Physcion-8-O-β-D-monoglucoside protects hepatocytes from TNF-α-mediated apoptosis by suppressing the PI3K/AKT /NF-κB signaling pathway

Physcion-8-O-β-D-monoglucoside protects hepatocytes from TNF-α-mediated apoptosis by suppressing the PI3K/AKT /NF-κB signaling 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 Article Physcion-8-O-β-D-monoglucoside protects hepatocytes from TNF-α-mediated apoptosis by suppressing the PI3K/AKT /NF-κB signaling pathway Zhihui Chen, Ting Chen, Zixin Chen, Wenchuan Luo, Wen Xu, Mei Huang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7542054/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Physcion-8-O-β-D-monoglucoside (PMG) is one of the active ingredients of Radix et Rhizoma Rhei, which had been used for treating liver disorders for hundreds of years in China. However, the hepatoprotective effects of PMG remain poorly understood. This study aimed to investigate the mechanism of the protection effects of PMG on Tumor necrosis factor-α (TNF-α)-induced hepatotoxicity. We developed both in vitro and in vivo models of liver injury to assess the protective effects of PMG against TNF-α-induced hepatotoxicity. The in vitro model employed TNF-α/actinomycin D in AML-12 cells, while the in vivo model utilized intraperitoneal injection of carbon tetrachloride (CCl 4 ) in mice. Interactions of PMG and TNFR1 (the receptor of TNF-α) were explored by molecular docking. AAV resuspension was administered before PMG treatment via intravenous injection to overexpress TNF-α in the CCl 4 -induced mice. Liver injury markers were examined, and the associated changes were detected using CCK8, Hoechst staining, Western blotting, and other molecular assays. PMG effectively reversed the morphological changes, restoring cell shape and structure in TNF-α injured cells. PMG protected against hepatotoxicity in vitro and in vivo . PMG and TNFR1 maintained robust binding activities. TNF-α overexpression counteracted the hepatoprotective effects of PMG, attenuating its influence on IL-6, AST, ALT, apoptosis, and the inactivation of the PI3K/AKT/NF-κB signaling pathway. PMG protected against TNF-α-induced hepatotoxicity by regulating the PI3K/AKT/NF-κB signaling pathway through TNF-α inhibition, suggesting that PMG holds potential as a novel therapeutic agent for acute liver injury. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Acute liver injury TNF-α PMG Hepatoprotection PI3K/AKT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Liver disease is a widespread clinical disease that poses a significant global healthcare challenge. Acute liver injury is the initiating condition of almost all the liver diseases, with persistent liver injury potentially leading to liver cirrhosis and liver cancer 1 . These liver diseases, which have varying etiologies, rapid progression, and serious consequences, will cause liver failure and hepatic encephalopathy if not treated promptly. Acute liver injury represents an inflammation-mediated process of hepatocellular damage, marked by hepatocyte necrosis and inflammation instigated by immune responses 2 . The immune response mediator, tumor necrosis factor-α (TNF-α), assumes a crucial role in inflammation through its interaction with receptors 3 . The TNF receptors are abundant in liver, makes the hepatocytes highly sensitive to TNF-α. TNF-α binding to its receptor, TNFR1, induces hepatocyte apoptosis and inflammatory responses, contributing significantly to the occurrence and progression of acute liver injury 4 , 5 . Modern pharmacological studies have showed the hepatoprotective effects of Radix et Rhizoma Rhei, a Chinese herb that has been widely used for centuries in treating liver disorders 6 , 7 . Aloe-Emodin and physcion are the main active ingredients of Radix et Rhizoma Rhei, both of them had been reported to exhibit effects on acute liver injury 8 , 9 . But poor oral bioavailability of these quinones limits the clinical applications of them. Physcion-8-O-β-D-monoglucoside (PMG) represents the glycosides form of physcion, demonstrating higher blood concentrations following oral administration compared to aloe-Emodin and physcion 10 . Our findings, obtained through surface plasmon resonance biosensor (SPR) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) analysis, demonstrate that PMG, a bioactive compound from Radix et Rhizoma Rhei, exhibits reversible binding to TNFR1 with a dissociation constant (Kd) of 376 nM. Additionally, PMG inhibited both cytotoxicity and apoptosis induced by TNF-α in L929 cells 11 . Whatever, the hepatoprotective effects of PMG in acute liver injury remains unexplored. Given the critical involvement of TNF-α and its receptor TNFR1 in liver damage, we examined the impact of PMG on acute liver injury induced by carbon tetrachloride (CCl 4 ) in mice, and found a reduction in pathological liver tissue injury, as well as decreased levels of nuclear factor kappa-B (NF-κB) p65, TNF-α, interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1) 12 . These promising findings prompted us to investigate the mechanisms by which PMG affects hepatocytes and explore the role of TNF-α acting in the present study. TNF-α, a pro-inflammatory cytokine predominantly secreted by activated monocytes and macrophages, exerts a wide range of biological activities. TNF-α upregulates inflammatory factors (NOS, IL-4, IL-6, and COX-2), thereby enhancing inflammation. It also promotes leukocyte adhesion to the vascular endothelium by upregulating the expression of VCAM-1 and ICAM-1. TNF-α involves the activation of the NF-kB signaling pathway, which subsequently increased the secretion of TNF-α, IL-1, IL-8, and NO, ultimately triggering an inflammatory cascade reaction. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway had been reported be involved in TNF-α-induced inflammatory-associated factors expression and subsequent activation of the downstream target NF-κB 13–15 . This study is aim to elucidate the contribution of TNF-α in the protective activities of PMG against acute liver injury. Firstly, the ability of PMG to mitigate TNF-α-induced hepatotoxicity was evaluated in vitro . Secondly, we explored the contribution of TNF-α in the protective effects of PMG on CCl 4 injury mice by TNF-α overexpression. Considering the important role of PI3K and its downstream effector Akt in TNF-α-mediated NF-κB activation, PMG as a novel chemical inhibitor is capable of effectively blocking the TNF-α-induced activation of the PI3K/AKT/NF-κB signaling cascade. Materials and methods Animals and cells Adult (25 g, 6–8 weeks of age) male ICR mice were acquired from Shanghai SLAC Laboratory Animal Co. Ltd (Production License: SCXK (Shanghai) 2022-0004). All animal studies were carried out in strict accordance with the ARRIVE guidelines. The experimental protocol received ethical approval from the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (ethics review number: FJTCM IACUC 2021097). The alpha mouse liver 12 (AML-12) cells were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China) and maintained in DMEM medium supplemented with 10% fetal bovine serum for optimal growth. Adeno-Associated Virus (AAV) Injections in Mice For in vivo gene transfer, mice were received a single tail vein injection of 100µl AAV resuspension 2 weeks before PMG treatment. AAV2/9-m-TNF (TNF AAV) and AAV2/9-Negative Control (NC AAV) were provided by Hanbio (Shanghai, China) at a titer of 1.1*10^12 vg/mL and 1.6*10^12 vg/mL respectively. Infection efficiency in the liver was verified by PCR and by Western blot. Animals and treatments The mouse model of acute liver injury was generated using intraperitoneal CCl₄ injection (0.1% in corn oil, 0.1mL/10g). Mice were grouped randomly (n = 8 per group): (1) control without any treatment, (2) CCl 4 , (3) PMG (20 mg/kg) + CCl 4 , PMG was intragastrically administered twice daily for 3 consecutive days before CCl 4 injection. (4) AAV-TNF + PMG + CCl 4 , (5) NC-AAV + PMG + CCl 4 , (6) 300 mg/kg bifendate (DDB, positive control group). After 16 h CCl 4 treatment, blood was collected via ocular extraction, after which the mice were euthanized by cervical dislocation, and the liver tissues were harvested. Cell administration Exponentially growing AML-12 cells were harvested and plated in complete medium. After 24 hours of incubation, the culture medium was removed, followed by 4 hours of serum starvation for synchronization. And then cells were pretreated with PMG (80 µM, 40 µM, 20 µM, 10 µM) for 6 hours. TNF-α (20 ng/mL) and actinomycin D (ActD) (10 ng/mL) were added after PMG treatment, and the cells were incubated for another 24 hours. Cell viability assay Following cell treatment, 10 µL of CCK-8 solution was added to each well of a 96-well plate, which was then incubated for 1 h at 37°C. The absorbance at 450 nm was measured using an Infinite 200PRO microplate reader. Detection of ALT and AST Serum samples were collected from animal experiments. Cell culture supernatants were collected from culture well plates at the indicated time points. ALT and AST activity levels were determined in both serum and supernatant using commercially available assay kits, according to the manufacturer’s instructions. Hoechst 33342 Staining The embedded liver tissues were cut into 4 µm slices. Then the slices were dewaxed in water, rinsed twice with PBS, and stained with Hoechst 33342 staining solution (diluted at 1:100). The number of positive cells were observed and counted under a laser scanning confocal microscope. Isolation of total protein and nucleoprotein The liver tissue was lysed in RIPA lysis buffer for total protein of extraction: total protein in the supernatant was collected after samples were centrifuged. Nucleoprotein were extracted according to the instructions of the nucleoprotein extraction kit (KeyGEN BioTECH, Nanjing, China): The tissue samples was homogenized in Buffer A and then centrifuged. The supernatant was removed, and Buffer C was added. After mixed thoroughly by vortexing, the samples were centrifuged, and the nucleoprotein in supernatant was collected. The concentration of the isolated protein was measured by BCA assay. Western blot analysis Protein samples were separated by 10% SDS-polyacrylamide gel, and then transferred onto polyvinylirdenediflouride (PVDF) membranes. The blocked membranes were incubated with the indicated primary antibodies: Anti-TNFα (1:1000; cat. No. CPA2174), anti- IL-6 (1:1000; cat. No. CPA4914), anti-AKT (1:1000; cat. No. CPA3481), anti-AKT (pT308) (1:1000; cat. No. CPA1032), anti-PI3K p85 alpha (1:1000; cat. No. CPA3286) and anti- PI3K p85 alpha (pY607) (1:1000; cat. No. CPA7142), anti-PCNA (1:2000; cat. No. CPA9205) and anti-β-actin (1:1000; cat. No. CPA9100) antibodies were obtained from Cohesion Biosciences Limited, and anti- Caspase-3 (1:1000 cat. No.9662), anti- NF-κBp65 (1:1000; cat. No. 8242), anti-IκB (1:1000; cat. No. 4814), anti- pIκB (Ser32) (1:1000; cat. No.2859) were purchased from Cell Signaling Technology, Inc.. Membranes were incubated with the appropriate secondary antibody second antibody on the next day. The bolts were visualized with enhanced chemiluminescence (ECL) staining. Image Lab 4.1 software was used to analyze gray values quantitatively. Quantitative real-time PCR (qRT-PCR) RNA samples were obtained from cell or liver tissue sources using Trizol reagent (Vazyme, China). After extraction, the purity of the RNA was evaluated. cDNA synthesis was generated with the QRT SuperMix for qPCR (+ gDNA wiper) kit following the manufacturer's protocol. Relative mRNA expression levels were determined using the 2-ΔΔCt method, with β-actin as a normalization control. The qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix (Vazyme) and the Applied Biosystems 7900HT real-time PCR system. The primer sequences are shown in Table 1. Molecular docking of the PMG and TNFR1 To further substantiate the interaction between PMG and TNFR1, we conducted a molecular docking analysis of these two components. Initially, we obtained the 3D protein structure of TNFR1 from the RCSB PDB database. The 2D structure of PMG was generated using ChemBioDraw. Subsequently, molecular docking was conducted with the prepared ligands and proteins utilizing the CDOCKER algorithm within Discovery Studio. Binding affinities of the ligands within the active site were determined using CDOCKER energy scores. Scoring functions, including hydrogen bond counts and distances, CDOCKER energy, and CDOCKER interaction energy, were initially calculated. Statistical analysis Data were analyzed statistically using SPSS 20.0 software (Chicago, IL, USA), with results presented as means ± SDs. A comparison of the results was performed with one-way ANOVA. Data with normal distribution and homogeneity of variance were compared by LSD test, and the other data were analyzed by Games-Howell test. A P value < 0.05 was considered statistically significant. Results PMG rehabilitates TNF-α-induced injury in AML-12 cells The AML-12 cell line, derived from normal mouse liver hepatocytes, serves as a valuable model for toxicology research. The in vitro model of TNF-α-induced hepatocytes injury model was used to investigate the hepatoprotection efficacy of PMG. Hepatotoxicity of AML-12 induced by TNF-α was sensitized by RNA synthesis inhibitors actinomycin D. As shown in Fig. 1 A, the normal AML-12 cells exhibited characteristic hepatocyte morphology, including the presence of peroxisomes and bile canalicular like structure. However, exposure to TNF-α, sensitized by actinomycin D, resulted in the irregular shape, cell shrinkage of AML-12 cells. Notably, PMG effectively reversed these morphological changes, restoring cell shape and structure. Cell viability, expressed as a percentage relative to control cells, was not significantly affected by the PMG exposure (Fig. 1 B). However, AML-12 cells treated with TNF-α (20 ng/mL) and actinomycin D (10 ng/mL) for 24 hours significantly decreased the cell survival rate to 72.1% compared to the control group. While treatment with 80µM PMG notably increased cell viability after 24 hours (Fig. 1 C). PMG at a concentration of 80 µM significantly protected cells against TNF-α-induced injury. Aspartate transaminase (ALT) and alanine transaminase (AST) are crucial indicators used to assess liver function and determine liver damage. The model group exhibited significantly elevated ALT and AST levels in culture supernatants compared to the control group ( P < 0.05). Both 80µM and 40µM PMG treatment significantly reduced ALT and AST levels ( P < 0.05) (Fig. 1 D), suggesting a protective effect against TNF-α-mediated injury in AML-12 cells. Treatment with TNF-α and actinomycin D induced a substantial increase in apoptosis in AML-12 cells, manifested by a marked upregulation of the apoptotic marker cleaved Caspase-3. Interestingly, a significant decrease in cleaved caspase-3 levels was observed in AML-12 cells following 24-hour treatment with either 80 µM or 40 µM PMG ( P < 0.01 or 0.05) (Fig. 1 F-G). Validation of the PMG–TNFR1 interaction by molecular docking Molecular docking was used to assess the binding interaction between PMG and TNFR1. The PDB code (3T6Q) of TNFR1 was acquired from RCSB. The binding energy of the PMG–TNFR1 interaction (CDOCKER ENERGY: 33.3411 kcal/mol) indicated robust binding activities (Fig. 2 A-B). The coordination of PMG to TNFR1 was stabilized by hydrogen bonds with residues (Fig. 2 C). The level of TNF-α and IL-6 in CCl 4 mice liver TNF-α, a pro-inflammatory cytokine, initiates the production of cytokines, including IL-6, which contribute to liver injury 16 . Our results showed significantly elevated protein and mRNA levels of TNF-α and IL-6 in CCl 4 -induced liver injury. However, pretreatment with PMG inhibited these increases. Furthermore, TNF-α overexpression was successfully achieved in the TNF-AAV group, in contrast to the NC-AAV group. Interestingly, the inhibitory effect of PMG on TNF-α and IL-6 production was reversed by TNF-α overexpression (Fig. 3 ). TNF-α overexpression influenced the effects of PMG on serum Alt and Ast activities in CCl 4 mice liver ALT and AST levels in serum were observed significantly increased in the CCl 4 group when compared to the control group. However, PMG pretreatment significantly reduced these activities. And the effects of PMG on serum AST and ALT were abolished in mice of TNF-α overexpression by AAV-TNF injection (Fig. 4 ), while there were no changes in the NC-AAV group. TNF-α overexpression blocked the inhibition of PMG on CCl 4 -caused hepatocyte necrosis The Hoechst 33342 staining assay were conducted to determine whether TNF-α overexpression inhibited the effects of PMG on cell apoptosis in CCl 4 -induced acute liver injury in mice. As shown in Fig. 5 , PMG inhibited CCl 4 -induced cell apoptosis and nuclear condensation in liver, while TNF-α overexpression blocked the effects of PMG (Fig. 5 A, B). Additionally, Caspase 3 is a critical executioner of apoptosis, the cleaved form of Caspase3 serves as a marker for activated Caspase-3 was analyzed by Western blot. The level of cleaved Caspase-3 expression in the CCl 4 group was significantly higher than the control group. However, treatment with PMG significantly reduced the expression of cleaved Caspase3. Interestingly, TNF-α overexpression blunted these effects (Fig. 5 C, D). TNF-α overexpression blocks the inhibition of PMG on the NFκB activation through PI3K/ Akt signaling pathway To elucidate the mechanism by which PMG protects against CCl₄-induced hepatocyte necrosis, the involvement of the PI3K/Akt/NF-κB signaling pathway was investigated. CCl₄ treatment resulted in IκB kinase complex degradation and NF-κB p65 nuclear translocation, thus indicating activation of the NF-κB pathway in the in vivo model of acute liver injury. PMG treatment markedly decreased IκB kinase phosphorylation and reduced the intranuclear expression of NF-κB p65. In summary, PMG suppressed the nuclear translocation of NF-κ B p65 and activation of the NF-κB pathway (Fig. 6 A-C). Furthermore, the roles of PI3K and Akt in the inhibition of PMG on NFκB activation were further investigated. PI3K and Akt phosphorylation increased in CCl4-induced mice. However, PMG inhibited not only NF-κB activation but also PI3K/Akt phosphorylation. Intriguingly, TNF-α overexpression abrogated the modulation of PMG on PI3K/Akt/NFκB signaling pathway resulting in increased PI3K and Akt phosphorylation (Fig. 7 ), as well as the activation of NFκB. Collectively, these data strongly support the conclusion that PMG inhibits TNF-α-induced hepatocyte necrosis via a mechanism dependent on the PI3K/Akt/NF-κB pathway. Discussion The inflammatory response is a key driver in liver injury progression. Different causes of liver injury create distinct inflammatory microenvironments within the liver, each characterized by rapid immune cell recruitment and the release of diverse inflammatory mediators. TNF-α, a multifaceted cytokine, plays a central role in this process, influencing immune system development, cell growth, and hepatocyte death 17 . While TNF-α initiates intracellular signaling cascades that promote apoptosis and accelerate hepatocyte death, it is also essential for hepatocyte proliferation during liver regeneration 18 , 19 . Although TNF-α inhibitors used in clinical settings can alleviate the liver injury, they also abolished the protective effects of TNF-α. TNF-α initiates diverse biological effects through signaling pathways activated by its two receptors, TNFR1 (p55) and TNFR2 (p75). TNFR1 signaling is responsible for the majority of TNF-α-induced effects, such as inflammatory responses and hepatocyte apoptosis, in the context of hepatitis 4 . Conversely, TNFR2 plays an important role in the recovery from hepatitis 20 . The unique superiorities of traditional Chinese medicine (TCM) lie in its two-way regulating functions and multi-channel, multi-target approach. Active ingredients in TCM have been found to block TNF-α production, and inhibit hepatocyte apoptosis to prevent the occurrence and development of liver injury 21 , 22 . DDB, a synthetic schisandrin C analog and clinically used hepatoprotective agent from Schisandra chinensis, serves as a positive control in hepatoprotective research 23 – 25 . The application of Radix et Rhizoma Rhei in the treatment of hepatic disorders dates back several centuries in traditional Chinese medicine. PMG, a compound found in the extract of Rheum officinale (one of the origins of TCM Rhizoma Rhei), binds to TNFR1 and inhibits TNF-α-induced cytotoxicity and apoptosis in L929 cells 11 . However, there are no studies targeting the impact of PMG on hepatocyte protection. Our in vitro results demonstrated that PMG protects AML-12 cells from TNF-α-induced damage. TNF-α-induced hepatotoxicity produced liver cell injury, as evidenced by the low cell viability, high AST and ALT level in supernatants, and high expression levels of cleaved Caspase 3, indicating that the TNF-α mediated hepatocyte apoptosis and hepatotoxicity were ameliorated by PMG. This discovery indicates that PMG may be a valuable lead compound in the development of new treatments for TNF-α-induced liver injury. CCl₄-induced liver injury in rodents serves as a well-established model for both acute and chronic disease. The regulatory effect of NF-κB signaling and anti-apoptotic pathway on TNF-α was observed in acute liver injured model induced by CCl 4 19, 26 . Our findings indicate that the CCl 4 operations resulted in increased levels of serum AST and ALT, along with elevated TNF-α and IL-6 expression, validating the successful induction of liver injury. The hepatoprotective effects of PMG were evident in the prevention of CCl₄-induced increases in serum AST and ALT, coupled with the attenuation of apoptosis by downregulating TNF-α, IL-6, and cleaved caspase-3. These data are consistent with the results of the in vitro experiments. Given that the liver is the organ with the highest uptake of plasmid DNA in the body 27 , tail vein injection appears to be rather specific for the liver. Overexpression of TNF-α, achieved via AAV-mediated gene delivery and confirmed by measuring mRNA and protein levels in mouse liver, abrogated the protective effects of PMG against CCl₄-induced liver injury. The regulatory effects of PMG on serum AST and ALT levels, and on the expression of IL-6 and cleaved caspase-3, were abolished by hepatic overexpression of TNF-α. The therapeutic efficacy of PMG in this model of acute liver injury appears to be attributable to its ability to effectively inhibit TNF-α/TNFR1-mediated hepatocytotoxicity and inflammatory responses, thereby preventing the development of liver failure. These data provide novel evidence on TNF-α function in the therapeutic effect of PMG in acute liver injury. TNF-R1, but not TNF-R2, possesses a death domain (DD) crucial for protein-protein interactions and the initiation of apoptosis 28 . This DD facilitates the recruitment of other DD-containing proteins, initiating a signaling cascade linking death receptors to caspase activation. TNF-α stimulation primarily activates this pathway through the RIP1/FADD/caspase-8 complex 29 . In hepatocytes, TNF-α binding to TNF-R1 activates caspase-8, leading to the activation of caspases-3 and − 7 and subsequent apoptosis 30 . Caspase-3, known as the executer of cell apoptosis, is a necessary killer proteinase in the cell apoptosis process and is more specific in the detection of apoptotic cells than Hoechst. Consistently, we demonstrated that PMG’s effects in inhibiting apoptosis relieved both TNF-α and CCl 4 induced hepatocytotoxicity and were responsible for its treatment of acute liver injury. TNF-α-induced cell death requires the canonical NF-κB signaling pathway 4 . This pathway centers on NF-κB, a nuclear transcription factor regulating genes crucial for apoptosis and inflammation. In its inactive form, cytoplasmic NF-κB is normally inhibited by IκB proteins. Phosphorylation of IκB by the IKK complex (IKK1/IKKa, IKK2/IKKb, and IKK3/IKKg) triggers IκB ubiquitination and degradation, releasing NF-κB dimers to translocate to the nucleus, bind DNA, and activate target genes 31 . TNF-α, a key activator, binds to its receptor, recruiting TRADD, which then recruits TRAF2 and NIK. NIK/MEKK1-mediated phosphorylation of IKK1 and IKK2 subsequently leads to IκB phosphorylation. Our experiments showed that PMG treatment markedly decreased IκB kinase phosphorylation and diminished the intranuclear expression of NF-κB. Overexpression of TNF-α, however, blocked the inhibition of PMG on the NFκB activation. TNF-α-induced NF-κB activation requires PI3K and its downstream target Akt 14 , 32 . PI3K is activated upon interaction with growth factor receptors or phosphorylated connexins, undergoing a conformational change. The resulting production of PIP3 recruits and activates Akt at the plasma membrane 33 . Then, as a downstream target of AKT, the NF-κB also shows increased activation. Our work demonstrated that CCl₄-induced acute liver injury is characterized by upregulation of the PI3K/Akt/NF-κB pathway, evidenced by increased PI3K and Akt phosphorylation. The protective effect of PMG on liver injury was associated with its suppression on PI3K/AKT/NF-κB signalling apoptosis in hepatocyte. Our findings suggest that PMG-regulated PI3K/AKT pathway was mediated by TNF-α. Overexpression of TNF-α significantly altered several key downstream events, including caspase-3 activation, PI3K and Akt phosphorylation, and NF-κB nuclear translocation, implying that induction of TNF-α was involved in the PMG regulation of PI3K/AKT/ NF-κB pathway. Therefore, our data revealed that TNF-α/PI3K/Akt/NF-κB axis was the main mechanism of PMG alleviates acute liver injury (Fig. 8 ). This study provides valuable insights into the therapeutic potential of PMG and underscores the importance of TNF-α in modulating this pathway. Conclusions In summary, PMG exerts a hepatoprotective effect against TNF-α-induced acute liver injury, primarily by suppressing TNF-α/PI3K/Akt/NF-κB signaling. These findings support the development of PMG as a novel therapeutic agent for acute liver injury. Abbreviations PMG, physcion-8-O-β-D-monoglucoside; TNF-α, tumor necrosis factor-α; CCl 4 , carbon tetrachloride; AST, Aspartate aminotransferase; ALT, alanine aminotransferase; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; NF-κB, nuclear factor kappa-B; ICAM-1, intercellular adhesion molecule-1; IL-6, interleukin-6. Declarations Declaration of interests The authors have no conflicts of interest to declare. Funding This work was supported by the National Natural Science Foundation of China under Grant (NO. 82204378); and the School Fund of Fujian University of Traditional Chinese Medicine under Grant (NO. X2019007-Tanlent; NO. XJC2022003). Author Contribution Zhihui Chen and Ting Chen: methodology, data curation, and Project administration, Zixin Chen, Wenchuan Luo and Wen Xu: investigation, software, and formal analysis, Mei Huang and Yuqin Zhang: conceptualization; Ru Jia and Ya Lin: resources, supervision; Lihong Nan and Yaping Chen: writing–original draft, funding acquisition. All authors reviewed the manuscript. Data Availability Data are available from the corresponding author on reasonable request. References Schümann, J. & Kammüller, M. in Encyclopedia of Immunotoxicology (ed Hans-Werner Vohr) 1-6 (Springer Berlin Heidelberg, 2005). Shojaie, L., Iorga, A. & Dara, L. Cell Death in Liver Diseases: A Review. International journal of molecular sciences 21 , doi:10.3390/ijms21249682 (2020). Chen, G. & Goeddel, D. V. TNF-R1 signaling: a beautiful pathway. Science 296 , 1634-1635, doi:10.1126/science.1071924 (2002). Cubero, F. J. et al. TNFR1 determines progression of chronic liver injury in the IKKgamma/Nemo genetic model. Cell Death Differ. 20 , 1580-1592, doi:10.1038/cdd.2013.112 (2013). Ding, W. X. & Yin, X. M. Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury. J. Cell. Mol. Med. 8 , 445-454, doi:10.1111/j.1582-4934.2004.tb00469.x (2004). Zhuang, T. et al. Hepatoprotection and hepatotoxicity of Chinese herb Rhubarb (Dahuang): How to properly control the "General (Jiang Jun)" in Chinese medical herb. Biomed. Pharmacother. 127 , 110224, doi:10.1016/j.biopha.2020.110224 (2020). Wang, J. B. et al. Hepatotoxicity or hepatoprotection? Pattern recognition for the paradoxical effect of the Chinese herb Rheum palmatum L. in treating rat liver injury. PLoS One 6 , e24498, doi:10.1371/journal.pone.0024498 (2011). Arosio, B. et al. Aloe-Emodin quinone pretreatment reduces acute liver injury induced by carbon tetrachloride. Pharmacol. Toxicol. 87 , 229-233, doi:10.1034/j.1600-0773.2000.d01-79.x (2000). Zhang, L. Y., Su, W. H., Xiong, Y. & Wang, D. Protective effect of physcione on acute liver injury in rats. Chinese Journal of Primary Medicine and Pharmacy 14 , 2014-2015 (2007). Cheng, W. H. Study on the pharmacokinetics of Polygonum multiflorum Thunb. , Beijing University of Chinese Medicine, (2020). Cao, Y. et al. Identification of a ligand for tumor necrosis factor receptor from Chinese herbs by combination of surface plasmon resonance biosensor and UPLC-MS. Anal. Bioanal. Chem. 408 , 5359-5367, doi:10.1007/s00216-016-9633-6 (2016). Chen, T., Chen, Y. P., Cao, Y. J. & Nan, L. H. The effects of Physcion-8-O-β-D-monoglucoside on acute liver injury induced by CCl4 in mice. Fujian Journal of Traditional Chinese Medicine 53 , 18-21+27, doi:10.13260/j.cnki.jfjtcm.012419 (2022). Xu, P., Wang, J., Yang, Z. W., Lou, X. L. & Chen, C. Regulatory roles of the PI3K/Akt signaling pathway in rats with severe acute pancreatitis. PLoS One 8 , e81767, doi:10.1371/journal.pone.0081767 (2013). Chen, H. W., Lin, A. H., Chu, H. C., Li, C. C. & Liu, K. L. Inhibition of TNF-α-Induced Inflammation by andrographolide via down-regulation of the PI3K/Akt signaling pathway. J. Nat. Prod. 74 , 2408-2413 (2011). Liu, Y. & Tie, L. Apolipoprotein M and sphingosine-1-phosphate complex alleviates TNF-alpha-induced endothelial cell injury and inflammation through PI3K/AKT signaling pathway. BMC Cardiovasc. Disord. 19 , 279, doi:10.1186/s12872-019-1263-4 (2019). Locksley, R. M., Killeen, N. & Lenardo, M. J. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104 , 487-501, doi:10.1016/s0092-8674(01)00237-9 (2001). Cai, L. et al. AMPK dependent protective effects of metformin on tumor necrosis factor-induced apoptotic liver injury. Biochem. Biophys. Res. Commun. 465 , 381-386, doi:10.1016/j.bbrc.2015.08.009 (2015). Zhao, S. et al. The concentration of tumor necrosis factor-alpha determines its protective or damaging effect on liver injury by regulating Yap activity. Cell Death Dis. 11 , 70, doi:10.1038/s41419-020-2264-z (2020). Dong, Y. et al. The protective or damaging effect of Tumor necrosis factor-alpha in acute liver injury is concentration-dependent. Cell & bioscience 6 , 8, doi:10.1186/s13578-016-0074-x (2016). Shibata, H. et al. The therapeutic effect of TNFR1-selective antagonistic mutant TNF-alpha in murine hepatitis models. Cytokine 44 , 229-233, doi:10.1016/j.cyto.2008.07.003 (2008). Ma, H. X. et al. Effect of Yinchen Siling Granule combined with Bone Mesenchymal Stem Cells Transplantation on NF-κB,iNOS and TNF-α in Rats with Acute Liver Failure. Journal of Emergency in Traditional Chinese Medicine 28 , 801-805, doi:10.3969/j.issn.1004-745X.2019.05.013 (2019). Jia, Y. J. et al. Effects of Gan-fu-kang on TNF-α mediated liver inflammatory injury and fibrosis in rats. Chinese Journal of Microecology 28 , 146-150, doi:10.13381/j.cnki.cjm.201602006 (2016). Yuan, W., Jian, F., &Rong, Y. Bifendate inhibits autophagy at multiple steps and attenuates oleic acid-induced lipid accumulation. Biochem. Biophys. Res. Commun. 631 , 115-123, doi:10.1016/j.bbrc.2022.09.067, (2022). Zou, J., Qi, F., Ye, L., &Yao, S. Protective role of grape seed proanthocyanidins against ccl4 induced acute liver injury in mice. Med. Sci. Monit. 22 , 880-889, doi:10.12659/MSM.895552, (2016). Li, H. et al. Bifendate indirectly inhibits alanine transaminase release without direct interaction in vitro and in vivo. Acta Poloniae Pharmaceutica - Drug Research 77 (4), doi:10.32383/appdr/125077, (2020). Li, J. et al. Hepatoprotective Effects of Heracleum candicans Against Carbon Tetrachloride-Induced Acute Liver Injury in Rats. Dose Response 19 , 15593258211029510, doi:10.1177/15593258211029510 (2021). Chen, X. & Calvisi, D. F. Hydrodynamic transfection for generation of novel mouse models for liver cancer research. Am. J. Pathol. 184 , 912-923, doi:10.1016/j.ajpath.2013.12.002 (2014). Wajant, H., Pfizenmaier, K. & Scheurich, P. Tumor necrosis factor signaling. Cell Death Differ. 10 , 45-65, doi:10.1038/sj.cdd.4401189 (2003). Zhou, H. et al. TNF-alpha Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection. Cell. Mol. Neurobiol. 42 , 1841-1857, doi:10.1007/s10571-021-01063-w (2022). Czaja, A. J. Targeting apoptosis in autoimmune hepatitis. Dig. Dis. Sci. 59 , 2890-2904, doi:10.1007/s10620-014-3284-2 (2014). Bottero, V., Withoff, S. & Verma, I. M. NF-kappaB and the regulation of hematopoiesis. Cell Death Differ. 13 , 785-797, doi:10.1038/sj.cdd.4401888 (2006). Kettritz, R. et al. Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils. J. Am. Soc. Nephrol. 13 , 1740-1749, doi:10.1097/01.asn.0000019411.36000.06 (2002). Ding, C. et al. Taxifolin, a novel food, attenuates acute alcohol-induced liver injury in mice through regulating the NF-kappaB-mediated inflammation and PI3K/Akt signalling pathways. Pharm. Biol. 59 , 868-879, doi:10.1080/13880209.2021.1942504 (2021). Additional Declarations No competing interests reported. Supplementary Files Supplementaryoriginalblots.pdf Cite Share Download PDF Status: Published Journal Publication published 09 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 30 Sep, 2025 Reviews received at journal 25 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers invited by journal 15 Sep, 2025 Editor assigned by journal 15 Sep, 2025 Editor invited by journal 12 Sep, 2025 Submission checks completed at journal 12 Sep, 2025 First submitted to journal 12 Sep, 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. 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-7542054","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":518174324,"identity":"c4471edf-73cd-49f8-84ef-44395a98cedb","order_by":0,"name":"Zhihui Chen","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhihui","middleName":"","lastName":"Chen","suffix":""},{"id":518174325,"identity":"3012b2a7-5660-4426-b4d7-4e12f8746f26","order_by":1,"name":"Ting Chen","email":"","orcid":"","institution":"Anyuan County People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Chen","suffix":""},{"id":518174326,"identity":"ffae752b-f157-48cf-bb9d-bb3eb27adbfb","order_by":2,"name":"Zixin Chen","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zixin","middleName":"","lastName":"Chen","suffix":""},{"id":518174327,"identity":"cce260d1-22a1-4b4e-9650-6d1cedede2b5","order_by":3,"name":"Wenchuan Luo","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenchuan","middleName":"","lastName":"Luo","suffix":""},{"id":518174328,"identity":"de4e7103-576f-4f4c-8181-d6eabee3a77b","order_by":4,"name":"Wen Xu","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Xu","suffix":""},{"id":518174329,"identity":"d650b8be-438e-4db0-ba8b-0e7c332acf9c","order_by":5,"name":"Mei Huang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Huang","suffix":""},{"id":518174330,"identity":"c98306c3-2533-4f6d-9245-0ce6d6832331","order_by":6,"name":"Yuqin Zhang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuqin","middleName":"","lastName":"Zhang","suffix":""},{"id":518174331,"identity":"a13874f2-8c52-4e3e-8b38-e5d64099749a","order_by":7,"name":"Ru Jia","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ru","middleName":"","lastName":"Jia","suffix":""},{"id":518174332,"identity":"5338d573-901c-43f9-a3aa-a2e86eae6885","order_by":8,"name":"Ya Lin","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Lin","suffix":""},{"id":518174333,"identity":"d9f39404-0cbf-4b99-a47a-a90794d5e686","order_by":9,"name":"Lihong Nan","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lihong","middleName":"","lastName":"Nan","suffix":""},{"id":518174334,"identity":"e97455b5-6248-41c2-a819-9d140293494b","order_by":10,"name":"Yaping Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIie3RMQrCMBSA4ZRAujxwjRT1CpWAOhS9ikFol3Z3bCnUpTjrLXoDUzK4CK6OitBdXdy0VgentKNg/iFDyEfIC0I63Q/WwkZ0nN6drhmKasMI60h7Ecf2JXUZCNmQ2Ltt0l4RyVOBGxJ04DEDcL3U2OfXA3I6mcDFUSWMFY/OMHKCFEls+chlmSBDW0Uwfd8SbJAk2EeSZwIIVRFCeWIBkR68yaOeAOTV86cfIuoJNaNqyP3yLczy7RlbSzJQkok0T6+v7AHNTzd/Pu4st3GhJF/3iXJ5jQo3O1/WChsf1el0uj/rCYH5TDX9HAIeAAAAAElFTkSuQmCC","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-09-05 08:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7542054/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7542054/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-38701-6","type":"published","date":"2026-03-09T15:58:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92064842,"identity":"0a032c45-6169-4fe9-9468-363d488116ce","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1807291,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscriptrevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/a2c1ffd9916f6d66b63db8b7.docx"},{"id":92064828,"identity":"b3ad1a4b-8ae3-4187-90d3-56b8b60650ad","added_by":"auto","created_at":"2025-09-24 08:45:30","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11221,"visible":true,"origin":"","legend":"","description":"","filename":"e9d9cb08464d4f84b1dcbcaecb85647c.json","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/3d26873e98a71d3d67a687ad.json"},{"id":92064830,"identity":"e413a093-fa08-45f4-ab34-3c8fe14d9abb","added_by":"auto","created_at":"2025-09-24 08:45:30","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":895292,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryoriginalblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/ec81fd85ae766c3c2f6d74ed.pdf"},{"id":92064843,"identity":"dd7830b7-7b8e-4c18-af4e-c28da8f3fbc9","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98853,"visible":true,"origin":"","legend":"","description":"","filename":"e9d9cb08464d4f84b1dcbcaecb85647c1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/edc113b9c8368a67156f41be.xml"},{"id":92065241,"identity":"6ee7d2b6-283f-4525-a0b1-10a9d1f48072","added_by":"auto","created_at":"2025-09-24 08:53:33","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":465378,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/a10fa43a7b301e219394c5a3.jpeg"},{"id":92065245,"identity":"61ba2e82-b159-4179-9cd9-b0de79527f4e","added_by":"auto","created_at":"2025-09-24 08:53:34","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106515,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/592d999a703a6cc8c7f85f8a.jpeg"},{"id":92064825,"identity":"7d2a0055-4ffe-4c21-98dc-04455f61219d","added_by":"auto","created_at":"2025-09-24 08:45:29","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116462,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/c93352c7786dada5fecf3778.jpeg"},{"id":92064835,"identity":"e73b3c61-a1a0-466c-a6de-ea3b90751583","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45535,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/629c21ced27aab7f8963d0e5.jpeg"},{"id":92064838,"identity":"d08c05da-a932-4a9a-a135-dc8f6368c1f3","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":691620,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/a8a4c708e9673d4f81d59ea4.jpeg"},{"id":92064839,"identity":"f9e68dba-637d-4729-bb1e-d7421350daa7","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67213,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/c2c6d66cdfed51f31ae11865.jpeg"},{"id":92064833,"identity":"5bc05b42-9948-49f5-b6b5-151154b8edc6","added_by":"auto","created_at":"2025-09-24 08:45:30","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115842,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/fe3f74e8a107808dcca3e27e.jpeg"},{"id":92065243,"identity":"fb812c51-fbff-4420-865b-54acd8ab8c8d","added_by":"auto","created_at":"2025-09-24 08:53:33","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147022,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/a81309dc0bcabfe8fa0a2633.jpeg"},{"id":92064837,"identity":"fc7ffff4-2cf0-4422-afb9-f7ad97cb5650","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75454,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/4585d041f511c5c172cf4db0.png"},{"id":92064854,"identity":"582fcd0d-682d-4492-8e9a-7559e5fa68be","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49923,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/8bbe8348a4f9f2db7bd55be5.png"},{"id":92064847,"identity":"f6361748-6874-4bf1-ac8c-f236a16235d4","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33763,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/c41a7581298a01bb18dc69a0.png"},{"id":92064852,"identity":"80073953-f85d-460c-9bbb-a3eb3856c9ce","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25156,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/53b45eb9cd4db60b34279289.png"},{"id":92064840,"identity":"0d52876d-883d-4889-be28-91f9a033dbc3","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116222,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/a3468ec9d10d56efd51bd772.png"},{"id":92064846,"identity":"d020f8c2-9d23-44d2-a533-2c0fb4a901f0","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42501,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/bff58ca0171cce1c01833c75.png"},{"id":92066240,"identity":"3c7eefff-543f-433a-9b3a-922881796ada","added_by":"auto","created_at":"2025-09-24 09:01:33","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40128,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/e1fabc67aad2acd8f6e741e3.png"},{"id":92064834,"identity":"bf3bff74-2a22-452a-b9b3-39aa3c973bc3","added_by":"auto","created_at":"2025-09-24 08:45:30","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46119,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/fe3f4ec8780d2485fbd5fce6.png"},{"id":92064851,"identity":"183c0cad-20ca-42f8-aafc-19407383d9ea","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96589,"visible":true,"origin":"","legend":"","description":"","filename":"e9d9cb08464d4f84b1dcbcaecb85647c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/62d9cb0f8dff8a736695a109.xml"},{"id":92064855,"identity":"4d00a17e-6155-43b8-9a0f-07844caef8cd","added_by":"auto","created_at":"2025-09-24 08:45:33","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107577,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/bb7b1a742ee5bc668ecefa1b.html"},{"id":92064848,"identity":"3ab8ee14-cb39-4a49-96e1-80702c5c8b2d","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1074035,"visible":true,"origin":"","legend":"\u003cp\u003eA. Brightfield microscopy (200× magnification) of TNF-α-induced AML-12 cells pretreated with PMG (10-80 μM) for 24 h. B. Effect of PMG (10-80 μM) on AML-12 cell viability. C. The inhibitory effect of PMG on TNF-α-mediated cell cytotoxicity. D. Effects of PMG on AST and ALT levels in the culture supernatant. E. The chemical structure of PMG. F-G. Western blot analysis of the Cleaved Caspase-3 and total Caspase-3 in AML-12 cells. All data are presented as the means ± SDs (n = 6). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the Control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the TNF-α/ActD group.\u003c/p\u003e\n\u003cp\u003ePMG attenuated TNF-α-induced injury in AML-12 cells.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/bf75418f38e64453f54946a3.png"},{"id":92064827,"identity":"604fcd28-78c4-4885-8434-0383cde43490","added_by":"auto","created_at":"2025-09-24 08:45:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":570886,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking analysis of PMG and TNFR1.\u003c/p\u003e\n\u003cp\u003eA. 2D interaction diagram of PMG in the active site of TNFR1. B. The PMG-TNFR1 complex. C. Hydrogen bond interaction diagram of PMG and TNFR1.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/f2e1e38b5386b562f2f9a345.png"},{"id":92064831,"identity":"352e4f9a-600d-43f6-9a75-c591ec89c8f0","added_by":"auto","created_at":"2025-09-24 08:45:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":679757,"visible":true,"origin":"","legend":"\u003cp\u003eTNF-α overexpressed in mice and the IL-6 levels changed evidently.\u003c/p\u003e\n\u003cp\u003emRNA levels and expression levels of proteins related to TNF-α overexpressed in CCl\u003csub\u003e4\u003c/sub\u003e mice were calculated. A-C. Western blot analysis of TNF-α and IL-6 in each treatment group. D-E. RT-qPCR measurements of TNF-α and IL-6 in mice treated with CCl\u003csub\u003e4\u003c/sub\u003e with or without AVV-TNF compared to untreated controls. All data are expressed as the means ± SDs (n = 6). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs the Control group；\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs the CCl\u003csub\u003e4 \u003c/sub\u003egroup, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the PMG group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/2e738a84eee8e093fcbc9f92.png"},{"id":92064836,"identity":"980313ce-4758-4e40-9968-eeec2a193143","added_by":"auto","created_at":"2025-09-24 08:45:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":525824,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of PMG on serum levels of aspartate transaminase (ALT) and alanine transaminase (AST).\u003cstrong\u003e \u003c/strong\u003eAll data are expressed as the means ± SDs (n = 8). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs the Control group；\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the CCl\u003csub\u003e4 \u003c/sub\u003egroup, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs the PMG group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/d0c4a7e71ac4a0d6168956a3.png"},{"id":92064850,"identity":"83e77489-4c09-486e-a06d-1f9a3ca7e199","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1095584,"visible":true,"origin":"","legend":"\u003cp\u003eTNF-α overexpression blocked the inhibition of PMG on CCl\u003csub\u003e4\u003c/sub\u003e-caused hepatocyte necroptosis.\u003c/p\u003e\n\u003cp\u003eA. The scanning images showed the morphology of cell apoptosis by Hoechst 33342(×400). The yellow arrow indicates the apoptotic cells were dyed blue. B. Analysis of apoptotic cells. C-D. Western blot analysis of the Cleaved Caspase-3 and total Caspase-3 in liver tissues of mice in each group. All data are expressed as the means ± SDs (n = 6). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs the Control group；\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the CCl\u003csub\u003e4 \u003c/sub\u003egroup, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the PMG group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/39f3d29c74e47f5203ce0a23.png"},{"id":92065244,"identity":"883d0dd4-0e5d-4683-b13f-29deb7ae0026","added_by":"auto","created_at":"2025-09-24 08:53:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":793836,"visible":true,"origin":"","legend":"\u003cp\u003eTNF-α overexpression blocks the inhibition of PMG on the NFκB activation.\u003c/p\u003e\n\u003cp\u003eA-B. Expressions of NF-κB p65 proteins in nucleus were measured by western blotting analysis. The internal standard for nucleoprotein was proliferating cell nuclear antigen (PCNA). C-D. Phosphorylation\u0026nbsp;of IκB in total protein were detected by western blotting analysis. The internal standard for total protein was β-Actin. All data are expressed as the means ± SDs (n = 6). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs the Control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the CCl\u003csub\u003e4 \u003c/sub\u003egroup, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the PMG group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/dd725ee0bca796171c3093d9.png"},{"id":92064845,"identity":"9a3d0a34-5a34-427a-b077-bbabe676075b","added_by":"auto","created_at":"2025-09-24 08:45:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":649240,"visible":true,"origin":"","legend":"\u003cp\u003eTNF-α overexpression reversed the regulation of PMG on the phosphorylation levels of PI3K and AKT in CCl\u003csub\u003e4\u003c/sub\u003e mice.\u003c/p\u003e\n\u003cp\u003eA. The phosphorylation levels of PI3K and AKT in liver tissues were detected by western blotting analysis. B-C. The relative optical densities of the levels of phosphorylated PI3K and AKT. All data are expressed as the means ± SDs (n = 6). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs the Control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the CCl\u003csub\u003e4 \u003c/sub\u003egroup, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05 vs the PMG group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/326f3f23c903e02e8bc1a7c5.png"},{"id":92065240,"identity":"01f65d3e-05b5-4495-8b31-7d8550766c4f","added_by":"auto","created_at":"2025-09-24 08:53:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":748466,"visible":true,"origin":"","legend":"\u003cp\u003eThe underlying mechanisms by which PMG attenuates hepatotoxicity by inhibiting TNF-α injury.\u003c/p\u003e\n\u003cp\u003eSchematic diagram illustrating the involvement of the TNF-α/PI3K/Akt/NF-κB signaling pathway in the effects of PMG on CCl\u003csub\u003e4\u003c/sub\u003e-induced acute liver injury.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/cba4cdb67c6287655b80d6ce.png"},{"id":104739338,"identity":"6f3823a3-262d-4914-91a1-77b2ea0978fe","added_by":"auto","created_at":"2026-03-16 16:03:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7063814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/768bf436-39a8-4e26-8659-d7cedbe52677.pdf"},{"id":92064826,"identity":"f15ed0e1-ed97-45a0-9ca4-fea2ae8b5a6b","added_by":"auto","created_at":"2025-09-24 08:45:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":895292,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryoriginalblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7542054/v1/3ef2a29c9ce45bdea7f234c0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physcion-8-O-β-D-monoglucoside protects hepatocytes from TNF-α-mediated apoptosis by suppressing the PI3K/AKT /NF-κB signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiver disease is a widespread clinical disease that poses a significant global healthcare challenge. Acute liver injury is the initiating condition of almost all the liver diseases, with persistent liver injury potentially leading to liver cirrhosis and liver cancer \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These liver diseases, which have varying etiologies, rapid progression, and serious consequences, will cause liver failure and hepatic encephalopathy if not treated promptly.\u003c/p\u003e\u003cp\u003eAcute liver injury represents an inflammation-mediated process of hepatocellular damage, marked by hepatocyte necrosis and inflammation instigated by immune responses\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The immune response mediator, tumor necrosis factor-α (TNF-α), assumes a crucial role in inflammation through its interaction with receptors\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The TNF receptors are abundant in liver, makes the hepatocytes highly sensitive to TNF-α. TNF-α binding to its receptor, TNFR1, induces hepatocyte apoptosis and inflammatory responses, contributing significantly to the occurrence and progression of acute liver injury\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eModern pharmacological studies have showed the hepatoprotective effects of Radix et Rhizoma Rhei, a Chinese herb that has been widely used for centuries in treating liver disorders\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Aloe-Emodin and physcion are the main active ingredients of Radix et Rhizoma Rhei, both of them had been reported to exhibit effects on acute liver injury\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. But poor oral bioavailability of these quinones limits the clinical applications of them. Physcion-8-O-β-D-monoglucoside (PMG) represents the glycosides form of physcion, demonstrating higher blood concentrations following oral administration compared to aloe-Emodin and physcion\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Our findings, obtained through surface plasmon resonance biosensor (SPR) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) analysis, demonstrate that PMG, a bioactive compound from Radix et Rhizoma Rhei, exhibits reversible binding to TNFR1 with a dissociation constant (Kd) of 376 nM. Additionally, PMG inhibited both cytotoxicity and apoptosis induced by TNF-α in L929 cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Whatever, the hepatoprotective effects of PMG in acute liver injury remains unexplored. Given the critical involvement of TNF-α and its receptor TNFR1 in liver damage, we examined the impact of PMG on acute liver injury induced by carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) in mice, and found a reduction in pathological liver tissue injury, as well as decreased levels of nuclear factor kappa-B (NF-κB) p65, TNF-α, interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1)\u003csup\u003e12\u003c/sup\u003e. These promising findings prompted us to investigate the mechanisms by which PMG affects hepatocytes and explore the role of TNF-α acting in the present study.\u003c/p\u003e\u003cp\u003eTNF-α, a pro-inflammatory cytokine predominantly secreted by activated monocytes and macrophages, exerts a wide range of biological activities. TNF-α upregulates inflammatory factors (NOS, IL-4, IL-6, and COX-2), thereby enhancing inflammation. It also promotes leukocyte adhesion to the vascular endothelium by upregulating the expression of VCAM-1 and ICAM-1. TNF-α involves the activation of the NF-kB signaling pathway, which subsequently increased the secretion of TNF-α, IL-1, IL-8, and NO, ultimately triggering an inflammatory cascade reaction. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway had been reported be involved in TNF-α-induced inflammatory-associated factors expression and subsequent activation of the downstream target NF-κB\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study is aim to elucidate the contribution of TNF-α in the protective activities of PMG against acute liver injury. Firstly, the ability of PMG to mitigate TNF-α-induced hepatotoxicity was evaluated \u003cem\u003ein vitro\u003c/em\u003e. Secondly, we explored the contribution of TNF-α in the protective effects of PMG on CCl\u003csub\u003e4\u003c/sub\u003e injury mice by TNF-α overexpression. Considering the important role of PI3K and its downstream effector Akt in TNF-α-mediated NF-κB activation, PMG as a novel chemical inhibitor is capable of effectively blocking the TNF-α-induced activation of the PI3K/AKT/NF-κB signaling cascade.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals and cells\u003c/h2\u003e\u003cp\u003eAdult (25 g, 6\u0026ndash;8 weeks of age) male ICR mice were acquired from Shanghai SLAC Laboratory Animal Co. Ltd (Production License: SCXK (Shanghai) 2022-0004). All animal studies were carried out in strict accordance with the ARRIVE guidelines. The experimental protocol received ethical approval from the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (ethics review number: FJTCM IACUC 2021097). The alpha mouse liver 12 (AML-12) cells were obtained from Procell Life Science\u0026amp;Technology Co., Ltd (Wuhan, China) and maintained in DMEM medium supplemented with 10% fetal bovine serum for optimal growth.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAdeno-Associated Virus (AAV) Injections in Mice\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e gene transfer, mice were received a single tail vein injection of 100\u0026micro;l AAV resuspension 2 weeks before PMG treatment. AAV2/9-m-TNF (TNF AAV) and AAV2/9-Negative Control (NC AAV) were provided by Hanbio (Shanghai, China) at a titer of 1.1*10^12 vg/mL and 1.6*10^12 vg/mL respectively. Infection efficiency in the liver was verified by PCR and by Western blot.\u003c/p\u003e\n\u003ch3\u003eAnimals and treatments\u003c/h3\u003e\n\u003cp\u003eThe mouse model of acute liver injury was generated using intraperitoneal CCl₄ injection (0.1% in corn oil, 0.1mL/10g). Mice were grouped randomly (n\u0026thinsp;=\u0026thinsp;8 per group): (1) control without any treatment, (2) CCl\u003csub\u003e4\u003c/sub\u003e, (3) PMG (20 mg/kg)\u0026thinsp;+\u0026thinsp;CCl\u003csub\u003e4\u003c/sub\u003e, PMG was intragastrically administered twice daily for 3 consecutive days before CCl\u003csub\u003e4\u003c/sub\u003e injection. (4) AAV-TNF\u0026thinsp;+\u0026thinsp;PMG\u0026thinsp;+\u0026thinsp;CCl\u003csub\u003e4\u003c/sub\u003e, (5) NC-AAV\u0026thinsp;+\u0026thinsp;PMG\u0026thinsp;+\u0026thinsp;CCl\u003csub\u003e4\u003c/sub\u003e, (6) 300 mg/kg bifendate (DDB, positive control group). After 16 h CCl\u003csub\u003e4\u003c/sub\u003e treatment, blood was collected via ocular extraction, after which the mice were euthanized by cervical dislocation, and the liver tissues were harvested.\u003c/p\u003e\n\u003ch3\u003eCell administration\u003c/h3\u003e\n\u003cp\u003eExponentially growing AML-12 cells were harvested and plated in complete medium. After 24 hours of incubation, the culture medium was removed, followed by 4 hours of serum starvation for synchronization. And then cells were pretreated with PMG (80 \u0026micro;M, 40 \u0026micro;M, 20 \u0026micro;M, 10 \u0026micro;M) for 6 hours. TNF-α (20 ng/mL) and actinomycin D (ActD) (10 ng/mL) were added after PMG treatment, and the cells were incubated for another 24 hours.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eFollowing cell treatment, 10 \u0026micro;L of CCK-8 solution was added to each well of a 96-well plate, which was then incubated for 1 h at 37\u0026deg;C. The absorbance at 450 nm was measured using an Infinite 200PRO microplate reader.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDetection of ALT and AST\u003c/h2\u003e\u003cp\u003eSerum samples were collected from animal experiments. Cell culture supernatants were collected from culture well plates at the indicated time points. ALT and AST activity levels were determined in both serum and supernatant using commercially available assay kits, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHoechst 33342 Staining\u003c/h3\u003e\n\u003cp\u003eThe embedded liver tissues were cut into 4 \u0026micro;m slices. Then the slices were dewaxed in water, rinsed twice with PBS, and stained with Hoechst 33342 staining solution (diluted at 1:100). The number of positive cells were observed and counted under a laser scanning confocal microscope.\u003c/p\u003e\n\u003ch3\u003eIsolation of total protein and nucleoprotein\u003c/h3\u003e\n\u003cp\u003eThe liver tissue was lysed in RIPA lysis buffer for total protein of extraction: total protein in the supernatant was collected after samples were centrifuged. Nucleoprotein were extracted according to the instructions of the nucleoprotein extraction kit (KeyGEN BioTECH, Nanjing, China): The tissue samples was homogenized in Buffer A and then centrifuged. The supernatant was removed, and Buffer C was added. After mixed thoroughly by vortexing, the samples were centrifuged, and the nucleoprotein in supernatant was collected. The concentration of the isolated protein was measured by BCA assay.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eProtein samples were separated by 10% SDS-polyacrylamide gel, and then transferred onto polyvinylirdenediflouride (PVDF) membranes. The blocked membranes were incubated with the indicated primary antibodies: Anti-TNFα (1:1000; cat. No. CPA2174), anti- IL-6 (1:1000; cat. No. CPA4914), anti-AKT (1:1000; cat. No. CPA3481), anti-AKT (pT308) (1:1000; cat. No. CPA1032), anti-PI3K p85 alpha (1:1000; cat. No. CPA3286) and anti- PI3K p85 alpha (pY607) (1:1000; cat. No. CPA7142), anti-PCNA (1:2000; cat. No. CPA9205) and anti-β-actin (1:1000; cat. No. CPA9100) antibodies were obtained from Cohesion Biosciences Limited, and anti- Caspase-3 (1:1000 cat. No.9662), anti- NF-κBp65 (1:1000; cat. No. 8242), anti-IκB (1:1000; cat. No. 4814), anti- pIκB (Ser32) (1:1000; cat. No.2859) were purchased from Cell Signaling Technology, Inc.. Membranes were incubated with the appropriate secondary antibody second antibody on the next day. The bolts were visualized with enhanced chemiluminescence (ECL) staining. Image Lab 4.1 software was used to analyze gray values quantitatively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eRNA samples were obtained from cell or liver tissue sources using Trizol reagent (Vazyme, China). After extraction, the purity of the RNA was evaluated. cDNA synthesis was generated with the QRT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) kit following the manufacturer's protocol. Relative mRNA expression levels were determined using the 2-ΔΔCt method, with β-actin as a normalization control. The qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix (Vazyme) and the Applied Biosystems 7900HT real-time PCR system. The primer sequences are shown in Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking of the PMG and TNFR1\u003c/h2\u003e\u003cp\u003eTo further substantiate the interaction between PMG and TNFR1, we conducted a molecular docking analysis of these two components. Initially, we obtained the 3D protein structure of TNFR1 from the RCSB PDB database. The 2D structure of PMG was generated using ChemBioDraw. Subsequently, molecular docking was conducted with the prepared ligands and proteins utilizing the CDOCKER algorithm within Discovery Studio. Binding affinities of the ligands within the active site were determined using CDOCKER energy scores. Scoring functions, including hydrogen bond counts and distances, CDOCKER energy, and CDOCKER interaction energy, were initially calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed statistically using SPSS 20.0 software (Chicago, IL, USA), with results presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs. A comparison of the results was performed with one-way ANOVA. Data with normal distribution and homogeneity of variance were compared by LSD test, and the other data were analyzed by Games-Howell test. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePMG rehabilitates TNF-α-induced injury in AML-12 cells\u003c/h2\u003e\u003cp\u003eThe AML-12 cell line, derived from normal mouse liver hepatocytes, serves as a valuable model for toxicology research. The \u003cem\u003ein vitro\u003c/em\u003e model of TNF-α-induced hepatocytes injury model was used to investigate the hepatoprotection efficacy of PMG. Hepatotoxicity of AML-12 induced by TNF-α was sensitized by RNA synthesis inhibitors actinomycin D.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the normal AML-12 cells exhibited characteristic hepatocyte morphology, including the presence of peroxisomes and bile canalicular like structure. However, exposure to TNF-α, sensitized by actinomycin D, resulted in the irregular shape, cell shrinkage of AML-12 cells. Notably, PMG effectively reversed these morphological changes, restoring cell shape and structure. Cell viability, expressed as a percentage relative to control cells, was not significantly affected by the PMG exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, AML-12 cells treated with TNF-α (20 ng/mL) and actinomycin D (10 ng/mL) for 24 hours significantly decreased the cell survival rate to 72.1% compared to the control group. While treatment with 80\u0026micro;M PMG notably increased cell viability after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). PMG at a concentration of 80 \u0026micro;M significantly protected cells against TNF-α-induced injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAspartate transaminase (ALT) and alanine transaminase (AST) are crucial indicators used to assess liver function and determine liver damage. The model group exhibited significantly elevated ALT and AST levels in culture supernatants compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both 80\u0026micro;M and 40\u0026micro;M PMG treatment significantly reduced ALT and AST levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), suggesting a protective effect against TNF-α-mediated injury in AML-12 cells.\u003c/p\u003e\u003cp\u003eTreatment with TNF-α and actinomycin D induced a substantial increase in apoptosis in AML-12 cells, manifested by a marked upregulation of the apoptotic marker cleaved Caspase-3. Interestingly, a significant decrease in cleaved caspase-3 levels was observed in AML-12 cells following 24-hour treatment with either 80 \u0026micro;M or 40 \u0026micro;M PMG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eValidation of the PMG\u0026ndash;TNFR1 interaction by molecular docking\u003c/h2\u003e\u003cp\u003eMolecular docking was used to assess the binding interaction between PMG and TNFR1. The PDB code (3T6Q) of TNFR1 was acquired from RCSB. The binding energy of the PMG\u0026ndash;TNFR1 interaction (CDOCKER ENERGY: 33.3411 kcal/mol) indicated robust binding activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). The coordination of PMG to TNFR1 was stabilized by hydrogen bonds with residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eThe level of TNF-α and IL-6 in CCl\u003csub\u003e4\u003c/sub\u003e mice liver\u003c/h2\u003e\u003cp\u003eTNF-α, a pro-inflammatory cytokine, initiates the production of cytokines, including IL-6, which contribute to liver injury\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Our results showed significantly elevated protein and mRNA levels of TNF-α and IL-6 in CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury. However, pretreatment with PMG inhibited these increases. Furthermore, TNF-α overexpression was successfully achieved in the TNF-AAV group, in contrast to the NC-AAV group. Interestingly, the inhibitory effect of PMG on TNF-α and IL-6 production was reversed by TNF-α overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTNF-α overexpression influenced the effects of PMG on serum Alt and Ast activities in CCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e \u003cb\u003emice liver\u003c/b\u003e\u003c/p\u003e\u003cp\u003eALT and AST levels in serum were observed significantly increased in the CCl\u003csub\u003e4\u003c/sub\u003e group when compared to the control group. However, PMG pretreatment significantly reduced these activities. And the effects of PMG on serum AST and ALT were abolished in mice of TNF-α overexpression by AAV-TNF injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), while there were no changes in the NC-AAV group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eTNF-α overexpression blocked the inhibition of PMG on CCl\u003csub\u003e4\u003c/sub\u003e-caused hepatocyte necrosis\u003c/h2\u003e\u003cp\u003eThe Hoechst 33342 staining assay were conducted to determine whether TNF-α overexpression inhibited the effects of PMG on cell apoptosis in CCl\u003csub\u003e4\u003c/sub\u003e-induced acute liver injury in mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, PMG inhibited CCl\u003csub\u003e4\u003c/sub\u003e-induced cell apoptosis and nuclear condensation in liver, while TNF-α overexpression blocked the effects of PMG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, Caspase 3 is a critical executioner of apoptosis, the cleaved form of Caspase3 serves as a marker for activated Caspase-3 was analyzed by Western blot. The level of cleaved Caspase-3 expression in the CCl\u003csub\u003e4\u003c/sub\u003e group was significantly higher than the control group. However, treatment with PMG significantly reduced the expression of cleaved Caspase3. Interestingly, TNF-α overexpression blunted these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTNF-α overexpression blocks the inhibition of PMG on the NFκB activation through PI3K/ Akt signaling pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the mechanism by which PMG protects against CCl₄-induced hepatocyte necrosis, the involvement of the PI3K/Akt/NF-κB signaling pathway was investigated. CCl₄ treatment resulted in IκB kinase complex degradation and NF-κB p65 nuclear translocation, thus indicating activation of the NF-κB pathway in the \u003cem\u003ein vivo\u003c/em\u003e model of acute liver injury. PMG treatment markedly decreased IκB kinase phosphorylation and reduced the intranuclear expression of NF-κB p65. In summary, PMG suppressed the nuclear translocation of NF-κ B p65 and activation of the NF-κB pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, the roles of PI3K and Akt in the inhibition of PMG on NFκB activation were further investigated. PI3K and Akt phosphorylation increased in CCl4-induced mice. However, PMG inhibited not only NF-κB activation but also PI3K/Akt phosphorylation. Intriguingly, TNF-α overexpression abrogated the modulation of PMG on PI3K/Akt/NFκB signaling pathway resulting in increased PI3K and Akt phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), as well as the activation of NFκB. Collectively, these data strongly support the conclusion that PMG inhibits TNF-α-induced hepatocyte necrosis via a mechanism dependent on the PI3K/Akt/NF-κB pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe inflammatory response is a key driver in liver injury progression. Different causes of liver injury create distinct inflammatory microenvironments within the liver, each characterized by rapid immune cell recruitment and the release of diverse inflammatory mediators. TNF-α, a multifaceted cytokine, plays a central role in this process, influencing immune system development, cell growth, and hepatocyte death\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. While TNF-α initiates intracellular signaling cascades that promote apoptosis and accelerate hepatocyte death, it is also essential for hepatocyte proliferation during liver regeneration\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although TNF-α inhibitors used in clinical settings can alleviate the liver injury, they also abolished the protective effects of TNF-α. TNF-α initiates diverse biological effects through signaling pathways activated by its two receptors, TNFR1 (p55) and TNFR2 (p75). TNFR1 signaling is responsible for the majority of TNF-α-induced effects, such as inflammatory responses and hepatocyte apoptosis, in the context of hepatitis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Conversely, TNFR2 plays an important role in the recovery from hepatitis\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe unique superiorities of traditional Chinese medicine (TCM) lie in its two-way regulating functions and multi-channel, multi-target approach. Active ingredients in TCM have been found to block TNF-α production, and inhibit hepatocyte apoptosis to prevent the occurrence and development of liver injury\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. DDB, a synthetic schisandrin C analog and clinically used hepatoprotective agent from Schisandra chinensis, serves as a positive control in hepatoprotective research\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e–\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe application of Radix et Rhizoma Rhei in the treatment of hepatic disorders dates back several centuries in traditional Chinese medicine. PMG, a compound found in the extract of \u003cem\u003eRheum officinale\u003c/em\u003e (one of the origins of TCM Rhizoma Rhei), binds to TNFR1 and inhibits TNF-α-induced cytotoxicity and apoptosis in L929 cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, there are no studies targeting the impact of PMG on hepatocyte protection. Our \u003cem\u003ein vitro\u003c/em\u003e results demonstrated that PMG protects AML-12 cells from TNF-α-induced damage. TNF-α-induced hepatotoxicity produced liver cell injury, as evidenced by the low cell viability, high AST and ALT level in supernatants, and high expression levels of cleaved Caspase 3, indicating that the TNF-α mediated hepatocyte apoptosis and hepatotoxicity were ameliorated by PMG. This discovery indicates that PMG may be a valuable lead compound in the development of new treatments for TNF-α-induced liver injury.\u003c/p\u003e\u003cp\u003eCCl₄-induced liver injury in rodents serves as a well-established model for both acute and chronic disease. The regulatory effect of NF-κB signaling and anti-apoptotic pathway on TNF-α was observed in acute liver injured model induced by CCl\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e19, 26\u003c/sup\u003e. Our findings indicate that the CCl\u003csub\u003e4\u003c/sub\u003e operations resulted in increased levels of serum AST and ALT, along with elevated TNF-α and IL-6 expression, validating the successful induction of liver injury. The hepatoprotective effects of PMG were evident in the prevention of CCl₄-induced increases in serum AST and ALT, coupled with the attenuation of apoptosis by downregulating TNF-α, IL-6, and cleaved caspase-3. These data are consistent with the results of the \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e\u003cp\u003eGiven that the liver is the organ with the highest uptake of plasmid DNA in the body\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, tail vein injection appears to be rather specific for the liver. Overexpression of TNF-α, achieved via AAV-mediated gene delivery and confirmed by measuring mRNA and protein levels in mouse liver, abrogated the protective effects of PMG against CCl₄-induced liver injury. The regulatory effects of PMG on serum AST and ALT levels, and on the expression of IL-6 and cleaved caspase-3, were abolished by hepatic overexpression of TNF-α. The therapeutic efficacy of PMG in this model of acute liver injury appears to be attributable to its ability to effectively inhibit TNF-α/TNFR1-mediated hepatocytotoxicity and inflammatory responses, thereby preventing the development of liver failure. These data provide novel evidence on TNF-α function in the therapeutic effect of PMG in acute liver injury.\u003c/p\u003e\u003cp\u003eTNF-R1, but not TNF-R2, possesses a death domain (DD) crucial for protein-protein interactions and the initiation of apoptosis\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This DD facilitates the recruitment of other DD-containing proteins, initiating a signaling cascade linking death receptors to caspase activation. TNF-α stimulation primarily activates this pathway through the RIP1/FADD/caspase-8 complex\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In hepatocytes, TNF-α binding to TNF-R1 activates caspase-8, leading to the activation of caspases-3 and − 7 and subsequent apoptosis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Caspase-3, known as the executer of cell apoptosis, is a necessary killer proteinase in the cell apoptosis process and is more specific in the detection of apoptotic cells than Hoechst. Consistently, we demonstrated that PMG’s effects in inhibiting apoptosis relieved both TNF-α and CCl\u003csub\u003e4\u003c/sub\u003e induced hepatocytotoxicity and were responsible for its treatment of acute liver injury.\u003c/p\u003e\u003cp\u003eTNF-α-induced cell death requires the canonical NF-κB signaling pathway\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This pathway centers on NF-κB, a nuclear transcription factor regulating genes crucial for apoptosis and inflammation. In its inactive form, cytoplasmic NF-κB is normally inhibited by IκB proteins. Phosphorylation of IκB by the IKK complex (IKK1/IKKa, IKK2/IKKb, and IKK3/IKKg) triggers IκB ubiquitination and degradation, releasing NF-κB dimers to translocate to the nucleus, bind DNA, and activate target genes\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. TNF-α, a key activator, binds to its receptor, recruiting TRADD, which then recruits TRAF2 and NIK. NIK/MEKK1-mediated phosphorylation of IKK1 and IKK2 subsequently leads to IκB phosphorylation. Our experiments showed that PMG treatment markedly decreased IκB kinase phosphorylation and diminished the intranuclear expression of NF-κB. Overexpression of TNF-α, however, blocked the inhibition of PMG on the NFκB activation.\u003c/p\u003e\u003cp\u003eTNF-α-induced NF-κB activation requires PI3K and its downstream target Akt \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. PI3K is activated upon interaction with growth factor receptors or phosphorylated connexins, undergoing a conformational change. The resulting production of PIP3 recruits and activates Akt at the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Then, as a downstream target of AKT, the NF-κB also shows increased activation. Our work demonstrated that CCl₄-induced acute liver injury is characterized by upregulation of the PI3K/Akt/NF-κB pathway, evidenced by increased PI3K and Akt phosphorylation. The protective effect of PMG on liver injury was associated with its suppression on PI3K/AKT/NF-κB signalling apoptosis in hepatocyte.\u003c/p\u003e\u003cp\u003eOur findings suggest that PMG-regulated PI3K/AKT pathway was mediated by TNF-α. Overexpression of TNF-α significantly altered several key downstream events, including caspase-3 activation, PI3K and Akt phosphorylation, and NF-κB nuclear translocation, implying that induction of TNF-α was involved in the PMG regulation of PI3K/AKT/ NF-κB pathway. Therefore, our data revealed that TNF-α/PI3K/Akt/NF-κB axis was the main mechanism of PMG alleviates acute liver injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This study provides valuable insights into the therapeutic potential of PMG and underscores the importance of TNF-α in modulating this pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, PMG exerts a hepatoprotective effect against TNF-α-induced acute liver injury, primarily by suppressing TNF-α/PI3K/Akt/NF-κB signaling. These findings support the development of PMG as a novel therapeutic agent for acute liver injury.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePMG, physcion-8-O-β-D-monoglucoside; TNF-α, tumor necrosis factor-α; CCl\u003csub\u003e4\u003c/sub\u003e, carbon tetrachloride; AST, Aspartate aminotransferase; ALT, alanine aminotransferase; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; NF-κB, nuclear factor kappa-B; ICAM-1, intercellular adhesion molecule-1; IL-6, interleukin-6.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of interests\u003c/h2\u003e\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Natural Science Foundation of China under Grant (NO. 82204378); and the School Fund of Fujian University of Traditional Chinese Medicine under Grant (NO. X2019007-Tanlent; NO. XJC2022003).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZhihui Chen and Ting Chen: methodology, data curation, and Project administration, Zixin Chen, Wenchuan Luo and Wen Xu: investigation, software, and formal analysis, Mei Huang and Yuqin Zhang: conceptualization; Ru Jia and Ya Lin: resources, supervision; Lihong Nan and Yaping Chen: writing\u0026ndash;original draft, funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSch\u0026uuml;mann, J. \u0026amp; Kamm\u0026uuml;ller, M. in \u003cem\u003eEncyclopedia of Immunotoxicology\u003c/em\u003e (ed Hans-Werner Vohr) 1-6 (Springer Berlin Heidelberg, 2005).\u003c/li\u003e\n\u003cli\u003eShojaie, L., Iorga, A. \u0026amp; Dara, L. Cell Death in Liver Diseases: A Review. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, doi:10.3390/ijms21249682 (2020).\u003c/li\u003e\n\u003cli\u003eChen, G. \u0026amp; Goeddel, D. V. TNF-R1 signaling: a beautiful pathway. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e296\u003c/strong\u003e, 1634-1635, doi:10.1126/science.1071924 (2002).\u003c/li\u003e\n\u003cli\u003eCubero, F. J.\u003cem\u003e et al.\u003c/em\u003e TNFR1 determines progression of chronic liver injury in the IKKgamma/Nemo genetic model. \u003cem\u003eCell Death Differ.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 1580-1592, doi:10.1038/cdd.2013.112 (2013).\u003c/li\u003e\n\u003cli\u003eDing, W. X. \u0026amp; Yin, X. M. Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury. \u003cem\u003eJ. Cell. Mol. Med.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 445-454, doi:10.1111/j.1582-4934.2004.tb00469.x (2004).\u003c/li\u003e\n\u003cli\u003eZhuang, T.\u003cem\u003e et al.\u003c/em\u003e Hepatoprotection and hepatotoxicity of Chinese herb Rhubarb (Dahuang): How to properly control the \u0026quot;General (Jiang Jun)\u0026quot; in Chinese medical herb. \u003cem\u003eBiomed. Pharmacother.\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 110224, doi:10.1016/j.biopha.2020.110224 (2020).\u003c/li\u003e\n\u003cli\u003eWang, J. B.\u003cem\u003e et al.\u003c/em\u003e Hepatotoxicity or hepatoprotection? Pattern recognition for the paradoxical effect of the Chinese herb Rheum palmatum L. in treating rat liver injury. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e24498, doi:10.1371/journal.pone.0024498 (2011).\u003c/li\u003e\n\u003cli\u003eArosio, B.\u003cem\u003e et al.\u003c/em\u003e Aloe-Emodin quinone pretreatment reduces acute liver injury induced by carbon tetrachloride. \u003cem\u003ePharmacol. Toxicol.\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 229-233, doi:10.1034/j.1600-0773.2000.d01-79.x (2000).\u003c/li\u003e\n\u003cli\u003eZhang, L. Y., Su, W. H., Xiong, Y. \u0026amp; Wang, D. Protective effect of physcione on acute liver injury in rats. \u003cem\u003eChinese Journal of Primary Medicine and Pharmacy\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 2014-2015 (2007).\u003c/li\u003e\n\u003cli\u003eCheng, W. H. \u003cem\u003eStudy on the pharmacokinetics of Polygonum multiflorum Thunb. \u003c/em\u003e, Beijing University of Chinese Medicine, (2020).\u003c/li\u003e\n\u003cli\u003eCao, Y.\u003cem\u003e et al.\u003c/em\u003e Identification of a ligand for tumor necrosis factor receptor from Chinese herbs by combination of surface plasmon resonance biosensor and UPLC-MS. \u003cem\u003eAnal. Bioanal. Chem.\u003c/em\u003e \u003cstrong\u003e408\u003c/strong\u003e, 5359-5367, doi:10.1007/s00216-016-9633-6 (2016).\u003c/li\u003e\n\u003cli\u003eChen, T., Chen, Y. P., Cao, Y. J. \u0026amp; Nan, L. H. The effects of Physcion-8-O-\u0026beta;-D-monoglucoside on acute liver injury induced by CCl4 in mice. \u003cem\u003eFujian Journal of Traditional Chinese Medicine \u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 18-21+27, doi:10.13260/j.cnki.jfjtcm.012419 (2022).\u003c/li\u003e\n\u003cli\u003eXu, P., Wang, J., Yang, Z. W., Lou, X. L. \u0026amp; Chen, C. Regulatory roles of the PI3K/Akt signaling pathway in rats with severe acute pancreatitis. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e81767, doi:10.1371/journal.pone.0081767 (2013).\u003c/li\u003e\n\u003cli\u003eChen, H. W., Lin, A. H., Chu, H. C., Li, C. C. \u0026amp; Liu, K. L. Inhibition of TNF-\u0026alpha;-Induced Inflammation by andrographolide via down-regulation of the PI3K/Akt signaling pathway. \u003cem\u003eJ. Nat. Prod.\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 2408-2413 (2011).\u003c/li\u003e\n\u003cli\u003eLiu, Y. \u0026amp; Tie, L. Apolipoprotein M and sphingosine-1-phosphate complex alleviates TNF-alpha-induced endothelial cell injury and inflammation through PI3K/AKT signaling pathway. \u003cem\u003eBMC Cardiovasc. Disord.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 279, doi:10.1186/s12872-019-1263-4 (2019).\u003c/li\u003e\n\u003cli\u003eLocksley, R. M., Killeen, N. \u0026amp; Lenardo, M. J. The TNF and TNF receptor superfamilies: integrating mammalian biology. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 487-501, doi:10.1016/s0092-8674(01)00237-9 (2001).\u003c/li\u003e\n\u003cli\u003eCai, L.\u003cem\u003e et al.\u003c/em\u003e AMPK dependent protective effects of metformin on tumor necrosis factor-induced apoptotic liver injury. \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e \u003cstrong\u003e465\u003c/strong\u003e, 381-386, doi:10.1016/j.bbrc.2015.08.009 (2015).\u003c/li\u003e\n\u003cli\u003eZhao, S.\u003cem\u003e et al.\u003c/em\u003e The concentration of tumor necrosis factor-alpha determines its protective or damaging effect on liver injury by regulating Yap activity. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 70, doi:10.1038/s41419-020-2264-z (2020).\u003c/li\u003e\n\u003cli\u003eDong, Y.\u003cem\u003e et al.\u003c/em\u003e The protective or damaging effect of Tumor necrosis factor-alpha in acute liver injury is concentration-dependent. \u003cem\u003eCell \u0026amp; bioscience\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 8, doi:10.1186/s13578-016-0074-x (2016).\u003c/li\u003e\n\u003cli\u003eShibata, H.\u003cem\u003e et al.\u003c/em\u003e The therapeutic effect of TNFR1-selective antagonistic mutant TNF-alpha in murine hepatitis models. \u003cem\u003eCytokine\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 229-233, doi:10.1016/j.cyto.2008.07.003 (2008).\u003c/li\u003e\n\u003cli\u003eMa, H. X.\u003cem\u003e et al.\u003c/em\u003e Effect of Yinchen Siling Granule combined with Bone Mesenchymal Stem Cells Transplantation on NF-\u0026kappa;B,iNOS and TNF-\u0026alpha; in Rats with Acute Liver Failure. \u003cem\u003eJournal of Emergency in Traditional Chinese Medicine\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 801-805, doi:10.3969/j.issn.1004-745X.2019.05.013 (2019).\u003c/li\u003e\n\u003cli\u003eJia, Y. J.\u003cem\u003e et al.\u003c/em\u003e Effects of Gan-fu-kang on TNF-\u0026alpha; mediated liver inflammatory injury and fibrosis in rats. \u003cem\u003eChinese Journal of Microecology\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 146-150, doi:10.13381/j.cnki.cjm.201602006 (2016).\u003c/li\u003e\n\u003cli\u003eYuan, W., Jian, F., \u0026amp;Rong, Y. Bifendate inhibits autophagy at multiple steps and attenuates oleic acid-induced lipid accumulation. \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e \u003cstrong\u003e631\u003c/strong\u003e, 115-123, doi:10.1016/j.bbrc.2022.09.067, (2022). \u003c/li\u003e\n\u003cli\u003eZou, J.,\u003cem\u003e \u003c/em\u003eQi, F., Ye, L., \u0026amp;Yao, S. Protective role of grape seed proanthocyanidins against ccl4 induced acute liver injury in mice. \u003cem\u003eMed. Sci. Monit.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 880-889, doi:10.12659/MSM.895552, (2016). \u003c/li\u003e\n\u003cli\u003eLi, H.\u003cem\u003e et al.\u003c/em\u003e Bifendate indirectly inhibits alanine transaminase release without direct interaction in vitro and in vivo. \u003cem\u003eActa Poloniae Pharmaceutica - Drug Research\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e(4), doi:10.32383/appdr/125077, (2020).\u003c/li\u003e\n\u003cli\u003eLi, J.\u003cem\u003e et al.\u003c/em\u003e Hepatoprotective Effects of Heracleum candicans Against Carbon Tetrachloride-Induced Acute Liver Injury in Rats. \u003cem\u003eDose Response\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 15593258211029510, doi:10.1177/15593258211029510 (2021).\u003c/li\u003e\n\u003cli\u003eChen, X. \u0026amp; Calvisi, D. F. Hydrodynamic transfection for generation of novel mouse models for liver cancer research. \u003cem\u003eAm. J. Pathol.\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 912-923, doi:10.1016/j.ajpath.2013.12.002 (2014).\u003c/li\u003e\n\u003cli\u003eWajant, H., Pfizenmaier, K. \u0026amp; Scheurich, P. Tumor necrosis factor signaling. \u003cem\u003eCell Death Differ.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 45-65, doi:10.1038/sj.cdd.4401189 (2003).\u003c/li\u003e\n\u003cli\u003eZhou, H.\u003cem\u003e et al.\u003c/em\u003e TNF-alpha Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection. \u003cem\u003eCell. Mol. Neurobiol.\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1841-1857, doi:10.1007/s10571-021-01063-w (2022).\u003c/li\u003e\n\u003cli\u003eCzaja, A. J. Targeting apoptosis in autoimmune hepatitis. \u003cem\u003eDig. Dis. Sci.\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 2890-2904, doi:10.1007/s10620-014-3284-2 (2014).\u003c/li\u003e\n\u003cli\u003eBottero, V., Withoff, S. \u0026amp; Verma, I. M. NF-kappaB and the regulation of hematopoiesis. \u003cem\u003eCell Death Differ.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 785-797, doi:10.1038/sj.cdd.4401888 (2006).\u003c/li\u003e\n\u003cli\u003eKettritz, R.\u003cem\u003e et al.\u003c/em\u003e Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1740-1749, doi:10.1097/01.asn.0000019411.36000.06 (2002).\u003c/li\u003e\n\u003cli\u003eDing, C.\u003cem\u003e et al.\u003c/em\u003e Taxifolin, a novel food, attenuates acute alcohol-induced liver injury in mice through regulating the NF-kappaB-mediated inflammation and PI3K/Akt signalling pathways. \u003cem\u003ePharm. Biol.\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 868-879, doi:10.1080/13880209.2021.1942504 (2021).\u003c/li\u003e\n\u003c/ol\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acute liver injury, TNF-α, PMG, Hepatoprotection, PI3K/AKT","lastPublishedDoi":"10.21203/rs.3.rs-7542054/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7542054/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhyscion-8-O-β-D-monoglucoside (PMG) is one of the active ingredients of Radix et Rhizoma Rhei, which had been used for treating liver disorders for hundreds of years in China. However, the hepatoprotective effects of PMG remain poorly understood. This study aimed to investigate the mechanism of the protection effects of PMG on Tumor necrosis factor-α (TNF-α)-induced hepatotoxicity. We developed both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models of liver injury to assess the protective effects of PMG against TNF-α-induced hepatotoxicity. The \u003cem\u003ein vitro\u003c/em\u003e model employed TNF-α/actinomycin D in AML-12 cells, while the \u003cem\u003ein vivo\u003c/em\u003e model utilized intraperitoneal injection of carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) in mice. Interactions of PMG and TNFR1 (the receptor of TNF-α) were explored by molecular docking. AAV resuspension was administered before PMG treatment via intravenous injection to overexpress TNF-α in the CCl\u003csub\u003e4\u003c/sub\u003e-induced mice. Liver injury markers were examined, and the associated changes were detected using CCK8, Hoechst staining, Western blotting, and other molecular assays. PMG effectively reversed the morphological changes, restoring cell shape and structure in TNF-α injured cells. PMG protected against hepatotoxicity \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. PMG and TNFR1 maintained robust binding activities. TNF-α overexpression counteracted the hepatoprotective effects of PMG, attenuating its influence on IL-6, AST, ALT, apoptosis, and the inactivation of the PI3K/AKT/NF-κB signaling pathway. PMG protected against TNF-α-induced hepatotoxicity by regulating the PI3K/AKT/NF-κB signaling pathway through TNF-α inhibition, suggesting that PMG holds potential as a novel therapeutic agent for acute liver injury.\u003c/p\u003e","manuscriptTitle":"Physcion-8-O-β-D-monoglucoside protects hepatocytes from TNF-α-mediated apoptosis by suppressing the PI3K/AKT /NF-κB signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 08:45:20","doi":"10.21203/rs.3.rs-7542054/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T07:59:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T14:13:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T03:35:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190596648313835325893734070011287232698","date":"2025-09-18T00:30:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155201619360813305891421749010307947000","date":"2025-09-16T03:09:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72852685303092068809093086775109947123","date":"2025-09-16T00:55:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T00:46:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-16T00:30:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-12T17:23:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T05:28:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-12T05:25:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f206add6-e4e7-40c0-a23b-7f16ba30b5a4","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55065119,"name":"Biological sciences/Cell biology"},{"id":55065120,"name":"Health sciences/Diseases"},{"id":55065121,"name":"Biological sciences/Drug discovery"},{"id":55065122,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-03-16T16:01:32+00:00","versionOfRecord":{"articleIdentity":"rs-7542054","link":"https://doi.org/10.1038/s41598-026-38701-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-09 15:58:17","publishedOnDateReadable":"March 9th, 2026"},"versionCreatedAt":"2025-09-24 08:45:20","video":"","vorDoi":"10.1038/s41598-026-38701-6","vorDoiUrl":"https://doi.org/10.1038/s41598-026-38701-6","workflowStages":[]},"version":"v1","identity":"rs-7542054","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7542054","identity":"rs-7542054","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}