not-yet-known not-yet-known not-yet-known unknown Schisandra Lignans Target GPX4 to Suppress Ferroptosis: A First-in-Class Natural Therapy for INH-Induced Liver Injury

not-yet-known not-yet-known not-yet-known unknown Schisandra Lignans Target GPX4 to Suppress Ferroptosis: A First-in-Class Natural Therapy for INH-Induced Liver Injury | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 28 February 2025 V1 Latest version Share on not-yet-known not-yet-known not-yet-known unknown Schisandra Lignans Target GPX4 to Suppress Ferroptosis: A First-in-Class Natural Therapy for INH-Induced Liver Injury Authors : Yuting Zheng , Huan Lan , Caihong Liu , Lin An , Peng Wu , Rong Zhang , Zhongqiu Liu , Jinjun Wu , and Caiyan Wang 0000-0002-8605-2340 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174072873.31515610/v1 240 views 140 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Isoniazid (INH), a cornerstone of tuberculosis therapy, is plagued by dose-limiting hepatotoxicity with limited therapeutic options. While Schisandra chinensis lignans exhibit hepatoprotective potential, their mechanistic interplay with ferroptosis—a newly recognized driver of INH-induced liver injury—remains unexplored. Here, we delineate a novel molecular axis by which Schisandrin A (SinA) and Schisandrin B (SinB) mitigate INH-induced hepatotoxicity through ferroptosis suppression. Using human hepatocyte models (L02, WRL68) and C57BL/6J mice, we demonstrate that SinA/SinB significantly attenuate hepatic injury markers (ALT/AST), iron overload, lipid peroxidation, and glutathione depletion. Crucially, both compounds rescued GPX4 expression by blocking its ubiquitin-proteasomal degradation, as evidenced by CETSA, DARTS, and Co-IP. Strikingly, GPX4 knockout abolished their protective effects, underscoring GPX4 as the pivotal target. ITC and SPR revealed high-affinity binding of SinA/SinB to GPX4, while molecular docking identified Lys31 and Lys90 as critical residues for SinB-GPX4 interaction. Mutagenesis studies confirmed that K31/K90 substitutions abolished SinB’s efficacy, highlighting a structure-dependent mechanism. Notably, in vivo administration of SinA/SinB restored hepatic GPX4 levels, suppressed ferroptotic markers (MDA, GSH), and ameliorated histopathological damage. This work not only uncovers GPX4 stabilization as a druggable strategy against ferroptosis but also positions SinA/SinB as first-in-class natural compounds targeting this pathway for INH-induced liver injury. Our findings bridge traditional medicine with modern mechanistic insights, offering a blueprint for developing standardized, multitargeted therapies to address drug-induced hepatotoxicity. Schisandra Lignans Target GPX4 to Suppress Ferroptosis: A First-in-Class Natural Therapy for INH-Induced Liver Injury Running title: Schisandra lignans alleviate INH-induced liver injury Authors and affiliations: Yuting Zheng 1, # , Huan Lan 1, # , Caihong Liu 1 , Lin An 1 , Peng Wu 1 , Rong Zhang 1 , Zhongqiu Liu 1, * , Jinjun Wu 1, * , Caiyan Wang 1, * 1 State Key Laboratory of Traditional Chinese Medicine Syndrome, International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China. # These authors contributed equally to this work. * Corresponding authors: Zhongqiu Liu, E-mail: [email protected] , Phone: +8620-39358061, Fax: +8620-39358071 Jinjun Wu, E-mail: [email protected] ,cn, phone: +8620-39358647, Fax: +8620-39358071 Caiyan wang, E-mail: [email protected] ,cn, phone: +86-020-39358875, Fax: +86-020-39358071 not-yet-known not-yet-known not-yet-known unknown Authors’ Email Aaddresses: [email protected] (Yuting Zheng). [email protected] (Huan Lan). [email protected] (Caihong Liu). [email protected] (Lin An). [email protected] (Peng Wu). [email protected] (Rong Zhang). [email protected] (Zhongqiu Liu), [email protected] (Jinjun Wu), [email protected] (Caiyan Wang). not-yet-known not-yet-known not-yet-known unknown Abstract Isoniazid (INH), a cornerstone of tuberculosis therapy, is plagued by dose-limiting hepatotoxicity with limited therapeutic options. While Schisandra chinensis lignans exhibit hepatoprotective potential, their mechanistic interplay with ferroptosis—a newly recognized driver of INH-induced liver injury—remains unexplored. Here, we delineate a novel molecular axis by which Schisandrin A (SinA) and Schisandrin B (SinB) mitigate INH-induced hepatotoxicity through ferroptosis suppression. Using human hepatocyte models (L02, WRL68) and C57BL/6J mice, we demonstrate that SinA/SinB significantly attenuate hepatic injury markers (ALT/AST), iron overload, lipid peroxidation, and glutathione depletion. Crucially, both compounds rescued GPX4 expression by blocking its ubiquitin-proteasomal degradation, as evidenced by CETSA, DARTS, and Co-IP. Strikingly, GPX4 knockout abolished their protective effects, underscoring GPX4 as the pivotal target. ITC and SPR revealed high-affinity binding of SinA/SinB to GPX4, while molecular docking identified Lys31 and Lys90 as critical residues for SinB-GPX4 interaction. Mutagenesis studies confirmed that K31/K90 substitutions abolished SinB’s efficacy, highlighting a structure-dependent mechanism. Notably, in vivo administration of SinA/SinB restored hepatic GPX4 levels, suppressed ferroptotic markers (MDA, GSH), and ameliorated histopathological damage. This work not only uncovers GPX4 stabilization as a druggable strategy against ferroptosis but also positions SinA/SinB as first-in-class natural compounds targeting this pathway for INH-induced liver injury. Our findings bridge traditional medicine with modern mechanistic insights, offering a blueprint for developing standardized, multitargeted therapies to address drug-induced hepatotoxicity. Keywords : INH-induced liver injury; Schisandrin A; Schisandrin B; Ferroptosis; GPX4; Ubiquitination Abbreviations Full name ALT Alanine aminotransferase AST Aspartate aminotransferase CETSA Cellular Thermal Shift Assay CHX Cycloheximide Co-IP Co-immunoprecipitation DARTS Drug affinity responsive target stability DDB Bifendate DMEM Dulbecco’s Modified Eagle Medium DSF Differential scanning fluorimetry FBS Fetal bovine serum GSH Glutathione GSSG Glutathione, oxydized GPX4 Glutathione peroxidase 4 INH Isoniazid Lipid ROS Lipid Reactive Oxygen Species MDA Malondialdehyde MG132 Z-Leu-Leu-Leu-al MTT 3-(4,5-dimethyl thiazol 2-yl)-2,5-diphenyl tetrazolium bromide NAC N-Acetyl-L-cysteine PBS Phosphate-buffered saline PPIX Protoporphyrin SinA Schisandrin A SinB Schisandrin B SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel SolA Schisandrol A SolB Schisandrol B SPR Surface plasmon resonance StnA Schisantherin A StnB Schisantherin B ITC Isothermal calorimetry Introduction Isoniazid (INH) is a frontline medication in tuberculosis treatment, but the administration of INH carries significant risks 1, 2, 3, 4 . Particularly, its hepatotoxicity, represents one of the most prevalent adverse reactions during anti-tuberculosis therapy, affecting approximately 48% patients 5, 6, 7, 8 . This hepatotoxicity leads to abnormal liver function and in severe cases, to hepatitis or liver failure 9, 10, 11, 12, 13 . Currently, treatments such as bicyclic alcohols, biphenyl diesters, tiopronin, adenosine methionine, and various vitamins are widely employed to address INH-induced liver damage 14, 15 . On the other hand, these therapeutic reagents often cause additional side effects, resulting in symptoms such as nausea, vomiting, and fatigue; they may also give rise to more serious complications 16 . Therefore, it is imperative to identify drugs with distinct targets that exhibit superior pharmacological effects while minimizing toxicities and side effects. This issue warrants urgent attention in clinical practice. Traditional Chinese medicines (TCMs) exhibit a wide range of applications and offer therapeutic benefits through multiple pathways and targets. Numerous medications and active constituents derived from TCMs have been validated for their hepatoprotective effects, acting at various stages of liver injury. Schisandra chinensis , a well-established hepatoprotective reagent in clinical practice, contains Schisandrin lignans as its primary chemical components, which demonstrate diverse pharmacological activities 17, 18, 19 . Previous studies have indicated that Schisandrin lignans exert protective effects against several types of liver injury. For instance, Schisandra lignans enhances the excretion of bile acids from both serum and liver, thereby preventing and treating cholestatic liver injury induced by bile acids 20, 21 . Furthermore, it has shown efficacy in mitigating chemical liver injuries caused by alcohol and carbon tetrachloride (CCl 4 ), not only alleviating pathological changes in hepatocytes but also inhibiting hepatocyte apoptosis and reducing the formation of liver fibrosis 22 . Pretreatment with Schisandrin A (SinA) significantly improves hepatic ischemia-reperfusion injury in mice. The hepatoprotective mechanisms associated with this compound are closely linked to the upregulation of antioxidant enzyme expression, scavenging reactive oxygen species (ROS), inhibiting the release of inflammatory cytokines, reducing inflammatory cell infiltration, downregulating pro-apoptotic gene expression, and promoting hepatocyte regeneration, etc 23, 24 . However, the effectiveness of Schisandrin lignans in ameliorating INH-induced liver injury remains underexplored. Therefore, further investigation into its underlying molecular mechanisms is warranted. Ferroptosis is a novel form of programmed cell death that differentiates itself from autophagy and apoptosis. This process is characterized by excessive iron-dependent lipid peroxidation, wherein the abnormal accumulation of lipid peroxides in the presence of Fe 2+ leads to membrane disruption and subsequent irreversible cell death 25, 26, 27 . Glutathione peroxidase 4 (GPX4) plays a pivotal role in regulating ferroptosis and its primary function involves converting intracellular lipid peroxides into their corresponding alcohols, thereby safeguarding cells against oxidative stress damage. Research has demonstrated that SinA inhibits ferroptosis in cells and mitigates acetaminophen-induced acute liver injury by suppressing ROS accumulation through the Nrf2 and NF-κB signaling pathways. Additionally, it upregulates the expression of GPX4 and SLC7A11 while reducing the production of oxidized lipids 28 . The mechanism underlying statin-induced hepatotoxicity may be associated with ferroptosis resulting from mitochondrial damage 29 . Furthermore, studies have reported a significant increase in lipid oxidative stress within hepatocytes during INH treatment, indicating an active ferroptosis process 30 . It has also been found that decreasing ROS levels and increasing glutathione (GSH) can reduce the occurrence of hepatocyte ferroptosis, thus ameliorating liver injury caused by INH 31,32,33,34 .However, the role of Schisandrin lignans in modulating ferroptosis during INH-induced liver injury remains unexplored. Consequently, this study conducts in vitro and in vivo experiments to investigate the efficacy of Schisandrin lignans in mitigating liver injury induced by INH and to elucidate the underlying molecular mechanisms. Findings of this research not only advances the development and application of Schisandrin lignans but also provides experimental evidence for the formulation of novel therapeutics aimed at ameliorating INH-induced liver injury. Results SinA and SinB mitigate INH-induced hepatocyte injury via attenuation of liver enzyme leakage and protoporphyrin IX accumulation As principal lignans of Schisandra chinensis, SinA and SinB were investigated for their hepatoprotective potential against INH-induced cytotoxicity. To establish a clinically relevant in vitro model, human hepatic L02 and WRL68 cell lines were exposed to escalating concentrations of INH (12.5-100 mM) for 24 hours. MTT viability assays revealed a dose-dependent cytotoxic profile, with 50 mM INH inducing approximately 50% cell viability reduction (IC50=66.34 mM for L02, 53.58 mM for WRL68; Fig. 1A-C), which was subsequently adopted as the optimal injury-inducing concentration for mechanistic studies. To systematically evaluate the cytoprotective effects of Schisandra lignans, six structurally related compounds (SinA, SinB, SolA, SolB, StnA, StnB) were administered at doses of 25, 50, and 100 μM respectively after INH challenge. ALT and AST levels, established biomarkers of hepatocyte membrane integrity, were quantified. Compared with the blank control group, the ALT/AST level in the INH group was significantly elevated, indicative of severe membrane damage (Fig. 1D-G). All tested lignans attenuated enzyme leakage to varying degrees, with SinA and SinB exhibiting superior efficacy by restoring ALT/AST to near-baseline levels, significantly outperforming other lignans. High doses of PPIX have certain hepatotoxicity and can cause cholestatic liver injury. INH can directly inhibit the activity of FECH. Inhibiting the activity of this enzyme will reduce the ability of PPIX to be converted into heme, leading to the accumulation of PPIX in cells and eventually causing INH-induced hepatotoxicity and liver injury. The content of PPIX was detected by laser confocal microscopy. Compared with the blank control group, the PPIX level in the INH group was significantly increased. Compared with the INH group, the six schisandrin lignans, NAc group and DDB group significantly reduced the PPIX content. Among them, SinA and SinB had the best effect. Based on these findings, SinA and SinB were selected for subsequent mechanistic exploration of their hepatoprotective pathways. SinA and SinB suppress INH-induced ferroptosis through dual regulation of iron homeostasis and lipid peroxidatio n To delineate the involvement of ferroptosis in INH-mediated hepatotoxicity, we systematically evaluated key hallmarks of this iron-dependent cell death pathway. Pharmacological intervention with 25, 50, 100 μM SinA/SinB (24 hours pretreatment) significantly attenuated INH-induced dysregulation of redox homeostasis in both L02 and WRL68 cells. MDA revealed a 1.48~1.92-fold (L02) and 1.54~1.99-fold (WRL68) increase in INH-treated cells compared to controls. MDA content in SinA high concentration groups was 1.04±0.04 (L02) and 1.18±0.09 (WRL68), and that in SinB group was 1.25±0.06 (L02) and 1.12±0.03(WRL68), respectively ( Fig. 2A-D ), indicating potent inhibition of membrane lipid oxidation. Concurrently, intracellular GSH depletion induced by INH reduced to 0.63~0.67-fold (L02) and 0.67~0.73-fold (WRL68) compared to controls. GSH content in SinA high concentration groups was 0.96±0.03 (L02) and 0.86±0.01 (WRL68), and that in SinB group was 0.95±0.02 (L02) and 0.94±0.03(WRL68), respectively ( Fig. 2E-H ). This dual modulation of oxidative stress markers suggests restoration of cellular antioxidant capacity. Ferroptosis execution critically depends on labile iron pool (LIP) accumulation. Using the FeRhoNox-1 fluorescent probe, confocal imaging of the Fe 2+ content showed its significant reduction in these groups compared to the INH group ( Fig. 2I, J ). To directly visualize lipid peroxidation dynamics, we employed C11-BODIPY⁵⁸¹/⁵⁹¹, a ratiometric probe exhibiting fluorescence spectral shift upon oxidation. Laser confocal microscopy revealed that, relative to the blank control group, L02 and WRL68 cells exhibited increased green fluorescence intensity (indicative of oxidized lipid ROS) and decreased red fluorescence intensity (indicative of reduced lipid ROS) following INH administration. Compared with the INH group, both SinA and SinB groups demonstrated a reduction in green fluorescence intensity and an increase in red fluorescence intensity ( Fig. 2K, L ). Meanwhile, through flow cytometry detection, it was found that compared with the blank control group (100%), the lipid ROS levels in L02 and WRL68 cells increased after administration of INH, with the contents being 132.68 ± 2.11% and 125.23 ± 0.55%, respectively. Compared with the INH group, the lipid ROS levels decreased in the SinA or SinB administration groups, with the contents being 108.71±0.18%, 108.58±0.54%, 112.23±0.45%, and 108.66±1.53%, respectively ( Fig. 2M, N ). SinA and SinB preserve mitochondrial integrity via restoration of membrane potential and cristae architecture Mitochondrial damage intensifies lipid peroxidation and ROS generation, promoting ferroptosis; while lipid peroxidation and ROS generation during ferroptosis also further damage mitochondria. Mitochondrial membrane potential is the main indicator for detecting mitochondrial damage. Through laser confocal detection, it was found that compared with the blank control group, L02 and WRL68 cells treated with INH could enhance the green light intensity (FITC monomer) and weaken the red light intensity (PE polymer). Compared with the INH group, the green light intensity was weakened, and the red-light intensity was enhanced in the SinA or SinB re-treatment groups ( Fig. 3A, B ). The effects of SinA and SinB on the mitochondrial morphology in L02 and WRL68 cells induced by INH were detected by transmission electron microscopy (TEM). The results showed that the mitochondrial morphology in the blank control group was normal, with no significant shrinkage or damage. Compared with the blank control group, the mitochondria in the INH group were shrunk, the density of the double-layer membrane increased, and the mitochondrial cristae decreased or disappeared. However, compared with the INH group, the mitochondrial damage in the SinA or SinB re-administration groups was significantly improved ( Fig. 3C, D ). GPX4 upregulation mediates SinA/SinB-dependent ferroptosis resistance GPX4 serves as the central regulator of ferroptosis by neutralizing lipid hydroperoxides, we investigated its expression dynamics. Western blot experiments demonstrated that compared with the blank control group (100%), the expression of GPX4 in the INH group was 43.68% to 46.25% (L02) and 53.76% to 63.56% (WRL68). The high-dose SinA treatment group restored GPX4 expression to 84.04±2.59% (L02) and 90.76±3.07% (WRL68), while in the SinB group, it was 98.34±2.58% (L02) and 100.51±3.56% (WRL68), respectively ( Fig. 4A-D ), correlating with attenuated lipid peroxidation. Immunofluorescence experiments demonstrated that the fluorescence expression of GPX4 decreased in L02 and WRL68 cells after modeling with INH, but it increased after the administration of SinA or SinB, further verifying that INH can down-regulate the expression of GPX4, while SinA and SinB can reverse this phenomenon ( Fig. 4E, F ). To establish GPX4’s causal role in SinA/SinB-mediated protection, siRNA-mediated GPX4 knockout (KO) models were generated (KO efficiency Intriguingly, GPX4 ablation completely abolished the hepatoprotective effects of SinA/SinB. These findings mechanistically position GPX4 as the indispensable mediator of SinA/SinB’s anti-ferroptotic activity. SinA and SinB regulate intracellular MDA, lipid peroxidation, and GSH levels through GPX4-dependent mechanisms Our data indicate that treatment with SinA or SinB can alleviate the increase in malondialdehyde (MDA) content induced by INH in wild-type cells, but the MDA level in GPX4 knockout cells is 1-2 times that of the wild type ( Fig. 5A-D ). Concordantly, SinA or SinB significantly reduced GSH levels compared to wild-type controls ( Fig. 5E-H ). To evaluate lipid peroxidation, we utilized the C11-BODIPY probe and observed that SinA or SinB increased the red/green fluorescence ratio (indicative of lipid peroxidation) in wild-type cells. Conversely, in GPX4-depleted cells, these compounds suppressed red fluorescence while enhancing green fluorescence, suggesting reduced oxidative stress ( Fig. 5I, J ; Fig. 6A, B ). Strikingly, GPX4 knockout abolished the protective effects of SinA or SinB on mitochondrial integrity ( Fig. 5K, L ). To further validate GPX4’s role, we overexpressed GPX4 in L02 and WRL68 cells ( Fig. 6C, D ), confirmed by immunofluorescence ( Fig. 6E, F ). In GPX4-overexpressing cells, SinA or SinB treatment reduced MDA levels by 50–100% compared to wild-type cells ( Fig. 6G-J ) and more effectively mitigated GSH depletion ( Fig. 6K-N ). Overexpression of GPX4 also restored the red fluorescence signal (reflecting lipid peroxidation) to near-normal levels in SinA- or SinB-treated cells ( Fig. 7A, B, E, F ) and significantly improved mitochondrial membrane potential ( Fig. 7C, D ).Collectively, these findings underscore GPX4 as a central mediator of SinA and SinB in counteracting ferroptosis and alleviating INH-induced hepatic injury by modulating oxidative stress and mitochondrial dysfunction. SinA and SinB upregulated GPX4 through inhibiting the ubiquitination-proteasomal pathway We next investigated the mechanisms by which SinA and SinB regulate GPX4. RT-qPCR analysis revealed no significant changes in GPX4 mRNA levels upon SinA or SinB treatment ( Fig. 8A-D ), excluding transcriptional regulation. However, CETSA and DARTS assays demonstrated enhanced thermal and proteolytic stability of GPX4 in SinA- or SinB-treated groups compared to the DMSO control ( Fig. 8E-H ), suggesting post-translational stabilization. Western blotting ruled out mitophagy-mediated stabilization, as LC3B-II/LC3B-I ratios remained unaffected ( Fig. S1A-D ). Notably, co-treatment with the translation inhibitor cycloheximide (CHX) did not abolish GPX4 upregulation ( Fig. 8I-L ), further supporting a degradation-related mechanism. Since ubiquitination often targets proteins for proteasomal degradation, we examined GPX4 stability using the proteasome inhibitor MG132. SinA or SinB synergistically increased GPX4 levels with MG132 ( Fig. 8M-P ), implying that these compounds attenuate GPX4 degradation. Consistently, Co-IP assays confirmed that SinA and SinB significantly reduced ubiquitination of GPX4 ( Fig. 8Q, R ). Collectively, these findings indicate that SinA and SinB stabilize GPX4 by inhibiting its ubiquitination, thereby blocking proteasomal degradation, rather than enhancing transcription or translation. SinA and SinB markedly improved INH-Induced liver injury via the ferroptosis pathway in vivo SinA and SinB significantly ameliorated INH-induced liver injury via modulation of the ferroptosis pathway in vivo . C57BL/6J mice were administered 150 mg/kg INH via oral gavage. Two hours of post-INH treatment, SinA (25, 50, 100 mg/kg), SinB (25, 50, 100 mg/kg), and the positive control DDB (5.35 mg/kg) were administered daily for 20 days, while the blank and INH-only groups received no further intervention ( Fig. 9A, B ). Compared to the INH group, SinA- and SinB-treated mice exhibited a marked reduction in liver index and serum levels of ALT and AST, indicating improved hepatic function ( Fig. 9C-F ). Histopathological analysis via HE staining revealed near-normal liver lobule architecture in SinA/SinB groups, with attenuated hepatocyte edema, minimal hyaline degeneration, and only slight nuclear shrinkage ( Fig. 9G ). Furthermore, SinA and SinB administration significantly suppressed lipid peroxidation, as evidenced by decreased MDA levels and elevated GSH content ( Fig. 9H, I ). Mechanistically, both compounds upregulated GPX4 expression, confirmed by quantitative analysis ( Fig. 9J ) and IHC, which demonstrated enhanced GPX4 protein levels in liver tissues ( Fig. 9K ). These findings underscore the therapeutic potential of SinA and SinB in mitigating ferroptosis-driven liver injury. SinA and SinB directly interacted with the target GPX4 We conducted molecular docking work to identify the preferred binding site and theoretical binding mode of Schisandra lignans to GPX4 using the Autodock1.1.2 software. All the ligands showed similar binding energies, ranging from -5.3 to -6.8 kcal/mol, but they bind to different sites of the protein. Particularly, SolA shows abundant interactions with GPX4, centering at Asn28, whereas SolB interacts with Lys99 and Met102 ( Fig. 10A-F ). In contrast, SinA mainly contacts Lys90 and SinB makes contacts with Lys31 and Lys90. Lastly, the binding sites for StnA is Arg33 and Asn146 and His15 for StnB. As shown in Fig. 10G-L , SPR indicated that the affinities of SolA, SolB, SinA, SinB, StnA and StnB were 94.4 μM, 5.18 μM, 1.6 μM, 863 nM,0.4 mM and 0.38 mM, respectively. Indeed, SinA and SinB bind much more strongly with GPX4. As shown in Fig. 10M and N, the CETSA indicated that SinA and SinB increased the thermal stability of GPX4. ITC assay further showed that the binding Kd values of SinA, SinB to GPX4 were 1.19 μM and 9.09 μM, respectively ( Fig. 10O and P ). We then analyzed the stability of GPX4 with SinA or SinB ( Fig. 10Q and R ). The melting temperature (Tm) of GPX4 with SinA and SinB were 51.59 °C and 52 °C compared to 46 °C for the GPX4 with DMSO. Correspondingly, DARTS showed that the digestion of GPX4 by protease E was attenuated in the presence of SinA or SinB ( Fig. 10S and T ). Furthermore, the ConSurf Server (https://consurf.tau.ac.il) was used for sequence conservation analysis of GPX4 ( Fig. 10U ). A multiple sequence alignment of GPX4 from different species was conducted. H. sapiens, M. musculus, P. troglodytes, R. norvegicus and S. scrofa respectively stand for Homo sapiens, Mus musculus, Pan troglodytes, Rattus norvegicus and Sus scrofa . The red background represents extremely conserved residues, and the red font represents relatively conserved residues. Meanwhile, through comparing GPX4 sequences from different species, we found that conserved Lys31 and Lys90 could play a key role ( Fig. 10V ). SinA or SinB could bind to the residues K31 and K90 in the GPX4 As shown in Fig. 11A-D, the CETSA indicated that SinA and SinB increased the thermal stability of GPX4, but not that of GPX4-K31R, GPX4-K90R, supporting direct interactions between the ligands and GPX4. To understand how Lys31 and Lys90 would impact the binding affinities, we examined the affinity changes of the K31R and K90R mutants through the ITC experiment and the resulting Kd values were 19.3 μM, 73.3 μM, 86.2 μM and 120 μM, respectively, consistent with DSF assay ( Fig. 11E-H ). Correspondingly, DARTS showed that the digestion of GPX4-K31R by protease E was significantly weakened in the presence of SinA ( Fig. 11I ). However, in the presence of SinB, the digestion of GPX4-K31R by protease E was not significantly reduced ( Fig. 11J ). In the presence of SinA or SinB, digestion of GPX4-K90R by protease E was not significantly diminished ( Fig. 11I and J ). We also analyzed the stability of GPX4-K31R and GPX4-K90R incubation with SinA or SinB, and these experiments showed that GPX4-K31R and GPX4-K90R did not show greater stability under drug incubation ( Fig. 11K-N ). Mutations in residues K31R and K90R of GPX4 attenuate the effect of SinA or SinB Since the massive production of lipid peroxidation is the direct cause of ferroptosis, we then determined the redox-associated indicators GSH and GSSG. INH apparently augmented the GSSG levels and reduced the GSH activities, whereas SinA or SinB treatment significantly reduced the oxidative damage ( Fig. 12A and B ). However, once the mutation of GPX4 to GPX4-K31R, the protective effect of SinA or SinB on oxidative damage was weakened. After the mutation of GPX4 to GPX4-K90R, the effect of SinB on oxidative damage was disappeared. In particular, the protective effect of SinA or SinB on GPX4-K31R/K90R disappeared completely. INH apparently augmented the MDA levels, whereas SinA or SinB treatment significantly reduced the the MDA levels ( Fig. 12C ). After the mutation of GPX4 to GPX4-K31R, the effect of SinB on decreasing MDA level was weakened. After the mutation of GPX4 to GPX4-K90R, the effect of SinA or SinB on decreasing MDA level disappeared. In addition, the effect of SinA or SinB on GPX4-K31R/K90R also completely disappeared. Lipid peroxides measurement in WRL68 cells by the C11 BODIPY reagent revealed that SinA and SinB significantly lowered the GFP/RFP ratios, suggesting decreases in lipid peroxidation. When GPX4 was mutated, their effect on improving ferroptosis in INH-induced WRL68 cells was diminished. In addition, they were unable to rescue ferroptosis in GPX4-K31R/K90R, suggesting that their inhibition of ferroptosis was dependent on GPX4 ( Fig. 12D-F ). Discussion Schisandra sphenanthera is a traditional Chinese medicinal herb with a rich historical background. In recent years, it has been the subject of extensive research concerning its therapeutic effects on various chronic liver diseases 7 . This plant is abundant in diverse chemical constituents, among which lignans are the most prevalent. These lignan compounds possess complex structures and exhibit a wide range of biological activities, including substantial antioxidative properties, calcium channel blocking capabilities, anti-tumor effects, hepatoprotective functions, choleretic activity, and cardiovascular protection. Research indicates that the lignans from Schisandra sphenanthera can effectively prevent and mitigate acetaminophen-induced liver damage by reducing GSH consumption in liver and inhibiting the metabolic activation process of acetaminophen mediated by cytochrome P450 enzymes 5 . Furthermore, these compounds alleviate inflammatory responses, prevent programmed cell death, and improve oxidative stress status by modulating the mitogen-activated protein kinase signaling pathway. Notably, they have shown significant efficacy in mitigating liver ischemia-reperfusion injury in rat models 16 . Particularly notable are two significant active substances extracted from Schisandra sphenanthera , SinA and SinB, which have drawn strong attention because of their distinct pharmacological properties. These components not only deepen the understanding of Schisandra sphenanthera ’s efficacy in accordance with traditional medical theories but also offer new perspectives for modern drug development. In this study, we demonstrate for the first time that Schisandra lignans inhibit ferroptosis in INH-induced liver injury, mainly by stabilising GPX4. Furthermore, we investigated the potential mechanisms of action of SinA and SinB in INH-induced liver injury. Ferroptosis, a newly recognized form of regulated cell death triggered by iron overload and the accumulation of lipid peroxidation products, has become a research hotspot. Our research represents the first instance in which INH has been shown to initiate the ferroptosis process in liver cells. Recent studies have revealed that the development of a GPX4 activator to inhibit ferroptosis is a challenging approach. Liu et al. proposed a potential GPX4 activator, compound C3, for the prevention of cell death and lipid peroxide accumulation induced by erastin. However, the clinical use of GPX4 activator has been limited, and the selectivity and druggability have been relatively poor. Importantly, our study discovered that SinA and SinB not only stabilise GPX4 to inhibit ferroptosis, but also bind to GPX4 residues K31 and K90. We believe that SinA and SinB, as GPX4 stabilizers, will play an important role in the treatment of diseases associated with ferroptosis in the future. During this process, excessive lipid peroxidation emerges as a significant biochemical hallmark of ferroptosis within these cells, accompanied by marked increases in MDA and superoxide dismutase levels 19 . Concurrently, we observed notable alterations in mitochondrial morphology, including swelling, vacuolization, and disintegration or dissolution of cristae structures. These morphological changes directly impair mitochondrial function, evidenced by decreased mitochondrial membrane potential and diminished respiratory capacity 18 . More importantly, our study further elucidated that INH significantly inhibits the expression of GPX4, both in vivo and in vitro , thereby inducing ferroptosis. This finding offers new insights into the understanding of the molecular mechanisms underlying INH-induced liver injury 17 . Building upon these observations, we investigated the effects of SinA and SinB on INH-induced ferroptosis. The experimental results demonstrated that these two compounds could significantly mitigate the extent of ferroptosis induced by INH while improving phenotypic manifestations associated with liver injury. This finding not only substantiates the potential role of SinA and SinB as anti-ferroptosis reagents but also provides valuable insights into the designs of novel hepatoprotective drugs. However, when we employed hepatocyte-specific GPX4 siRNA to silence GPX4, we observed that SinA and SinB were ineffective in reversing the ferroptosis induced by INH, suggesting that GPX4 may serve as a critical target of SinA and SinB. In summary, this study not only elucidates a novel mechanism underlying INH-induced liver injury through ferroptosis but also demonstrates the potential role of SinA and SinB in mitigating ferroptosis and thus greatly enhancing our understanding of the pharmacological properties of Schisandra sphenanthera . Ferroptosis is a recently identified regulated form of cell death, characterized by excessive iron accumulation and lipid peroxidation. It distinguishes itself from other forms of cell death, such as apoptosis, necrosis, and autophagy, through its unique morphological features, chemical reactions, and genetic markers. A notable ultrastructural characteristic of ferroptosis is the alteration in mitochondrial morphology. It has been documented that ferroptosis plays a significant role in various diseases, including liver injury, cancer, and cardiovascular disorders, etc. The regulation of ferroptosis primarily hinges on the competition between the ferroptosis execution system and the antioxidant defense mechanisms. The SLC7A11/GSH/GPX4 signaling axis represents one of the classic antioxidant pathways involved in this process. Extracellular cysteine enters cells via SLC7A11 and is subsequently converted into cysteine to facilitate glutathione synthesis. GPX4 utilizes reduced glutathione as a cofactor to detoxify lipid peroxides into lipid alcohols, hence inhibiting ferroptosis. Inhibition of SLC7A11 or GPX4 activity or impairment in GSH synthesis can precipitate ferroptosis. Ferroptosis has ebeen reported to play a critical role in the pathogenesis of both acute and chronic liver diseases including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, and hepatocellular carcinoma. Furthermore, ferroptosis contributes to diabetic-related hepatic pathological damage by exerting pathogenic effects. Thus, it presents a promising avenue for therapeutic intervention. In specific pathological liver diseases or stages of disease, such as viral infections, genetic disorders, and metabolic liver diseases, the types of cells that undergo iron deposition in the liver and their roles in pathological progression may vary significantly. Based on identified ferroptosis targets (such as SLC7A11, transferrin, ferritin, Nrf2), along with their specificity across different liver pathologies, considerable efforts have been directed towards drug development. Although substantial progress has been made in elucidating the role of ferroptosis in liver pathology, further clarification is required regarding the clinical responses to ferroptosis-targeted treatments. Our study revealed that in mouse liver cells stimulated by INH, there was a significant inhibition of GPX4 expression and a reduction in GSH levels. This ultimately led to excessive lipid peroxidation, mitochondrial damage, and cell death. To gain insights into the mechanisms through which SinA and SinB inhibit ferroptosis in liver cells by targeting GPX4, we evaluated the effects of these two compounds on ferroptosis within hepatic cells. This investigation utilized L02 and WRL68 cell lines to establish an in vitro model of INH-induced ferroptosis. The results indicated that the occurrence of ferroptosis within liver cells was associated with decreased levels of GPX4 protein. Treatment with SinA and SinB effectively inhibited ROS activity while reducing mRNA levels of key genes associated with ferroptosis markers in hepatic cells. Additionally, these treatments upregulated the expression of GPX4—a critical regulatory protein for ferroptosis—and consequently inhibited its progression. Furthermore, overexpression of GPX4 protein mitigated lipid peroxidation-induced damage within hepatocytes and enhanced antioxidant signaling pathway activity. In addition, through molecular docking and thermal shift experiments, we visualized the binding patterns of Schisandra lignans with GPX4, supporting GPX4 as a direct pharmacological target for both SinA and SinB. Additionally, it suggested that the hydroxymethyl group in SinB forms hydrogen bonding interactions with Lys31 and Lys90. Notably, SinB plays a crucial role in maintaining the active state of GPX4 and may contribute to the stabilization of GPX4, awaiting further experimental validation. Differential scanning fluorimetry (DSF), CETSA, and DARTs experiments indicate that SinB enhances its structural stability by binding to Lys31 and Lys90, exhibiting stronger binding affinities than that to Lys90. To validate the contribution from the sites to GPX4’s binding, we substituted Lys31 with Arginine and also replaced Lys90 with Arginine. Under these mutant backgrounds, the targeted regulatory effect of SinB on GPX4 was diminished. This study not only provides new insights into the structure-function relationship between SinB and GPX4 but also offers significant support for developing novel agonists targeting GPX4. Although our research has uncovered the promising therapeutic effect of SinA and SinB during INH-induced liver injury, the pharmacokinetic properties of Schisandra lignans still present much more challenges for clinical translation. For example, Schisandra lignans exhibit relatively low oral bioavailability due to poor solubility, extensive first-pass metabolism, and potential efflux by drug transporters like P-glycoprotein. These factors limit their systemic exposure and therapeutic efficacy. Additionally, their distribution in tissues is often uneven, with some lignans showing preferential accumulation in the liver, which may be advantageous for hepatoprotective effects but could limit their utility in targeting other organs. In the future, advanced drug delivery systems, such as nanoparticles or lipid-based formulations, could be explored to enhance bioavailability and achieve more targeted tissue distribution. In addition, the INH-induced liver injury model, while valuable for studying drug-induced hepatotoxicity, has significant limitations, particularly its inability to replicate the complex environment of tuberculosis (TB) combination therapy. Future research should focus on developing more comprehensive models that incorporate multi-drug regimens to better mimic clinical conditions. In conclusion, our study has indicated that SinA and SinB are capable of mitigating INH-induced liver injury through the inhibition of ferroptosis, with GPX4 as their target. Further investigations have revealed that SinA and SinB reduce the ubiquitination levels of GPX4 and interact with residues 31 and 90 of GPX4, thereby hindering its proteolytic degradation. In light of these novel findings, SinA and SinB hold promise as potential therapeutic options for the clinical management of INH-induced liver injury. not-yet-known not-yet-known not-yet-known unknown Materials and methods Chemicals and reagents INH (CAS 54-85-3) was procured from Sigma-Aldrich (St Louis, MO, USA). SinA (CAS 61281-38-7), Schisandrin B (SinB, CAS 61281-37-6), Schisandrol A (SolA, CAS 7432-28-2), Schisandrol B (SolB, CAS 58546-54-6), Schisantherin A (StnA, CAS 58546-56-8), and Schisantherin B (StnB, CAS 58546-55-7) were all acquired from Chengdu Manster Biotechnology Co., Ltd (Chengdu, China). The purities of these compounds were determined to be >98% by the HPLC method. N-Acetyl-L-cysteine (NAC, CAS 616-91-1) was purchased from Aladdin (Shanghai, China). Bifendate (DDB, CAS 73536-69-3) was obtained from Sichuan Weikeqi Pharmaceutical Co., Ltd (Sichuan, China). High-glucose Dulbecco’s Modified Eagle Medium (DMEM), RPMI-1640 medium, and penicillin-streptomycin were procured from Gibco (Carlsbad, CA, USA). Trypsin and fetal bovine serum (FBS) were acquired from ExCell Bio (Jiangsu, China). Phosphate-buffered saline (PBS) was obtained from Nanjing BioChannel Biotechnology Co., Ltd (Nangjing, China). 3-(4,5-dimethyl thiazol 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Shanghai Acmec Biochemical Technology Co., Ltd (Shanghai, China). Kits for aspartate aminotransferase (AST), and alanine aminotransferase (ALT), and GSH were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The malondialdehyde (MDA) kit and Hoechst 33342 were acquired from Beyotime Biotechnology (Shanghai, China). The FeRhoNox-1 fluorescent probe was obtained from Glpbio (Monclair, CA, USA). BODIPY™ 581/591 C11 was bought from Thermo Fisher Scientific (Waltham, MA, USA). JC-1 dye was acquired from Yeasen (Shanghai, China). DAPI stain was bought from Servicebio (Wuhan, China). Antibodies to GPX4 and β-actin were obtained from Abmart Shanghai Co., Ltd (Shanghai, China). Cell culture L02 cells were cultivated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin solution (Invitrogen, Carlsbad, CA, USA). Likewise, WRL68 cells were cultivated in DMEM high-glucose medium that also contained 10% FBS and 1% penicillin-streptomycin solution. Both cell lines were maintained in an incubator with 5% CO 2 at 37 °C under saturated humidity. Subculturing was initiated when cell confluence exceeded 80%, followed by medium replacement one day after subculturing. Animals and experimental design The animal experiments in this study complied with animal care laws and guidelines. All procedures were approved by the Guangzhou University of Chinese Medicine Laboratory Animal Ethics Committee (N0.20230811). C57BL/6 mice, aged four-six weeks, were obtained from the Guangdong Animal Testing Center. The mice were housed under standard conditions, with a temperature of 25 ± 2 °C and a relative humidity of 50 ± 5%. A 12-hours on/off lighting cycle was implemented using incandescent lamps. The mice were provided with ad libitum access to feed and water throughout the study. SPF-grade male C57BL/6 mice were randomly divided into nine groups, including a normal control group, an INH group, an INH+SinA (Low) group, an INH+SinA (Middle) group, an INH+SinA (High) group, an INH+SinB (Low) group, an INH+SinB (Middle) group, an INH+SinB (High) group and an INH+DDB group with 10 mice in each group. Except for the normal control group, which received intragastric administrations of saline, all other groups received 150 mg/kg of INH daily for 20 consecutive days to establish a mouse liver injury model. Two hours after each INH administration, the normal control group and INH group received saline intragastrically, the positive control group received 5.35 mg/kg of DDB, and the SinA and SinB groups received 25, 50, and 100 mg/kg of the respective drugs. All mice were gavaged with an equal volume of 0.1 ml/10g of the respective treatments daily for 20 consecutive days. MTT assay L02 and WRL68 cells were inoculated into 96-well plates at a density of 5× 10 3 cells per well. Once the cells adhered to the plate, they were treated with INH at concentrations of 12.5, 25, 50, and 100 mM, with 100 μL/well and six replicate wells for each concentration. Cell viability was determined by adding MTT (0.5 mg/mL) to each well and incubated for 4 h at 37 °C. Subsequently, formazan crystal products were dissolved in dimethylsulfoxide (DMSO) for 10 min and absorbance value was measured at 570 nm with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). ALT or AST assay The ALT and AST levels in the cells were determined using ALT and AST assay kits. According to the instructions, cellular samples were collected and incubated with the respective ALT and AST working solutions at 37 °C. The absorbance values were then measured at 510 nm using a microplate reader. Protoporphyrin (PPIX) content determination Owing to the absence of endogenous precursors for protoporphyrin IX, such as 5-aminolevulinic acid (ALA) (A0325, TCI, Shanghai, China), in cells, exogenous 5-ALA was added to stimulate the production of protoporphyrin. L02 and WRL68 cells were inoculated into confocal dishes at a density of 1.5× 10 4 cells per dish. Once the cells adhered to the dish, modeling and drug administration were carried out. After the incubation, the cells were incubated with 1 mM 5-ALA in the dark for 4 hours. Imaging of the cells was subsequently conducted using a laser scanning confocal microscope (Leica TCS SP8 X, Leica, Wetzlar, Germany). Fe 2+ content detection The intracellular Fe²⁺ content was determined using the FeRhoNox-1 (Fe 2+ indicator) fluorescent probe. L02 and WRL68 cells were inoculated into confocal dishes at a density of 1.5× 10 4 cells per dish. The cells were washed with PBS after the incubation and then incubated with 5 μM FeRhoNox-1 working solution in the dark for 1 hour. The cells were washed three times with PBS and subsequently incubated with Hoechst 33342 for 10 minutes. After another three washes, serum-free medium was added to the cells. Images were obtained using a laser scanning confocal microscope with an excitation wavelength of 532 nm or 543 nm and an emission wavelength of approximately 570 nm. Mitochondrial membrane potential analysis Mitochondrial membrane potential was evaluated using the JC-1 staining method. Cells were inoculated with 50 μg/mL of JC-1 staining working solution for 30 minutes at 37 °C in the dark. JC-1 monomer fluorescence (green) was detected at an excitation wavelength of 514 nm, while JC-1 aggregate fluorescence (red) was detected at an emission wavelength of 590 nm. Images were obtained using a laser scanning confocal microscope. Immunofluorescence analysis The cells were fixed in 4% paraformaldehyde for 20 minutes and then permeabilized with 0.5% Triton X-100 for 30 minutes. After being washed by PBS, the cells were blocked with 5% BSA for 30 minutes at room temperature, washed 3 more times with PBS, and probed with the GPX4 antibody overnight at 4 °C. The cells were incubated with the secondary antibody conjugated to Dylight-594 for 2 hours at room temperature. After washing 3 times with PBS, the cells were then incubated with 20 mg/mL DAPI for 20 minutes. Images were captured (594 nm/618 nm for Dylight-594; 488 nm/519 nm for GFP and 340 nm/488 nm for DAPI) using confocal microscopy. HE staining The liver tissue was fixed in 4% paraformaldehyde for no less than 24 hours, followed by dehydration through an ethanol gradient and clarification with xylene before immersing the tissue in wax. Subsequently, the tissue was embedded in paraffin and 4-μm thin sections were made. The sections were placed on slides and baked in a slide warmer for 2 hours, then dewaxed with xylene and rehydrated through an ethanol gradient, followed by immersion in distilled water. The sections were stained with hematoxylin for 2 minutes, differentiated with 1% hydrochloric acid in alcohol, and counterstained with 0.5% eosin for 10 seconds. They were then dehydrated through an ethanol gradient, immersed in xylene, air-dried, and mounted with neutral resin. Finally, images of the sections were captured under optical microscopes at 10 x and 40 x magnifications. IHC staining Immunohistochemical staining for GPX4 was conducted on 4-μm paraffin-embedded liver tissue sections. The sections were initially dewaxed with xylene and rehydrated through an ethanol gradient. Antigen retrieval was accomplished by incubating the sections in a citrate buffer. Subsequently, the sections were blocked with 5% BSA and then incubated with the anti-GPX4 antibody at 4 °C overnight. After the primary antibody incubation was completed, the sections were incubated with the secondary antibody, followed by DAB staining for visualization. The staining reaction was terminated by rinsing in running water when the color development was fulfilled. The nuclei were then stained with hematoxylin. Finally, images of the sections were captured under optical microscopes at 20 x and 40 x magnifications. Western blotting Cells were homogenized with the ice-cold RIPA buffer for 30 minutes to provide whole cell lysates. Concentrations of total proteins were determined using an enhanced BCA protein assay kit (DQ111-01, TransGen Biotech, Beijing, China). Lysates were separated by 8%-12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% non-fat milk, probed with primary antibody (1:1000 diluted) for 2 hours, and further incubated with the secondary antibody (1:3000 diluted) for 1 hour. Then, membranes were washed and developed with an enhanced chemiluminescence (ECL) substrate. Images were captured using a Tanon 5200 Imaging Analysis System (Tanon, Shanghai, China). Relative protein expressions were shown by densitometry analysis using the Image J software. not-yet-known not-yet-known not-yet-known unknown RT-qPCR assay L02 and WRL68 cells were seeded into 6-well plates at a density of 2.0× 105 cells per well. Once cells adhered to the plate, modeling and drug administration were performed. Following the completion of the incubation period, the total RNA from the cells was extracted using the Trizol method. Subsequently, cDNA was generated from the total RNA utilizing a reverse transcription kit (RR037A, TaKaRa, Japan) in accordance with the provided instructions. Real-time PCR was conducted as outlined employing a SYBR® Green Pro Taq HS qPCR Kit (RR430S, Accurate Biology, Hunan, China). The GPX4 primers utilized were as followed forward primer 5’-GTGGATGAAGATCCAACCC-3’ and reverse primer 5’-TTGTCGATGAGGAACTTGG-3’; whereas GADPH primers used were: forward primer 5’-GAGTCAACGGATTTGGTCGT-3’ and reverse primer 5’- GACAAGCTTCCCGTTCTCAG-3’. not-yet-known not-yet-known not-yet-known unknown siRNA Interference and Plasmid Transfection The siRNA sequences targeting GPX4 (siGPX4) and the control siRNA (siCon) were custom synthesized by Suzhou Genepharma Co., Ltd (Suzhou, China). The sense sequences for GPX4 siRNA-1 (siGPX4-1), siRNA-2 (siGPX4-2) and siRNA-3 (siGPX4-3) are 5’-CAGGGAGUAACGAAGAGAU-3’, 5’-GTGGATGAAGATCCAACCCAA-3’ and 5’-GCACATGGTTAACCTGGACAA-3’ respectively. The overexpressing plasmid of GPX4 (pcDNA3.1-GPX4) and the control vector (pcDNA3.1) were obtained from Obio Technology (Shanghai) Corp., Ltd. All siRNAs and plasmids were transfected into the cells using Lipofectamine™ 3000 Transfection Reagent following the manufacturer’s instructions for a duration of 48 hours. not-yet-known not-yet-known not-yet-known unknown Co-immunoprecipitation (Co-IP) analysis Cells were lysed using pre-chilled NP40 (BL653A, Biosharp, Anhui, China), and the supernatant obtained after centrifugation was quantified. A portion of this supernatant was used as Input for subsequent Western blot experiments. The remaining total cellular protein, quantified to 250 μg, was mixed with either the GPX4 antibody (1:200) or normal IgG (1:200) and incubated on a rocker at 4 °C for 2 hours. Following the incubation, 40 μL of agarose beads were added and incubated overnight on a rocker at 4 °C. After the overnight incubation, the mixture was centrifuged, and the beads were washed three times with PBS. Subsequently, 70 μL of 1×SDS-PAGE Sample Loading Buffer was added, and the samples were heated at 100 °C for 5 minutes. Ubiquitination was then detected by Western blot. Molecular docking analysis Molecular docking was conducted with AutoDock Vina 1.1.2, preparing the receptor protein with PyMol 2.52 by removing water molecules, ions, and small molecules, and setting up a docking box to encompass the entire protein. The highest scoring docked conformation was identified as the binding conformation and visualized using PyMol 2.5.2. The GPX4 (PDB: 6HN3). The 3D structure of SinA and SinB were downloaded from Pubchem, and their energies were minimized under the MMFF94 force field. The compound CIDs are as follows：SinA (155256) and SinB (108130). not-yet-known not-yet-known not-yet-known unknown Surface plasmon resonance (SPR) assay The interaction between GPX4 and the compounds SinA or SinB was assessed using the BiacoreX100 system (GE Healthcare, Uppsala, Sweden) at 25 °C. Recombinant human GPX4 protein was immobilized on a CM5 sensor chip using an amine coupling kit (GE Healthcare, Buckinghamshire, UK), achieving immobilization levels of approximately 8876 RU (Response Units). The compounds were then injected as analytes at various concentrations into a PBS buffer (0.5% DMSO), serving as the running buffer. Binding affinity studies involved injecting analytes at specified concentrations with a flow rate of 30 μL/min, allowing 150 seconds each for contact and dissociation phases. The chip was subsequently washed with the running buffer between analyses. Preparation of recombinant proteins The cDNA encoding the human cytosolic GPX4 (c-GPX4) was amplified by RT-PCR, and the resulting fragment was ligated into the pET28α (+) vector. The expression strategy was designed in such a way that the complete primary structure of GPX4 (except the starting Met) was retained. An additional tail of 11 amino acids, which included a hexa-histidine-tag and was introduced to allow its effective purification. The final N-terminal sequence of the recombinant protein reads M-R-G-S-H-H-H-H-G-S-A-C, in which the last C corresponds to the initial M of the native enzyme. GPX4 was expressed in E. coli Codonplus, and the cells were cultured in the Luria Bertani medium at 37 °C to an optical density of 0.6-0.8 at 600 nm, then induced with 1 mM Isopropyl-β-D-thiogalactopyranoside for 8 hours at 25 °C. The protein was purified by nickel affinity chromatography, and concentrated using an Amicon filter cartridge (Merck Millipore, USA). The protein was further purified by a Sephadex G-200 size exclusion column (MicroCal, GE Healthcare, USA) and concentrated to about 5 mg/mL using a 21-kDa concentrator. SDS-PAGE was used to determine the purity of GPX4. Isothermal titration calorimetry (ITC) assay The binding interactions of western blotting with both wild-type GPX4 and the GPX4-K31R, GPX4-K90R mutants were analyzed by an EAQ-ITC instrument (MicroCal, GE Healthcare, USA) at 25 °C. SinA or SinB was prepared in a buffer containing 20 mM Tris-HCl (pH 7.5) and 150 mmol/L NaCl. Then, 0.1 mM SinA or SinB was injected 19 times in 60-μl aliquots from the syringe into a sample cell containing 300 μl of 0.4 mM GPX4. The resulting data were processed using the Origin 7.0 software, fitting to a two-site binding model to determine thermodynamic parameters such as enthalpy change ( ΔH ), entropy change ( ΔS ), and the equilibrium dissociation constant ( KD ). Cellular thermal shift assay (CETSA) Purified recombinant GPX4 (6 mg/mL) was incubated with SinA or SinB (100 μmol/L) for 30 minutes at 25 °C. The supernatant was evenly divided into 6 tubes according to 18 μg per tube and heated at 45, 50, 55, 60, 65 and 70 °C, respectively for 10 minutes, after which the supernatant was obtained by centrifugation for western blotting analysis. Drug affinity responsive target stability (DARTS) assay Purified recombinant GPX4 (6 mg/mL) was diluted 1:10 with the TNC buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM CaCl 2 ) and treated by SinA or SinB of various concentrations, or DMSO as a control for 1 hour at room temperature. Pronase (25 μg/mL) was then added and incubated for 30 minutes at 37 °C. The reactions were stopped by adding the SDS-PAGE loading buffer, and the samples were analyzed via western blotting using an anti-GPX4 antibody. GSH detection The GSH and glutathione, oxydized (GSSG) were measured using the GSH and GSSG assay kits. Following treatment, the cells were rinsed with PBS and then collected by centrifugation, and the supernatant was discarded. Cell lysis was achieved by a protein removal reagent three times the volume of the cell pellet, followed by two cycles of freeze-thawing using liquid nitrogen and a water bath at 37 °C. The lysates were then cooled for 5 minutes at 4 °C and centrifuged at 10,000 g for 10 minutes. The clear supernatant obtained was used to measure the total GSH levels. To quantify GSSG, the supernatant samples were treated with a diluted GSH clearing aid (20 μL/100 μL of the sample) and GSH clearing working solution (4 μL/100 μL of the sample), followed by an incubation at 25 ºC for 60 minutes. The absorbance was measured at 412 nm after an additional 25 minutes. The concentration of GSH in the samples was determined by comparing the absorbance values to a standard curve provided with the kit. The concentration of reduced GSH was calculated by subtracting twice the amount of GSSG from the total glutathione measured: GSH=Total Glutathione – 2 × GSSG. not-yet-known not-yet-known not-yet-known unknown Measurement of MDA content To determine the level of malondialdehyde, a marker of lipid peroxidation and the MDA assay kit were utilized. Following the drug treatment, the cells were lysed using 100 μL of the protein lysis buffer. According to the instructions provided with the kit, the MDA detection solution was prepared and combined with the cell lysate. This mixture was then heated to 100 °C for 15 minutes, allowed to cool, and centrifuged at 10,000 g for 10 minutes. A volume of 200 μL of the supernatant was pipetted into a 96-well plate, and the absorbance was measured at 532 nm. The concentration of MDA was calculated based on a standard curve and normalized to the protein concentration in the samples. Intracellular BODIPY 581/591 C11 staining Lipid peroxidation within cells was determined using the BODIPY 581/591 C11 probe. The cells were plated at a density of 2.0× 10 4 cells/well in confocal dishes. Following treatment, the cells were incubated with 50 μL of Hank’s Balanced Salt Solution (HBSS) containing 40 nM BODIPY 581/591 C11 for 30 minutes at 37 °C. Fluorescence microscopy images were obtained using a laser scanning confocal microscope. The ratio of green fluorescent protein (GFP) green to red fluorescence protein (RFP), which reflects the oxidation of BODIPY 581/591 C11, was analyzed and quantified using the Image J software. not-yet-known not-yet-known not-yet-known unknown Statistical analysis The data were expressed as mean ± SD. Statistical analysis was performed using Student’s t-test and one-way ANOVA test in SPSS19.0 software. Graphs were created using GraphPad Prism 6.0 software. A significance level of P < 0.05 was considered statistically significant. 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Deng Y, Chu X, Li Q, Zhu G, Hu J, Sun J et al. Xanthohumol ameliorates drug-induced hepatic ferroptosis via activating Nrf2/xCT/GPX4 signaling pathway. Phytomedicine 2024; 126 : 155458. Figure legends Fig. 1. SinA and SinB repaired ALT, AST and PPIX content in vitro . (A) Flowchart of drug screening in cells. (B-C) Effects of INH on the cell viability of L02 and WRL68 cells as determined by MTT assay (n = 6). (D-G) Evaluation of the degree of INH-induced liver injury improvement by SinA, SinB, SolA, SolB, StnA, StnB, NAC, and DDB in L02 and WRL68 cells using ALT and AST kits (n = 3). (H-I) Detection of the alleviation of INH-induced PPIX accumulation by SinA, SinB, SolA, SolB, StnA, StnB, Nac, and DDB in L02 and WRL68 cells using laser scanning confocal microscopy (scale bar: 25 μm, n = 3). Data are mean ± SD. ** P < 0.01, *** P < 0.001 vs. control group, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. INH group. Fig. 2. SinA and SinB could significantly inhibit intracellular Fe 2+ accumulation and lipid peroxidation. (A-D) Measurement of MDA levels in L02 and WRL68 cells (n = 3). (E-H) Measurement of GSH levels in L02 and WRL68 cells (n = 3). (I-J) Detection of Fe²⁺ content in L02 and WRL68 cells using laser scanning confocal microscopy (scale bar: 50 μm, n = 3). (K-L) Detection of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in L02 and WRL68 cells using laser scanning confocal microscopy (scale bar: 50 μm, n = 3). (M-N) Flow cytometry analysis of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in L02 and WRL68 cells (n = 3). Data are mean ± SD. ** P < 0.01, *** P < 0.001 vs. control group, ## P < 0.01, ### P < 0.001 vs. INH group. Fig. 3. SinA and SinB could maintain mitochondrial homeostasis. (A-B) Detection of mitochondrial membrane potential levels in L02 and WRL68 cells using laser scanning confocal microscopy (scale bar: 50 μm, n = 3). (C-D) Observation of mitochondrial morphology in L02 and WRL68 cells by TEM (upper scale bar: 2.0 μm, lower scale bar: 1.0 μm, n = 5). Data are mean ± SD. *** P < 0.001 vs. control group, ### P < 0.001 vs. INH group. Fig. 4. SinA and SinB upregulated the expression of GPX4 protein. (A-D) Western blot analysis of GPX4 protein expression levels in L02 and WRL68 cells. (E-F) Laser scanning confocal microscopy for detection of GPX4 fluorescence intensity in L02 and WRL68 cells (scale bar: 50 μm, n = 3). (G-H) Western blot assessment of GPX4 knockdown efficiency in L02 and WRL68 cells. (I-J) Laser scanning confocal microscopy for verification of GPX4 knockdown efficiency in L02 and WRL68 cells (scale bar: 50 μm, n = 3). Data are mean ± SD. *** P < 0.001 vs. control group, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. INH group. Fig. 5. GPX4 knockout weakened SinA and SinB to inhibit lipid peroxidation and mitochondrial damage. (A-D) Measurement of MDA levels in L02 and WRL68 cells after GPX4 knockdown (n = 3). (E-H) Measurement of GSH levels in L02 and WRL68 cells after GPX4 knockdown (n = 3). (I-J) Detection of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in L02 and WRL68 cells after GPX4 Knockdown using laser scanning confocal microscopy (scale bar: 50 μm, n = 3). (K-L) Detection of mitochondrial membrane potential levels in L02 and WRL68 cells after GPX4 Knockdown using laser scanning confocal microscopy (scale bar: 50 μm, n = 3). Data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding groups. Fig. 6. GPX4 overexpression enhanced SinA and SinB to restrain the elevation of MDA content and the reduction of GSH content. (A-B) Flow cytometry analysis of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in L02 and WRL68 cells after GPX4 knockdown (n = 3). (C-D) Western blot assessment of GPX4 overexpression efficiency in L02 and WRL68 cells. (E-F) Laser scanning confocal microscopy for verification of GPX4 overexpression efficiency in L02 and WRL68 cells (scale bar: 50 μm, n = 3). (G-J) Measurement of MDA levels in L02 and WRL68 cells after GPX4 overexpression (n = 3). (K-N) Measurement of GSH levels in L02 and WRL68 cells after GPX4 overexpression (n = 3). Data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding groups. Fig. 7. GPX4 overexpression enhanced SinA and SinB to inhibit lipid peroxidation and mitochondrial damage. (A-B) Laser scanning confocal microscopy detection of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in L02 and WRL68 cells after GPX4 overexpression (scale bar: 50 μm, n = 3). (C-D) Laser scanning confocal microscopy detection of mitochondrial membrane potential levels in L02 and WRL68 cells after GPX4 overexpression (scale bar: 50 μm, n = 3). (E-F) Flow cytometry analysis of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in L02 and WRL68 cells after GPX4 overexpression (n = 3). Data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding groups. Fig. 8. SinA and SinB supressed the ubiquitination and degradation of GPX4. (A-D) RT-qPCR analysis of GPX4 mRNA levels in L02 and WRL68 cells (n = 3). (E-F) DARTS assay to evaluate the binding affinity of GPX4 in L02 and WRL68 cells. (G-H) CETSA to assess the binding affinity of GPX4 in L02 and WRL68 cells. (I-L) Western blot analysis of the effects of SinA and SinB on GPX4 expression levels in L02 and WRL68 cells in the presence of CHX. (M-P) Western blot analysis of the effects of SinA and SinB on GPX4 expression levels in L02 and WRL68 cells in the presence of MG132. (Q-R) Western blot analysis of GPX4 ubiquitination levels in L02 and WRL68 cells under the influence of SinA and SinB. Data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding groups. N.S. not significant. Fig. 9. SinA and SinB could markedly improve INH-induced liver injury via the ferroptosis pathway in vivo . (A) Flowchart of animal drug administration. (B) Images of mouse livers from various groups. (C) Weight curve of mice (n = 6-10). (D) Liver index of mice (n = 6-10). (E) ALT levels in mouse liver tissue (n = 6). (F) AST levels in mouse liver tissue (n = 6). (G) HE staining of mouse liver tissue (scale bar: 200 μm, n = 3). (H) MDA Levels in mouse liver tissue (n = 6). (I) GSH levels in mouse liver tissue (n = 6). (J) Western blot analysis of GPX4 expression in mouse liver tissue. (K) IHC staining of GPX4 in mouse liver tissue (scale bar: 100 μm, n = 3). Data are mean ± SD. * P < 0.05, *** P < 0.001 vs. control group, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. INH group. Fig. 10. Interaction analysis of SinA and SinB with GPX4. ( A-F ) The SinA, SinB, SolA, SolB, StnA, StnB and GPX4 interaction diagram. Hydrogen bonds are shown as black dotted lines. (G-L) The affinities of GPX4 binding with different components were detected by Biacore. (M-N) CETSA experiments of SinA and SinB with GPX4 protein. (O-P) Investigation of binding affinity of different ligand and GPX4. Representative ITC titration profiles of GPX4 with two ligands. The top graphs represent the raw ITC thermograms, and the bottom graphs represent the fitted binding isotherms. The corresponding Kd value has been marked in the figure. ITC experiments of SinA and SinB with GPX4 (Q-R) Thermal stabilities of GPX4 with two different ligands by DSF assay. GPX4 represents the group that GPX4 protein without the peptide, and is colored in black. The GPX4 protein incubated with are colored in Black, red and blue, respectively. The ΔTm values are summarized on the table and represent the Tm values of the peptide-added group minus that of the GPX4 group (n = 3). (S-T) DARTs experiments of SinA and SinB with GPX4 protein. (U) Sequence conservation analysis of GPX4. (V) Multiple sequence alignment of GPX4 from different species. H. sapiens , M. musculus , P. troglodytes, R. norvegicus and S. scrofa represent Homo sapiens , Mus musculus , Pan troglodytes and Rattus norvegicus, Sus scrofa , respectively. The red background represents extremely conserved residues, and the red font represents relatively conserved residues. Data are mean ± SD. ** P < 0.01, *** P < 0.01 vs. corresponding groups. Fig. 11. SinB directly interacted with GPX4 in K31 and K90. (A-D) CETSA experiments of SinA and SinB with GPX4-K31R, GPX4-K90R protein. (E-H) Investigation of binding affinity of different ligand and GPX4-K31R or GPX4-K90R protein. Representative ITC titration profiles of GPX4-K31R or GPX4-K90R protein with two ligands. The top graphs represent the raw ITC thermograms, and the bottom graphs represent the fitted binding isotherms. The corresponding Kd value has been marked in the figure. ITC experiments of SinA and SinB with GPX4-K31R or GPX4-K90R protein. (I-J) DARTs experiments of SinA and SinB with GPX4-K31R, GPX4-K90R protein. (K-N) Thermal stabilities of GPX4-K31R or GPX4-K90R with two different ligands by DSF assay. GPX4-K31R or GPX4-K90R represents the group that GPX4-K31R or GPX4-K90R protein without the peptide, and is colored in black. The GPX4-K31R or GPX4-K90R protein incubated with are colored in Black, red and blue, respectively. The ΔTm values are summarized on the table and represent the Tm values of the peptide-added group minus that of the GPX4-K31R or GPX4-K90R group ( n=3 ). Data are mean ± SD. * P < 0.05 vs. corresponding groups. N.S. not significant. Fig. 12. SinB directly interacted with GPX4 in K31 and K90. (A) GSH levels in WRL68 cell (B) GSSG levels in WRL68 cell. (C) MDA levels in WRL68 cell (D) Laser scanning confocal microscopy detection of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in WRL68 Cells after GPX-K31R overexpression. (E) Laser scanning confocal microscopy detection of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in WRL68 cells after GPX4-K90R overexpression. (F) Laser scanning confocal microscopy detection of C11-BODIPY and oxidized C11-BODIPY fluorescence levels in WRL68 Cells after GPX-K31R overexpression (scale bar: 50 μm, n = 3). Data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding groups. N.S. not significant. not-yet-known not-yet-known not-yet-known unknown Supplementary Information Supplementary Fig. S1. SinA and SinB did not stabilize GPX4 through the mitochondrial autophagy pathway. (A-D) Western blot analysis of LC3BII/LC3BI expression in L02 and WRL68 cells (n = 3). Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 82274214 and 82322074), Research Foundation of Education Bureau of Guangdong Province (Grant No. 2022ZDZX2010), Science and Technology Program of Guangzhou (Grant No. 202201011420), and the grant of Department of education of Guangdong Province (Grant No. 2023KTSCX024). not-yet-known not-yet-known not-yet-known unknown Author Contribution Statement Yuting Zheng : Investigation, Methodology, Formal analysis, Writing – original draft. Huan Lan : Investigation, Formal analysis, Writing – review & editing. Caihong Liu, Lin An, Peng Wu and Zhang Rong : Investigation, Formal analysis. Zhongqiu Liu and Jinjun Wu : Conceptualization, Writing – review & editing, Supervision. Caiyan Wang : Conceptualization, Writing – review & editing, Supervision, Funding acquisition. Conflict of Interest Statement The authors declare that they have no conflict of interest. Ethics Statement All Institutional and National Guidelines for the care and use of animals were followed. Data Availability Statement All data generated or analyzed during this study are included in this article. Information & Authors Information Version history V1 Version 1 28 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords hepatopharmacology lipids toxicology Authors Affiliations Yuting Zheng Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Huan Lan Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Caihong Liu Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Lin An Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Peng Wu Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Rong Zhang Guangzhou University of Chinese Medicine View all articles by this author Zhongqiu Liu Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Jinjun Wu Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Caiyan Wang 0000-0002-8605-2340 [email protected] Guangzhou University of Chinese Medicine International Institute for Translational Chinese Medicine View all articles by this author Metrics & Citations Metrics Article Usage 240 views 140 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yuting Zheng, Huan Lan, Caihong Liu, et al. not-yet-known not-yet-known not-yet-known unknown Schisandra Lignans Target GPX4 to Suppress Ferroptosis: A First-in-Class Natural Therapy for INH-Induced Liver Injury. 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