment with different concentrations of PNS (0,250, 500, and 1000 μM) and Viability chart of HaCaT cells exposed to UVB radiation and treated with varying concentrations (0,250, 500, and 1000 μM) of PNS. (F) The cell viability graph of HaCaT cells was assessed after treatment with PNS for various time intervals (0, 24, 48, 72, and 96 h). (G and H) The impact of PNS on apoptosis of HaCaT cells was assessed by flow cytometry using the Annexin V-FITC/PI method to determine cell apoptosis rate. The experiments were conducted using a minimum of three biologically independent samples (n≥3). Error bars represent the mean±standard deviation (S.D.) values (C, D). Statistical significance was determined using Student's t-test, with *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 denoting different levels of significance.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/9e1e418f3fde5bd436fbe1ea.png"},{"id":84245711,"identity":"03371690-644f-46f1-a01f-d7637938f21b","added_by":"auto","created_at":"2025-06-09 16:40:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5443526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe activation of the PI3K/AKT/mTOR signaling pathway is inhibited by PNS upon UVB irradiation, while it also regulates the expression of apoptosis and autophagy proteins. \u003c/strong\u003e(A and B) The effect of UVB and PNS on the expression of PI3K/AKT/mTOR pathway proteins. (C and D) The effect of UVB and PNS on the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase 3. (E and F) The effect of UVB and PNS on autophagy in HaCaT cells. (G and H) Treatment with concentrations of 250μM and 500μM PNS, followed by cell irradiation, a significant upregulation in the expression level of NAT10 protein was observed. (I and J) Immunofluorescence detection of LC3 II in HaCaT cells (×200). The expression levels of target genes, including mTOR, PI3K, AKT1, Bax, Bcl-2, Caspase 3, p62 and Beclin-1 were normalized to the expression levels of housekeeping proteins GAPDH and β-actin. The experiments were conducted using a minimum of three biologically independent samples. Error bars represent the mean ± S.D. (A, B, C, G). Statistical significance was determined by Student's t-test, with *P \u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, and ****P\u0026lt;0.0001 denoting different levels of significance.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/7bd7cd915b94133dc4f897db.png"},{"id":84245715,"identity":"9ea47008-4faa-4ecc-b95b-ba56d318621f","added_by":"auto","created_at":"2025-06-09 16:40:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10172138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe administration of NGR1 effectively alleviates UVB-induced sunburn injury in nude mice. \u003c/strong\u003e(A)Visualization map of docking between NGR1 and target molecules. (B) The photographs depict mouse skin samples from various experimental groups following exposure to UVB radiation. (C) Microscopic images of skin sections stained with H\u0026amp;E were acquired from the control group, UVB irradiation group, ATRT positive drug-treated group, and NGR1-treated group. (D) The levels of TNF-α, IL-1β, IL-6, and IL-10 in the serum of UVB-irradiated nude mice were determined through ELISA analysis following NGR1 treatment. (E) ELISA analysis showed that after NGR1 treatment, the levels of MDA, SOD, T-AOC, and CAT in the serum of UVB-irradiated nude mice were determined. The experiments were conducted using a minimum of three biologically independent samples (n≥3). Error bars represent the mean±standard deviation (S.D.) values (D, E). Statistical significance was determined using Student's t-test, with *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 denoting different levels of significance.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/2db20a77a5f3a8382b3d9eae.png"},{"id":84245712,"identity":"3cf9b532-d79c-47fd-9e29-7920f7d7a962","added_by":"auto","created_at":"2025-06-09 16:40:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5134870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe presence of NGR1 enhances the survival rate of HaCaT cells under UVB irradiation, while inhibiting apoptosis of these cells under UVB irradiation and regulating the degradation of the RNA acetylase NAT10. \u003c/strong\u003e(A) Viability diagram of HaCaT cells after treatment with different concentrations of NGR1 (0,250, 500, and 1000 μM) and viability chart of HaCaT cells exposed to UVB radiation and treated with varying concentrations (0,250, 500, and 1000 μM) of NGR1. (B) The cell viability graph of HaCaT cells was assessed after treatment with NGR1 for various time intervals (0, 24, 48, 72, and 96 h). (C and D) The impact of NGR1 on apoptosis of HaCaT cells was assessed by flow cytometry using the Annexin V-FITC/PI method to determine cell apoptosis rate. (E and F) Immunofluorescence detection of NAT10 in HaCaT cells (×400). All data were performed on n≥3 biologically independent samples. Error bars are shown as mean±S.D. (A, B). p-values by Student's t-test, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/43ef8150495fb5519418049d.png"},{"id":84246242,"identity":"9be1ef01-7ac6-4f54-9427-6b0002cd38ff","added_by":"auto","created_at":"2025-06-09 16:48:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3961307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe study involved conducting differential proteomics analysis and in vitro validation on different experimental groups after the knockdown of NAT10. \u003c/strong\u003e(A) The sample overview images obtained from protein quantification across different experimental groups were comparatively analyzed, with a comprehensive statistical evaluation of the number of peptides and proteins identified and quantified in each individual sample. (B) A heatmap is generated based on quantitative correlation coefficients. (C) The volcano plot depicts the distinct protein expression patterns between the UVB group and the NGR1 group. (D) The Principal Component Analysis (PCA) plot demonstrates the differential protein expression between the UVB group and NGR1 group. (E) The quantitative heatmap illustrates the variation in protein expression between the UVB group and NGR1 group. (F-H) The bubble chart depicts the enrichment of GO terms for proteins identified in the UVB and NGR1 groups, encompassing biological processes (BP), cellular components (CC), and molecular functions (MF). (I) The bar chart depicts the KEGG pathway annotations of proteins identified in the UVB and NGR1 groups. (J) The bar chart illustrates the COG annotations of proteins identified in the UVB and NGR1 groups. (K) The bar chart depicts the Pfam annotations of proteins identified in the UVB and NGR1 groups. (L) The protein interaction network diagrams will be generated based on the log2 fold change (FC) values for the proteins identified in both the UVB and NGR1 groups. (M) The protein interaction network diagrams will be generated for the proteins identified in the UVB and NGR1 groups, based on their degree values.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/87bef31ab30015997c27d7c3.png"},{"id":84244952,"identity":"45bfd349-b538-4293-b0a4-0d908d9e41b4","added_by":"auto","created_at":"2025-06-09 16:32:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3251956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of NAT10 is downregulated by UVB radiation via autophagic pathways, and it has been observed that NGR1 effectively counteracts the dose-dependent downregulation of RNA acetyl transferase NAT10 induced by UVB through autophagy receptors P62 and NBR1.\u003c/strong\u003e (A)The effect of NAT10 on the apoptosis of 293T cells irradiated by UVB was analyzed by flow cytometry. (B and C) After treatment with concentrations of 250μM and 500μM NGR1, followed by cell irradiation, a significant upregulation in the expression level of NAT10 protein was observed. (D and E) Upon administration of NGR1 at various concentrations (0, 250, 500, and 1000μM), a significant dose-dependent modulation of NAT10 protein expression in HaCaT cells was observed. (F and G) The proteasome inhibitor MG132 did not demonstrate any impact on the UVB-induced downregulation of NAT10, whereas the lysosomal function inhibitor Bafilomycin A1 (BfnA1) effectively impeded the UVB-induced downregulation of NAT10. (H and I) The genetic inhibition of autophagy through the deletion of essential autophagy genes ATG5 or ATG7 in HaCaT cells effectively abrogated the UVB-induced downregulation of NAT10. (J) The HaCaT cells were subjected to co-immunoprecipitation using control species-matched IgG and anti-NAT10 antibodies, followed by immunoblot analysis of NBR1, P62, and NAT10. (K) The molecular mechanism underlying the therapeutic effects of PNS and NGR1 on UVB-induced skin sunburn injury. The experiments were conducted using a minimum of three biologically independent samples. The error bars represent the mean ± S.D. (A, B, C, I). Statistical significance was determined using Student's t-test, with *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, and ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001 indicating different levels of significance.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/5f77dad813887613620375f8.png"},{"id":98813856,"identity":"c4c8faf4-6f5d-4840-bfe0-bda2f24febb4","added_by":"auto","created_at":"2025-12-22 16:05:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":48801252,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/0f7c51fb-da9f-4511-a356-d7b905db314c.pdf"},{"id":84246243,"identity":"b5edf65b-b27c-49f8-a651-761867e9f217","added_by":"auto","created_at":"2025-06-09 16:48:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2678589,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/6ed4b25fafb68bb780d3fe21.docx"},{"id":84244940,"identity":"03df90c1-75ac-49f5-b72b-7c896f509573","added_by":"auto","created_at":"2025-06-09 16:32:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":166010,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/7b9178dafe17a392b84eaf6a.docx"},{"id":84244944,"identity":"6d570670-29d1-49a5-a9cd-8a409e46ef7e","added_by":"auto","created_at":"2025-06-09 16:32:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":656247,"visible":true,"origin":"","legend":"","description":"","filename":"Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-6678920/v1/4f897fde145fefd4356d9281.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Notoginsenoside R1 Mitigates UVB-induced Skin Sunburn Injury through Modulation of N4-acetylcytidine and Macroautophagy","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003eThe combination of PNS and NGR1 effectively mitigates UVB-induced skin sunburn injury in a nude mice model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003ePNS effectively inhibits the activation of the PI3K/Akt/mTOR signaling pathway in response to UVB irradiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003eThe peripheral nervous system regulates the expression levels of apoptotic proteins and autophagy-related proteins in response to UVB irradiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003eThe autophagic degradation of NAT10 under UVB stress was facilitated by NGR1 through the involvement of NBR1 and p62.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eUVB, the most biologically active component of solar radiation, is characterized by its high energy and strong penetrative capabilities. Exposure to UVB induces skin damage, which elicits a complex biological response marked by systemic changes in skin morphology and function. This damage mechanism encompasses structural remodeling of the skin, characterized by epidermal hyperplasia, degradation of the extracellular matrix resulting in reduced skin elasticity, and the onset of cellular damage along with inflammatory cascades. Chronic exposure to UVB not only induces direct damage to skin tissues but also expedites skin aging processes and markedly elevates the risk of cutaneous malignancies.\u003c/p\u003e \u003cp\u003eThe process of skin aging involves both endogenous and exogenous factors, manifesting as phenotypic features such as roughness, pigmentation, and reduced skin elasticity. Exogenous aging is attributed to environmental exposure[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], while endogenous aging occurs gradually over time. The term \"(Ultraviolet B) UVB-induced skin sunburn injury of the skin\" refers to the thickening of the skin and reduction in its elasticity caused by exposure to sunlight, resulting in significant cellular damage and inflammation factors. This condition is commonly associated with pronounced wrinkles, pigmentation issues, laxity, and sagging [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Excessive UVB exposure can lead to the development of skin lesions, accelerated sunburn injury, and even an increased risk of skin tumorigenesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].Therefore, the search for and development of drugs to repair skin photo-damage has received significant attention from researchers both domestically and internationally.\u003c/p\u003e \u003cp\u003eIn recent years, with an increasing number of reports on the beneficial effects of natural remedies on skin health, there has been a growing trend among individuals to develop natural drug treatments for skin UVB damage. Many effective ingredients found in natural remedies, such as flavonoids, isoflavones, and ginsenosides, possess properties that protect the skin[\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Within the field of skincare, saponin compounds are widely recognized as safe and effective natural anti-aging agents. Many studies have demonstrated the significant impact of saponin compounds on skin care and repair. For instance, ginsenoside Rg1 has been found to attenuate UVB-induced glucocorticoid resistance in keratinocytes through the Nrf2/HDAC2 signaling pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Treatment with ginsenoside Rc can effectively inhibit ROS production and prevent the elevation of pro-MMP-2 and \u0026minus;\u0026thinsp;9 levels in UVB-induced HaCaT keratinocytes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The presence of ginsenoside Rc in keratinocytes plays a crucial role in protecting the skin from photooxidative stress caused by UV radiation, as it acts as an effective anti-sunburn injury agent and enhances barrier function [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePanax Notoginseng Saponins (PNS) extract is derived from \u003cem\u003ePanax Notoginseng (Burk.) F.H.Chen\u003c/em\u003e, a member of the Araliaceous family. PNS, a complex bioactive compound derived from plants in the Araliaceous ginseng family, has attracted considerable attention in medical research. Its diverse pharmacological properties, including promotion of hematopoietic function, modulation of immune responses, alleviation of inflammation, and inhibition of aging processes, have positioned it as a key focus in clinical studies. This component was officially incorporated into the 2020 edition of the Chinese Pharmacopoeia and has exhibited substantial clinical value in the treatment of traumatic diseases. In the realm of skin injuries, PNS has demonstrated promising application potential, especially in mitigating UVB-induced skin damage. Nevertheless, further in-depth scientific investigation is warranted to clarify its precise cellular repair mechanisms. Notoginsenoside R1 (NGR1), a specific component of Panax Notoginseng, is a crucial constituent of Panax Notoginseng total saponins. It demonstrates a wide range of benefits, encompassing blood pressure reduction, heart rate decrease, analgesic effects provision, inflammation combatting, and aging processes delay. The isolated Ginsenoside C-Mx from Panax Notoginseng leaves' total saponins could inhibit intracellular ROS, MMP-1, and Interleukin-6 (IL-6) expression induced by UVB radiation. It effectively reverses the degradation of type I procollagen caused by UVB exposure through regulation of the TGF-β/Smad signaling pathway[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The application of Ginsenoside C-Y significantly decreases the levels of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-α) in the body after UVB exposure. Additionally, it restrains matrix metalloproteinase-1 (MMP-1) production, promotes the synthesis of type I collagen, inhibits melanin secretion and tyrosinase activity, and reduces the amount of melanin in the skin. These effects contribute to the inhibition of skin photodamage and prevention of excessive skin pigmentation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Ginsenoside Rk3 significantly enhances the activities of hydroxyproline, Superoxide Dismutase (SOD), and GSH-PX enzymes in mouse skin tissue and blood, while reducing the expression levels of malondialdehyde, MMP-1, MMP-3, IL-6, Interleukin-1β (IL-1β), and TNF-α. This leads to suppression of inflammation and skin sunburn injury, thereby providing protection for skin health [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Research has demonstrated that saponin components derived from Panax Notoginseng are extensively utilized in diseases related to skin injuries. Although the potential efficacy of PNS against UVB-induced skin sunburn injury has been discovered, currently its repair mechanism remains unclear.\u003c/p\u003e \u003cp\u003eThe regulation of molecular functions is significantly influenced by RNA modifications, and accumulating evidence increasingly suggests that targeting the pathways governed by RNA modifications holds great promise in the field of cancer therapy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The N\u003csup\u003e4\u003c/sup\u003e-acetylcytidine (ac\u003csup\u003e4\u003c/sup\u003eC) modification is a prevalent chemical alteration found in mRNA, playing a pivotal role in the regulation of mRNA stability and translation. However, the precise impact of ac\u003csup\u003e4\u003c/sup\u003eC modification on diseases remains elusive[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The N-acetyltransferase-like protein (NAT10) is the primary eukaryotic RNA enzyme responsible for catalyzing the production of ac\u003csup\u003e4\u003c/sup\u003eC, exhibiting both acetyltransferase activity and RNA-binding capability. Despite its crucial role as the sole recognized ac\u003csup\u003e4\u003c/sup\u003eC \"writer\" protein, the impact of NAT10 on disease progression remains to be elucidated. Drawing on a review of the literature and preliminary laboratory findings, it has been demonstrated that the NAT10 protein undergoes degradation under UVB-induced conditions. This process not only influences the formation of UVB-induced photoproducts, such as cyclobutane pyrimidine dimers (CPDs), but also contributes to the regulation of UVB-induced DNA damage and carcinogen-related repair mechanisms[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we unveil the mechanism through which NAT10-mediated mRNA ac\u003csup\u003e4\u003c/sup\u003eC modification regulates UVB-induced skin damage.\u003c/p\u003e \u003cp\u003eNetwork pharmacology is an interdisciplinary field that merges principles from systems biology and network informatics. In recent years, this approach has gained widespread application in the development of novel therapeutics, with a particular focus on elucidating the synergistic interactions among multiple components, pathways, and targets[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The integration of vast amounts of information enables the discovery of novel drug targets and molecular mechanisms[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Network pharmacology facilitates the comprehensive analysis of multi-component drugs' effects on human physiology, enabling the identification of therapeutic targets for effective drug components, thereby augmenting drug efficacy while minimizing adverse reactions[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Presently, network pharmacology is increasingly employed to investigate the treatment potential of traditional Chinese medicine across diverse diseases.\u003c/p\u003e \u003cp\u003eThe aim of this study was to investigate the therapeutic efficacy of PNS and NGR1 in mitigating skin UVB-induced skin sunburn injury and elucidate its underlying mechanism. Initially, liquid chromatography-mass spectrometry was employed to analyze the chemical composition of PNS. The subsequent step involved the utilization of network pharmacology to predict potential targets and molecular mechanisms associated with PNS in mitigating skin aging caused by UVB light, followed by preliminary experimental validation. The aim of this article is to investigate the therapeutic mechanisms of PNS and NGR1 in treating UVB-induced skin sunburn injury and provide supportive data for the development of products targeting such treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eChemical Reagent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe specific preparation method for PNS is primarily outlined in the dedicated chapter on PNS in the 2020 edition of the Chinese Pharmacopoeia. The procedure involves crushing Panax Notoginseng into coarse powder, extracting it with 70% ethanol, filtering the mixture, concentrating the filtration under reduced pressure, passing it through a column of non-polar or weakly polar styrene-type macro-porous adsorption resin, washing with water, and discarding the washings. The resin is subsequently subjected to elution with 80% ethanol, followed by concentration under reduced pressure, decolorization, further concentration, and refinement to obtain the extract. Finally, the extract is dried to yield the ultimate product. Notoginsenoside R1 is one of the active components isolated from the total saponins of PNS. As a unique active component of Panax Notoginseng, it holds an important position within the total saponins. PNS with a purity of over 75% was procured from the China Food and Drug Research Institute, batch number: 110870-202105. The purities of the substances Notoginsenoside R1 (NGR1) (C\u003csub\u003e47\u003c/sub\u003eH\u003csub\u003e80\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003e(110745-202322). Rg1 (C\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e72\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e) (110703-202436), Rb1 (C\u003csub\u003e54\u003c/sub\u003eH\u003csub\u003e92\u003c/sub\u003eO\u003csub\u003e23\u003c/sub\u003e) (110704-202331), Rd (C\u003csub\u003e48\u003c/sub\u003eH\u003csub\u003e82\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e) (111818-202305), and Re (C\u003csub\u003e48\u003c/sub\u003eH\u003csub\u003e82\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e) (110754-202330) are all greater than 95% and were obtained from the same institute. All-Trans Retinoic Acid (ATRA) was sourced from Sigma-Aldrich, located in St. Louis, Missouri, USA. The formic acid used, which was of mass spectrometry grade, was also provided by Sigma-Aldrich, St. Louis, MO, USA. Chromatic-grade acetonitrile and methanol were purchased from Fisher Scientific, based in Pittsburgh, Pennsylvania, USA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUHPLC-Q-Orbitrap-MS/MS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreparation of PNS Sample Solution: Accurately weigh 10 mg of PNS and subject it to ultrasonic extraction using 10 mL of 70% (v/v) methanol in water for 15 minutes at ambient temperature. Filter the resulting mixture through a 0.22 \u0026mu;m membrane filter. Place the filtered solution into a vial for subsequent analysis by Ultra-High-Performance Liquid Chromatography coupled with Quadrupole Orbitrap Mass Spectrometry (UHPLC-Q-Orbitrap-MS/MS).\u003c/p\u003e\n\u003cp\u003eChromatographic separation was carried out using an Ultimate 3000 ultra-high performance liquid chromatography system (Thermo, San Jose, CA, USA) equipped with a Supelco C18 column (dimensions: 3.0 \u0026times; 50 mm, particle size: 2.7 \u0026mu;m; supplied by Sigma-Aldrich). The column temperature was set at 35\u0026deg;C. The mobile phases consisted of acetonitrile (phase A) and water (phase B). The separation of the experimental samples was achieved using a gradient elution program as follows: 80% solvent B from 0 to 20 minutes, decreasing to 54% B from 20 to 45 minutes, further reduced to 45% B from 45 to 55 minutes, held at 45% B from 55 to 60 minutes, then increased back to 80% B from 60 to 65 minutes, and maintained at this concentration for an additional 5 minutes. The sample injection volume was set at 5 \u0026mu;L, with a flow rate of 0.4 mL per minute.\u003c/p\u003e\n\u003cp\u003eMass spectrometric analysis was performed using a Q-Orbitrap-MS/MS instrument (Thermo, San Jose, CA, USA) with an electrospray ionization source operated in negative ion mode. The ion source parameters were configured as follows: sheath gas flow at 40 arbitrary units, auxiliary gas flow at 10 arbitrary units, and sweep gas flow at 1 arbitrary unit. The S-Lens RF level was set to 55%. The capillary voltage was configured at -3.5 kV, and the capillary temperature was maintained at 350\u0026deg;C. Full MS data were collected in centroid mode over an m/z range of 150 to 1,500 Da, employing a resolution of 70,000. The automatic gain control (AGC) target was set to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e, and the maximum injection time was 100 ms. For tandem mass spectrometry, data were acquired in Full-MS/ddMS\u003csup\u003e2\u003c/sup\u003e mode with the following parameters: a resolution of 17,000, an AGC target of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e, a maximum injection time of 50 ms, a loop count of 5, a Top N value of 5, an isolation window of 4.0 m/z, and stepped collision energies of 35, 45, and 55.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUHPLC-Q-Trap-MS/MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreparation of the PNS sample solution: Accurately weigh 1 mg of PNS at room temperature, followed by subjecting it to ultrasonic extraction using 10 mL of a 70% (v/v) methanol/water solution for a duration of 15 minutes. Subsequently, filter the resulting mixture through a 0.22 \u0026mu;m membrane filter to obtain a purified PNS sample solution. To prepare the standard solution, accurately weigh appropriate amounts of NGR1, Rg1, Rb1, Re, and Rd. Under controlled room temperature conditions, subject them to ultrasonic extraction using 1 mL of a 70% (v/v) methanol/water solution for a duration of 15 minutes. This process will result in the creation of a mixed solution with a concentration of 100 ng/mL, Similarly, this mixture is also passed through a 0.22 \u0026mu;m membrane filter for filtration. The filtered solutions will then be transferred to dedicated sample vials for subsequent analysis using a Triple Quadrupole Mass Spectrometer (UHPLC-Q-Trap-MS/MS).\u003c/p\u003e\n\u003cp\u003eUsing a quadrupole mass spectrometer (QTRAP6500; ABSciex, Framingham, MA), chromatographic separation was performed on an ultra-high-performance liquid chromatography (UPLC) system in the reversed-phase mode using a Waters ACQUITY BEHC 18 column (length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 \u0026mu;m, manufactured by Waters Corporation in Milford, MA, USA). Mobile phases A and B consist of water with 0.1 mmol/L ammonium acetate and acetonitrile respectively at a flow rate of 0.35 mL/min and maintained at a temperature of 35\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe multi-component quantification of PNS was achieved using a gradient separation scheme, which involved the following steps: mobile phase B concentration started at 30% from 0 to 2.0 minutes, increased to 37% up to 5.0 minutes, further rose to 42% at 9.0 minutes, adjusted to 47% at 14.0 minutes, reached a concentration of mobile phase B of 51.0% at 17.0 minutes and swiftly increased between 20.0 and 21.0minutes until it reached 95%, before returning back to the initial concentration of 30% during 22-23 minutes.\u003c/p\u003e\n\u003cp\u003eThe operational parameters of the mass spectrometer were optimized with the following specific settings: ion spray voltage set to -4.5 kV in negative ion mode; ion source temperature maintained at 550 \u0026deg;C; gas 1 pressure set to 20 psi; gas 2 pressure adjusted to 30 psi; and curtain gas pressure regulated at 10 psi. During the analysis, the multiple reaction monitoring (MRM) mode was employed for precise quantification of each analyte of PNS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human keratinocytes (HaCaT) (Catalog number: ATCC CRL-2404) and embryonal kidney 293T cells (293T) (Catalog number: ATCC CRL-3216) were obtained from Proteintech (Proteintech, Wuhan, China). The cell lines were maintained in DMEM (Gibico) enriched with 10% fetal bovine serum (FBS, Gibico), along with penicillin (at a concentration of 100 units per milliliter) and streptomycin (at a concentration of 100 micrograms per milliliter), all sourced from Gibico, USA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale BALB/c-nude mice, aged 6 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). The animals were housed under specific pathogen-free conditions for one week to acclimate. The environmental conditions included a temperature range of 15-25\u0026deg;C and relative humidity between 40% and 65%. A 12-hour light/dark cycle was maintained, and the mice had unrestricted access to both food and water. Forty nude mice were divided into five groups: the control group without treatment (Sham), the UVB-induced model group treated with cream base only (UVB, cream base), The positive control group is treated with a 0.1% ATRA cream and exposed to UVB radiation (UVB, 0.1% ATRA), the low-dose PNS-treated group using 1.5% PNS cream along with UVB exposure (UVB, PNS-Low), and the high-dose PNS-treated group using 3.0% PNS cream along with UVB exposure (UVB, PNS-High). The low-dose NBR1-treated group received 1.5% NBR1 cream in conjunction with UVB exposure (UVB, NBR1-Low), while the high-dose NBR1-treated group received 3.0% NBR1 cream along with UVB exposure (UVB, NBR1-High). After receiving UVB irradiation at a dose of 80 mJ/cm\u0026sup2;, administered 5-6 times per week for one week, the nude mice underwent a fasting period of 12 hours and were subsequently anesthetized following the final drug administration. Blood samples and skin tissues were collected for analysis. The animal experiment was conducted in accordance with the approved protocol of the Institutional Animal Care and Use Committee, which was authorized by the Animal Ethics Committee of Changchun University of Chinese Medicine. The registration number is 2024299, and the specific date is June 13, 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and Eosin (H\u0026amp;E) Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSkin samples were collected from each group of nude mice for histological examination, following established protocols. Briefly, the skin samples were fixed in 4% neutral formaldehyde for 24 hours, embedded in paraffin under vacuum, and sectioned into 10\u0026mu;m-thick slices. These sections were deparaffinized using xylene and then subjected to a series of ethanol rehydration and dehydration steps before being stained with hematoxylin and eosin. Identical cross-sections from each sample on three slides were selected, and four different microscopic fields (at\u0026times;200 magnification) per slide were photographed. Stained images were acquired using an optical microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked Immunosorbent Assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of PNS and NBR1 on UVB-induced inflammatory and antioxidant factors, serum samples were collected from nude mice in the control group, model group, positive control group, PNS treatment group, and NBR1 treatment group. The expression levels of inflammatory factors including TNF-\u0026alpha;, IL-1\u0026beta;, IL-6 and Interleukin-10 (IL-10)(Jiangsu Enzyme-linked Immunosorbent Assay Industry Co., Ltd., Batch Numbers MM-013M1, MM0040M1, MM0163M1, MM-0176M1) and antioxidant factors MOD, SOD, Total Antioxidant Capacity (T-AOC), Catalase Micrococcus lysodeikticus (CAT) (Nanjing Jiancheng Bioengineering Institute, Batch Numbers A003-1, A001-1, A015, A007-1) were analyzed following the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUVB Irradiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell UVB radiation was performed in accordance with our previously established protocols [29]. Following two rinses with phosphate-buffered saline (1\u0026times;PBS buffer, Invitrogen), the cells were subjected to UVB irradiation (20 mJ/cm\u0026sup2;, unless otherwise indicated) using a UV Stratalinker 2400 device fitted with UVB lamps (Stratagene). For control groups, mock irradiation was performed. The UVB dosage was consistently measured using a Goldilux UV meter equipped with a UVB sensor (Oriel Instruments).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the cytotoxic impact of UVB exposure on HaCaT keratinocytes and examine the possible protective roles of PNS and NBR1, we employed the CCK-8 assay to determine cell viability. Cells were plated in a 96-well plate at a concentration of 1\u0026times;10^4 cells per well and incubated beforehand for 24 hours. Following this, the cells were exposed to different concentrations of PNS and NBR1 (0, 250, 500, 1000 \u0026mu;M) for a period of 24 hours. Subsequently, the cells were subjected to UVB irradiation and then incubated with various concentrations of PNS and NBR1 (0, 250, 500, 1000 \u0026mu;M) for a period of 24 hours. Additionally, PNS and NBR1 were administered at a fixed concentration of 250 \u0026mu;M but across different time points (0, 24, 48, 72, and 96 hours). Following these treatments, 10 \u0026mu;L of CCK-8 solution (Beyotime, Shanghai, China) was added to each well, and the plates were incubated for 4 hours under conditions of 37\u0026deg;C with 5% CO2 in a humidified environment. Finally, the absorbance was recorded at 450 nm using a Tecan Infinite1000 microplate reader (Tecan, Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescent Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter UVB irradiation, separate treatments of PNS and NBR1 were administered, HaCaT cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded onto cell climbing membranes in 6-well plates for a duration of 24 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized using PBS containing 0.2% Triton X-100 for a period of 20 minutes. Subsequently, the cells were incubated with a solution of 3% bovine serum albumin for 30 minutes to block non-specific binding sites. Following this, the cells were subjected to overnight incubation at a temperature of 4\u0026deg;C with Microtubule-associated proteins 1A/1B light chain 3B (LC3-II) antibody (diluted at a ratio of 1:75), followed by co-incubation with the corresponding fluorescent secondary antibody. Finally, DAPI staining was performed using a concentration of 500 ng/mL for a duration of five minutes, and cell staining was observed utilizing an Olympus fluorescence inverted microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain protein extracts, cells were first washed with cold PBS and then lysed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The resulting protein solutions were subjected to sonication and subsequently centrifuged at 13,000 RPM for 20 minutes at 4\u0026deg;C. Protein concentrations were determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Following quantification, the samples underwent heat treatment at 70\u0026deg;C for a duration of 10 minutes. The protein levels were then evaluated by performing SDS-polyacrylamide gel electrophoresis, which was subsequently followed by immunoblot analysis. The antibodies utilized in this study are listed as follows:\u003c/p\u003e\n\u003cp\u003eAnti-GAPDH (Proteintech, Cat#10494, 1:2000),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnti-\u0026beta;-actin (Bioss, bs-0061R, 1:2000),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnti-AKT1 (Servicebio, GB13011, 1:500),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnti-PI3K (Proteintech, Cat#27921, 1:500),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnti-mTOR (Proteintech, Cat#66888, 1:500),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnti-Bax (Proteintech, Cat#68111, 1:500),\u003c/p\u003e\n\u003cp\u003eAnti-Bcl-2(Proteintech, Cat #68103, 1:500),\u003c/p\u003e\n\u003cp\u003eAnti -Caspase 3 (Proteintech, Cat #19677, 1:500)\u003c/p\u003e\n\u003cp\u003eAnti-P62 (Abcam, ab207305, 1:1000)\u003c/p\u003e\n\u003cp\u003eAnti-Beclin-1 (Abcam, ab207612, 1:1000)\u003c/p\u003e\n\u003cp\u003eAnti-NAT10 (Abcam, ab194297, 1:2000)\u003c/p\u003e\n\u003cp\u003eAnti-NBR1 (Abcam, ab55474, 1:1000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing flow cytometry (FACSCalibur, Becton Dickinson, USA) for the flow cytometric analysis of cells. After knocking down NAT10 using siRNA, 293T cells were pre-treated with a UVB dose of 20mJ/cm\u0026sup2;. The cells were then collected 24 hours later for further analysis. HaCaT cells were first pretreated with a UVB dose of 20 mJ/cm\u0026sup2;. Following this, the cells were incubated for 24 hours in the presence or absence of PNS and NGR1. After incubation, the cells were collected, washed with PBS, and resuspended in 0.5 mL of binding buffer. To differentiate between early apoptotic cells (which stain positive for annexin V (AV) but negative for propidium iodide (PI); AV\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e-\u003c/sup\u003e) and late apoptotic or necrotic cells (which stain positive for both AV and PI; AV\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e), dual-color flow cytometry analysis was conducted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Evaluation of The Degradation Pathway of NAT10\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 24 hours of UVB irradiation, HaCaT cells were subjected to treatment with either MG132 (MG, 10 \u0026mu;M) or bafilomycin A1 (BafA1, 50 ng/mL) for a duration of 6 or 24 hours. Another group of cells was left untreated. Subsequently, immunoblot analysis was performed on both the treated and untreated cells to detect NAT10 and GAPDH proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA Transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe siRNAs utilized were as follows: siRNA-ATG7 (sc-41447), siRNA-ATG5 (sc-41445), and siRNA-control (sc-37007) obtained from Santa Cruz Biotechnology. Each of these was transfected into distinct cells using a specialized medium for siRNA transfection (sc-36868) in conjunction with a specific transfection reagent for siRNAs (sc-29528), in accordance with the guidelines supplied by the manufacturer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Application of NAT10 Pulldown Combined with Proteomic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn NAT10 pull-down experiments, the Universal Magnetic Beads Co-Immunoprecipitation (Co-IP) Kit (Proteintech, Cat#54002) was utilized in accordance with the provided instructions. The sample was treated with a reaction solution containing 1% SDC, 100 mM Tris-HCl (pH 8.5), 10 mM TCEP, and 40 mM CAA. Incubation was performed at 95\u0026deg;C for 10 minutes to achieve thorough denaturation, reduction, and alkylation of the proteins. The mixture obtained was then processed through centrifugation, after which the supernatant was diluted with an equivalent volume of ddH\u003csub\u003e2\u003c/sub\u003eO. For the overnight digestion at 37\u0026deg;C, trypsin was introduced in a proportion of 1:50 (enzyme to protein, by weight). The next day, the pH was adjusted to 6.0 using TFA to halt the digestion. Following another centrifugation step (12,000 g for 15 minutes), peptide purification was carried out on the supernatant using a custom-made SDB-RPS desalting column. The peptides that were eluted were then dried under vacuum and stored at -20\u0026deg;C for subsequent use.\u003c/p\u003e\n\u003cp\u003eThe analysis of the samples was conducted using a timsTOF Pro instrument (Bruker Daltonics), an integrated device that combines trapped ion mobility spectrometry (TIMS) with quadrupole time-of-flight mass spectrometry. This timsTOF Pro system was connected to an UltiMate 3000 RSLC nano liquid chromatography system (Thermo) equipped with a Captive Spray nano ion source, provided by Bruker Daltonics. Peptide samples were introduced into a C18 Trap column (75 \u0026micro;m \u0026times; 2 cm, 3 \u0026micro;m bead size, 100 \u0026Aring; pore diameter, Thermo) and subsequently separated using a reversed-phase C18 analytical column (75 \u0026micro;m \u0026times; 15 cm, 1.7 \u0026micro;m bead size, 100 \u0026Aring; pore diameter, IonOpticks). The separation gradient was created using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nL per minute. Mass spectrometry analysis was conducted in diaPASEF mode with the capillary voltage configured at 1500 volts. Both MS and MS/MS spectra were recorded within an m/z range of 100 to 1,700, while ion mobility scans were performed over a range of 0.6 to 1.6 Vs/cm\u0026sup2;. The accumulation time and ramp time were each set to 50 milliseconds. The diaPASEF acquisition method was configured on the m/z-ion mobility plane utilizing the timsControl software from Bruker Daltonics. Collision energy varied linearly with ion mobility, starting at 59 eV when 1/K0 equaled 1.6 Vs/cm\u0026sup2; and decreasing to 20 eV when 1/K0 reached 0.6 Vs/cm\u0026sup2;.\u003c/p\u003e\n\u003cp\u003eThe DIA raw data were processed using DIA-NN (version 1.8.2 beta 11) in a library-independent manner. The spectral files were then matched against the Human protein sequence database (obtained from Uniprot on August 7, 2024, which includes 20,654 entries). The search parameters largely adhered to the default configurations, with several adjustments: options for precursor ion generation were activated to create an in silico-predicted spectral library; the enzyme Trypsin/P was utilized, permitting up to 2 missed cleavages. Carbamidomethyl modification on cysteine residues was defined as a fixed alteration, whereas oxidation on methionine residues and N-terminal acetylation of proteins was treated as variable modifications. Both the mass tolerance and MS1 accuracy were configured to 15 ppm, with match-between-runs (MBR) and heuristic protein inference enabled. To ensure reliable identifications, the precursor false discovery rate (FDR) was controlled at 1%. Protein intensities were normalized utilizing the MaxLFQ algorithm.\u003c/p\u003e\n\u003cp\u003eThe protein-level quantification analysis was conducted using the \u0026apos;pg_matrix.tsv\u0026apos; file derived from the DIA-NN search outcomes. Further bioinformatics analyses were carried out in the R statistical programming environment. To address the missing values, we employed a random Gaussian distribution centered at the mass spectrometer\u0026apos;s detection limit. This distribution was adjusted with a downshift of 1.8 times the standard deviation and a width of 0.25 times the standard deviation. For valid value filtering, we required that at least 50% of the values within each group be non-missing. Significantly differentially expressed proteins (DEPs) were identified using P-values from Student\u0026rsquo;s T-tests and specified fold change thresholds. Functional annotations were performed using several databases, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), EggNOG, Pfam, and UniProt for subcellular localization. To determine significantly enriched terms and pathways in the regulated set, a Fisher\u0026apos;s Exact Test was applied. Additionally, protein-protein interaction networks were built and evaluated utilizing the STRING database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-IP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells underwent two washes with chilled PBS, were collected on ice using a cell scraper, and then centrifuged at 4\u0026deg;C for 5 minutes at 1000 rpm. Following this, the supernatant was removed. Next, cold Co-IP lysis buffer (Proteintech, Cat#PR20037) was introduced, and the cell pellet was carefully resuspended through repeated pipetting until no aggregates remained. The sample was first incubated at 4\u0026deg;C for a period of 30 minutes, then subjected to centrifugation at 13,000 revolutions per minute (rpm) for 15 minutes at the same temperature. Following this step, the resulting supernatant was carefully transferred into a fresh tube.\u003c/p\u003e\n\u003cp\u003eThe reaction tubes were each supplemented with 20 \u0026mu;L of magnetic beads and an optimized amount of immune precipitation antibody (the appropriate dilution of the antibody was determined). The mixture was incubated at 4\u0026deg;C on a rotator for 4 hours. After incubation, the supernatant was removed using a magnetic stand. The beads were incubated with pre-prepared cell lysis buffer at 4\u0026deg;C overnight. Following the overnight incubation, the supernatant was discarded to eliminate non-binding proteins, and the beads were washed with PBS 2-3 times. Subsequently, the magnetic bead-bound immunocomplexes were resuspended in 25 \u0026mu;L of 5\u0026times;Laemmli buffer, subjected to boiling for 5-10 minutes, and subsequently analyzed through immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNetwork Pharmacology Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe saponin composition of PNS was determined by UHPLC-Q-Orbitrap-MS/MS. After being identified, the SMILES codes corresponding to each saponin were retrieved from the PubChem database. Subsequently, these SMILES were inputted into the Swiss Target Prediction platform (http://www.swisstargetprediction.ch/) to identify potential targets of these saponins, selecting proteins with a probability ＞0. By integrating reported targets from published studies, the final targets of the saponins were identified.\u003c/p\u003e\n\u003cp\u003eUsing the search terms \u0026quot;skin sunburn injury\u0026quot; \u0026quot;skin inflammation\u0026quot; and \u0026quot;skin cancer\u0026quot; as keywords, we conducted searches in three databases: GeneCards database (https://www.genecards.org/), OMIM database (https://www.omim.org/), and DrugBank database (https://www.drugbank.ca/) to retrieve targets associated with skin sunburn injury. Subsequently, we employed Draw Venn (http://bioinformatics.psb.ugent.be/webtools/Venn/) to generate a Venn diagram for integrating the disease targets, aiming to identify potential targets for skin photodamage.\u003c/p\u003e\n\u003cp\u003eThe construction of protein-protein interaction (PPI) networks was performed, and the gene targets that overlapped in both datasets were imported into the STRING database (http://string-db.org). The species selection in the operation interface was restricted to \u0026quot;Homo sapiens\u0026quot;, with a confidence score threshold set at \u0026ge;0.9. Utilizing Cytoscape 3.7.2 (https://www.cytoscape.org/), we established a potential key target network and systematically analyzed its network parameters.\u003c/p\u003e\n\u003cp\u003eTo investigate the impact of the peripheral nervous system (PNS) on signaling pathways and gene functions in skin UVB light damage, we utilized the DAVID database (https://david.ncifcrf.gov/) for GO functional analysis and KEGG pathway enrichment analysis. The obtained results were visualized using R version 3.4.1, where we focused on the top 10 outcomes from GO analysis and the top 30 outcomes from KEGG analysis, with significance levels set at FDR \u0026lt; 0.05 and P \u0026lt; 0.05. We then developed ginsenoside-target-pathway (G-T-P) networks to graphically illustrate the interactions between compounds, their targets, and associated pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eObtain the high-resolution crystal structure of the target protein from the Protein Data Bank (RCSB, https://www.rcsb.org). Use PyMOL 2.5.2 software to remove any non-protein atoms and solvent molecules, then save the cleaned structure in \u0026quot;pdb\u0026quot; format. Extract the three-dimensional structure of the compound from the PubChem database and leverage Open Babel 3.1.1 software to transform the file into either \u0026quot;mol2\u0026quot; or \u0026quot;pdb\u0026quot; format. Combine the pre-processed protein and compound structures within AutoDock 4.2.6 for molecular docking analysis, selecting the conformation with the lowest binding energy as the result. A docking energy less than 0 kcal/mol suggests potential molecular interaction, while a value below -5 kcal/mol indicates a strong binding affinity. Finally, use PyMOL software to visually analyze the docking results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data were presented as mean\u0026plusmn;standard deviation (S.D.). Statistical significance of inter-group differences was assessed using one-way analysis of variance, followed by Dunnett\u0026apos;s test or Tukey-Kramer multiple comparison test to analyze differences between the groups of the Means. SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA) was employed for statistical analysis. A P-value ＜ 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSaponin Composition Analysis of PNS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe composition of PNS was comprehensively analyzed utilizing UHPLC-Q-Orbitrap-MS/MS, with the total ion chromatogram depicted \u003cstrong\u003e(Figure 1A and Supplementary Figure 1)\u003c/strong\u003e. Through meticulous high-resolution mass spectrometry analysis of individual ions, subsequent fragmentation data, and comparative studies with entries in the PNS database, in conjunction with pertinent literature references, a definitive identification was made for a total of 16 saponin compounds. Extensive details regarding these constituents are provided \u003cstrong\u003e(Table 1 and 2)\u003c/strong\u003e. The total content of five components, namely NGR1, ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rd, and ginsenoside Re, was determined in accordance with the methods specified in the 2020 edition of the Chinese Pharmacopoeia, the chromatogram of the reference was shown in \u003cstrong\u003e(Figure 1B)\u003c/strong\u003e.Quantitative analysis of NGR1, Rg1, Rb1, Re, and Rd was performed using UHPLC-Q-Trap-MS/MS \u003cstrong\u003e(Figure 1C and 1D)\u003c/strong\u003e, the analysis revealed a combined content of 84.13%, establishing them as crucial constituents within PNS, therefore, we selected these 5 saponins for follow-up network pharmacological analysis. The UHPLC-Q-Orbitrap-MS/MS spectra were summarized, and potential fragmentation pathways were analyzed to identify four saponins: NGR1, ginsenoside Rd, ginsenoside Re, and ginsenoside Rg1 \u003cstrong\u003e(Figure 1J, 1K, 1M, 1N, 1O, 1P and 1Q)\u003c/strong\u003e. while the specific contents of 16 ginsenosides were determined by HPLC \u003cstrong\u003e(Table 7)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein-Protein Interaction (PPI) Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted a search in the PubChem database to retrieve the SMILES identifiers of the 5 crucial saponin compounds found in PNS, namely NGR1, ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rd, and ginsenoside Re. Subsequently, these identifiers were imported into the Swiss Target Prediction platform for predictive analysis, resulting in a total of 198 target predictions \u003cstrong\u003e(Figure 1E)\u003c/strong\u003e. Through GeneCards, OMIM, and DrugBank databases, we retrieved a total of 31,465 target genes associated with UVB-induced skin sunburn injury \u003cstrong\u003e(Figure 1F)\u003c/strong\u003e. After thorough screening and standardization using Uniprot to eliminate duplicates, we ultimately identified 19,689 unique targets. By intersecting the 198 drug-derived targets with the 19,689 UVB-induced skin sunburn injury targets, we obtained a final set of 94 overlapping targets \u003cstrong\u003e(Figure 1G)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe imported the 94 common targets into the STRING database, resulting in the construction of a Protein-Protein Interaction (PPI) network consisting of 82 nodes and 1,015 edges \u003cstrong\u003e(Supplementary Figure 2)\u003c/strong\u003e. The initial network was obtained by applying a condition where the parameter degree was set to be greater than or equal to the median value (\u0026ge;23). The network is characterized by highly interconnected nodes, including AKT serine/threonine kinase 1(AKT1), IL6, Bcl-2-Associated X protein (Bax), B-Cell Lymphoma 2 (Bcl-2), CXCL8, FN1, MAPK8, IL1B, HSP90AA1 and GSK3B, etc. These proteins have been identified as the top 11 hubs in the network based on their Degree, BC and DC values \u003cstrong\u003e(Table 3)\u003c/strong\u003e. Consequently, these proteins represent potential targets for mitigating skin UVB-induced sunburn injury pathways in the PNS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConducting Gene Ontology (GO) and KEGG Pathway Enrichment Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DAVID 6.8 platform was utilized to conduct GO functional enrichment and KEGG pathway enrichment analyses on 94 targets associated with ginsenoside and skin sunburn injury. The bar charts illustrate the top 10 results for each of the three GO enrichment categories\u003cstrong\u003e\u0026nbsp;(Figure 1H)\u003c/strong\u003e. Additionally, the bubble plot illustrates the enrichment of the top 20 KEGG pathways, providing a schematic diagram of how PNS mediates prevention of UVB-induced skin sunburn injury through multiple signaling pathways\u003cstrong\u003e\u0026nbsp;(Figure 1I)\u003c/strong\u003e. Through KEGG analysis, we have identified that total saponins derived from Panax Notoginseng may potentially participate in 30 signaling pathways associated with the treatment of UVB-induced skin damage. The major signaling pathways encompass the PI3K-AKT, Notch, Rap1, neurotrophins, thyroid hormone, Ras, JAK-STAT, and MAPK signaling cascades. The PI3K-AKT signaling pathway, Notch signaling pathway, and Rap1 signaling pathway exhibit greater significance in comparison to the remaining pathways. These pathways encompass 16 crucial PPI network target points such as GF, Cytokine, ECM, ITGA, ITGB, AKT, HSP90, Bcl-2, and NF-kB. Moreover, considering the number and proportion of genes involved in each pathway, the PI3K-AKT pathway emerges as the most plausible candidate for repairing UVB-induced skin sunburn injury \u003cstrong\u003e(Table 4)\u003c/strong\u003e. These findings suggest that the potential mechanism underlying PNS-mediated repair of UVB-induced skin sunburn injury may be associated with the PI3K-AKT signaling pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the enrichment analysis from the GO and KEGG databases, it is suggested that PNS may implement its therapeutic benefits in treating UVB-induced skin sunburn injuries via the activation of the PI3K-AKT and mTOR signaling pathways. We employed the \u0026quot;Path-view\u0026quot; package in R to visualize the PI3K-AKT and mTOR signaling pathways\u003cstrong\u003e\u0026nbsp;(Supplementary Figure 3 and 4)\u003c/strong\u003e. These findings suggest that the potential mechanism underlying PNS-mediated repair of UVB-induced skin sunburn injury may be associated with modulation of the PI3K-AKT-mTOR signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGinsenoside-Target-Pathway (G-T-P) Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain a deeper comprehension of the interconnections among ginsenosides, targets, and biological pathways in PNS, we constructed a comprehensive ginsenoside-target-pathway (G-T-P) network \u003cstrong\u003e(Supplementary Figure 5)\u003c/strong\u003e. Additionally, a summary was provided for the analysis of the 16 saponins in PNS and their associated pathways in relation to UVB-induced protein damage\u003cstrong\u003e\u0026nbsp;(Table 5 and Supplementary Figure 6)\u003c/strong\u003e. This intricate network comprises 65 nodes consisting of 5 compounds, 50 targets, and 10 pathways. The green nodes represent potential targets, the purple nodes symbolize ginsenosides, the yellow nodes depict signaling pathways, while the connecting lines signify their interactions. The larger and opaquer the nodes are, the higher their degree of association. The illustrated G-T-P network demonstrates that each ginsenoside interacts with numerous targets implicated in diverse pathways \u003cstrong\u003e(Supplementary Figure 5)\u003c/strong\u003e, suggesting a multi-target and multi-pathway mechanism in PNS for mitigating UVB-induced skin sunburn injury. The degrees of Rela, Jun, Stat3, IL2, Fos and Bcl-2 are 11, 11, 11, 10, 10 and 10 respectively. This indicates a close association between these proteins and the therapeutic efficacy of PNS in repairing UVB-induced skin sunburn injury. It highlights the significant role played by the PI3K-AKT pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Administration of PNS Effectively Alleviates UVB-induced Skin Sunburn Injury in A Nude Mice Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe H\u0026amp;E staining results revealed pronounced skin sunburn injury nude mice following UVB irradiation, characterized by an increase in epidermal thickness and evident signs of sunburn injury on the dorsal skin. However, treatment with PNS significantly alleviated epidermal hypertrophy and effectively restored UVB-induced skin sunburn injury \u003cstrong\u003e(Figure 2A and 2B)\u003c/strong\u003e. The treatment with PNS significantly reduced the levels of pro-inflammatory cytokines TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6 in UVB-exposed nude mice, while also increasing the expression of the anti-inflammatory cytokine IL-10 \u003cstrong\u003e(Figure 2C)\u003c/strong\u003e. Additionally, PNS significantly augmented the expression of antioxidant enzymes SOD, T-AOC, and CAT in nude mice subjected to UVB irradiation, while concurrently suppressing MOD expression \u003cstrong\u003e(Figure 2D)\u003c/strong\u003e. The findings suggest that PNS possess the ability to effectively counteract UVB-induced skin sunburn injury in nude mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Application of PNS Enhances the Survival Rate of HaCaT Cells under UVB Radiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter treatment with various concentrations of PNS (0, 250, 500, and 1000 \u0026mu;M), HaCaT cells demonstrated no significant cytotoxicity as indicated by cell viability exceeding 95% \u003cstrong\u003e(Figure 2E)\u003c/strong\u003e. Compared to the control group, HaCaT cells exhibited cell death and a decrease in cell viability to 50% following irradiation with 20 mJ/cm\u003csup\u003e2\u003c/sup\u003e of UVB. Subsequent treatment with different concentrations of PNS (0, 250, 500, and 1000 \u0026mu;M) significantly enhanced cell viability, demonstrating a pronounced dose-dependent effect \u003cstrong\u003e(Figure 2E)\u003c/strong\u003e. Following exposure to PNS for various time periods (0, 24, 48, 72 and 96 h), HaCaT cells exhibited minimal cytotoxic effects, as indicated by cell viability exceeding 95% \u003cstrong\u003e(Figure 2F)\u003c/strong\u003e. The findings suggest that the presence of PNS enhances the viability of HaCaT cells under UVB radiation exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Application of PNS Mitigates UVB-induced Apoptosis in HaCaT Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of PNS on the apoptosis of HaCaT cells under UVB irradiation was assessed through flow cytometry. Flow cytometry analysis revealed that the apoptotic rate of HaCaT cells under UVB irradiation was 4.62%. After treatment with 250\u0026mu;M and 500\u0026mu;M PNS, the percentages of apoptotic cells were merely 3.63% and 1.86%, respectively, indicating a significant inhibition of cellular apoptosis \u003cstrong\u003e(Figure 2G and 2H)\u003c/strong\u003e. These findings suggest that PNS effectively inhibit apoptosis in HaCaT cells under UVB irradiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Activation of the PI3K/AKT/mTOR Signaling Pathway under UVB Irradiation is Inhibited by PNS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe network pharmacology analysis reveals that PNS exhibits therapeutic potential in repairing UVB-induced skin sunburn injury, possibly through the modulation of multiple signaling pathways including PI3K-AKT, Proteoglycans in cancer, Pathways in cancer, Notch and Rap1 signaling pathways. The PI3K/AKT/mTOR signaling pathway exhibits significant enrichment in KEGG analysis and may serve as a pivotal pathway. We observed a substantial upregulation of Phosphatidylinositol-3-kinase (PI3K), AKT1, and mTOR proteins in HaCaT cells upon UVB irradiation. The expression of PI3K, AKT1, and mTOR proteins were significantly downregulated after irradiation when treated with 250 and 500 \u0026mu;M PNS \u003cstrong\u003e(Figure 3A and 3B)\u003c/strong\u003e. This suggests that PNS effectively inhibits the activation of the PI3K/AKT/mTOR signaling pathway under UVB irradiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Expressions of Apoptotic Proteins are Regulated by PNS in Response to UVB Irradiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe network pharmacology findings suggest that PNS may play a role in regulating cellular apoptosis for the repair of UVB-induced skin sunburn injury. Our experimental results further validate the inhibitory effect of PNS on apoptosis in HaCaT cells exposed to UVB irradiation. Further verification revealed that PNS (250, 500\u0026mu;M) upregulates the expression of anti-apoptotic factor Bcl-2 protein and downregulates pro-apoptotic factors Bax and Cysteine-aspartic proteases 3 (Caspase 3) proteins in HaCaT cells on UVB irradiation\u003cstrong\u003e\u0026nbsp;(Figure 3C and 3D)\u003c/strong\u003e. This demonstrates that PNS regulates apoptotic protein expression on UVB irradiation, thereby inhibiting cell apoptosis and playing a role in repairing UVB-induced skin sunburn injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Expressions of Autophagy-related Proteins are Regulated by PNS in Response to UVB Irradiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe network pharmacology findings suggest that PNS may be involved in the regulation of autophagy for repairing UVB-induced skin sunburn injury. This study aims to investigate the impact of PNS on autophagy in HaCaT cells exposed to UVB radiation. The protein expression of autophagy markers LC3-II, Beclin 1 coiled-coil domain autophagy regulator (Beclin-1), and Sequestosome 1 (P62) were assessed using Western blot and immunofluorescence techniques. Our findings revealed that high doses of PNS significantly upregulated the expression of P62 protein in UVB-irradiated HaCaT cells while downregulating the expression of Beclin-1 protein \u003cstrong\u003e(Figure 3E and 3F)\u003c/strong\u003e. The immunofluorescence results demonstrated that high doses of PNS significantly downregulated the expression of LC3-II in UVB-irradiated HaCaT cells \u003cstrong\u003e(Figure 3I and 3J)\u003c/strong\u003e. PNS modulates the expression of autophagy-related proteins on UVB irradiation, thereby inhibiting cellular autophagy and exerting a reparative effect on UVB-induced skin sunburn injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Downregulation of RNA Acetylase NAT10 Induced by UVB can be Effectively Reversed in a Dose-dependent Manner by PNS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe observed a significant reduction in NAT10 protein expression in HaCaT cells following exposure to UVB irradiation. Treatment of irradiated cells with 250 and 500 \u0026mu;M PNS led to a marked restoration of NAT10 protein expression \u003cstrong\u003e(Figure 3G and 3H)\u003c/strong\u003e. These findings indicate that PNS effectively mitigates the UVB-induced downregulation of RNA acetyltransferase NAT10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNGR1 has the Most Optimal Interaction Mode for Treating UVB-Induced Skin Sunburn Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the binding activity between targets and components, this study selected the top five core targets with the highest degree values in the protein-protein interaction network and their corresponding chemical components for molecular docking analysis (Table 6). The results demonstrated that all docking modes exhibited binding energies lower than -5 kJ/mol, with hydrogen bonds formed between the ligands and receptors. These findings suggest that the corresponding protein targets and chemical components possess strong binding affinity. The docking results were visually analyzed using PyMOL software. It was observed that NGR1 exhibited a significant docking effect on the core targets (top 5 in degree value ranking) within the protein-protein interaction network, with its docking RMSD values ranking among the highest. The binding energies of these core targets were all lower than -5.0 kJ/mol, and clear hydrogen-bond interactions were identified \u003cstrong\u003e(Figures 4A)\u003c/strong\u003e. Through in-depth analysis, it was determined that NGR1 demonstrated the most optimal binding affinity during the process of PNS treating UVB-induced skin damage. Consequently, it can be reasonably inferred that NGR1 plays a pivotal role in the therapeutic efficacy of PNS in mitigating UVB-induced damage. In the visualization results, green dotted lines represent hydrogen bonds, the orange-yellow color indicates the chemical component structure, and blue highlights the binding sites of target proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Administration of NGR1 Effectively Alleviates UVB-induced Sunburn Injury in A Nude Mice Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe H\u0026amp;E staining results demonstrated significant skin sunburn injury in nude mice following UVB irradiation, as evidenced by increased epidermal thickness and prominent signs of sunburn on dorsal skin. Notably, treatment with NGR1 markedly attenuated epidermal hypertrophy and efficiently mitigated UVB-induced skin sunburn injury \u003cstrong\u003e(Figure 4B and 4C)\u003c/strong\u003e. The treatment with NBR1 significantly decreased the levels of pro-inflammatory cytokines, including TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6, in UVB-exposed nude mice, while concurrently enhancing the expression of the anti-inflammatory cytokine IL-10 \u003cstrong\u003e(Figure 4D)\u003c/strong\u003e. Additionally, NBR1 significantly enhanced the expression of antioxidant enzymes, including SOD, T-AOC, and CAT, in nude mice exposed to UVB irradiation, while simultaneously inhibiting MOD expression \u003cstrong\u003e(Figure 4E)\u003c/strong\u003e. These findings indicate that NBR1 has the potential to effectively alleviate UVB-induced skin sunburn injury in nude mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Application of NGR1 Significantly Enhances the Survival Rate of HaCaT Cells Exposed to UVB Radiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter treatment with various concentrations of NGR1 (0, 250, 500, and 1000 \u0026mu;M), HaCaT cells showed no significant cytotoxicity, as evidenced by cell viability remaining above 95% \u003cstrong\u003e(Figure 5A)\u003c/strong\u003e. In contrast, exposure to 20 mJ/cm\u0026sup2; UVB irradiation resulted in a marked decrease in cell viability to approximately 50% compared to the control group. Subsequent treatment with various concentrations of NGR1 (0, 250, 500, and 1000 \u0026mu;M) significantly increased cell viability in a dose-dependent manner \u003cstrong\u003e(Figure 5A)\u003c/strong\u003e. Upon exposure to NGR1 for different time intervals (0, 24, 48, 72, and 96 h), HaCaT cells demonstrated minimal cytotoxicity, as evidenced by cell viability remaining above 95% \u003cstrong\u003e(Figure 5B)\u003c/strong\u003e. These results indicate that NGR1 treatment enhances the viability of HaCaT cells under UVB radiation exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Application of NGR1 Inhibits UVB-induced HaCaT Cells Apoptosis and Regulates the RNA Acetylase NAT10.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of NGR1 on the apoptosis of HaCaT cells under UVB irradiation was evaluated using flow cytometry. Flow cytometry analysis demonstrated that the apoptotic rate of HaCaT cells exposed to UVB irradiation was 4.62%. Following treatment with 250 \u0026mu;M and 500 \u0026mu;M NGR1, the percentages of apoptotic cells decreased to 3.09% and 1.30%, respectively, indicating a significant reduction in cellular apoptosis \u003cstrong\u003e(Figure 5C and 5D)\u003c/strong\u003e. The immunofluorescence results demonstrated that high doses of NGR1 significantly downregulated the expression of NAT10 in UVB-irradiated HaCaT cells \u003cstrong\u003e(Figure 5E and 5F)\u003c/strong\u003e. These results indicate that NGR1 can effectively inhibit the apoptosis of HaCaT cells under UVB irradiation and regulate the down - regulation of NAT10 induced by UVB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Proteomic Analysis of Distinct Cohorts following NAT10 Affinity Purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compare the proteomes of different groups, we conducted NAT10 pull-down treatments on the KB group, UVB group, and UVB-induced group separately with NGR1 (250\u0026mu;M) drug administration, followed by comprehensive proteomic analysis. We performed a combined analysis of three replicates from all groups and successfully identified a total of 44,618 peptides corresponding to 6,398 distinct proteins \u003cstrong\u003e(Figure 6A)\u003c/strong\u003e. By calculating the Pearson correlation coefficient (R) between each pair of samples, we evaluated the quantitative reproducibility among replicate samples. Subsequently, a heatmap was generated based on the quantitative correlation coefficients to visually depict the consistency in measurements across samples\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Figure 6B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential Proteomic Analysis of Distinct Cohorts Following NAT10 Pull-down\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe detection signal intensities of each peptide were obtained through a database search of mass spectrometry raw data, and the quantitative information of the corresponding proteins was calculated. Following normalization of the results, we compared the quantities of the same proteins among different samples. By comparing the proteomic data of the UVB group and the NGR1 group, we constructed volcano plots to visually depict differential protein expression \u003cstrong\u003e(Figure 6C)\u003c/strong\u003e, performed principal component analysis (PCA) to analyze sample clustering patterns \u003cstrong\u003e(Figure 6D)\u003c/strong\u003e, and generated quantitative heatmaps to illustrate protein expression differences between the two groups \u003cstrong\u003e(Figure 6E)\u003c/strong\u003e. Through our comparative analysis, we have identified several autophagy-related proteins, namely p62, NBR1, ATG5, MTOR, and MAP1LC3B2 among the differential proteins. This discovery suggests that NGR1 may counteract the downregulation of RNA acetyltransferase NAT10 induced by UVB through leveraging these autophagy proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Proteome was Subjected to Annotation and Enrichment Analysis Across Distinct Groups after Performing NAT10 Pull-down\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed Gene Ontology (GO) annotation analysis on all identified proteins, elucidating their molecular functions (MF) \u003cstrong\u003e(Figure 6H)\u003c/strong\u003e, cellular locations (CC) \u003cstrong\u003e(Figure 6G)\u003c/strong\u003e, and biological processes (BP) \u003cstrong\u003e(Figure 6F)\u003c/strong\u003e. Additionally, we conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the identified proteins \u003cstrong\u003e(Figure 6I)\u003c/strong\u003e. The results clearly demonstrate a close association between these proteins and the mTOR pathway, a well-established pathway involved in the regulation of autophagy. This further confirms that NGR1 mediates the downregulation of acetyltransferase NAT10 under UVB through mechanisms related to autophagy regulation. COG (Clusters of Orthologous Groups of proteins, orthologous protein clusters) is constructed based on classifying coding proteins from complete genomes of bacteria, algae, and eukaryotes according to their evolutionary relationships. We conducted COG annotation analysis on all identified proteins from both the UVB and NGR1 groups \u003cstrong\u003e(Figure 6J)\u003c/strong\u003e.\u0026nbsp;The findings suggest a close association between these proteins and the \u0026quot;Inorganic ion transport and metabolism\u0026quot; pathway. Additionally, we performed Pfam protein domain annotation analysis on all identified proteins from the UVB and NGR1 groups, with the results depicted in the figure\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Figure 6K)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Analysis of PPI Networks after NAT10 Pull-down was Conducted Across Different Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe utilized the STRING database to analyze the differential proteins between the UVB and NGR1 groups, subsequently constructing protein interaction network diagrams. These two diagrams were generated based on the log2 FC value and Degree value, respectively \u003cstrong\u003e(Figure 6L and 6M)\u003c/strong\u003e. In the diagrams, each node represents a differential protein, and the lines connecting the nodes indicate established or potentially predicted interactions between the proteins. The color of the nodes corresponds to the differential fold change or other scores of the proteins, while the size of the nodes reflects their connectivity within the interaction network. The associations we have discovered involve proteins such as PLG, MRPS35, GAK, MRPS5, EIF3I, and F13A1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Deficiency of NAT10 Increases UVB-Induced Cell Apoptosis Efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of NAT10 on UVB radiation-triggered apoptosis in 293T cells was evaluated using flow cytometry. The results demonstrated that the apoptosis rate increased to 6.00% in cells treated with siRNA-mediated NAT10 knockdown, compared to 3.64% in the control group\u003cstrong\u003e\u0026nbsp;(Figure 7A)\u003c/strong\u003e. This finding indicates that depletion of the RNA acetyltransferase NAT10 enhances UVB-induced apoptosis in 293T cells, highlighting its critical role in modulating UVB-induced skin damage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Downregulation of NAT10 by UVB Occurs through the Process of Autophagy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our initial investigations, we assessed the impact of UVB irradiation on the abundance of NAT10 in keratinocytes. Our findings revealed that exposure to UVB radiation leads to a downregulation of NAT10 expression in HaCaT cells. To further elucidate the underlying mechanism behind this downregulation, we examined the roles of two crucial protein degradation pathways: the proteasome and the autophagy-lysosome system. The proteasome inhibitor MG132 did not exert any influence on the UVB-induced downregulation of NAT10. In contrast, bafilomycin A1 (BafA1), a lysosome function inhibitor, effectively blocked the UVB-induced downregulation of NAT10 \u003cstrong\u003e(Figure 7F and 7G)\u003c/strong\u003e. This implies that UVB-induced autophagy may play a role in mediating the downregulation of NAT10. To test this hypothesis, we genetically suppressed autophagy by deleting the essential autophagy genes ATG5 or ATG7 in HaCaT cells, resulting in effective inhibition of UVB-induced NAT10 downregulation \u003cstrong\u003e(Figure 7H and 7I)\u003c/strong\u003e. Collectively, these findings suggest that autophagy mediates the UVB-induced downregulation of NAT10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Downregulation of RNA Acetylase NAT10 Mediated by UVB can be Effectively Reversed in A Dose-dependent Manner by NGR1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe observed a significant decrease in NAT10 protein expression in HaCaT cells upon exposure to UVB irradiation. The treatment of irradiated cells with 250 and 500 \u0026mu;M NGR1 resulted in a significant increase in NAT10 protein expression \u003cstrong\u003e(Figure 7B and 7C)\u003c/strong\u003e. After administering various concentrations of NGR1 (0, 250, 500, and 1000 \u0026mu;M), we observed a dose-dependent regulation of NAT10 protein expression in HaCaT cells by NBR1\u003cstrong\u003e(Figure 7D and 7E)\u003c/strong\u003e. The results suggest that NGR1 effectively counteracts the UVB-induced downregulation of RNA acetyltransferase NAT10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Autophagic Degradation of NAT10 under UVB Stress was Mediated by NGR1 through The Involvement of NBR1 and p62\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the autophagic receptor responsible for UVB-induced downregulation of NAT10, based on proteomics results, our specific focus was directed towards elucidating the roles played by autophagic receptors P62 (also known as SQSTM1) and NBR1. After exposing HaCaT cells to UVB irradiation, we administered NGR1 at concentrations of 250 \u0026mu;M and 1000 \u0026mu;M, respectively. Co-immunoprecipitation analysis revealed that NAT10 interacted with both p62 and NBR1, indicating that NGR1 facilitated the decrease in NAT10 abundance through its association with NBR1 and p62\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Figure 7J)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to current environmental factors, the process of ozone depletion has commenced, leading to the formation of ozone holes that can result in a diminished protective effect against UVB radiation[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consequently, the augmented influx of UVB radiation reaching the Earth's surface may induce various dermatological conditions and suppress immune response[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The investigation into the molecular mechanisms underlying UVB-induced damage to organisms and its protective effects hold both theoretical and practical significance.\u003c/p\u003e \u003cp\u003eExposure to ultraviolet radiation can result in damage to the dermal extracellular matrix, including collagen and elastin proteins, within the skin. This is histologically characterized by excessive epidermal damage, abnormal proliferation of keratinocytes, and focal melanocyte hyperplasia, among other effects [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Exposure to UVB radiation can lead to detrimental alterations in skin tissue. These changes compromised integrity of the skin barrier, augmented cutaneous thickness and wrinkling, as well as diminished elasticity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Notable transformations involve irregular thickening of the epidermal layer, excessive hyperkeratosis within the stratum corneum, flattening at the junction between epidermis and dermis, along with vanishing dermal papillae. In the dermis, there is often infiltration of inflammatory cells, accompanied by irregular arrangement of elastic fibers characterized by fragmentation or aggregation into clusters, alterations in the structure and quantity of collagen fibers, as well as abnormal accumulation of elastin[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, significant degenerative changes are observed in skin collagen. The collagen fibers exhibit fragmentation and disordered arrangement, while the micro vessels display twisted and dilated characteristics. Additionally, there is evidence of surrounding infiltration by inflammatory cells, along with focal proliferation of melanocytes[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Skin sunburn injury is a multifaceted process that involves various intricate mechanisms, including but not limited to cell apoptosis, oxidative stress-induced damage, matrix metalloproteinases activation, and cellular autophagy impairment [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe most potent active components extracted from Panax Notoginseng are PNS, which have been widely used in clinical applications [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The primary individual components in PNS are predominantly NGR1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, and ginsenoside Re. Previous studies have demonstrated the potent anti-inflammatory properties of ginsenoside Rb1, ginsenoside Rg1, and NGR1[\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].The ginsenosides Rg1, Re, Rb1, and Rd derived from Panax Notoginseng have been extensively utilized for their anti-aging, anti-cancer, and immune-modulating properties. However, the current development and utilization of PNS and specific ingredients NGR1 remain relatively limited.\u003c/p\u003e \u003cp\u003eNGR1, a representative monomeric component of PNS, is distinguished by its well-defined chemical structure and properties. Unlike the complex mixture of PNS components, utilizing NGR1 as a single ingredient provides significant advantages in quality control. The high purity of NGR1 ensures consistent drug dosages, which enhances both the safety and efficacy of clinical treatments. Unlike the potential allergic reactions associated with PNS, which are often unpredictable, the adverse effects of NGR1 are more predictable and manageable. Existing studies have demonstrated no significant toxic side effects at standard doses. The literature confirms that NGR1 exerts significant protective and reparative effects on UVB-induced skin damage through multiple mechanisms, such as anti-inflammatory activity, antioxidant properties, promotion of DNA damage repair, and protection of dermal fibroblasts.\u003c/p\u003e \u003cp\u003eThe PI3K/AKT/mTOR signaling pathway is a crucial regulatory pathway that governs cellular apoptosis and autophagy, intricately linked to the progression of diverse malignancies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The PI3K enzyme functions as an intracellular kinase, whereas AKT operates as a serine/threonine kinase. PI3K interacts with growth factor receptors to regulate the conformation of the AKT protein, thereby playing a pivotal role in cellular signaling pathways. The subsequent activation of apoptotic proteins, including phosphorylated Bad and Caspase 9, is triggered by this initial activation, thereby regulating the process of apoptosis. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The UVB-induced PI3K/AKT signaling pathway can suppress autophagy through mTOR[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], with UVRAG typically serving as an initiator of autophagy. Suppression of UVRAG levels leads to activation inhibition of autophagy[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The transcriptional activity of AMPK, Sesn2, TSC2, and UVRAG is influenced by UVB, thereby interfering with the process of autophagy in the body. In summary, UVB exposure can induce autophagy in organisms, which is regulated by various UVB-mediated signaling pathways. The dynamic process of autophagy is typically tightly controlled. Upon initiation of autophagy, multiple signaling pathways converge on the same target, specifically the complex comprising mammalian target of rapamycin complex 1 (mTORC1) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], consisting of TOR, Raptor, GβL/mLST8, PRAS40, and DEPTOR. Among these components, mTOR plays a particularly significant role. Therefore, the upregulation of LC3-II protein expression induces the formation of autophagosomes that merge with lysosomes, thereby facilitating the elimination of damaged organelles. In our study, we observed a reduction in PI3K, AKT, and mTOR protein expression upon treatment with PNS, suggesting that PNS exerts inhibitory effects on the activation of the PI3K/AKT/mTOR signaling pathway.\u003c/p\u003e \u003cp\u003eWe constructed a PPI network of the primary active ingredients in PNS and identified shared targets associated with UVB-induced skin sunburn injury. Subsequently, we conducted GO enrichment analysis and KEGG pathway analysis to elucidate the functions of their key genes. Later, we conducted a series of in vitro and in vivo experiments to further validate that the total saponins from Panax Notoginseng exert their effects by effectively inhibiting the PI3K/AKT/mTOR pathway. Our study revealed that PNS significantly downregulates the expression of PI3K, AKT, and mTOR proteins, thereby indicating its potent ability to inhibit the activation of the PI3K/AKT/mTOR signaling pathway. Furthermore, we investigated the pharmacological effects of PNS to identify a safe and efficacious therapeutic mechanism and pathway for the treatment of UVB-induced skin sunburn injury. Following treatment with PNS, no significant cytotoxicity was observed in keratinocytes. In this study, protein expression levels were utilized as primary indicators of UVB-induced damage. Our findings suggest that PNS not only significantly attenuates UVB-induced apoptosis, but also modulates the levels of P62, Beclin-1, and LC3-II. Thus, PNS exhibits a regulatory effect on UVB-induced autophagy. Considering its established effects on other pathologies, PNS holds promise as a potential candidate for preventing UVB-induced skin sunburn injury.\u003c/p\u003e \u003cp\u003eRNA acetylation, specifically referring to the N\u003csup\u003e6\u003c/sup\u003e-acetyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) modification of RNA, is a prevalent chemical modification in RNA molecules that holds significant biological implications. However, the mechanism and regulatory factors of ac\u003csup\u003e4\u003c/sup\u003eC acetylation, a recently discovered RNA chemical modification, are still in the exploratory stage. Specifically, the modulating factors of this modification remain unclear, with only one key enzyme identified so far - NAT10 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The N-acetyltransferase 10 (NAT10) enzyme plays a crucial role in catalyzing RNA acetylation modifications, and its aberrant expression or dysfunction has been closely linked to the pathogenesis and progression of various diseases, including cancer and [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Manipulating the activity or expression level of NAT10 may offer novel strategies and tools for the prevention and treatment of these conditions.\u003c/p\u003e \u003cp\u003eWe have demonstrated several innovations in the treatment of UVB-induced skin sunburn injury using PNS and NGR1. Our research findings indicate that PNS effectively neutralizes free radicals generated by UVB radiation and suppresses the release of inflammatory factors, thereby ameliorating UVB-induced skin sunburn injury. The study proposes that modulation of the PI3K/AKT/mTOR signaling pathway and inhibition of apoptosis and autophagy through involvement of the PNS play a crucial role in attenuating UVB-induced skin sunburn injury. To ensure scientific validity and reliability, our research design encompasses animal and cell models along with multiple evaluation metrics, offering valuable insights for potential clinical applications of other natural drugs. Our study also investigates the degradation pathway of NAT10 induced by UVB radiation and confirms the interaction between NGR1 and autophagy receptors p62 and NBR1 in regulating NAT10's degradation. These findings not only offer a novel perspective on the response of skin cells to ultraviolet radiation but also present potential targets for the development of innovative strategies to prevent and treat UVB-induced skin damage. Through these investigations, our objective is to comprehensively elucidate the role of NAT10 in UVB-mediated skin injury, thus establishing a robust foundation for future clinical applications. Due to their natural origin, PNS and NGR1 demonstrate exceptional tolerance and market potential, positioning them as a significant innovation in the advancement of contemporary skincare products.\u003c/p\u003e \u003cp\u003eIn summary, both PNS and NGR1 effectively mitigate UVB-induced apoptosis and autophagy in cells. Importantly, the protective effects of PNS were partially mediated through the PI3K/AKT/mTOR signaling pathway, while NGR1 primarily exerted its effects by modulating the RNA acetyltransferase NAT10. However, it remains a subject for further investigation whether PNS and NGR1 are involved in other mechanisms during the process of UVB-induced skin photodegradation. Moreover, PNS and NGR1 possess significant research value and application potential as potential agents for mitigating chronic skin inflammation or preventing UVB-induced skin carcinogenesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have demonstrated several innovations in the treatment of UVB-induced skin sunburn injury using PNS. Our research findings indicate that PNS effectively neutralizes free radicals generated by UVB radiation and suppresses the release of inflammatory factors, thereby ameliorating UVB-induced skin sunburn injury. The study proposes that modulation of the PI3K/AKT/mTOR signaling pathway and inhibition of apoptosis and autophagy through involvement of the peripheral nervous system play a crucial role in attenuating UVB-induced skin sunburn injury. To ensure scientific validity and reliability, our research design encompasses animal and cell models along with multiple evaluation metrics, offering valuable insights for potential clinical applications of other natural drugs. For the specific component NGR1, our primary focus was on its effects on the RNA acetyltransferase NAT10 \u003cb\u003e(Fig.\u0026nbsp;7K)\u003c/b\u003e. Through experimental validation, we not only discovered that NGR1 can regulate the downregulation of NAT10 under UVB-mediated conditions but also revealed that NGR1 controls the degradation of NAT10 under UVB stress through autophagic receptors p62 and NBR1. The naturally derived ingredients PNS and NGR1 demonstrate exceptional tolerance and market potential, positioning them as significant innovations in the advancement of contemporary skincare products.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePanax Notoginseng Saponins (PNS)\u003c/p\u003e\n\u003cp\u003eUltra-high-performance Liquid Chromatography Coupled with Quadrupole Orbitrap Mass Spectrometry (UHPLC-Q-Orbitrap-MS/MS)\u003c/p\u003e\n\u003cp\u003eHematoxylin and Eosin (HE)\u003c/p\u003e\n\u003cp\u003eGlyceraldehyde-3-phosphate Dehydrogenase (GAPDH)\u003c/p\u003e\n\u003cp\u003eInterleukin-1\u0026beta; (IL-1\u0026beta;)\u003c/p\u003e\n\u003cp\u003eInterleukin-6 (IL-6)\u003c/p\u003e\n\u003cp\u003eInterleukin-10 (IL-10)\u003c/p\u003e\n\u003cp\u003eCatalase Micrococcus Lysodeikticus (CAT)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTotal Antioxidant Capacity (T-AOC)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSuperoxide Dismutase (SOD)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSequestosome 1 (P62)\u003c/p\u003e\n\u003cp\u003eBeclin 1 Coiled-coil Domain Autophagy Regulator (Beclin-1)\u003c/p\u003e\n\u003cp\u003eMicrotubule-associated Protein Light Chain 3B (LC3-II)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAKT Serine/Threonine Kinase 1(AKT1)\u003c/p\u003e\n\u003cp\u003ePhosphatidylinositol-3-kinase (PI3K)\u003c/p\u003e\n\u003cp\u003eMammalian Target of Rapamycin (mTOR)\u003c/p\u003e\n\u003cp\u003eBcl-2-associated X Protein (Bax)\u003c/p\u003e\n\u003cp\u003eB-cell Lymphoma 2 (Bcl-2)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCysteine-aspartic Proteases 3 (Caspase 3)\u003c/p\u003e\n\u003cp\u003eNotoginsenoside R1(NGR1)\u003c/p\u003e\n\u003cp\u003eAll-trans Retinoic Acid (ATRA)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLiquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).\u003c/p\u003e\n\u003cp\u003eN-acetyltransferase 10 (NAT10)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was endorsed by National Natural Science Foundation of China, 82104494; Shanghai Pujiang Program, China, 23PJ1412300; The Project of \u0026quot;Taking on Challenges and Accepting Responsibilities\u0026quot; of The Seventh People\u0026apos;s Hospital of Shanghai University of Traditional Chinese Medicine, QYCXZY250303.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCredit Author Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShuyun Liang, Xiaokang Liu, Yuting Yang and Fangyuan Zhang Wrote the original draft preparation, collected the data and drew the figures; Zizhao Yang, Tong Zhang, Xiaobo Sun, and Dean Guo provided the editing and writing assistance and suggestions; Zizhao Yang and Jiyu Gong approved the final version of manuscript for publication. All the authors have made great contributions during the periods of the manuscript\u0026apos;s initiation and submission. The data were exclusively generated internally, with no involvement of external paper mills. All authors willingly assume responsibility for all aspects of the work to ensure its integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that they possess no known conflicting financial interests or personal relationships that could have potentially influenced the findings presented in this paper. The animal experiment was conducted in accordance with the approved protocol of the Institutional Animal Care and Use Committee, which was authorized by the Animal Ethics Committee of Changchun University of Chinese Medicine. The registration number is 2024299, and the specific date is June 13, 2024. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEl-Domyati M, Attia S, Saleh F, Brown D, Birk DE, Gasparro F, Ahmad H, Uitto J. Intrinsic aging vs. photoaging: a comparative histopathological, immunohistochemical, and ultrastructural study of skin. Exp Dermatol. 2002;11(5):398\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilchrest BA. Skin aging and photoaging: an overview. J Am Acad Dermatol. 1989;21(3 Pt 2):610\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Duan E. Fighting against Skin Aging: The Way from Bench to Bedside. Cell Transplant. 2018;27(5):729\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmaro-Ortiz A, Yan B, D'Orazio JA. Ultraviolet radiation, aging and the skin: prevention of damage by topical cAMP manipulation. 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Signal Transduct Target therapy. 2022;7(1):334.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalmus G, Larrieu D, Barros AC, Collins C, Abrudan M, Demir M, Geisler NJ, Lelliott CJ, White JK, Karp NA, et al. Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome. Nat Commun. 2018;9(1):1700.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao L, He Y, Li SJ, Yu XM, Liu ZC, Liang YY, Yang H, Yang J, Zhang GG, Deng CM, et al. Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner. Cell Res. 2023;33(5):355\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 7 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Panax Notoginseng Saponins (PNS), Panax Notoginsenoside R1 (NGR1), N4-acetyltransferase 10 (NAT10), UVB Irradiation, PI3K/AKT/mTOR Pathway, Autophagy.","lastPublishedDoi":"10.21203/rs.3.rs-6678920/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6678920/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePanax Notoginseng Saponins (PNS) have exhibited therapeutic effects in the repair of skin photoaging induced by UVB radiation; however, the precise mechanism of action remains to be elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is designed to utilize network pharmacology prediction methods to explore the mechanisms by which PNS repair UVB-induced skin photoaging. Furthermore, the chemical composition of PNS was characterized using UHPLC-Q-Orbitrap-MS/MS. An in-depth analysis of the pharmacodynamics of a specific component of PNS, Notoginsenoside R1 (NGR1), was also performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQualitative and quantitative analyses were conducted utilizing UHPLC-Q-orbitrap-MS/MS and UHPLC-Q-Trap-MS/MS to investigate the chemical constituents of PNS. Furthermore, network pharmacology predictions were employed to explore the key targets and mechanisms by which PNS mitigates UVB-induced skin sunburn injury. Furthermore, a nude mouse model was employed to validate the therapeutic efficacy of PNS and its constituent NGR1, whereas HaCaT cells were utilized to elucidate their target mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was designed to thoroughly examine the fundamental mechanisms responsible for the efficacy of PNS in alleviating UVB-induced skin sunburn injury. Additionally, 16 primary saponin components within PNS were identified and subjected to quantitative analysis. Network pharmacology methodologies were utilized to identify 49 key targets of PNS in alleviating UVB-induced skin photoaging. Administration of PNS and NGR1 ameliorates UVB-induced photoaging symptoms through the reduction of inflammation, enhancement of antioxidant defense, inhibition of PI3K/AKT/mTOR signaling pathway activation, and regulation of cellular homeostasis proteins. Furthermore, it provides protection against apoptosis in HaCaT cells by upregulating essential cellular homeostasis proteins, such as p62, while concurrently downregulating autophagy-related proteins, including Beclin-1 and LC3-II. NAT10 expression is reduced by UVB radiation; however, this reduction can be reversed by the administration of drugs PNS and NGR1. The autophagy pathway, which is regulated by NBR1 and p62, is likely involved in the degradation of NAT10 under both physiological and UVB-induced conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential of PNS and NGR1 in skin sunburn injury therapies is evidenced by their capability to mitigate UVB-induced skin damage via the inhibition of PI3K/AKT/mTOR signaling pathway activation, the reduction of cellular apoptosis and autophagy, and the enhancement of RNA acetyl transferase NAT10 regulation. The findings not only lay robust groundwork for subsequent clinical trials of PNS and NGR1 ointment but also furnish compelling evidence to elucidate the therapeutic mechanisms of PNS and NGR1 in the prevention and treatment of UVB-induced skin sunburn injury.\u003c/p\u003e","manuscriptTitle":"Notoginsenoside R1 Mitigates UVB-induced Skin Sunburn Injury through Modulation of N4-acetylcytidine and Macroautophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 16:32:04","doi":"10.21203/rs.3.rs-6678920/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-28T03:48:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-26T09:18:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T09:11:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126511870178048285729020725662133701779","date":"2025-06-12T02:32:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142512229164728639164586786463705508944","date":"2025-06-09T04:49:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-06T08:52:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-19T04:31:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-19T04:28:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2025-05-16T08:35:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f925419-cc81-42f6-8cc4-b917c936fa1a","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T15:59:38+00:00","versionOfRecord":{"articleIdentity":"rs-6678920","link":"https://doi.org/10.1186/s13020-025-01270-3","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2025-12-18 15:57:11","publishedOnDateReadable":"December 18th, 2025"},"versionCreatedAt":"2025-06-09 16:32:04","video":"","vorDoi":"10.1186/s13020-025-01270-3","vorDoiUrl":"https://doi.org/10.1186/s13020-025-01270-3","workflowStages":[]},"version":"v1","identity":"rs-6678920","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6678920","identity":"rs-6678920","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}