Intense pulsed light rejuvenates UVB-induced photo-aging in human keratinocytes and guinea pig skin by inhibition of ERK-AP1-MMP pathway

Intense pulsed light rejuvenates UVB-induced photo-aging in human keratinocytes and guinea pig skin by inhibition of ERK-AP1-MMP pathway | 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 Study protocol Intense pulsed light rejuvenates UVB-induced photo-aging in human keratinocytes and guinea pig skin by inhibition of ERK-AP1-MMP pathway Congcong Liu, Wenzhi Hu, Qingsong Bai, Mingmin Lu, Jiayi Xiang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4589602/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Skin photo-aging is mainly caused by ultraviolet (UV) irradiation. Intense pulsed light (IPL) is a widely used nonablative treatment for photo-aging, while the mechanism is unclear. Here, we investigate the anti-photoaging effects of IPL and the underlying mechanism. This study demonstrated that UV-triggered extracellular signal-regulated kinases (ERK) together with c-jun NH2-terminal kinase (JNK) while IPL suppressed ERK but activated JNK in human skin keratinocytes (hKCs). The different ERK / JNK expression patterns induced by UV and IPL resulted in different c-fos / c-jun(AP-1) phosphorylation, CyclinD1, and matrix metalloproteinase (MMPs) expression. Furtherly, treatment of hKCs with ERK inhibitor (PD98059) revealed that a certain dose of IPL at 17 Jcm 2 (IPL17) significantly promoted c-fos / c-jun phosphorylation by inhibiting the ERK pathway. IPL17 inhibited MMPs expression in guinea pig skin and promoted c-fos / c-jun phosphorylation, epidermal proliferation, and collagen remodeling in vivo. These findings indicated that ERK was involved in IPL rejuvenation by regulating c-fos / c-jun / CyclinD1 / MMPs, providing a potential target for skin rejuvenation. Ultraviolet Intense pulsed light Photoaging Matrix metalloproteinase Extracellular signal-regulated kinases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Skin photo-aging, characterized by increased laxity and fine wrinkles, affects not only external appearance but also psychological and social effects [1–3]. Our skin is a barrier against radiant, chemical, and physical damage. The damaged factors, including ultraviolet radiation (UV), climate change, sleep deprivation, and air pollution, induce skin aging. Among them, UV is a major cause [4]. The spectrum of solar radiation reaching the Earth consists of UV, visible light, and infrared radiation [5]. Different wavelengths have varying effects. UV (100-400nm) can cause skin sunburn, inflammation, aging, and cancer [6, 7]. Intense pulsed light (IPL) has been confirmed with anti-aging effects in clinical practice, whereas the mechanism is unclear [8, 9]. Mitogen-activated protein kinase (MAPK) mainly consists of extracellular signal-regulated kinases (ERK), p38 mitogen-activated protein kinase (p38), and c-jun NH2-terminal kinase (JNK), which are critical for cell proliferation, differentiation, and apoptosis. Many studies have demonstrated that MAPKs play an important role in UV-induced skin photo-aging [10, 11]. However, several studies showed that IPL and lasers in the visible light and near-infrared range could also activate the MAPK pathway and promote skin rejuvenation [12–15]. It has been demonstrated that IPL can activate interleukin (IL), transforming growth factor-β (TGF-β), together with MAPK pathways [16–18]. Activator protein 1(AP-1), composed of c-jun and c-fos, is a critical downstream target of the MAPK pathway that mediates cell proliferation and collagen remodeling [19]. We hypothesized that MAPKs are involved in UV-induced skin photo-aging and IPL rejuvenation. We studied the effects of UV and 17 J / cm 2 of IPL (IPL17) on expression patterns of MAPKs, AP-1, cell proliferation, and matrix metalloproteinase (MMPs) production in human skin keratinocytes (hKCs) and guinea pig skin. The results showed that IPL17 could rejuvenate ultraviolet-irradiated human keratinocytes and guinea pig skin in MMP production and cell proliferation by regulating ERK / AP-1. Materials and methods Materials Human Keratinocyte Growth Supplement (HKGS) / fetal bovine serum (FBS) were procured through Gibco™ (NY, USA). Trypsin-EDTA (0.25%), dimethyl sulfoxide (DMSO), RIPA buffer, Dulbecco's Modified Eagle's Medium (DMEM), BCA proteomic assay, penicillin-streptomycin solution (SP), phosphate-buffered saline (PBS), and goat anti-murine IgG-HRP were obtained by Solarbio (Beijing, China). Goat anti-rabbit IgG-HRP was obtained by ZSGB-BIO (Beijing, China). EpiLife Medium, CCK8 Assay Kit was provided by Dojindo(Kumamoto, Japan). PD98059 was purchased from MedChemExpress (USA). Phosphorylated c-fos(p-c-fos), phosphorylated p38(p-p38), p-JNK, together with CyclinD1 (for Western blot), MMP-1, MMP-9(for immunohistochemistry), and phosphorylated c-jun (p-c-jun, for Western blot / immunohistochemistry) were acquired through Cell Signaling Technology™ (USA). MMP-1, MMP-2, MMP-9, Phosphorylated ERK(p-ERK) and GAPDH (for Western blot), p-c-fos (for immunohistochemistry), and MMP-3(for Western blot and immunohistochemistry) were acquired through Abcam™ (Cambridge, UK). Cellular handling / Curluring Primary human epidermal keratinocytes (hKCs) were isolated from foreskin biopsies from juvenile donors. HKCs were used in passages 1–4. Cell isolation and culture protocol was performed according to a previous study [20]. Different intervention methods created three categories of hKCs: control group (without UV irradiation), UV-irradiated group, and UV + IPL-irradiated group. Animals Thirty white guinea pigs (male, 8 weeks old, weighing 300-400g each) were obtained from Beijing Keyu Animal Breeding Center. The dorsal skin of each guinea pig was shaved before IPL / UV irradiation. Three groups of guinea pigs were randomly selected: the control group (not exposed to UV irradiation), the UV-irradiated group, and the UV + IPL-irradiated group (n = 10). The Air Force Specialty Medical Center's (Beijing, China) Ethics Committee authorized the protocols. The guinea pigs were anesthetized by intraperitoneal injection with sodium pentobarbital for skin biopsy. UV Irradiation A Philips ultraviolet-B (UVB) lamp (Netherlands) emits in the 310–311 nm range. A UV radiometer measured the UVB intensity. Before UVB irradiation, the medium was replaced with PBS. To explore the dosage effect of UVB in hKCs, the UVB dose was 50, 100, and 200 mJ / cm 2 . The UVB dose was 100 mJ / cm 2 to explore the time effect of UVB irradiation. The cells were gathered 24 hours following UVB irradiation. The irradiation method was improved for animal experiments based on relevant reports [21]. The guinea pigs were treated with a daily exposure to 100 mJ / cm 2 of UVB radiation for a period of a 10-weeks. The cumulative exposure dose of UVB was 7.0 J / cm 2 . IPL Irradiation The guinea pigs were irradiated by an intense pulsed light device (M22, Lumenis Ltd., Israel.). All treatments were performed using wavelength 590–1200 nm, spot size 15×35 mm, pulse duration 12 ms, pulse delay 12 ms, and two pulses. In the dosage-effect experiment, the dose of IPL irradiation on hKCs was 10, 17, and 23 J / cm 2 . In a time-effect experiment on hKCs, the IPL dose was 17 J / cm 2 . For animal experiments, the dorsal skin of guinea pigs was irradiated with 17J / cm 2 of IPL once every 3 weeks for 3 consecutive times to emulate the clinical IPL rejuvenation process. Specimens were taken on the first, seventh, and 14th day after the last IPL irradiation. Staining Using staining kits, Masson's Trichrome's and Sirius Red Stain-steps were done in line with routine protocols. Slices were cut at 4µm thickness. Leica of Germany's light microscopy was used to capture the photographs. Immunohistochemistry (IHC) IHC protocol (HRP-Peroxide-DAB) was used in this study. After dewaxing and antigen retrieval, the serum and endogenous peroxidase sections were blocked. Primary antibodies include p-c-fos (1:100), p-c-jun (1:150), MMP-1(1:150), MMP-3(1:400), and MMP-9(1:400) were left to incubate at 4°C overnight along with the sections. Next, the biotinylated secondary antibodies were left to incubate at room temperature for 20 minutes. Lastly, the DAB (3,3-diaminobenzidine) color was developed, and the nuclei were stained with DAPI. Transmission Electron Microscope (TEM) The tissues were fixed using a solution containing 2.5% glutaraldehyde and then postfixed with an osmium tetroxide solution (1%). The next step involved dehydrating tissue portions using a graded ethanol series and embedding them in epoxy resin. The copper grids were used to mount ultrathin slices, stained with lead citrate and uranyl acetate. CCK-8 Assay Cell viability was measured through CCK-8 assay (Cell Counting Kit-8). The HKCs were placed within a 96-well plate (5000 cells / well). The hKCs were incubated with a 10% CCK-8 solution (120 minutes / 37°C / 5% CO 2 ) on the first, third, and fifth day after UV or IPL irradiation. The microplate reader used to measure the absorbance value was Bio-Rad 680, USA, and the measurement was taken at 450 nm. Western Blot Analysis With the RAPI buffer, the hKCs were lysed. PVDF membranes were used to transfer protein that had undergone 10% SDS-PAGE separation. Primary antibodies were exposed to membranes (4°C / overnight). Antibodies were diluted 1:1000 within 5% non-fat milk and included p-ERK, p-p38, phosphorylated JNK (p-JNK), p-c-jun, and p-c-fos, as well as MMP-1, MMP-2, MMP-3, MMP-9, CyclinD1 together with GAPDH. Secondary antibodies were applied to the membranes, remained together within ambient temperature, followed by placing into incubation for an hour. Lastly, an ECL Western blot detection method was used to find the protein bands. MMP-2 and MMP-9 Gel Zymography Gel zymography was used following the manufacturer's instructions. Gelatin zymography gels comprise 10% polyacrylamide gels separating gel with 1 mg / ml gelatin. Protein extracts containing active MMPs were gained from the supernatant of hKCs. The samples were denatured in SDS buffer, and 20µg of protein was used for SDS-PAGE. After staining with Coomassie Brilliant Blue, the gel was decolorized followed by its photographing with a gel imaging device. MMP activity was seen as stainless bands on the blue-stained gelatin backdrop. Evaluation of images Photographs of HE / TEM / Masson's trichrome / Sirius red stain-steps, immunohistochemistry, and Western blotting were assessed through Image J® (v. 1.50, NIH, USA). The quantifications of epidermal thickness, collagen fibers, MAPKs, MMPs, and AP-1 were performed blindly. Statistical analyses These were conducted using GraphPad Prism V8.2.0 (San Diego, USA). Mean ± standard deviation (SD) summarized datasets. One-way analysis of variance (ANOVA) performed cohort comparative statistical analyses. This study considered statistical significance to be achieved when p-value was < 0.05 (#), < 0.01 (##), or < 0.001 (###) in comparison with control group. Additionally, significance was denoted by * when the p-value was < 0.05, ** when < 0.01, and *** when < 0.001 compared to the UV-irradiated group. Results Effects of UV and IPL on MAPKs / AP-1 expression in hKCs Dosage-influence experiments showed that neither the control nor the UV-irradiated groups had p-p38 levels significantly altered. P-ERK and phosphorylation of JNK (p-JNK) were notably elevated in a dose-dependent manner following UV irradiation at doses of 50, 100, and 200 mJ/cm 2 (Fig. 1 A). Time-effect experiments demonstrated that p-p38 levels showed no significant changes after 100 mJ / cm 2 UV irradiation. However, p-ERK and p-JNK peaked at 15 minutes, increased 2–3 folds, respectively, and then recovered to baseline at 120 minutes (Fig. 1 B). After different doses of IPL irradiation, the p-p38 level showed no significant change, and the p-JNK was increased dose-dependently (17J / cm 2 and 23J / cm 2 ). It demonstrated a non-linear relationship between p-ERK and IPL irradiation. At a dose of 17 J / cm 2 , IPL irradiation inhibited the ERK pathway. However, at a dose of 23 J / cm 2 , it notably activated ERK phosphorylation (Fig. 1 C). The ERK activation pattern following IPL irradiation differed from that of UV irradiation. In the time-effect experiment, IPL17 immediately inhibited p-ERK and gradually recovered to a normal level in 120 minutes. The p-JNK peaked at 15 minutes and restored to its normal level in 120 minutes (Fig. 1 D). The effect of UV and IPL irradiation on p-c-fos / p-c-jun was assessed through Western blot analysis. The findings indicate that the phosphorylated c-fos (p-c-fos) was considerably inhibited when exposed to 50, 100, and 200 mJ / cm 2 doses of UV radiation. However, phosphorylation of c-jun (p-c-jun) was considerably raised to more than 7-fold following 200 mJ / cm 2 of UV irradiation (Fig. 1 E). P-c-fos showed no significant changes after different doses of IPL irradiation. Interestingly, among different doses of IPL irradiation, 17 J / cm 2 dose dramatically increased the p-c-jun level 5-fold (Fig. 1 F). These results demonstrated that UV and IPL resulted in different ERK / JNK and c-fos / c-jun expression patterns, which resulted in different expression patterns of MMPs and CyclinD1. Effects of UV and IPL on hKCs proliferation and MMPs expression The activation of CyclinD1 by AP-1 is crucial for the proliferation of cells. The Western blot technique evaluated proteomic expression level for CyclinD1. Twenty-four hours after UV irradiation, CyclinD1 was significantly down-regulated dose-dependently. However, CyclinD1 was increased after IPL irradiation (Fig. 2 A). Cell proliferation assay by CCK-8 assay showed that doses of 100 and 200 mJ / cm 2 notably decreased cell viability on the first and third day. In contrast, after IPL17 treatment, the cell viability was significantly increased on the first day (Fig. 2 B). CCK-8 assay showed consistent results with that of Western blot, in which UV suppressed hKCs proliferation while IPL promoted hKCs proliferation, especially at the dose of 17 J / cm 2 . MMPs, a critical downstream target gene of AP-1, were reported to participate in the degradation of extracellular matrix (ECM) and collagen remodeling. Hence, we detected the MMP levels in UV and IPL-irradiated hKCs. Western blot showed that UV irradiation promoted significant elevation for MMP-1, MMP-2, MMP-3, and MMP-9 in a dose-dependent manner (Fig. 2 C). Nevertheless, IPL irradiation resulted in a non-linear response in MMP levels. IPL irradiation remarkably reduced the level of MMP-1 and MMP-3 at a dose of 17 J / cm 2 . At a dose of 23 J / cm 2 , markedly MMP-1 / MMP-3 upregulation was observed. There were no significant changes in MMP-2 and MMP-9 expression levels among IPL-irradiated groups and the control (Fig. 2 D). The gelatin zymography assay determined MMP-2 and MMP-9 expression profiles following UV / IPL exposure. Twenty-four hours after UV irradiation, MMP-2 and MMP-9 functions were significantly enhanced, while there were no significant variations in MMP-2 and MMP-9 expression after IPL irradiation (Fig. 2 E). It has been known that cellular microvilli number partly represents the secretory activity of hKCs. It participates in ECM degradation, remodeling and homeostasis [22]. Furtherly, we examined the subcellular structure of hKCs via TEM. Compared with the control, there were more microvilli in hCKs after UV irradiation or IPL17 treatment. This result implied that UV triggered the cell secretory function and might participate in remodeling the ECM in the subepidermal dermis (Fig. 2 F). IPL17 rejuvenated ultraviolet-irradiated hKCs in MMP production via ERK / AP-1 IPL17 showed a special photobiomodulation effect on ERK that differed from other doses. Furtherly, we investigate the effects and mechanism of IPL17 on ERK, c-fos / c-jun, and the downstream MMPs. The data showed that IPL17 significantly suppressed p-ERK stimulated by UV. Moreover, IPL17 significantly restored UV-related p-c-fos downregulation and increased UV-induced p-c-jun upregulation (Fig. 3 A, B, C). Compared with the UV-treated group, UV + IPL irradiation significantly upregulated CyclinD1 together with downregulating MMP-1, MMP-2, and MMP-3 expression levels in hKCs. MMP-9 expression levels showed no significant difference between the UV + IPL and UV groups (Fig. 3 E, F, G). When treated hKCs with PD98059 at a concentration of 20µM[23], p-c-fos increased significantly, negatively correlated to the level of p-ERK (Fig. 3 H, I, G, K). It was found that PD98059 resulted in p-c-fos upregulation in hKCs, with similar effects to that of IPL17 irradiation. These results implied that IPL alleviated UV-induced photo-aging via ERK / AP-1 pathway. IPL17 on UV-irradiated guinea pig skin collagen fibers and epidermal thickness The epidermis's thickness was considerably less in the group exposed to UV radiation than in the control group. However, UV + IPL irradiation significantly increased the epidermal thickness from the seventh day as compared with the control, as well as the UV-irradiated group (Fig. 4 A). The UV + IPL-irradiated group demonstrated a remarkable reproduction of collagen fibers in the dermis (Fig. 4 B, C). TEM revealed that collagen fiber was rearranged with a dense distribution, and the average diameter of fibers was larger than the UV-irradiated group (Fig. 4 D). These results demonstrated IPL could repair UV-damaged skin from the epidermis to the dermis IPL17 influence over AP-1 / MMPs expression-profiles within guinea pig skin Compared with the control, p-c-fos significantly decreased following UV irradiation. Compared to the group exposed to UV radiation, p-c-fos increased dramatically on the first, seventh, and 14th day following IPL irradiation (Fig. 5 A). P-c-jun showed a significant elevation following UV irradiation as compared with the control. Furtherly, IPL irradiation increased the p-c-jun expression level on the first, seventh, and 14th day after UV irradiation (Fig. 5 B). In comparison to the control, UV dramatically boosted the MMP-1, MMP-3, together with MMP-9 expression. IPL, however, downregulated MMP-1 and MMP-3 within guinea pig skin exposed to UV radiation. However, nil discernible variation within MMP-9 expression profiles across UV-irradiated and IPL groups (Fig. 5 C, D, E). These results indicated that IPL irradiation enhances collagen deposition by suppressing MMPs. Discussion Skin aging is typically identified by wrinkles, a reduction in epidermal thickness, a decrease in skin elasticity, and changes in pigmentation [24]. The endogenous combined with exogenous factors lead to skin aging [25, 26]. Intrinsic factors, such as genes, age, and hormones, cause irreversible aging and physiological alterations. In contrast, extrinsic aging usually occurs at exposed sites, including the hands and face, which affects people's physical and mental health [27]. Popular treatments against skin photo-aging include surgery, chemical peeling, and mechanical abrasion. These therapies have proved effective but also shown obvious side effects, such as pigmentation and a long recovery period. Non-invasive treatments, including microneedles and phototherapies, have been used in skin rejuvenation, promoting collagen and elastin production and increasing water content [28–30]. Accumulating studies have demonatrated advantages of IPL rejuvenation in efficacy and safety [15]. Nevertheless, the underlying mechanism is still unclear. The study aimed to examine the impact and mechanism of photobiomodulation using IPL on both hKCs cell lines and guinea pig skin. MAPKs have been shown mediate UV-induced skin aging [31, 32]. The activated MAPKs regulate heterodimeric c-fos / c-jun, consisting of AP-1, to maintain skin homeostasis by targeting genes, including cyclin D1 and MMPs. Previous studies demonstrated UV and downstream MAPKs cascade impairment of cell functions and tissue degradation [33–35]. Our results showed that both UV and IPL triggered the MAPK signaling pathway, but their patterns differed. UV exposure caused phosphorylation of ERK and JNK in dose-dependent ways, without selectivity. This resulted in a decrease in p-c-fos combined with an increase in p-c-jun. Such unbalances between p-c-jun and p-c-fos directly affected downstream AP-1, Cyclin D1, and MMPs. However, IPL, particularly at 17 J / cm 2 dose, promoted p-JNK and suppressed p-ERK significantly, leading to a stable p-c-fos / increased p-c-jun level. Consequently, IPL17 promoted Cyclin D1 up-regulation and greatly downregulated MMP-1 / MMP-3. Hence, MAPK / ERK seemed to be a key checkpoint in the skin aging process proved by previous studies [36]. Examining whether MAPK / ERK inhibitors may reverse the UV-induced aging processes would be interesting. We found that with the treatment of 20 µM PD98059, a MAPK / ERK inhibitor [23, 37], the decrease of p-c-fos was rescued after UV treatment. This result supported our hypothesis that 17 J / cm 2 of IPL can alleviate skin photo-aging through inhibition of the MAPK / ERK signaling pathway (Fig. 6 ). The results were also consistent with previous studies that certain herbal extracts could prevent UV-induced skin photo-aging via ERK / AP-1 pathway, reducing MMPs production and enhancing collagen deposition [32, 38, 39]. The therapeutic effect of IPL was also notably in vivo. IPL inhibited the phosphorylation of ERK (p-ERK) but activated the p-c-jun, improved epidermal proliferation, and suppressed dermal MMP-1 and MMP-3 to reduce collagen destruction. Special staining showed that UV-caused collagen loss, fragmentation, and breakage of collagen fibers were remarkably alleviated by IPL17 irradiation. Although guinea pig experiments cannot reveal the IPL rejuvenation mechanism in detail, the results confirmed that IPL17 inhibited MMP expression and promoted c-fos / c-jun phosphorylation, epidermal proliferation, and collagen remodeling in vivo. Conclusion In conclusion, our study demonstrated that 17 J / cm 2 of IPL rejuvenates ultraviolet-irradiated human keratinocytes and guinea pig skin in MMP production and cell proliferation via ERK / AP-1 pathway. Additionally, we provided a potential target and underlying mechanism for IPL rejuvenation. Declarations Conflict of interest The authors declare no conflicts of interest. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 82373460, 82003321). Author Contribution Congcong Liu, Wenzhi Hu, and Qingsong Bai wrote the main manuscript text. Mingmin Lu, Jiayi Xiang, and Lina Tan prepared the figures. Ye Tao and Kui Ma performed the statistical analysis and reviewed the manuscript. 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Propolis Suppresses UV-Induced Photoaging in Human Skin through Directly Targeting Phosphoinositide 3-Kinase. Nutrients, 2020, 12 (12): 3790. X. Duan, T. Wu, T. Liu, H. Yang, X. Ding, Y. Chen, Y. Mu. Vicenin-2 ameliorates oxidative damage and photoaging via modulation of MAPKs and MMPs signaling in UVB radiation exposed human skin cells. J. Photochem. Photobiol. B, 2019, 190 76-85. L. Subedi, T. H. Lee, H. M. Wahedi, S. H. Baek, S. Y. Kim. Resveratrol-Enriched Rice Attenuates UVB-ROS-Induced Skin Aging via Downregulation of Inflammatory Cascades. Oxid. Med. Cell. Longev., 2017, 2017 8379539. H. Lee, J. Sung, Y. Kim, H. S. Jeong, J. Lee. Protective Effects of Unsaponifiable Matter from Perilla Seed Meal on UVB-induced Damages and the Underlying Mechanisms in Human Skin Fibroblasts. Antioxidants (Basel), 2019, 8 (12): 644. L. Wang, J. Y. Oh, W. Lee, Y. J. Jeon. Fucoidan isolated from Hizikia fusiforme suppresses ultraviolet B-induced photodamage by down-regulating the expressions of matrix metalloproteinases and pro-inflammatory cytokines via inhibiting NF-κB, AP-1, and MAPK signaling pathways. Int. J. Biol. Macromol., 2021, 166 751-759. Yu Yao, Zhang Xia, Liu Fengzhen, Zhu Peiying, Zhang Liping, Peng You, Yan Xinyu, Li Yin, Hua Peng, Liu Caiyue, Li Qingfeng, Zhang Liang. A stress-induced miR-31–CLOCK–ERK pathway is a key driver and therapeutic target for skin aging. Nature Aging, 2021, 1 (9): Y. Li, H. Ding, L. Liu, Y. Song, X. Du, S. Feng, X. Wang, X. Li, Z. Wang, X. Li, J. Li, J. Wu, G. Liu. Non-esterified Fatty Acid Induce Dairy Cow Hepatocytes Apoptosis via the Mitochondria-Mediated ROS-JNK/ERK Signaling Pathway. Front Cell Dev Biol, 2020, 8 245. Y. He, Y. Hu, X. Jiang, T. Chen, Y. Ma, S. Wu, J. Sun, R. Jiao, X. Li, L. Deng, W. Bai. Cyanidin-3-O-glucoside inhibits the UVB-induced ROS/COX-2 pathway in HaCaT cells. J. Photochem. Photobiol. B, 2017, 177 24-31. S. H. Xuan, N. H. Lee, S. N. Park. Atractyligenin, a terpenoid isolated from coffee silverskin, inhibits cutaneous photoaging. J. Photochem. Photobiol. B, 2019, 194 166-173. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4589602","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Study protocol","associatedPublications":[],"authors":[{"id":321762462,"identity":"0c7b8d10-5980-4e23-9ce1-e6dffc1835c5","order_by":0,"name":"Congcong Liu","email":"","orcid":"","institution":"Air Force Medical Center, Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Congcong","middleName":"","lastName":"Liu","suffix":""},{"id":321762463,"identity":"25ba6fe7-c0be-4ef2-8e2a-8c65f7ff8d0d","order_by":1,"name":"Wenzhi Hu","email":"","orcid":"","institution":"Air Force Medical Center, Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenzhi","middleName":"","lastName":"Hu","suffix":""},{"id":321762464,"identity":"f85f6aac-6d0a-4ed2-b699-1c5b2c6a4fbe","order_by":2,"name":"Qingsong Bai","email":"","orcid":"","institution":"Air Force Medical Center, Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qingsong","middleName":"","lastName":"Bai","suffix":""},{"id":321762465,"identity":"e50ce828-8bc5-467b-9342-c6d3f2afbf27","order_by":3,"name":"Mingmin Lu","email":"","orcid":"","institution":"Air Force Medical Center, Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingmin","middleName":"","lastName":"Lu","suffix":""},{"id":321762466,"identity":"99f86944-88ba-4e67-98a6-46ca579bd7d4","order_by":4,"name":"Jiayi Xiang","email":"","orcid":"","institution":"Graduate School of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Xiang","suffix":""},{"id":321762467,"identity":"9dc50143-03cd-470d-bc33-36bde0f5ea5c","order_by":5,"name":"Lina Tan","email":"","orcid":"","institution":"Air Force Medical Center, Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Tan","suffix":""},{"id":321762468,"identity":"f8868d9f-4f51-423f-9e07-b4171c2677fe","order_by":6,"name":"Ye Tao","email":"","orcid":"","institution":"Air Force Medical Center, Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Tao","suffix":""},{"id":321762471,"identity":"e6e9218f-c023-4554-b57b-b7151a624c4c","order_by":7,"name":"Kui Ma","email":"","orcid":"","institution":"The Fourth Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kui","middleName":"","lastName":"Ma","suffix":""},{"id":321762472,"identity":"0f912739-296f-4b20-b617-22407ca5e34a","order_by":8,"name":"Lixia Zhang","email":"","orcid":"","institution":"The Fourth Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lixia","middleName":"","lastName":"Zhang","suffix":""},{"id":321762473,"identity":"824a8977-3965-47e5-9005-164819b7512f","order_by":9,"name":"Weijie Gu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYLCCBB4JBjZm9oMPEipqSNDCx86TbPDgzDESbJLjZzCTfNjCTFilfESO4YMHMhZybMwMaRWJDWwM/O3dCXi1GN7IMTYAOsyYjZnx2I3EHTIMEmfObsCvZUbuNgmglsQ2oC03Es+wMRhI5BLUsv0HUEs9UItZAUgjQS3yErnbQCGWAPSLGQNRWgx43n8GOcywjZknWSLhzDEegn6Rb09L/Pizp05evv/4wY8/Kmrk+Nt7CdhyAEgw9iAEePAqB9vSACJ/EFQ3CkbBKBgFIxkAAP8AQfJaLFF9AAAAAElFTkSuQmCC","orcid":"","institution":"Air Force Medical Center, Air Force Medical University","correspondingAuthor":true,"prefix":"","firstName":"Weijie","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2024-06-16 12:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4589602/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4589602/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59580815,"identity":"73b54851-23aa-486a-a0be-7c7f95daa303","added_by":"auto","created_at":"2024-07-03 12:27:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":280486,"visible":true,"origin":"","legend":"\u003cp\u003eUV and IPL-induced MAPK / AP-1 phosphorylation in hKCs was assessed through Western blot. (\u003cstrong\u003eA\u003c/strong\u003e) Dosage-influence by UV upon MAPK phosphorylation. The hKCs were treated with UV at 50, 100, and 200 mJ / cm\u003csup\u003e2\u003c/sup\u003e. (\u003cstrong\u003eB\u003c/strong\u003e) Time-effect of UV on MAPKs phosphorylation. The phosphorylation of MAPKs at 0, 15, 30, 60, and 120 minutes following 100 mJ / cm\u003csup\u003e2\u003c/sup\u003e UV irradiation. (\u003cstrong\u003eC\u003c/strong\u003e) Dosage-effect of IPL irradiation (10, 17, and 23J / cm\u003csup\u003e2\u003c/sup\u003e) on phosphorylation of MAPKs. (\u003cstrong\u003eD\u003c/strong\u003e) Time-effect of IPL (17 J / cm\u003csup\u003e2\u003c/sup\u003e) on MAPKs phosphorylation. (\u003cstrong\u003eE\u003c/strong\u003e) Dosage-effect of UV on p-c-fos / p-c-jun. (\u003cstrong\u003eF\u003c/strong\u003e) The IPL dosage effect on p-c-fos / p-c-jun. An internal control, GAPDH, was utilized. The data is presented in mean ± standard deviation (SD).\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4589602/v1/7e9d44f77f64e076781f03e4.jpg"},{"id":59581166,"identity":"f6f30623-ba57-4c0e-8ec9-be3b7726587c","added_by":"auto","created_at":"2024-07-03 12:35:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":304348,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of UV and IPL irradiation on hKCs proliferation and MMPs expression were examined by Western blot and CCK-8 assay. (\u003cstrong\u003eA\u003c/strong\u003e) Dosage-effect of UV and IPL on CyclinD1 expression measured by Western blot. (\u003cstrong\u003eB\u003c/strong\u003e) Effects of UV and IPL on hKCs proliferation measured by CCK-8 assay. (\u003cstrong\u003eC\u003c/strong\u003e) Dosage-effect of UV on hKCs MMPs expression. (\u003cstrong\u003eD\u003c/strong\u003e) Dosage-effect of IPL on MMPs expression. (\u003cstrong\u003eE\u003c/strong\u003e) Effects of UV and IPL on MMP-2 and MMP-9 examined by Gelatin zymography assay. (\u003cstrong\u003eF\u003c/strong\u003e) This is an image of the ultrastructure of hKCs, which was detected using TEM. The scale bar in the photo is 500nm. Datasets reflected mean ± standard deviation (SD). Statistically significant thresholds consisted of: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 when compared with control group.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4589602/v1/86087125cf0a779c1b513be8.jpg"},{"id":59579937,"identity":"8356f190-8de2-4d55-b0f6-dcf4eb0791a5","added_by":"auto","created_at":"2024-07-03 12:11:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":644911,"visible":true,"origin":"","legend":"\u003cp\u003eIPL17 rejuvenated ultraviolet-irradiated hKCs in MMPs production via ERK / AP-1 pathway. (\u003cstrong\u003eA-G\u003c/strong\u003e) The expression profiles for p-ERK, p-c-fos, p-c-jun, CyclinD1, together with MMPs in hKCs after UV (100 mJ / cm\u003csup\u003e2\u003c/sup\u003e) and IPL (17 J / cm\u003csup\u003e2\u003c/sup\u003e) irradiation. (\u003cstrong\u003eH-K\u003c/strong\u003e) Expression profiles for p-ERK, p-c-fos, and p-c-jun within PD98059+UV treated hKCs. An internal control, GAPDH, was utilized.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4589602/v1/061f301b9b0c8dc414f05023.jpg"},{"id":59580261,"identity":"c5bcf377-d46b-4d4f-bff1-ef1784a6743a","added_by":"auto","created_at":"2024-07-03 12:19:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":387744,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of UV and IPL on epidermal thickness, collagen content, and ultrastructure in guinea skin. (\u003cstrong\u003eA\u003c/strong\u003e) HE staining of the guinea skin and the epidermal thickness. (\u003cstrong\u003eB\u003c/strong\u003e) Masson's trichrome stain for collagen fiber density. (\u003cstrong\u003eC\u003c/strong\u003e) Area density of collagen fibers by Sirius Red stain (scale bar = 100 μm). (\u003cstrong\u003eD\u003c/strong\u003e) Arrangement and average diameter of collagen fibers by TEM (scale bar = 500nm).\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4589602/v1/02caf83b07b2070e76d9c6ad.jpg"},{"id":59581729,"identity":"8d8251cb-c62a-48a3-8568-9acc4be09bdb","added_by":"auto","created_at":"2024-07-03 12:43:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":493324,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry examined the Effect of UV and IPL on AP-1 and MMPs in guinea skin. (\u003cstrong\u003eA-E\u003c/strong\u003e) The samples received staining with p-c-fos, p-c-jun, together with MMPs antibodies, and photomicrographs were taken. Optical density for samples was assessed through Image J®. Datasets reflected mean ± the standard deviation (SD). Meanwhile, #, ##, and ### identified statistical significance through \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u0026lt;0.01, and \u0026lt;0.001, accordingly, in comparison with control group. *, **, together with *** suggest statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u0026lt;0.001, \u0026lt;0.001) compared to the UV-irradiated group.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4589602/v1/4fefcfa2ee2e53c42a1a5faa.jpg"},{"id":59579932,"identity":"6e859b8b-c97a-4360-b523-0761037deaf5","added_by":"auto","created_at":"2024-07-03 12:11:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116253,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic model of the signaling pathway in the UVB and IPL-induced cyclinD1/MMPs expression that is regulated by MAPKs / AP-1\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4589602/v1/0eebb56ac56cded110e294ec.jpg"},{"id":73263229,"identity":"2c77aec4-4eab-4fc2-8b00-4eb855dda7b5","added_by":"auto","created_at":"2025-01-08 09:53:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2715933,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4589602/v1/e5506e58-d8f0-4f29-aff9-3d7bea8cfaa0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intense pulsed light rejuvenates UVB-induced photo-aging in human keratinocytes and guinea pig skin by inhibition of ERK-AP1-MMP pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkin photo-aging, characterized by increased laxity and fine wrinkles, affects not only external appearance but also psychological and social effects [1\u0026ndash;3]. Our skin is a barrier against radiant, chemical, and physical damage. The damaged factors, including ultraviolet radiation (UV), climate change, sleep deprivation, and air pollution, induce skin aging. Among them, UV is a major cause [4]. The spectrum of solar radiation reaching the Earth consists of UV, visible light, and infrared radiation [5]. Different wavelengths have varying effects. UV (100-400nm) can cause skin sunburn, inflammation, aging, and cancer [6, 7]. Intense pulsed light (IPL) has been confirmed with anti-aging effects in clinical practice, whereas the mechanism is unclear [8, 9].\u003c/p\u003e \u003cp\u003eMitogen-activated protein kinase (MAPK) mainly consists of extracellular signal-regulated kinases (ERK), p38 mitogen-activated protein kinase (p38), and c-jun NH2-terminal kinase (JNK), which are critical for cell proliferation, differentiation, and apoptosis. Many studies have demonstrated that MAPKs play an important role in UV-induced skin photo-aging [10, 11]. However, several studies showed that IPL and lasers in the visible light and near-infrared range could also activate the MAPK pathway and promote skin rejuvenation [12\u0026ndash;15]. It has been demonstrated that IPL can activate interleukin (IL), transforming growth factor-β (TGF-β), together with MAPK pathways [16\u0026ndash;18]. Activator protein 1(AP-1), composed of c-jun and c-fos, is a critical downstream target of the MAPK pathway that mediates cell proliferation and collagen remodeling [19].\u003c/p\u003e \u003cp\u003eWe hypothesized that MAPKs are involved in UV-induced skin photo-aging and IPL rejuvenation. We studied the effects of UV and 17 J / cm\u003csup\u003e2\u003c/sup\u003e of IPL (IPL17) on expression patterns of MAPKs, AP-1, cell proliferation, and matrix metalloproteinase (MMPs) production in human skin keratinocytes (hKCs) and guinea pig skin. The results showed that IPL17 could rejuvenate ultraviolet-irradiated human keratinocytes and guinea pig skin in MMP production and cell proliferation by regulating ERK / AP-1.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eHuman Keratinocyte Growth Supplement (HKGS) / fetal bovine serum (FBS) were procured through Gibco\u0026trade; (NY, USA). Trypsin-EDTA (0.25%), dimethyl sulfoxide (DMSO), RIPA buffer, Dulbecco's Modified Eagle's Medium (DMEM), BCA proteomic assay, penicillin-streptomycin solution (SP), phosphate-buffered saline (PBS), and goat anti-murine IgG-HRP were obtained by Solarbio (Beijing, China). Goat anti-rabbit IgG-HRP was obtained by ZSGB-BIO (Beijing, China). EpiLife Medium, CCK8 Assay Kit was provided by Dojindo(Kumamoto, Japan). PD98059 was purchased from MedChemExpress (USA). Phosphorylated c-fos(p-c-fos), phosphorylated p38(p-p38), p-JNK, together with CyclinD1 (for Western blot), MMP-1, MMP-9(for immunohistochemistry), and phosphorylated c-jun (p-c-jun, for Western blot / immunohistochemistry) were acquired through Cell Signaling Technology\u0026trade; (USA). MMP-1, MMP-2, MMP-9, Phosphorylated ERK(p-ERK) and GAPDH (for Western blot), p-c-fos (for immunohistochemistry), and MMP-3(for Western blot and immunohistochemistry) were acquired through Abcam\u0026trade; (Cambridge, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCellular handling / Curluring\u003c/h2\u003e \u003cp\u003ePrimary human epidermal keratinocytes (hKCs) were isolated from foreskin biopsies from juvenile donors. HKCs were used in passages 1\u0026ndash;4. Cell isolation and culture protocol was performed according to a previous study [20]. Different intervention methods created three categories of hKCs: control group (without UV irradiation), UV-irradiated group, and UV\u0026thinsp;+\u0026thinsp;IPL-irradiated group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThirty white guinea pigs (male, 8 weeks old, weighing 300-400g each) were obtained from Beijing Keyu Animal Breeding Center. The dorsal skin of each guinea pig was shaved before IPL / UV irradiation. Three groups of guinea pigs were randomly selected: the control group (not exposed to UV irradiation), the UV-irradiated group, and the UV\u0026thinsp;+\u0026thinsp;IPL-irradiated group (n\u0026thinsp;=\u0026thinsp;10). The Air Force Specialty Medical Center's (Beijing, China) Ethics Committee authorized the protocols. The guinea pigs were anesthetized by intraperitoneal injection with sodium pentobarbital for skin biopsy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eUV Irradiation\u003c/h2\u003e \u003cp\u003eA Philips ultraviolet-B (UVB) lamp (Netherlands) emits in the 310\u0026ndash;311 nm range. A UV radiometer measured the UVB intensity. Before UVB irradiation, the medium was replaced with PBS. To explore the dosage effect of UVB in hKCs, the UVB dose was 50, 100, and 200 mJ / cm\u003csup\u003e2\u003c/sup\u003e. The UVB dose was 100 mJ / cm\u003csup\u003e2\u003c/sup\u003e to explore the time effect of UVB irradiation. The cells were gathered 24 hours following UVB irradiation. The irradiation method was improved for animal experiments based on relevant reports [21]. The guinea pigs were treated with a daily exposure to 100 mJ / cm\u003csup\u003e2\u003c/sup\u003e of UVB radiation for a period of a 10-weeks. The cumulative exposure dose of UVB was 7.0 J / cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIPL Irradiation\u003c/h2\u003e \u003cp\u003eThe guinea pigs were irradiated by an intense pulsed light device (M22, Lumenis Ltd., Israel.). All treatments were performed using wavelength 590\u0026ndash;1200 nm, spot size 15\u0026times;35 mm, pulse duration 12 ms, pulse delay 12 ms, and two pulses. In the dosage-effect experiment, the dose of IPL irradiation on hKCs was 10, 17, and 23 J / cm\u003csup\u003e2\u003c/sup\u003e. In a time-effect experiment on hKCs, the IPL dose was 17 J / cm\u003csup\u003e2\u003c/sup\u003e. For animal experiments, the dorsal skin of guinea pigs was irradiated with 17J / cm\u003csup\u003e2\u003c/sup\u003e of IPL once every 3 weeks for 3 consecutive times to emulate the clinical IPL rejuvenation process. Specimens were taken on the first, seventh, and 14th day after the last IPL irradiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStaining\u003c/h2\u003e \u003cp\u003eUsing staining kits, Masson's Trichrome's and Sirius Red Stain-steps were done in line with routine protocols. Slices were cut at 4\u0026micro;m thickness. Leica of Germany's light microscopy was used to capture the photographs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eIHC protocol (HRP-Peroxide-DAB) was used in this study. After dewaxing and antigen retrieval, the serum and endogenous peroxidase sections were blocked. Primary antibodies include p-c-fos (1:100), p-c-jun (1:150), MMP-1(1:150), MMP-3(1:400), and MMP-9(1:400) were left to incubate at 4\u0026deg;C overnight along with the sections. Next, the biotinylated secondary antibodies were left to incubate at room temperature for 20 minutes. Lastly, the DAB (3,3-diaminobenzidine) color was developed, and the nuclei were stained with DAPI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscope (TEM)\u003c/h2\u003e \u003cp\u003eThe tissues were fixed using a solution containing 2.5% glutaraldehyde and then postfixed with an osmium tetroxide solution (1%). The next step involved dehydrating tissue portions using a graded ethanol series and embedding them in epoxy resin. The copper grids were used to mount ultrathin slices, stained with lead citrate and uranyl acetate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 Assay\u003c/h2\u003e \u003cp\u003eCell viability was measured through CCK-8 assay (Cell Counting Kit-8). The HKCs were placed within a 96-well plate (5000 cells / well). The hKCs were incubated with a 10% CCK-8 solution (120 minutes / 37\u0026deg;C / 5% CO\u003csub\u003e2\u003c/sub\u003e) on the first, third, and fifth day after UV or IPL irradiation. The microplate reader used to measure the absorbance value was Bio-Rad 680, USA, and the measurement was taken at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eWith the RAPI buffer, the hKCs were lysed. PVDF membranes were used to transfer protein that had undergone 10% SDS-PAGE separation. Primary antibodies were exposed to membranes (4\u0026deg;C / overnight). Antibodies were diluted 1:1000 within 5% non-fat milk and included p-ERK, p-p38, phosphorylated JNK (p-JNK), p-c-jun, and p-c-fos, as well as MMP-1, MMP-2, MMP-3, MMP-9, CyclinD1 together with GAPDH. Secondary antibodies were applied to the membranes, remained together within ambient temperature, followed by placing into incubation for an hour. Lastly, an ECL Western blot detection method was used to find the protein bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMMP-2 and MMP-9 Gel Zymography\u003c/h2\u003e \u003cp\u003eGel zymography was used following the manufacturer's instructions. Gelatin zymography gels comprise 10% polyacrylamide gels separating gel with 1 mg / ml gelatin. Protein extracts containing active MMPs were gained from the supernatant of hKCs. The samples were denatured in SDS buffer, and 20\u0026micro;g of protein was used for SDS-PAGE. After staining with Coomassie Brilliant Blue, the gel was decolorized followed by its photographing with a gel imaging device. MMP activity was seen as stainless bands on the blue-stained gelatin backdrop.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of images\u003c/h2\u003e \u003cp\u003ePhotographs of HE / TEM / Masson's trichrome / Sirius red stain-steps, immunohistochemistry, and Western blotting were assessed through Image J\u0026reg; (v. 1.50, NIH, USA). The quantifications of epidermal thickness, collagen fibers, MAPKs, MMPs, and AP-1 were performed blindly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThese were conducted using GraphPad Prism V8.2.0 (San Diego, USA). Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) summarized datasets. One-way analysis of variance (ANOVA) performed cohort comparative statistical analyses. This study considered statistical significance to be achieved when p-value was \u0026lt;\u0026thinsp;0.05 (#), \u0026lt;\u0026thinsp;0.01 (##), or \u0026lt;\u0026thinsp;0.001 (###) in comparison with control group. Additionally, significance was denoted by * when the p-value was \u0026lt;\u0026thinsp;0.05, ** when \u0026lt;\u0026thinsp;0.01, and *** when \u0026lt;\u0026thinsp;0.001 compared to the UV-irradiated group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of UV and IPL on MAPKs\u003c/b\u003e / \u003cb\u003eAP-1 expression in hKCs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDosage-influence experiments showed that neither the control nor the UV-irradiated groups had p-p38 levels significantly altered. P-ERK and phosphorylation of JNK (p-JNK) were notably elevated in a dose-dependent manner following UV irradiation at doses of 50, 100, and 200 mJ/cm\u003csup\u003e2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTime-effect experiments demonstrated that p-p38 levels showed no significant changes after 100 mJ / cm\u003csup\u003e2\u003c/sup\u003e UV irradiation. However, p-ERK and p-JNK peaked at 15 minutes, increased 2\u0026ndash;3 folds, respectively, and then recovered to baseline at 120 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAfter different doses of IPL irradiation, the p-p38 level showed no significant change, and the p-JNK was increased dose-dependently (17J / cm\u003csup\u003e2\u003c/sup\u003e and 23J / cm\u003csup\u003e2\u003c/sup\u003e). It demonstrated a non-linear relationship between p-ERK and IPL irradiation. At a dose of 17 J / cm\u003csup\u003e2\u003c/sup\u003e, IPL irradiation inhibited the ERK pathway. However, at a dose of 23 J / cm\u003csup\u003e2\u003c/sup\u003e, it notably activated ERK phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The ERK activation pattern following IPL irradiation differed from that of UV irradiation. In the time-effect experiment, IPL17 immediately inhibited p-ERK and gradually recovered to a normal level in 120 minutes. The p-JNK peaked at 15 minutes and restored to its normal level in 120 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe effect of UV and IPL irradiation on p-c-fos / p-c-jun was assessed through Western blot analysis. The findings indicate that the phosphorylated c-fos (p-c-fos) was considerably inhibited when exposed to 50, 100, and 200 mJ / cm\u003csup\u003e2\u003c/sup\u003e doses of UV radiation. However, phosphorylation of c-jun (p-c-jun) was considerably raised to more than 7-fold following 200 mJ / cm\u003csup\u003e2\u003c/sup\u003e of UV irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). P-c-fos showed no significant changes after different doses of IPL irradiation. Interestingly, among different doses of IPL irradiation, 17 J / cm\u003csup\u003e2\u003c/sup\u003e dose dramatically increased the p-c-jun level 5-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results demonstrated that UV and IPL resulted in different ERK / JNK and c-fos / c-jun expression patterns, which resulted in different expression patterns of MMPs and CyclinD1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects of UV and IPL on hKCs proliferation and MMPs expression\u003c/h2\u003e \u003cp\u003eThe activation of CyclinD1 by AP-1 is crucial for the proliferation of cells. The Western blot technique evaluated proteomic expression level for CyclinD1. Twenty-four hours after UV irradiation, CyclinD1 was significantly down-regulated dose-dependently. However, CyclinD1 was increased after IPL irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cell proliferation assay by CCK-8 assay showed that doses of 100 and 200 mJ / cm\u003csup\u003e2\u003c/sup\u003e notably decreased cell viability on the first and third day. In contrast, after IPL17 treatment, the cell viability was significantly increased on the first day (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). CCK-8 assay showed consistent results with that of Western blot, in which UV suppressed hKCs proliferation while IPL promoted hKCs proliferation, especially at the dose of 17 J / cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMMPs, a critical downstream target gene of AP-1, were reported to participate in the degradation of extracellular matrix (ECM) and collagen remodeling. Hence, we detected the MMP levels in UV and IPL-irradiated hKCs. Western blot showed that UV irradiation promoted significant elevation for MMP-1, MMP-2, MMP-3, and MMP-9 in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Nevertheless, IPL irradiation resulted in a non-linear response in MMP levels. IPL irradiation remarkably reduced the level of MMP-1 and MMP-3 at a dose of 17 J / cm\u003csup\u003e2\u003c/sup\u003e. At a dose of 23 J / cm\u003csup\u003e2\u003c/sup\u003e, markedly MMP-1 / MMP-3 upregulation was observed. There were no significant changes in MMP-2 and MMP-9 expression levels among IPL-irradiated groups and the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The gelatin zymography assay determined MMP-2 and MMP-9 expression profiles following UV / IPL exposure. Twenty-four hours after UV irradiation, MMP-2 and MMP-9 functions were significantly enhanced, while there were no significant variations in MMP-2 and MMP-9 expression after IPL irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). It has been known that cellular microvilli number partly represents the secretory activity of hKCs. It participates in ECM degradation, remodeling and homeostasis [22]. Furtherly, we examined the subcellular structure of hKCs via TEM. Compared with the control, there were more microvilli in hCKs after UV irradiation or IPL17 treatment. This result implied that UV triggered the cell secretory function and might participate in remodeling the ECM in the subepidermal dermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIPL17 rejuvenated ultraviolet-irradiated hKCs in MMP production via ERK / AP-1\u003c/h2\u003e \u003cp\u003eIPL17 showed a special photobiomodulation effect on ERK that differed from other doses. Furtherly, we investigate the effects and mechanism of IPL17 on ERK, c-fos / c-jun, and the downstream MMPs. The data showed that IPL17 significantly suppressed p-ERK stimulated by UV. Moreover, IPL17 significantly restored UV-related p-c-fos downregulation and increased UV-induced p-c-jun upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, C). Compared with the UV-treated group, UV\u0026thinsp;+\u0026thinsp;IPL irradiation significantly upregulated CyclinD1 together with downregulating MMP-1, MMP-2, and MMP-3 expression levels in hKCs. MMP-9 expression levels showed no significant difference between the UV\u0026thinsp;+\u0026thinsp;IPL and UV groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F, G). When treated hKCs with PD98059 at a concentration of 20\u0026micro;M[23], p-c-fos increased significantly, negatively correlated to the level of p-ERK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I, G, K). It was found that PD98059 resulted in p-c-fos upregulation in hKCs, with similar effects to that of IPL17 irradiation. These results implied that IPL alleviated UV-induced photo-aging via ERK / AP-1 pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIPL17 on UV-irradiated guinea pig skin collagen fibers and epidermal thickness\u003c/h2\u003e \u003cp\u003eThe epidermis's thickness was considerably less in the group exposed to UV radiation than in the control group. However, UV\u0026thinsp;+\u0026thinsp;IPL irradiation significantly increased the epidermal thickness from the seventh day as compared with the control, as well as the UV-irradiated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The UV\u0026thinsp;+\u0026thinsp;IPL-irradiated group demonstrated a remarkable reproduction of collagen fibers in the dermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). TEM revealed that collagen fiber was rearranged with a dense distribution, and the average diameter of fibers was larger than the UV-irradiated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results demonstrated IPL could repair UV-damaged skin from the epidermis to the dermis\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIPL17 influence over AP-1 / MMPs expression-profiles within guinea pig skin\u003c/h2\u003e \u003cp\u003eCompared with the control, p-c-fos significantly decreased following UV irradiation. Compared to the group exposed to UV radiation, p-c-fos increased dramatically on the first, seventh, and 14th day following IPL irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). P-c-jun showed a significant elevation following UV irradiation as compared with the control. Furtherly, IPL irradiation increased the p-c-jun expression level on the first, seventh, and 14th day after UV irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In comparison to the control, UV dramatically boosted the MMP-1, MMP-3, together with MMP-9 expression. IPL, however, downregulated MMP-1 and MMP-3 within guinea pig skin exposed to UV radiation. However, nil discernible variation within MMP-9 expression profiles across UV-irradiated and IPL groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D, E). These results indicated that IPL irradiation enhances collagen deposition by suppressing MMPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSkin aging is typically identified by wrinkles, a reduction in epidermal thickness, a decrease in skin elasticity, and changes in pigmentation [24]. The endogenous combined with exogenous factors lead to skin aging [25, 26]. Intrinsic factors, such as genes, age, and hormones, cause irreversible aging and physiological alterations. In contrast, extrinsic aging usually occurs at exposed sites, including the hands and face, which affects people's physical and mental health [27]. Popular treatments against skin photo-aging include surgery, chemical peeling, and mechanical abrasion. These therapies have proved effective but also shown obvious side effects, such as pigmentation and a long recovery period. Non-invasive treatments, including microneedles and phototherapies, have been used in skin rejuvenation, promoting collagen and elastin production and increasing water content [28\u0026ndash;30]. Accumulating studies have demonatrated advantages of IPL rejuvenation in efficacy and safety [15]. Nevertheless, the underlying mechanism is still unclear.\u003c/p\u003e \u003cp\u003eThe study aimed to examine the impact and mechanism of photobiomodulation using IPL on both hKCs cell lines and guinea pig skin. MAPKs have been shown mediate UV-induced skin aging [31, 32]. The activated MAPKs regulate heterodimeric c-fos / c-jun, consisting of AP-1, to maintain skin homeostasis by targeting genes, including cyclin D1 and MMPs. Previous studies demonstrated UV and downstream MAPKs cascade impairment of cell functions and tissue degradation [33\u0026ndash;35]. Our results showed that both UV and IPL triggered the MAPK signaling pathway, but their patterns differed. UV exposure caused phosphorylation of ERK and JNK in dose-dependent ways, without selectivity. This resulted in a decrease in p-c-fos combined with an increase in p-c-jun. Such unbalances between p-c-jun and p-c-fos directly affected downstream AP-1, Cyclin D1, and MMPs. However, IPL, particularly at 17 J / cm\u003csup\u003e2\u003c/sup\u003e dose, promoted p-JNK and suppressed p-ERK significantly, leading to a stable p-c-fos / increased p-c-jun level. Consequently, IPL17 promoted Cyclin D1 up-regulation and greatly downregulated MMP-1 / MMP-3. Hence, MAPK / ERK seemed to be a key checkpoint in the skin aging process proved by previous studies [36]. Examining whether MAPK / ERK inhibitors may reverse the UV-induced aging processes would be interesting. We found that with the treatment of 20 \u0026micro;M PD98059, a MAPK / ERK inhibitor [23, 37], the decrease of p-c-fos was rescued after UV treatment. This result supported our hypothesis that 17 J / cm\u003csup\u003e2\u003c/sup\u003e of IPL can alleviate skin photo-aging through inhibition of the MAPK / ERK signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results were also consistent with previous studies that certain herbal extracts could prevent UV-induced skin photo-aging via ERK / AP-1 pathway, reducing MMPs production and enhancing collagen deposition [32, 38, 39]. The therapeutic effect of IPL was also notably in vivo. IPL inhibited the phosphorylation of ERK (p-ERK) but activated the p-c-jun, improved epidermal proliferation, and suppressed dermal MMP-1 and MMP-3 to reduce collagen destruction. Special staining showed that UV-caused collagen loss, fragmentation, and breakage of collagen fibers were remarkably alleviated by IPL17 irradiation. Although guinea pig experiments cannot reveal the IPL rejuvenation mechanism in detail, the results confirmed that IPL17 inhibited MMP expression and promoted c-fos / c-jun phosphorylation, epidermal proliferation, and collagen remodeling in vivo.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study demonstrated that 17 J / cm\u003csup\u003e2\u003c/sup\u003e of IPL rejuvenates ultraviolet-irradiated human keratinocytes and guinea pig skin in MMP production and cell proliferation via ERK / AP-1 pathway. Additionally, we provided a potential target and underlying mechanism for IPL rejuvenation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 82373460, 82003321).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCongcong Liu, Wenzhi Hu, and Qingsong Bai wrote the main manuscript text. Mingmin Lu, Jiayi Xiang, and Lina Tan prepared the figures. Ye Tao and Kui Ma performed the statistical analysis and reviewed the manuscript. Weijie Gu and Lixia Zhang designed the study and supervised the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. L. Cohen, A. Rivkin, S. Dayan, A. Shamban, W. P. 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B, 2019, 194 166-173.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ultraviolet, Intense pulsed light, Photoaging, Matrix metalloproteinase, Extracellular signal-regulated kinases","lastPublishedDoi":"10.21203/rs.3.rs-4589602/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4589602/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSkin photo-aging is mainly caused by ultraviolet (UV) irradiation. Intense pulsed light (IPL) is a widely used nonablative treatment for photo-aging, while the mechanism is unclear. Here, we investigate the anti-photoaging effects of IPL and the underlying mechanism. This study demonstrated that UV-triggered extracellular signal-regulated kinases (ERK) together with c-jun NH2-terminal kinase (JNK) while IPL suppressed ERK but activated JNK in human skin keratinocytes (hKCs). The different ERK / JNK expression patterns induced by UV and IPL resulted in different c-fos / c-jun(AP-1) phosphorylation, CyclinD1, and matrix metalloproteinase (MMPs) expression. Furtherly, treatment of hKCs with ERK inhibitor (PD98059) revealed that a certain dose of IPL at 17 Jcm\u003csup\u003e2\u003c/sup\u003e (IPL17) significantly promoted c-fos / c-jun phosphorylation by inhibiting the ERK pathway. IPL17 inhibited MMPs expression in guinea pig skin and promoted c-fos / c-jun phosphorylation, epidermal proliferation, and collagen remodeling in vivo. These findings indicated that ERK was involved in IPL rejuvenation by regulating c-fos / c-jun / CyclinD1 / MMPs, providing a potential target for skin rejuvenation.\u003c/p\u003e","manuscriptTitle":"Intense pulsed light rejuvenates UVB-induced photo-aging in human keratinocytes and guinea pig skin by inhibition of ERK-AP1-MMP pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 12:11:21","doi":"10.21203/rs.3.rs-4589602/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa95d587-6f06-44e7-a021-dbe7d1ab8fd1","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-08T09:53:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 12:11:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4589602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4589602","identity":"rs-4589602","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}