Loganin ameliorates acetaminophen-induced acute liver injury via targeting TOP1MT-mediated hepatocyte apoptosis and ferroptosis

Loganin ameliorates acetaminophen-induced acute liver injury via targeting TOP1MT-mediated hepatocyte apoptosis and ferroptosis | 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 Loganin ameliorates acetaminophen-induced acute liver injury via targeting TOP1MT-mediated hepatocyte apoptosis and ferroptosis Dou Niu, Yue Yang, Xiaobo Yu, Teng Hui, Meng Wang, Jigang Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3994000/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acetaminophen (APAP) overdose can cause severe liver injury, and new drugs are urgent needed for effective treatment. Small molecules in Chinese medicine have long been a treasured reservoir for drugs screening. Here, we reported that loganin (LOG), an active ingredient in Corni Fructus, exerts hepatoprotective effects as indicated by potently alleviated liver damages in APAP-induced liver injury (AILI) murine model. LOG reversed the decreased SOD, GSH and CAT levels, and reduced lipid peroxidation, ROS production, and iron overload and hence reduced apoptosis/ferroptosis of hepatocytes of AILI models, as apoptosis/ferroptosis inducers abolished, whereas their inhibitors enhanced the effect of LOG. Through the activity-based proteome profiling (ABPP) clickable alkyne-tagged LOG probe, mitochondrial topoisomerase I (TOP1MT) was captured as a direct target of LOG, which was further validated by CETSA and ITC assays. Deficiency of TOP1MT significantly compromised the effects of LOG on H2O2-induced oxidative stress cell model via regulating downstream apoptosis/ferroptosis regulators Bax, Bcl-2, NRF2, GSH, SLC7A11, and GPX4. Consistently, LOG effect was greatly eliminated in AILI mice once the endogenous hepatic TOP1MT was knocked-down by AAV-TOP1MT shRNA. Thus, TOP1MT might be a potential target for AILI treatment and LOG represents one of the most promising candidate drugs or lead compounds. Biological sciences/Drug discovery/Target identification Biological sciences/Chemical biology/Mechanism of action Acetaminophen Loganin Liver injury TOP1MT Ferroptosis Activity-based proteome profiling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction As the most popular and top-selling analgesic and antipyretic drug used worldwide, Acetaminophen (APAP) is considered safe and effective when taken at the recommended dosage 1 . Once overdose occurred, however, the overwhelming of phase II metabolizing enzymes often results in excessive deposition of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) and depletes the crucial antioxidant glutathione (GSH) 2 . In turn, it leads to mitochondrial oxidative stress, impaired mitochondrial function and hepatocyte death or even liver failure 3, 4 . Until now, APAP overdose is the predominant form of drug-induced liver injury (DILI) and major cause of acute liver failure worldwide 5 . Although N-acetyl cysteine (NAC) has been recommended by FDA as the first-line drug to treat DILI, the potential adverse effects and a relatively narrow therapeutic window greatly limited its clinical use 6 . If the early critical stages of APAP-induced liver injury (AILI) are missed, liver transplantation becomes the only viable option to improve patient survival 7 . Therefore, there is an urgent need for strengthening the incomplete understanding of its pathogenesis, changing unsatisfactory therapies status, and developing alternative therapeutic strategies that can effectively intervene at various stages of AILI and provide better outcomes for patients 8 . Ferroptosis is a novel form of programmed cell death characterized by the iron-dependent accumulation of lipid peroxidation (LPO) within cellular membranes. This process has been broadly implicated in a wide spectrum of diseases including neurodegeneration, ischemia-reperfusion injury, cancer, and other pathological conditions 9 . Erastin, a well-known inducer of ferroptosis, exerts its effects by inhibiting system Xc−, thereby suppressing cellular cysteine uptake and depleting GSH, which plays vital roles in antioxidant defense mechanisms against reactive oxygen species (ROS). The enzyme glutathione peroxidase 4 (GPX4) utilizes GSH to repair damaged lipids and convert toxic lipid hydroperoxides into non-toxic lipid alcohols. However, depletion of GSH disrupts the function of GPX4, leading to the accumulation of lipid peroxides and subsequent initiation of apoptotic and ferroptotic cell death 10 . As the major organ for lipid metabolism, it is not surprising that mounting evidence reported that ferroptosis plays critical roles in various hepatic diseases 11, 12, 13 . However, whether it is involved in AILI and targeting ferroptosis can emerge as a novel and promising therapeutic approach for AILI has almost completely unknown, except only rare studies demonstrated that ferroptosis is related with APAP-induced primary hepatocytes death 14 . A wide array of valuable natural products has been extensively utilized worldwide as preventive and therapeutic agents for various diseases, including liver injury 15 . Loganin (LOG), an iridoid glycoside compound ( Figure S1A and S1B ), is one of the core bioactive components of Corni Fructus with multiple biological properties, such as anti-apoptotic 16 , anti-inflammatory 17 , anti-oxidant 18 and neuroprotective activities 19 . Accumulating evidence has reported that LOG can be used for the treatment of diabetes 20 , lung injury 21 , renal injury 17 and liver injury 22 . However, the molecular mechanisms of the action of LOG still remain unclear. In this study, we demonstrated that LOG effectively protects against AILI by inhibiting oxidative stress and ROS production, and resulted apoptosis and ferroptosis, via directly targeting mitochondrial topoisomerase I (TOP1MT) and enhancing its antioxidant activities. Silencing of TOP1MT in HepG2 cells strengthen H 2 O 2 -induced accumulation of ROS, LPO and Fe 2+ /Fe 3+ , key markers of apoptosis and ferroptosis. Consistently, LOG effect was significantly impaired in the AILI model in liver-specific TOP1MT-knockdown mice. Taken together, our data reveal that LOG ameliorates AILI via directly binding and stabilizing TOP1MT, therefore promoting antioxidant proteins expression and inhibiting apoptosis and ferroptosis of hepatocytes. These findings provide new insights for the pathogenesis of AILI and suggest TOP1MT as a potential biomarker and therapeutic target for AILI. Results and discussion LOG ameliorates APAP-induced liver injury through inhibiting oxidative stress in vivo To investigate the protective effects of LOG on APAP-induced liver injury, the mice were subjected to the experimental procedures outlined in Fig. 1 A. As shown in Fig. 1 B, no statistically significant differences in body weights were observed between the initial and final measurements, except that an apparent weight loss was found in the APAP group. As to the survival rate of the mice, APAP exposure resulted in half of death, whereas administration of 160 mg/kg LOG significantly improved the lethal effect of APAP (Fig. 1 C). Observation of tissue appearance shows obvious necrotic spots and tissue hardening of the liver (Fig. 1 D). In addition, APAP mice got high pathological scores which was significantly decreased in the high dose LOG-treated mice ( p < 0.05) (Fig. 1 E). H&E staining results revealed that APAP induced extensive liver damage including destruction of liver architecture, hepatocyte ballooning degeneration, and ubiquitous centrilobular hepatocyte death, which were attenuated significantly in LOG treatment group (Fig. 1 F-G). Consistently, LOG administration at different doses (80 and 160 mg/kg) significantly improved the liver function of APAP-treated mice as indicated by the potently reduced serum ALT and AST levels (Fig. 1 H). Considering oxidative stress play pivotal roles in cytotoxic drugs associated hepatotoxicity, we measured the antioxidant enzymes activities in homogenized liver tissues, and we found that LOG treatment reversed the decrease of glutathione (GSH) and promoted the activities of superoxide dismutase (SOD) and catalase (CAT), which are all dysregulated key lipid peroxidation markers in liver injuries (Fig. 1 I) 23 . As a member of the cytochromes P450 (CYPs) phase I enzymes, CYP2E1 is extensively involved in the metabolism of various endogenous and exogenous compounds like APAP 24 . Our immunofluorescence data showed that CYP2E1 expression was strongly induced by APAP and apparently inhibited by LOG treatment (Fig. 1 J). Emerging evidence demonstrated that CYP2E1 can induce oxidative stress and augment tissue injury 25 . Given rise to these aforementioned enzymes have important roles in resisting ROS and free radicals 26 , we measured the ROS production in the mice, and the results showed that ROS is highly produced in APAP-treated group but significantly reversed in LOG group (Fig. 1 K). Taken together, these results collectively imply that oxidative stress is engaged in APAP-induced liver injury, which might be involved in the hepatoprotective mechanism of LOG. Loganin suppresses APAP-induced hepatocyte apoptosis and ferroptosis The excessive accumulation of lipid peroxides and ROS often leads to cell death. Considering the obvious hepatocyte death and lipid peroxidation as mentioned above, we wonder which kind of cell death is present in AILI. As shown in Fig. 2 A-B, TUNEL staining showed that APAP induced elevated hepatocyte apoptosis which was greatly reversed by LOG administration. In addition, transmission electron microscopy (TEM) observation suggested that ferroptosis, an iron-dependent programmed cell death 27 , was also potently induced by APAP, as indicated by the classic morphological abnormalities in mitochondria (Fig. 2 C). To confirm these, we measured the molecular signatures of the tissues. As expected, Western blot data showed that the proapoptotic gene Bax was upregulated, whereas the antiapoptotic Bcl-2 was apparently downregulated by APAP, but LOG treatment potently opposed these effects (Fig. 2 D-E). As to ferroptosis-related genes, Nrf2, a central transcription factor that regulates the antioxidant stress response 28 , was strongly suppressed by APAP but significantly increased by LOG. The expression levels of SLC7A11 and GPX4, two key negative regulators involved in the ferroptosis process, were found to be significantly decreased in the liver of APAP-treated mice (Fig. 2 D and 2 F). Therefore, these findings provide evidence that the inhibition of ROS-triggered hepatocyte apoptosis and ferroptosis mediated the hepatoprotective effects of LOG. Loganin suppresses HO-induced hepatocyte apoptosis and ferroptosis To further confirm the hepatoprotective effects of LOG on oxidative stress induced liver cell apoptosis and ferroptosis, a widely used H 2 O 2 -induced oxidative stress hepatocyte injury model was established in HepG2 cell line 29, 30, 31 . Considering ROS should be a central hub in the process of apoptosis and ferroptosis 32 , we firstly assessed the ROS levels in HepG2 cells. Our data demonstrated that ROS production was significantly increased in the H 2 O 2 -induced group, which was significantly reversed in the presence of LOG, indicating that LOG treatment effectively regulated and attenuated the excessive ROS production induced by H 2 O 2 (Fig. 3 A and 3 B). As depicted in Fig. 3 C-D, exposure to H 2 O 2 resulted in an elevated proportion of annexin V/PI-positive apoptotic cells. However, LOG exhibited a significant dose-dependent reversal of apoptosis in HepG2 cells. To confirm the inhibitory effects of LOG on hepatocyte ferroptosis, we assessed the ferroptosis negative regulatory protein levels of Nrf2, GPX4, and SLC7A11. In comparison to the H 2 O 2 -induced group, LOG treatment led to an upregulation in the levels of SLC7A11, Nrf2, and GPX4 (Fig. 3 E-F). As ferrous ions can rapidly trigger Fenton-like reaction to produce ROS and regulate the intercellular LPO as well, we thus conducted iron and LPO assessment (Fig. 3 G-H). As expected, elevated Fe 2+ /Fe 3+ and LPO was detected in H 2 O 2 -stimulated HepG2 cells, which was potently recovered in the presence of LOG, indicating an iron and lipid peroxidation homeostasis maintenance properties of LOG. Visualization of lipid droplets by C11-BODIPY staining further proved the effects of LOG (Fig. 3 I). Additionally, TEM analysis of HepG2 cells revealed that LOG-treatment increased the number of mitochondrial cristae and recovered mitochondrial morphology, indicating the potential role of LOG in preserving mitochondrial integrity (Fig. 3 J). Together, these data suggested that LOG exerts hepatoprotective effects via preventing oxidative stress-induced hepatocyte apoptosis and ferroptosis. Induction of apoptosis and ferroptosis abolishes the hepatoprotective effect of LOG To further investigate the role of apoptosis and ferroptosis in the hepatoprotective effect of LOG on AILI, H 2 O 2 -induced HepG2 oxidative stress model was treated by LOG in the absence or in the presence of apoptosis and ferroptosis inducers and inhibitors. As shown in Fig. 4 A-B, the apoptosis inducer CHX apparently reversed the elevated Bcl-2 protein levels ( p < 0.01) and decreased Bax protein levels ( p < 0.0001) induced by LOG. Notably, CHX significantly enhanced H 2 O 2 -induced Bax expression and almost completely blocked the effects of LOG on Bax suppression which was comparable to the level in untreated model cells. Similar data were obtained by flow cytometry analysis and CHX potently counteracted the protective effect of LOG on oxidative stress-induced HepG2 cell apoptosis (Fig. 4 C). On the contrary, co-administration of apoptosis inhibitor ZVAD further increased the expression of Bcl-2 induced by LOG treatment ( p < 0.01), although no apparent effects on Bax production was found, this may be due to strong protective effects of LOG (Fig. 4 D-F). Similar trends were found in ferroptosis inducer and inhibitor, in which the ferroptosis inducer erastin significantly reversed the elevated protein levels of Nrf2 ( p < 0.01), GPX4 ( p < 0.01), and SLC7A11 ( p < 0.05) induced by LOG (Fig. 4 G-H), whereas the ferroptosis inhibitor Fer-1 potently recovered H 2 O 2 -suppresed Nrf2 ( p < 0.01), GPX4 ( p < 0.05), and SLC7A11 ( p 0.05), which strongly indicates that LOG might exerts hepatoprotective effects through similar strength and signal pathways as Fer. These data collectively support the hypothesis that the anti-apoptotic and anti-ferroptotic activities of LOG is associated with its protective effects against AILI. Loganin directly targets mitochondrial topoisomerase I (TOP1MT) To identify the direct target and action mechanism of LOG, a clickable alkyne tag was designed and synthesized as LOG probe (LOG-P) (Fig. 5 A). The LOG-P exhibited similar cellular potency with unmodified LOG, suggesting that the attached alkyne tag in LOG-P does not interfere with the natural activity of LOG (Fig. 5 B). A click chemistry reaction between TAMRA-azide and the alkyne tag in LOG-P was performed to assess the subcellular localization of LOG 33 , and LOG is found to be mainly distributed in cytoplasm and nucleus (Fig. 5 C). Subsequently, incubation of HepG2 cells with LOG-P exhibited that the labelled LOG can enrich numerous proteins in a dose- (Fig. 5 D) and time-dependent (Fig. 5 E) manner, in which 100 µM LOG-P and 3-h incubation enriched most target proteins. Once HepG2 cells were incubated with LOG and LOG-P simultaneous, a remarkable competition for binding targets was observed (Fig. 5 F), indicating that LOG-P can specifically enrich the same targets as his unmodified parent compound LOG. Then, HepG2 cell lysates were incubated with 100 µM LOG-P in the presence or absence of excessive LOG as a competitor to raise the specificity of target screening (Fig. 5 G). The probe labeled proteins were affinity-purified in batches, separated by SDS-PAGE, and identified by LC-MS/MS. The functional distribution of LOG-P enriched targets was assessed using Gene Ontology (GO) analysis (Fig. 5 H). Additionally, pathway analysis using KEGG orthology (KO) further elucidated the signaling pathways and molecular interactions involved in the hepatoprotective effects of LOG (Fig. 5 I). Finally, 34 proteins were enriched in the LOG-P group compared to the “Compete” group (Fig. 5 J). Among them, a ferroptosis-associated protein TOP1MT drew our strong attention and preferred to study further 34, 35 . Virtual molecular docking revealed that LOG predominantly occupies the Lys493, Lys532, Thr501, Val502, Asp533, Ala499, and Gln421 residues of TOP1MT (Fig. 6 A). As expected, TOP1MT level was significantly reduced upon H 2 O 2 stimulation but greatly recovered by LOG treatment (Fig. 6 B-C). A pull-down assay indicated direct interaction between TOP1MT and LOG (Fig. 6 D). Additionally, the co-localization of LOG and TOP1MT in HepG2 cells provided further evidence that LOG can directly target TOP1MT (Fig. 6 E). An isothermal titration calorimetry (ITC) assay was performed to prove the direct binding of LOG to TOP1MT in vitro after the human recombinant TOP1MT proteins from E. coli (BL21) was firstly prepared. The results showed that LOG exhibited binding to TOP1MT in an approximate 1:1 stoichiometry (N = 0.983 ± 3.6e-2) and binding affinities was detected to be K d = 1.02e-6 ± 492e-6 µM. The binding thermodynamic parameters between LOG and TOP1MT was also demonstrated both enthalpy and entropy compensation (ΔG = -8.18 kcal/mol, ΔH = -80 ± 4.03 kcal/mol, -TΔS = 71.8 kcal/mol) (Fig. 6 F). Then, a cellular thermal shift assay (CETSA) was conducted to further confirm the direct occupation of TOP1MT by LOG. As shown in Fig. 6 G-H, a dramatic shift in the TOP1MT melting curve was observed as compared with the DMSO group and control gene β-actin, indicating that LOG could stabilize TOP1MT in HepG2 cell lysates. Altogether, these findings collectively demonstrated that LOG may attenuate AILI by directly targeting and stabilizing TOP1MT in hepatocytes. TOP1MT deficiency exacerbates ferroptosis and apoptosis in H 2 O 2 -induced HepG2 oxidative stress model We wonder whether and how TOP1MT is involved in LOG-attenuated liver injury. Given that both apoptosis and ferroptosis are closely related to mitochondrial function and play crucial roles in LOG-regulated AILI, TOP1MT was knocked down (KD) by specific siRNA in H 2 O 2 -induced HepG2 oxidative stress model and mitochondrial functions were detected. Firstly, DCFH-DA fluorescence dye was used to evaluate total intracellular ROS level. A significant increase of dichlorofluorescein (DCF) fluorescence intensity can be seen in cells exposed to H 2 O 2 , which was potently blocked by LOG addition. However, TOP1MT KD almost completely shielded this effect of LOG (Fig. 7 A). Then, the mitochondrial membrane potential (MMP) was measured by the lipophilic cationic probe JC-1. As shown in Fig. 7 B, H 2 O 2 exposure significantly decreased MMP of HepG2 cells compared with that in control group as indicated by remarkably reduced potential-dependent accumulation of JC-1. Interestingly, LOG effectively prevented HepG2 mitochondrial depolarization only in the absence of TOP1MT siRNA. Finally, lipid ROS was detected by C11 BODIPY and results showed that H 2 O 2 promoted lipid peroxidation and LOG reversed this effect in a TOP1MT-dependent manner (Fig. 7 C). Therefore, TOP1MT is extensively involved in LOG-regulated mitochondria function in H 2 O 2 -induced HepG2 oxidative stress model. To further validate the role of TOP1MT in LOG-regulated hepatocytotoxicity, apoptosis and ferroptosis-related genes were detected. We found that the inhibition of apoptosis and ferroptosis of H 2 O 2 -treated HepG2 cells by LOG was apparently weakened in TOP1MT deficient cells as indicated by decreased Bcl-2, Nrf2, GPX4, and SLC7A11, and increased Bax (Fig. 7 D-E), as well as the substantial increase in the levels of lipid peroxidation (LPO) and Fe 2+ /Fe 3+ (Fig. 7 F-G). In addition, TEM results showed an obvious ferroptosis of TOP1MT knockdown HepG2 cells exposed to H 2 O 2 which can not be saved by LOG pretreatment (Fig. 7 H). These findings indicate that LOG targeted TOP1MT plays a crucial role in hepatocytotoxicity via regulating the expression of Nrf2, GPX4, SLC7A11, Bcl-2, and Bax, and its deficiency results in elevated oxidative stress, disrupted iron homeostasis, dysregulated mitochondria function, and hepatocyte apoptosis and ferroptosis. Knockdown of TOP1MT abolished the hepatoprotective efficacy of LOG and aggravates ferroptosis and apoptosis in AILI mice Inspired by the results in vitro , the effect of TOP1MT on the hepaprotective activity of LOG was further investigated in AILI mice. We generated liver-specific TOP1MT knockdown mice by i.v. injection of adeno associated virus (AAV)-encoded TOP1MT shRNA (AAV-shRNA) into the C57BL/6J mice, and scramble shRNA (AAV-shNC) was used as a negative control (Fig. 8 A). Two weeks later, AILI model was established by APAP administration and treated with LOG as aforementioned. As shown in Fig. 8 B, administration of LOG did not affect body weight of the mice during the experimental period, as well as in control. However, TOP1MT KD mice exhibited an apparent reduction in body weight. Observation of tissue appearance shows more necrotic spots and tissue hardening of the liver in APAP and APAP + LOG + TOP1MT shRNA groups (Fig. 8 C). Similar trends were obtained in pathological scores as indicated by higher scores in APAP and APAP + LOG + TOP1MT shRNA groups. Although TOP1MT didn’t aggravate the pathological score of APAP model mice, it did significantly weaken the protective effect of LOG on AILI mice (Fig. 8 D). H&E staining data showed that APAP-induced liver damages was strongly attenuated by LOG, while this protective effects of LOG almost completely lost in TOP1MT KD mice (Fig. 8 E-F). In addition, the liver inflammation and oxidative stress markers including ALT, AST, CAT, GSH, and SOD were significantly disturbed by APAP, but potently improved by LOG treatment ( p < 0.01) and returned to normal levels. However, TOP1MT deficiency thoroughly deprived the anti-inflammatory and anti-oxidative properties of LOG, as indicated by the extremely blunted recoveries of these markers in the APAP + LOG + shRNA group (Fig. 8 G). We next investigated whether LOG exhibit the therapeutic effects on the liver by targeting TOP1MT. Notably, TOP1MT deficiency resulted in a significant upregulation of CYP2E1 even in the presence of LOG, suggesting increased oxidative stress and liver damage (Fig. 9 A). In addition, the hepatoprotective effects of LOG largely expired in TOP1MT KD mice and apparent hepatocyte apoptosis and ferroptosis can be seen as evidenced by TUNEL staining (Fig. 9 B) and TEM observation (Fig. 9 C). Consistently, the anti-apoptotic and anti-ferroptotic protein levels of Bcl-2, Nrf2, GPX4, and SLC7A11 were significantly decreased in APAP-treated TOP1MT KD mice, which can’t be effectively retrieved by LOG as in the wild type C57BL/6J mice. An opposite trend in the expression of proapoptotic protein Bax was observed, where its level was found to be elevated in the TOP1MT KD AILI model group compared to that in control group (Fig. 9 D-E). Taken together, these results indicated that TOP1MT KD exacerbates liver inflammation and impairs liver function, compromises the anti-apoptotic and anti-ferroptotic effects of LOG on AILI in mice. Thus, TOP1MT plays an indispensable role and could be a potential therapeutic target in AILI. Conclusions In this study, we found that LOG, a bioactive compound found in Corni Fructus which has garnered considerable attention due to its promising effects in the treatment of diverse ailments, showed strong protective effect against AILI. LOG administration potently reversed acute hepatocyte damages via recovering dysregulated ALT, AST, CAT, GSH, SOD, and CYP2E1 levels, excessive oxidative stress, lipid peroxidation and ROS production in AILI murine model and H 2 O 2 -induced cellular oxidative stress model. Through the use of an activity-based protein profiling (ABPP) strategy, we identified TOP1MT (mitochondrial topoisomerase I) as the most potential target of LOG, which was validated by virtual molecular docking, pull-down, CETSA and ITC assays. Finally, LOG inhibits oxidative stress-induced hepatocyte apoptosis and ferroptosis by binding and stabilizing TOP1MT and hence regulated the downstream apoptotic- and ferroptotic-associated Bax, Bcal-2, Nrf2, GPX-4, and SLC7A11 genes. In summary, our study demonstrates that LOG exerts hepatoprotective effects by inhibiting ferroptosis and apoptosis in both in vivo and in vitro models of AILI through regulating the TOP1MT/oxidative stress axis. Stabilization and maintenance of TOP1MT activity might be a tempting strategy for AILI treatment and LOG represents one of the most promising candidate drugs or lead compounds. Materials and methods Reagents Loganin (purity > 98%) was purchased from Bethealth People Biomedical Technology (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Japan). N-Acetyl-L-cysteine was obtained from Sigma Aldrich (USA). TAMRA-azide, Biotin-azide and THPTA are all purchased from ClickChemistryTools (Scottsdale, AZ). NaVc and CuSO 4 are purchased from Sigma Aldrich (Milwaukee, WI, USA). High capacity neutravidin agarose beads, TEAB and sequencing grade modified trypsin, Pierce™ Quantitative Fluorometric Peptide Assay Kit, and BODIPY™ 581/591 C11 were from Thermo Fisher Scientific (Waltham, MA, USA). Oasis HLB Extraction Cartridge was obtained from Waters (Milford, MA, USA). Specific primary antibodies against TOP1MT (ab121681), Nrf2 (ab137550), SLC7A11 (ab175186), and GPX4 (ab125066) were from Abcam (Shanghai, China). Specific primary antibodies against Bax (AG1208), Bcl-2 (AF0060), and β-actin (AF0003), and DCFH-DA (S0033) were from Beyotime Biotechnology (Shanghai China). LPO assay kit (A106-1-2), and Fe 2+ /Fe 3+ iron assay kit (A039-2-1) were from Nanjing Jiancheng biotechnology (Nanjing, China). Cell culture HepG2 cell was purchased from the Chinese Academy of Medical Sciences (Beijing, China) and cultured in Dulbecco’s modified Eagle’s medium (Hyclone, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) as well as 100 U/mL penicillin and streptomycin (Gibco, USA), and maintained at 37℃ with 5% CO 2 humidified atmosphere. Cells were passaged regularly when grown to approximately 80% confluence. Detection of cell viability HepG2 cells were seeded in 96-well plate at the density of 8000 cells/well for 24 h, and then treated with different concentrations of LOG and LOG probe (LOG-P) for 24 h. The cell viability was measured by CCK-8 kit according to the manufacturer’s instructions. Establishment of APAP-induced liver injury model in mice C57BL/6 male mice (19–22 g) obtained from Beijing Sibafu Biotechnology Co., LTD (Beijing, China) were maintained with standard conditions. All mice were adapted for 7 d before experiments. A total of 50 mice were randomly divided into 5 groups, 10 mice per group. They were given normal saline (vehicle, control), APAP (model), APAP + 80 mg/kg LOG (L-LOG), APAP + 160 mg/kg LOG (H-LOG), and APAP + 200 mg/kg silymarin (positive control), respectively. For APAP administration, mice were injected intraperitoneally with 400 mg/kg APAP once after 1 h of mice were administrated with LOG the last time. LOG was dissolved in normal saline, and injected i.g. to mice once a day for 10 successive days. After anaesthesia, blood and liver samples were taken. A section of liver tissue was preserved in 4% paraformaldehyde, while the remaining liver tissue was rapidly frozen in liquid N 2 for later use. All procedures were followed by the institutional animal care committee of Shaanxi Normal University, and approved by institutional guidelines and regulations (No. SYXK-2021-003). Biochemical and histological assays The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), catalase (CAT) superoxide dismutase (SOD), and glutathione (GSH) were assessed by kits from Jiancheng Biotechnology (Nanjing, China) adhering to the instructions. Liver tissues were sectioned and histological changes and liver injuries were examined using Hematoxylin-eosin (H&E), Hoechst, and TUNEL staining, as well as CYP2E1 immunofluorescence staining. Western blotting Proteins were extracted with RIPA buffer containing phenylmethylsulfonyl fluoride, separated by SDS-PAGE, and electro-transferred onto PVDF membranes. Then, samples were incubated with corresponding primary and secondary antibodies. The specific protein band was visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate in the ChemiDoc Imaging Systems (Bio-Rad, USA). Protein level was quantified by Image J, and normalized to β-actin loading control. Cellular imaging HepG2 cells were cultured in 4-chamber glass bottom dishes and treated with LOG-P at different concentrations with or without LOG. After 4 h, cells were washed with PBS trice and fixed with 4% paraformaldehyde for 20 min at room temperature (RT), and then permeabilized with 0.5% Triton X-100. Freshly prepared clicked reaction cocktail (1 mM NaVc, 100 mM THPTA, 1 mM CuSO 4 and 50 µM TAMRA-N3) were added into cells and reacted with shake for 2 h at RT. Then, the cells were washed with PBS gently. For co-localization experiments, cells were fixed, permeabilized and blocked with 5% BSA. Then, the cells were incubated with primary antibodies (1:500) at 4℃ overnight, and gently washed with PBS, followed by incubation with secondary fluorescent antibody (Alexa Fluor 488, Beyotime, China) (1:500) for 2 h, and unbound antibodies were removed. Finally, the microscopic images were captured using a confocal microscope (Leica SP8). Detection of mitochondrial transmembrane potential (MTP) JC-1 dye (C2003, Beyotime) was used to measure the MTP of HepG2 cells. In brief, HepG2 cells were treated as indicated and JC-1 (10 µM) was added at 37°C for 20 min, followed by measurement of JC-1 aggregate fluorescence (red) (Ex/Em = 488/583 nm) and JC-1 monomer fluorescence (green) (Ex/Em = 488/ 525 nm) under a fluorescence microscope (Leica SP8). Flow cytometry determination HepG2 cell apoptosis was measured with the annexin V-FITC/PI apoptosis detection kit (Cat. No 556547, BD Biosciences) according to the manufacture's instruction. Briefly, the cells were treated with LOG and washed with PBS, resuspended in binding buffer gently, incubated with annexin-V-FITC and PI respectively in the dark for 10 min and detected by flow cytometry (BD Accuri C6). The data were analyzed using Flowjo Software. In situ and in vitro fluorescence labeling Fluorescence labeling experiments were performed according to standard protocol 36 . HepG2 cells were seeded in 6-well plates and allowed to proliferate up to 80% confluence. Then, LOG-P or DMSO was added for 2 h, followed by washing the cells twice with prechilled PBS. After collecting the cells with centrifugation, total proteins were extracted with RIPA buffer containing protease inhibitor and quantified with a BCA kit. Thereafter, equal amounts of cell lysates from different groups were incubated with the click chemistry reaction (1 mM NaVc, 100 mM THPTA, 1 mM CuSO 4 and 50 µM TAMRA-azide) at RT for 2 h with vigorous shaking, and labelled proteins were precipitated by prechilled acetone at -20℃ overnight. After that, the samples were separated by SDS-PAGE, and visualized in a laser scanner (Azure Sapphire RGBNIR, USA). Finally, the gel was stained with Instant Blue Coomassie Protein Stain (ab119211, Abcam). LOG targets identification by pull-down and LC-MS/MS assay The direct targets of LOG were screened by pull-down and LC-MS/MS strategy as previously described with optimization 36 . HepG2 cells were treated with competitors for 1 h, followed by incubation with the LOG-P (5 mM) or DMSO for 4 h further, the soluble proteins were then extracted to carry out the click reaction as aforementioned. After airdried and thoroughly dissolved with 1.5% SDS in PBS. The supernatant sample was added to 50 µL of streptavidin beads. The mixture was left to incubate for 4 h at RT before the beads were washed with 5 mL of PBS containing 1% SDS (thrice), 0.1% SDS (once), 6 M urea (thrice), and PBS (twice) sequentially. To validate targets via LC-MS/MS, the proteins enriched by streptavidin beads were separated using SDS-PAGE. The relevant bands were excised into small pieces and washed with 25 mM NH 4 HCO 3 buffer and 50% acetonitrile (in 25 mM NH 4 HCO 3 ). Then, the samples were sequentially dehydrated via Speedvac, reduced with dithiothreitol (DTT), and alkylated by iodoacetamide (IAA) to eliminate free hydroxyl groups. Finally, peptide samples were obtained by digesting the samples with trypsin at 37℃ overnight, desalted with C18 column and analyzed with LC-MS/MS. Target protein analysis and gene ontology (GO) enrichment Based on the results of the DMSO (control) group, the LOG + LOG-P (compete) group and the LOG-P (treatment) group, GO enrichment analysis was performed on the targeted proteins utilizing the “clusterprofiler” package (version 3.18.1). Cellular thermal shift assay (CETSA) To monitor LOG target engagement in HepG2 cell lysates, a cellular thermal shift assay was performed. In brief, lysates were taken from 2 × 10 6 HepG2 cells, diluted in PBS, and then separated into aliquots. Each aliquot was incubated with LOG (10 µM) or DMSO in PCR-tubes for 30 min at RT, and then heated to different temperatures individually as indicated using the Veriti thermal cycler from Applied Biosystems/Life Technologies. After removing the aggregated proteins by centrifugation, the supernatants were collected and subjected to Western blot analysis. Isothermal titration calorimetry ITC experiments were conducted using a MicroCal PEAQ-ITC Isothermal Titration Calorimeter (Malvern Panalytical) as described previously with minor modifications 16 . In summary, LOG and recombinant protein (TOP1MT) were immersed in ITC buffer (20 mM Bis-Tris, 150 mM NaCl, 2 mM DTT). LOG (200 µM) was then titrated against 20 µM of proteins over 13 injections of 2 µL of LOG solution at a rate of 2 s/µL at 150 s time intervals. The assay was executed at 25℃ with agitation at 750 rpm. The data generated was analyzed with the aid of the MicroCal PEAQ-ITC Analysis Software Setup. Additionally, the composite model was utilized to analyze three control titrations. These consisted of ( 1 ) titrating 1 into the buffer, ( 2 ) titrating buffer into the proteins and ( 3 ) titrating buffer into another buffer. Liver histopathology quantitively assay Liver morphology was scored by blind on 0 to 3 scale: 0, none; 1, few number of inflammatory and apoptosis cells; 2, tissue perivascular infiltration; 3, centrilobular necrosis and apoptosis. H&E staining was used to estimate the extent of inflammation, the specimen was observed under a light microscope, using ImageJ software to calculate the number of liver cell. AAV-shRNA-mediated TOP1MT knockdown in mice HBAAV2/8-TBG-mIR30-m-TOP1mt-LUC and HBAAV2/8-TBG-LUC (Control) viral plasmids were purchased from Hanheng Biology Company (Shanghai, China). Then, 6-8-week old male C57BL/6J mice were dosed with HBAAV2/8-TBG-mIR30-m-TOP1mt-LUC and HBAAV2/8-TBG-LUC (Control) at 10 12 viral particles/mouse. The mice were divided into 5 groups: Control, Control + HBAAV2/8-TBG-LUC, APAP (400mg/kg), APAP + HBAAV2/8-TBG-mIR30-m-TOP1mt-LUC, APAP + LOG and APAP + LOG + HBAAV2/8-TBG-mIR30-m-TOP1mt-LUC. After 7 days of adaptive feeding, each mouse was injected with corresponding 100 µL AAV-shRNA and the following treatment were as mentioned above. Statistical analysis All data were analyzed by using GraphPad Prism 9.0. Error bars indicate the standard deviations of the outcomes from three separate experiments, unless otherwise specified. For statistical analysis of significant differences between groups, we employed a One-way analysis of variance (ANOVA) unless otherwise noted. Throughout the study, p < 0.05 was considered statistically significant. Declarations Acknowledgements This work was supported by the National Key Technologies Research and Development Program for Modernization of Traditional Chinese Medicine (Grant No. 2017YFC1701300), the Key Research and Development Projects of Shaanxi Province, China (Grant No. 2020ZDLSF05-10 and 2021ZDLSF04-04), Special project of Shaanxi Administration of traditional Chinese Medicine (Grant No. 2021-QYZL-03), Open Fund Project of Key Science and Technology Innovation Platform of Central Universities (Grant No. GK202205001, GK202205010), Innovation Ability Improvement Plan Project of Hebei Province (Grant No. 225A2501D). Author contributions Jiefang Kang and Xiaochang Xue designed the research, supervised the project and applied the grant proposals that supported this work. Dou Niu carried out most of the experiments. Jigang Wang designed and synthesized the LOP-P probe and guided the LOP targets screening. Yue Yang, Xiaobo Yu, Teng Hui and Meng Wang finished the knockdown animal model experiments. Dou Niu and Xiaochang Xue analyzed the data, drafted the manuscript; All the authors have read and approved the final manuscript. Conflicts of interest The authors declare that they have no competing interests. References Yan M, Huo Y, Yin S, Hu H. Mechanisms of acetaminophen-induced liver injury and its implications for therapeutic interventions. Redox biology 17 , 274-283 (2018). Qian H , et al. Dual roles of p62/SQSTM1 in the injury and recovery phases of acetaminophen-induced liver injury in mice. Acta Pharmaceutica Sinica B 11 , 3791-3805 (2021). Qiu Y, Benet LZ, Burlingame AL. Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. J Biol Chem 273 , 17940-17953 (1998). Jaeschke H , et al. 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Journal of Biological Chemistry 285 , 24609-24619 (2010). Jaeschke H , et al. Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls. Acta Pharm Sin B 11 , 3740-3755 (2021). Yang WS, Stockwell BR. Ferroptosis: Death by Lipid Peroxidation. Trends Cell Biol 26 , 165-176 (2016). Saeedi BJ , et al. Gut-Resident Lactobacilli Activate Hepatic Nrf2 and Protect Against Oxidative Liver Injury. Cell Metab 31 , 956-968.e955 (2020). Ahmed MME , et al. Aldo-Keto Reductase-7A Protects Liver Cells and Tissues From Acetaminophen-Induced Oxidative Stress and Hepatotoxicity. Hepatology 54 , 1322-1332 (2011). Marques PE , et al. Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology 56 , 1971-1982 (2012). Li GD , et al. A bioactive ligand-conjugated iridium(III) metal-based complex as a Keap1-Nrf2 protein-protein interaction inhibitor against acetaminophen-induced acute liver injury. Redox Biology 48 , (2021). Zhang Z , et al. Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells. Autophagy 14 , 2083-2103 (2018). Agard NJ, Prescher JA, Bertozzi CR. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 126 , 15046-15047 (2004). Tadokoro T , et al. Mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity. JCI Insight 5 , (2020). Kraft VAN , et al. GTP Cyclohydrolase 1/Tetrahydrobiopterin Counteract Ferroptosis through Lipid Remodeling. ACS Cent Sci 6 , 41-53 (2020). Wang J , et al. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum. Nat Commun 6 , 10111 (2015). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarydata.docx Figure S1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3994000","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":279065980,"identity":"6d029dea-c613-4113-ace4-2e5ded115bf9","order_by":0,"name":"Dou Niu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACCQbGBgYGAwY5KJ+ZSC0HDBiMSdECBAcYGBIbiNYiOftw8+cPBXfS58/uPSbBUGGd2MB+9gBeLdJ8iW0SBwye5W64cy5NguFMemIDT14CXi1yPIxtQL8czt0gkWMmwdh2OLFBgseAkJbmD0At6fIzQFr+EaFFmoexAeiwwwkMN0BaGojQItnD2CZxxuCw4YYbOcYWCcfSjdt4cvBrkTjD/vhDxZ/D8kCHGd74UGMt289+Br8WVJAAxGwkqB8Fo2AUjIJRgAMAAM9UQkGiOjuhAAAAAElFTkSuQmCC","orcid":"","institution":"Shaanxi Normal University","correspondingAuthor":true,"prefix":"","firstName":"Dou","middleName":"","lastName":"Niu","suffix":""},{"id":279065981,"identity":"61cd222f-16ec-4032-a968-ada21b6748f4","order_by":1,"name":"Yue Yang","email":"","orcid":"","institution":"Shaanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Yang","suffix":""},{"id":279065982,"identity":"45bbc6da-afbd-4961-85c0-a18689398636","order_by":2,"name":"Xiaobo Yu","email":"","orcid":"","institution":"Shaanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaobo","middleName":"","lastName":"Yu","suffix":""},{"id":279065983,"identity":"cb2c2f6d-8bb1-43f7-bf26-7f8f9ccd0326","order_by":3,"name":"Teng Hui","email":"","orcid":"","institution":"Shaanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Teng","middleName":"","lastName":"Hui","suffix":""},{"id":279065984,"identity":"da83389e-ba89-44dc-9110-6093cfa276c7","order_by":4,"name":"Meng Wang","email":"","orcid":"","institution":"Shaanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Wang","suffix":""},{"id":279065985,"identity":"89e4f5d5-0ddd-4d4f-93cc-e0cfdd4dbe04","order_by":5,"name":"Jigang Wang","email":"","orcid":"","institution":"Southern University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jigang","middleName":"","lastName":"Wang","suffix":""},{"id":279065986,"identity":"da8ceb3f-1f98-4c44-8760-c56fdccbfba7","order_by":6,"name":"Xiaochang Xue","email":"","orcid":"https://orcid.org/0000-0001-9875-9821","institution":"Shaanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaochang","middleName":"","lastName":"Xue","suffix":""},{"id":279065987,"identity":"e21943ce-a625-4fa6-987b-fc1dc265a977","order_by":7,"name":"Jiefang Kang","email":"","orcid":"","institution":"Shaanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jiefang","middleName":"","lastName":"Kang","suffix":""}],"badges":[],"createdAt":"2024-02-27 13:20:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3994000/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3994000/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52672910,"identity":"60801079-2d9d-474b-afc3-7edde4af08b5","added_by":"auto","created_at":"2024-03-14 10:33:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1307482,"visible":true,"origin":"","legend":"\u003cp\u003eLoganin attenuates APAP-induced hepatoxicity \u003cem\u003ein vivo\u003c/em\u003e. (A) Schematic illustration of the establishment of AILI mouse model and intervention with loganin. (B) The weight change of mice before and after APAP/LOG administration. (C) The survival rate of mice. (D) Morphological changes of liver in mice. (E) Liver pathology was scored on a 0 to 3 scale. (F) Histopathological examination after H\u0026amp;E staining. Upper bar = 500 μm, bottom bar = 100 μm. (G) Quantitative analysis of the H\u0026amp;E staining data using Image J. (H) Serum ALT and AST were measured. (I) CAT, GSH and SOD in liver homogenates were detected. (J) The expression levels of liver CYP2E1 in mice were determined by immunofluorescence. Bar = 50 μm. (K) The level of ROS was determined by flowcytometry. \u003csup\u003ens\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 compared with the APAP group or between indicated groups.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/61517e2c424bc73bce2c53ac.png"},{"id":52673301,"identity":"a8d594e0-994c-4c2c-b8f5-74163e1ce30b","added_by":"auto","created_at":"2024-03-14 10:41:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1122709,"visible":true,"origin":"","legend":"\u003cp\u003eLoganin prevent APAP-induced hepatocyte apoptosis and ferroptosis in mice. (A) The mice livers were stained by TUNEL. Bar = 50 μm. (B) Statistical analyses of total apoptotic cell numbers in (A). (C) Mitochondrial morphology of liver tissues was observed by transmission electron microscopy. (D) The protein expression of Bax, Bcl2, Nrf2, GPX4 and SLC7A11 were analyzed by Western blot. (E) Quantitative analysis of relative band intensity of Bax and Bcl2. (F) Quantitative analysis of relative band intensity of Nrf2, GPX4 and SLC7A11. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with the APAP group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/f7e0b8265aeda5e2932ba2db.png"},{"id":52672907,"identity":"b9ef55f4-7c38-4da8-aab8-3a0f506ff750","added_by":"auto","created_at":"2024-03-14 10:33:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1232810,"visible":true,"origin":"","legend":"\u003cp\u003eLoganin ameliorates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HepG2 cell apoptosis and ferroptosis. (A) ROS was detected using kit and observed using a confocal laser scanning microscope. (B) Quantitative analysis of the data in (A). (C) Cell apoptosis was detected using flow cytometry. (D) Quantitative analysis of the data in (C). (E) The protein expression of Nrf2, GPX4 and SLC7A11 was analyzed by Western blot. (F) Quantitative analysis of the data in (E). (G) The measurement of LPO levels in HepG2 cells. (H) The measurement of Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e level in HepG2 cells. (I) Confocal images showing the immunocytochemical assay with the BODIPY 581/591 C11 marker. Peroxidized lipids are shown in green, and nonperoxidized lipids are shown in red. (J) Mitochondrial morphology of HepG2 cell line was observed by transmission electron microscopy. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group. Bar = 50 μm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/5c8c5d228c9479f7a8cb0b98.png"},{"id":52672915,"identity":"54d9036f-3d9c-49b0-83de-97a3b24577e8","added_by":"auto","created_at":"2024-03-14 10:33:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":814029,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of apoptosis and ferroptosis abolishes the protective effects of LOG on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress model in HepG2 cells. (A) The expression of Bcl2 and Bax in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated HepG2 cells in the absence or presence of aopotosis inducer CHX was detected by Western blot. (B) Quantitative analysis of the data in (A). (C) HepG2 cells were treated as indicated and cell apoptosis was detected using flow cytometry. (D) The expression of Bcl2 and Bax in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated HepG2 cells in the absence or presence of apoptosis inhibitor ZVAD was detected by Western blot. (E) Quantitative analysis of the data in (D). (F) HepG2 cells were treated as indicated and cell apoptosis was detected using flow cytometry. (G) The expression of Nrf2, GPX4 and SLC7A11 in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated HepG2 cells with or without ferroptosis inducer erastin was detected by Western blot. (H) Quantitative analysis of the data in (G). (I) HepG2 cells were treated as indicated and the expression of Nrf2, GPX4 and SLC7A11 was detected by Western blot. (J) Quantitative analysis of the data in (I). \u003csup\u003ens\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 compared between indicated groups.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/c2ee4f2feac7f7976f2b4fe5.png"},{"id":52672911,"identity":"7efd3718-b1b5-481d-8e29-0c885dbe19ae","added_by":"auto","created_at":"2024-03-14 10:33:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":646416,"visible":true,"origin":"","legend":"\u003cp\u003eScreening and identification of loganin targets in heptocyte via ABPP in combination with LC–MS/MS. (A) The synthetic route of LOG-P. (B) The effect of LOG and LOG-P on HepG2 cell viability. (C) Immunofluorescence staining of the distribution of LOG-P in HepG2 cells (Bar = 25 μm). (C) \u003cem\u003eIn situ\u003c/em\u003e protein labelling with LOG-P in a dose-dependent manner in HepG2 cells. (D) \u003cem\u003eIn situ\u003c/em\u003e protein labelling with LOG-P in a time-dependent manner in HepG2 cells. (E) The competition of \u003cem\u003ein situ\u003c/em\u003eprotein labeling with LOG-P by LOG in HepG2 cells. (F) The pull down experiment of LOG-P. (G) Venn diagrams of control, LOG-P and LOG + LOG-P. (H) The functional distribution of LOG-P by GO level. (I) Gene pathway analysis using KEGG orthology (KO). (J) Venn diagram illustrating the overlap and unique elements among control, LOG-P and LOG + LOG-P.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/7998b4d1bc7e99db9c160efb.png"},{"id":52673321,"identity":"33051d3b-905b-4972-b917-d5ed11b91b24","added_by":"auto","created_at":"2024-03-14 10:41:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":682322,"visible":true,"origin":"","legend":"\u003cp\u003eLoganin directly binds to and stabilize TOP1MT. (A) Binding model of LOG with TOP1MT by molecular docking. (B) Western blot was used to verify that loganin can reverse H2O2 suppressed expression of TOP1MT. (C) Quantitative analysis of the data in (B). (D) Pull-down followed by immunoblotting to verify LOG directly targeting to TOP1MT \u003cem\u003ein situ\u003c/em\u003e. (E) Immunofluorescence staining of TOP1MT and LOG-P clicked with a red fluorescence dye TAMRA (Bar = 25 μm). (F) ITC analysis of the LOG and TOP1MT\u0026nbsp; interaction. Raw data for 13 sequential injections (2 μL per injection) of a solution of LOG (200 μmol/L) into a solution of TOP1MT (20 μmol/L). (I) “Net” heat effects obtained by subtracting the dilution heat from the reaction heat, which was fitted by using the “one set of sites” binding model. N (sites) = 0.983 ± 3.6e-2 KD (M) = 1.02e-6 ± 492e-6, ∆H (kJ/mol) = -80 ± 4.03, Offset (kJ/mol) = -3.71 ± 8.25, ∆G (kJ/mol) = -8.18, -T∆S (kJ/mol) = 71.8. (G) CETSA–WB experiment to further confirm the interaction between LOG and TOP1MT. (H) Quantitative analysis of CETSA relative band intensity. \u003csup\u003ens\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 compared between indicated groups.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/5f6dc4d7cd7a63115aed2a06.png"},{"id":52672917,"identity":"5681dd7f-5070-422c-ad3e-b33e0c53f942","added_by":"auto","created_at":"2024-03-14 10:33:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":953831,"visible":true,"origin":"","legend":"\u003cp\u003eTOP1MT mediates protective effects of loganin on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced hepatocyte apoptosis and ferroptosis. (A) ROS was detected using kit and observed using a confocal laser scanning microscope. Bar = 25 μm (B) JC-1 staining of HepG2 cells, green fluorescence images of the same field for JC-1 monomer distribution, and red fluorescence images for JC-1 multimer distribution. Bar = 25 μm (C) Confocal images showing the immunocytochemical assay with the BODIPY 581/591 C11 marker. Peroxidized lipids are shown in green, and nonperoxidized lipids are shown in red. Bar = 25 μm (D) The expression of Bax, Bcl-2, Nrf2, GPX4 and SLC7A11 in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated HepG2 in the absence or presence of LOG and TOP1MT siRNA was detected by Western blot. (E) Quantitative assay of the data in (D). (F) The measurement of LPO levels in HepG2 cells. (G) The measurement of Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e level in HepG2 cells. (H) HepG2 cells were treated as indicated and mitochondrial morphology was observed by transmission electron microscopy. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 compared between indicated groups.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/663d39440ac6aad4ed54582d.png"},{"id":52672914,"identity":"5abecf71-9928-4aba-ad68-0584cc1bf636","added_by":"auto","created_at":"2024-03-14 10:33:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1215657,"visible":true,"origin":"","legend":"\u003cp\u003eTOP1MT knockdown abolished the hepatoprotective efficacy of LOG in AILI mice. (A) The scheme of AAV injection and loganin administration. (B) The body weight of mice at the beginning or end of the experiments. (C) Morphological changes of liver in mice. (D) Liver pathology was scored on a 0 to 3 scale. (E) Histopathological examination with H\u0026amp;E staining. Upper bar = 500 μm, bottom bar = 100 μm. (F) Quantitative analysis of the H\u0026amp;E staining data using Image J. (G) Serum ALT, AST and CAT, GSH and SOD in liver were determined. \u003csup\u003ens\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 compared between indicated groups.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/2bb47f7def09c17fd3e1dc8c.png"},{"id":52672913,"identity":"80fb1743-1c08-4d5b-98a9-cfe7ba9ee358","added_by":"auto","created_at":"2024-03-14 10:33:21","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1346904,"visible":true,"origin":"","legend":"\u003cp\u003eTOP1MT knockdown weakens LOG protective effects and aggravates ferroptosis and apoptosis in AILI mice. (A) The expression levels of CYP2E1 in the liver of AILI mice were determined by immunofluorescence. (B) Hepatocytes apoptosis in the AILI mice were detected by TUNEL staining. (C) Mitochondrial morphology of liver tissues was observed by transmission electron microscopy. (D) The protein expression of Bax, Bcl2, Nrf2, GPX4 and SLC7A11 were analyzed by Western blot.\u0026nbsp; (E) Quantitative analysis of the data in (D). \u003csup\u003ens\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared between indicated groups. Bar = 50 μm.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/70420a6806dfed13ea651d0e.png"},{"id":54906592,"identity":"8522520f-7de3-41d6-aa91-7f2610136903","added_by":"auto","created_at":"2024-04-18 11:48:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9107152,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/eec5233f-5504-4689-a68d-96a2eb0cd125.pdf"},{"id":52672908,"identity":"00835ba7-3b60-4de7-8b8d-3c3559d96cfe","added_by":"auto","created_at":"2024-03-14 10:33:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":171742,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-3994000/v1/8e0c065eb5cc09cef69cdb5f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Loganin ameliorates acetaminophen-induced acute liver injury via targeting TOP1MT-mediated hepatocyte apoptosis and ferroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs the most popular and top-selling analgesic and antipyretic drug used worldwide, Acetaminophen (APAP) is considered safe and effective when taken at the recommended dosage\u003csup\u003e1\u003c/sup\u003e. Once overdose occurred, however, the overwhelming of phase II metabolizing enzymes often results in excessive deposition of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) and depletes the crucial antioxidant glutathione (GSH)\u003csup\u003e2\u003c/sup\u003e. In turn, it leads to mitochondrial oxidative stress, impaired mitochondrial function and hepatocyte death or even liver failure\u003csup\u003e3, 4\u003c/sup\u003e. Until now, APAP overdose is the predominant form of drug-induced liver injury (DILI) and major cause of acute liver failure worldwide\u003csup\u003e5\u003c/sup\u003e. Although N-acetyl cysteine (NAC) has been recommended by FDA as the first-line drug to treat DILI, the potential adverse effects and a relatively narrow therapeutic window greatly limited its clinical use\u003csup\u003e6\u003c/sup\u003e. If the early critical stages of APAP-induced liver injury (AILI) are missed, liver transplantation becomes the only viable option to improve patient survival\u003csup\u003e7\u003c/sup\u003e. Therefore, there is an urgent need for strengthening the incomplete understanding of its pathogenesis, changing unsatisfactory therapies status, and developing alternative therapeutic strategies that can effectively intervene at various stages of AILI and provide better outcomes for patients\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFerroptosis is a novel form of programmed cell death characterized by the iron-dependent accumulation of lipid peroxidation (LPO) within cellular membranes. This process has been broadly implicated in a wide spectrum of diseases including neurodegeneration, ischemia-reperfusion injury, cancer, and other pathological conditions\u003csup\u003e9\u003c/sup\u003e. Erastin, a well-known inducer of ferroptosis, exerts its effects by inhibiting system Xc\u0026minus;, thereby suppressing cellular cysteine uptake and depleting GSH, which plays vital roles in antioxidant defense mechanisms against reactive oxygen species (ROS). The enzyme glutathione peroxidase 4 (GPX4) utilizes GSH to repair damaged lipids and convert toxic lipid hydroperoxides into non-toxic lipid alcohols. However, depletion of GSH disrupts the function of GPX4, leading to the accumulation of lipid peroxides and subsequent initiation of apoptotic and ferroptotic cell death\u003csup\u003e10\u003c/sup\u003e. As the major organ for lipid metabolism, it is not surprising that mounting evidence reported that ferroptosis plays critical roles in various hepatic diseases\u003csup\u003e11, 12, 13\u003c/sup\u003e. However, whether it is involved in AILI and targeting ferroptosis can emerge as a novel and promising therapeutic approach for AILI has almost completely unknown, except only rare studies demonstrated that ferroptosis is related with APAP-induced primary hepatocytes death\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA wide array of valuable natural products has been extensively utilized worldwide as preventive and therapeutic agents for various diseases, including liver injury\u003csup\u003e15\u003c/sup\u003e. Loganin (LOG), an iridoid glycoside compound (\u003cb\u003eFigure S1A\u003c/b\u003e and \u003cb\u003eS1B\u003c/b\u003e), is one of the core bioactive components of Corni Fructus with multiple biological properties, such as anti-apoptotic\u003csup\u003e16\u003c/sup\u003e, anti-inflammatory\u003csup\u003e17\u003c/sup\u003e, anti-oxidant\u003csup\u003e18\u003c/sup\u003e and neuroprotective activities\u003csup\u003e19\u003c/sup\u003e. Accumulating evidence has reported that LOG can be used for the treatment of diabetes\u003csup\u003e20\u003c/sup\u003e, lung injury\u003csup\u003e21\u003c/sup\u003e, renal injury\u003csup\u003e17\u003c/sup\u003e and liver injury\u003csup\u003e22\u003c/sup\u003e. However, the molecular mechanisms of the action of LOG still remain unclear.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that LOG effectively protects against AILI by inhibiting oxidative stress and ROS production, and resulted apoptosis and ferroptosis, via directly targeting mitochondrial topoisomerase I (TOP1MT) and enhancing its antioxidant activities. Silencing of TOP1MT in HepG2 cells strengthen H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced accumulation of ROS, LPO and Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e, key markers of apoptosis and ferroptosis. Consistently, LOG effect was significantly impaired in the AILI model in liver-specific TOP1MT-knockdown mice. Taken together, our data reveal that LOG ameliorates AILI via directly binding and stabilizing TOP1MT, therefore promoting antioxidant proteins expression and inhibiting apoptosis and ferroptosis of hepatocytes. These findings provide new insights for the pathogenesis of AILI and suggest TOP1MT as a potential biomarker and therapeutic target for AILI.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eLOG ameliorates APAP-induced liver injury through inhibiting oxidative stress in vivo\u003c/h2\u003e\n\u003cp\u003eTo investigate the protective effects of LOG on APAP-induced liver injury, the mice were subjected to the experimental procedures outlined in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, no statistically significant differences in body weights were observed between the initial and final measurements, except that an apparent weight loss was found in the APAP group. As to the survival rate of the mice, APAP exposure resulted in half of death, whereas administration of 160 mg/kg LOG significantly improved the lethal effect of APAP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Observation of tissue appearance shows obvious necrotic spots and tissue hardening of the liver (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, APAP mice got high pathological scores which was significantly decreased in the high dose LOG-treated mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). H\u0026amp;E staining results revealed that APAP induced extensive liver damage including destruction of liver architecture, hepatocyte ballooning degeneration, and ubiquitous centrilobular hepatocyte death, which were attenuated significantly in LOG treatment group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF-G). Consistently, LOG administration at different doses (80 and 160 mg/kg) significantly improved the liver function of APAP-treated mice as indicated by the potently reduced serum ALT and AST levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e\n\u003cp\u003eConsidering oxidative stress play pivotal roles in cytotoxic drugs associated hepatotoxicity, we measured the antioxidant enzymes activities in homogenized liver tissues, and we found that LOG treatment reversed the decrease of glutathione (GSH) and promoted the activities of superoxide dismutase (SOD) and catalase (CAT), which are all dysregulated key lipid peroxidation markers in liver injuries (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI)\u003csup\u003e23\u003c/sup\u003e. As a member of the cytochromes P450 (CYPs) phase I enzymes, CYP2E1 is extensively involved in the metabolism of various endogenous and exogenous compounds like APAP\u003csup\u003e24\u003c/sup\u003e. Our immunofluorescence data showed that CYP2E1 expression was strongly induced by APAP and apparently inhibited by LOG treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Emerging evidence demonstrated that CYP2E1 can induce oxidative stress and augment tissue injury\u003csup\u003e25\u003c/sup\u003e. Given rise to these aforementioned enzymes have important roles in resisting ROS and free radicals\u003csup\u003e26\u003c/sup\u003e, we measured the ROS production in the mice, and the results showed that ROS is highly produced in APAP-treated group but significantly reversed in LOG group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eK). Taken together, these results collectively imply that oxidative stress is engaged in APAP-induced liver injury, which might be involved in the hepatoprotective mechanism of LOG.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eLoganin suppresses APAP-induced hepatocyte apoptosis and ferroptosis\u003c/h2\u003e\n\u003cp\u003eThe excessive accumulation of lipid peroxides and ROS often leads to cell death. Considering the obvious hepatocyte death and lipid peroxidation as mentioned above, we wonder which kind of cell death is present in AILI. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, TUNEL staining showed that APAP induced elevated hepatocyte apoptosis which was greatly reversed by LOG administration. In addition, transmission electron microscopy (TEM) observation suggested that ferroptosis, an iron-dependent programmed cell death\u003csup\u003e27\u003c/sup\u003e, was also potently induced by APAP, as indicated by the classic morphological abnormalities in mitochondria (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). To confirm these, we measured the molecular signatures of the tissues. As expected, Western blot data showed that the proapoptotic gene Bax was upregulated, whereas the antiapoptotic Bcl-2 was apparently downregulated by APAP, but LOG treatment potently opposed these effects (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). As to ferroptosis-related genes, Nrf2, a central transcription factor that regulates the antioxidant stress response\u003csup\u003e28\u003c/sup\u003e, was strongly suppressed by APAP but significantly increased by LOG. The expression levels of SLC7A11 and GPX4, two key negative regulators involved in the ferroptosis process, were found to be significantly decreased in the liver of APAP-treated mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). Therefore, these findings provide evidence that the inhibition of ROS-triggered hepatocyte apoptosis and ferroptosis mediated the hepatoprotective effects of LOG.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eLoganin suppresses HO-induced hepatocyte apoptosis and ferroptosis\u003c/h2\u003e\n\u003cp\u003eTo further confirm the hepatoprotective effects of LOG on oxidative stress induced liver cell apoptosis and ferroptosis, a widely used H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress hepatocyte injury model was established in HepG2 cell line\u003csup\u003e29, 30, 31\u003c/sup\u003e. Considering ROS should be a central hub in the process of apoptosis and ferroptosis\u003csup\u003e32\u003c/sup\u003e, we firstly assessed the ROS levels in HepG2 cells. Our data demonstrated that ROS production was significantly increased in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced group, which was significantly reversed in the presence of LOG, indicating that LOG treatment effectively regulated and attenuated the excessive ROS production induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC-D, exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e resulted in an elevated proportion of annexin V/PI-positive apoptotic cells. However, LOG exhibited a significant dose-dependent reversal of apoptosis in HepG2 cells. To confirm the inhibitory effects of LOG on hepatocyte ferroptosis, we assessed the ferroptosis negative regulatory protein levels of Nrf2, GPX4, and SLC7A11. In comparison to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced group, LOG treatment led to an upregulation in the levels of SLC7A11, Nrf2, and GPX4 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). As ferrous ions can rapidly trigger Fenton-like reaction to produce ROS and regulate the intercellular LPO as well, we thus conducted iron and LPO assessment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). As expected, elevated Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e and LPO was detected in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated HepG2 cells, which was potently recovered in the presence of LOG, indicating an iron and lipid peroxidation homeostasis maintenance properties of LOG. Visualization of lipid droplets by C11-BODIPY staining further proved the effects of LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI). Additionally, TEM analysis of HepG2 cells revealed that LOG-treatment increased the number of mitochondrial cristae and recovered mitochondrial morphology, indicating the potential role of LOG in preserving mitochondrial integrity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Together, these data suggested that LOG exerts hepatoprotective effects via preventing oxidative stress-induced hepatocyte apoptosis and ferroptosis.\u003c/p\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eInduction of apoptosis and ferroptosis abolishes the hepatoprotective effect of LOG\u003c/h2\u003e\n\u003cp\u003eTo further investigate the role of apoptosis and ferroptosis in the hepatoprotective effect of LOG on AILI, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HepG2 oxidative stress model was treated by LOG in the absence or in the presence of apoptosis and ferroptosis inducers and inhibitors. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, the apoptosis inducer CHX apparently reversed the elevated Bcl-2 protein levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and decreased Bax protein levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) induced by LOG. Notably, CHX significantly enhanced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced Bax expression and almost completely blocked the effects of LOG on Bax suppression which was comparable to the level in untreated model cells. Similar data were obtained by flow cytometry analysis and CHX potently counteracted the protective effect of LOG on oxidative stress-induced HepG2 cell apoptosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). On the contrary, co-administration of apoptosis inhibitor ZVAD further increased the expression of Bcl-2 induced by LOG treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), although no apparent effects on Bax production was found, this may be due to strong protective effects of LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD-F).\u003c/p\u003e\n\u003cp\u003eSimilar trends were found in ferroptosis inducer and inhibitor, in which the ferroptosis inducer erastin significantly reversed the elevated protein levels of Nrf2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GPX4 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and SLC7A11 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) induced by LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG-H), whereas the ferroptosis inhibitor Fer-1 potently recovered H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-suppresed Nrf2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GPX4 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and SLC7A11 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) production, and the effects were comparable to those of LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI-J). Interestingly, Fer and LOG co-administration not show any bonus effect on these antioxidant proteins (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), which strongly indicates that LOG might exerts hepatoprotective effects through similar strength and signal pathways as Fer.\u003c/p\u003e\n\u003cp\u003eThese data collectively support the hypothesis that the anti-apoptotic and anti-ferroptotic activities of LOG is associated with its protective effects against AILI.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eLoganin directly targets mitochondrial topoisomerase I (TOP1MT)\u003c/h2\u003e\n\u003cp\u003eTo identify the direct target and action mechanism of LOG, a clickable alkyne tag was designed and synthesized as LOG probe (LOG-P) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). The LOG-P exhibited similar cellular potency with unmodified LOG, suggesting that the attached alkyne tag in LOG-P does not interfere with the natural activity of LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). A click chemistry reaction between TAMRA-azide and the alkyne tag in LOG-P was performed to assess the subcellular localization of LOG\u003csup\u003e33\u003c/sup\u003e, and LOG is found to be mainly distributed in cytoplasm and nucleus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Subsequently, incubation of HepG2 cells with LOG-P exhibited that the labelled LOG can enrich numerous proteins in a dose- (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) and time-dependent (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE) manner, in which 100 \u0026micro;M LOG-P and 3-h incubation enriched most target proteins. Once HepG2 cells were incubated with LOG and LOG-P simultaneous, a remarkable competition for binding targets was observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF), indicating that LOG-P can specifically enrich the same targets as his unmodified parent compound LOG.\u003c/p\u003e\n\u003cp\u003eThen, HepG2 cell lysates were incubated with 100 \u0026micro;M LOG-P in the presence or absence of excessive LOG as a competitor to raise the specificity of target screening (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). The probe labeled proteins were affinity-purified in batches, separated by SDS-PAGE, and identified by LC-MS/MS. The functional distribution of LOG-P enriched targets was assessed using Gene Ontology (GO) analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). Additionally, pathway analysis using KEGG orthology (KO) further elucidated the signaling pathways and molecular interactions involved in the hepatoprotective effects of LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI). Finally, 34 proteins were enriched in the LOG-P group compared to the \u0026ldquo;Compete\u0026rdquo; group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Among them, a ferroptosis-associated protein TOP1MT drew our strong attention and preferred to study further \u003csup\u003e34, 35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eVirtual molecular docking revealed that LOG predominantly occupies the Lys493, Lys532, Thr501, Val502, Asp533, Ala499, and Gln421 residues of TOP1MT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). As expected, TOP1MT level was significantly reduced upon H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation but greatly recovered by LOG treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). A pull-down assay indicated direct interaction between TOP1MT and LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Additionally, the co-localization of LOG and TOP1MT in HepG2 cells provided further evidence that LOG can directly target TOP1MT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003eAn isothermal titration calorimetry (ITC) assay was performed to prove the direct binding of LOG to TOP1MT \u003cem\u003ein vitro\u003c/em\u003e after the human recombinant TOP1MT proteins from \u003cem\u003eE. coli\u003c/em\u003e (BL21) was firstly prepared. The results showed that LOG exhibited binding to TOP1MT in an approximate 1:1 stoichiometry (N\u0026thinsp;=\u0026thinsp;0.983\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6e-2) and binding affinities was detected to be K\u003csub\u003ed\u003c/sub\u003e = 1.02e-6\u0026thinsp;\u0026plusmn;\u0026thinsp;492e-6 \u0026micro;M. The binding thermodynamic parameters between LOG and TOP1MT was also demonstrated both enthalpy and entropy compensation (\u0026Delta;G = -8.18 kcal/mol, \u0026Delta;H = -80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.03 kcal/mol, -T\u0026Delta;S\u0026thinsp;=\u0026thinsp;71.8 kcal/mol) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Then, a cellular thermal shift assay (CETSA) was conducted to further confirm the direct occupation of TOP1MT by LOG. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG-H, a dramatic shift in the TOP1MT melting curve was observed as compared with the DMSO group and control gene \u0026beta;-actin, indicating that LOG could stabilize TOP1MT in HepG2 cell lysates.\u003c/p\u003e\n\u003cp\u003eAltogether, these findings collectively demonstrated that LOG may attenuate AILI by directly targeting and stabilizing TOP1MT in hepatocytes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eTOP1MT deficiency exacerbates ferroptosis and apoptosis in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HepG2 oxidative stress model\u003c/h2\u003e\n\u003cp\u003eWe wonder whether and how TOP1MT is involved in LOG-attenuated liver injury. Given that both apoptosis and ferroptosis are closely related to mitochondrial function and play crucial roles in LOG-regulated AILI, TOP1MT was knocked down (KD) by specific siRNA in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HepG2 oxidative stress model and mitochondrial functions were detected. Firstly, DCFH-DA fluorescence dye was used to evaluate total intracellular ROS level. A significant increase of dichlorofluorescein (DCF) fluorescence intensity can be seen in cells exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which was potently blocked by LOG addition. However, TOP1MT KD almost completely shielded this effect of LOG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Then, the mitochondrial membrane potential (MMP) was measured by the lipophilic cationic probe JC-1. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure significantly decreased MMP of HepG2 cells compared with that in control group as indicated by remarkably reduced potential-dependent accumulation of JC-1. Interestingly, LOG effectively prevented HepG2 mitochondrial depolarization only in the absence of TOP1MT siRNA. Finally, lipid ROS was detected by C11 BODIPY and results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e promoted lipid peroxidation and LOG reversed this effect in a TOP1MT-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). Therefore, TOP1MT is extensively involved in LOG-regulated mitochondria function in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HepG2 oxidative stress model.\u003c/p\u003e\n\u003cp\u003eTo further validate the role of TOP1MT in LOG-regulated hepatocytotoxicity, apoptosis and ferroptosis-related genes were detected. We found that the inhibition of apoptosis and ferroptosis of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated HepG2 cells by LOG was apparently weakened in TOP1MT deficient cells as indicated by decreased Bcl-2, Nrf2, GPX4, and SLC7A11, and increased Bax (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD-E), as well as the substantial increase in the levels of lipid peroxidation (LPO) and Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF-G). In addition, TEM results showed an obvious ferroptosis of TOP1MT knockdown HepG2 cells exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e which can not be saved by LOG pretreatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH).\u003c/p\u003e\n\u003cp\u003eThese findings indicate that LOG targeted TOP1MT plays a crucial role in hepatocytotoxicity via regulating the expression of Nrf2, GPX4, SLC7A11, Bcl-2, and Bax, and its deficiency results in elevated oxidative stress, disrupted iron homeostasis, dysregulated mitochondria function, and hepatocyte apoptosis and ferroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of TOP1MT abolished the hepatoprotective efficacy of LOG and aggravates ferroptosis and apoptosis in AILI mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInspired by the results \u003cem\u003ein vitro\u003c/em\u003e, the effect of TOP1MT on the hepaprotective activity of LOG was further investigated in AILI mice. We generated liver-specific TOP1MT knockdown mice by i.v. injection of adeno associated virus (AAV)-encoded TOP1MT shRNA (AAV-shRNA) into the C57BL/6J mice, and scramble shRNA (AAV-shNC) was used as a negative control (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). Two weeks later, AILI model was established by APAP administration and treated with LOG as aforementioned. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB, administration of LOG did not affect body weight of the mice during the experimental period, as well as in control. However, TOP1MT KD mice exhibited an apparent reduction in body weight. Observation of tissue appearance shows more necrotic spots and tissue hardening of the liver in APAP and APAP\u0026thinsp;+\u0026thinsp;LOG\u0026thinsp;+\u0026thinsp;TOP1MT shRNA groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC). Similar trends were obtained in pathological scores as indicated by higher scores in APAP and APAP\u0026thinsp;+\u0026thinsp;LOG\u0026thinsp;+\u0026thinsp;TOP1MT shRNA groups. Although TOP1MT didn\u0026rsquo;t aggravate the pathological score of APAP model mice, it did significantly weaken the protective effect of LOG on AILI mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). H\u0026amp;E staining data showed that APAP-induced liver damages was strongly attenuated by LOG, while this protective effects of LOG almost completely lost in TOP1MT KD mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE-F). In addition, the liver inflammation and oxidative stress markers including ALT, AST, CAT, GSH, and SOD were significantly disturbed by APAP, but potently improved by LOG treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and returned to normal levels. However, TOP1MT deficiency thoroughly deprived the anti-inflammatory and anti-oxidative properties of LOG, as indicated by the extremely blunted recoveries of these markers in the APAP\u0026thinsp;+\u0026thinsp;LOG\u0026thinsp;+\u0026thinsp;shRNA group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG).\u003c/p\u003e\n\u003cp\u003eWe next investigated whether LOG exhibit the therapeutic effects on the liver by targeting TOP1MT. Notably, TOP1MT deficiency resulted in a significant upregulation of CYP2E1 even in the presence of LOG, suggesting increased oxidative stress and liver damage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA). In addition, the hepatoprotective effects of LOG largely expired in TOP1MT KD mice and apparent hepatocyte apoptosis and ferroptosis can be seen as evidenced by TUNEL staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB) and TEM observation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC). Consistently, the anti-apoptotic and anti-ferroptotic protein levels of Bcl-2, Nrf2, GPX4, and SLC7A11 were significantly decreased in APAP-treated TOP1MT KD mice, which can\u0026rsquo;t be effectively retrieved by LOG as in the wild type C57BL/6J mice. An opposite trend in the expression of proapoptotic protein Bax was observed, where its level was found to be elevated in the TOP1MT KD AILI model group compared to that in control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD-E).\u003c/p\u003e\n\u003cp\u003eTaken together, these results indicated that TOP1MT KD exacerbates liver inflammation and impairs liver function, compromises the anti-apoptotic and anti-ferroptotic effects of LOG on AILI in mice. Thus, TOP1MT plays an indispensable role and could be a potential therapeutic target in AILI.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we found that LOG, a bioactive compound found in Corni Fructus which has garnered considerable attention due to its promising effects in the treatment of diverse ailments, showed strong protective effect against AILI. LOG administration potently reversed acute hepatocyte damages via recovering dysregulated ALT, AST, CAT, GSH, SOD, and CYP2E1 levels, excessive oxidative stress, lipid peroxidation and ROS production in AILI murine model and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cellular oxidative stress model. Through the use of an activity-based protein profiling (ABPP) strategy, we identified TOP1MT (mitochondrial topoisomerase I) as the most potential target of LOG, which was validated by virtual molecular docking, pull-down, CETSA and ITC assays. Finally, LOG inhibits oxidative stress-induced hepatocyte apoptosis and ferroptosis by binding and stabilizing TOP1MT and hence regulated the downstream apoptotic- and ferroptotic-associated Bax, Bcal-2, Nrf2, GPX-4, and SLC7A11 genes. In summary, our study demonstrates that LOG exerts hepatoprotective effects by inhibiting ferroptosis and apoptosis in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models of AILI through regulating the TOP1MT/oxidative stress axis. Stabilization and maintenance of TOP1MT activity might be a tempting strategy for AILI treatment and LOG represents one of the most promising candidate drugs or lead compounds.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eLoganin (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) was purchased from Bethealth People Biomedical Technology (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Japan). N-Acetyl-L-cysteine was obtained from Sigma Aldrich (USA). TAMRA-azide, Biotin-azide and THPTA are all purchased from ClickChemistryTools (Scottsdale, AZ). NaVc and CuSO\u003csub\u003e4\u003c/sub\u003e are purchased from Sigma Aldrich (Milwaukee, WI, USA). High capacity neutravidin agarose beads, TEAB and sequencing grade modified trypsin, Pierce\u0026trade; Quantitative Fluorometric Peptide Assay Kit, and BODIPY\u0026trade; 581/591 C11 were from Thermo Fisher Scientific (Waltham, MA, USA). Oasis HLB Extraction Cartridge was obtained from Waters (Milford, MA, USA). Specific primary antibodies against TOP1MT (ab121681), Nrf2 (ab137550), SLC7A11 (ab175186), and GPX4 (ab125066) were from Abcam (Shanghai, China). Specific primary antibodies against Bax (AG1208), Bcl-2 (AF0060), and β-actin (AF0003), and DCFH-DA (S0033) were from Beyotime Biotechnology (Shanghai China). LPO assay kit (A106-1-2), and Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e iron assay kit (A039-2-1) were from Nanjing Jiancheng biotechnology (Nanjing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHepG2 cell was purchased from the Chinese Academy of Medical Sciences (Beijing, China) and cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Hyclone, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) as well as 100 U/mL penicillin and streptomycin (Gibco, USA), and maintained at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere. Cells were passaged regularly when grown to approximately 80% confluence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetection of cell viability\u003c/h2\u003e \u003cp\u003eHepG2 cells were seeded in 96-well plate at the density of 8000 cells/well for 24 h, and then treated with different concentrations of LOG and LOG probe (LOG-P) for 24 h. The cell viability was measured by CCK-8 kit according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of APAP-induced liver injury model in mice\u003c/h2\u003e \u003cp\u003eC57BL/6 male mice (19\u0026ndash;22 g) obtained from Beijing Sibafu Biotechnology Co., LTD (Beijing, China) were maintained with standard conditions. All mice were adapted for 7 d before experiments. A total of 50 mice were randomly divided into 5 groups, 10 mice per group. They were given normal saline (vehicle, control), APAP (model), APAP\u0026thinsp;+\u0026thinsp;80 mg/kg LOG (L-LOG), APAP\u0026thinsp;+\u0026thinsp;160 mg/kg LOG (H-LOG), and APAP\u0026thinsp;+\u0026thinsp;200 mg/kg silymarin (positive control), respectively. For APAP administration, mice were injected intraperitoneally with 400 mg/kg APAP once after 1 h of mice were administrated with LOG the last time. LOG was dissolved in normal saline, and injected i.g. to mice once a day for 10 successive days. After anaesthesia, blood and liver samples were taken. A section of liver tissue was preserved in 4% paraformaldehyde, while the remaining liver tissue was rapidly frozen in liquid N\u003csub\u003e2\u003c/sub\u003e for later use. All procedures were followed by the institutional animal care committee of Shaanxi Normal University, and approved by institutional guidelines and regulations (No. SYXK-2021-003).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical and histological assays\u003c/h2\u003e \u003cp\u003eThe levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), catalase (CAT) superoxide dismutase (SOD), and glutathione (GSH) were assessed by kits from Jiancheng Biotechnology (Nanjing, China) adhering to the instructions. Liver tissues were sectioned and histological changes and liver injuries were examined using Hematoxylin-eosin (H\u0026amp;E), Hoechst, and TUNEL staining, as well as CYP2E1 immunofluorescence staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eProteins were extracted with RIPA buffer containing phenylmethylsulfonyl fluoride, separated by SDS-PAGE, and electro-transferred onto PVDF membranes. Then, samples were incubated with corresponding primary and secondary antibodies. The specific protein band was visualized using SuperSignal\u0026trade; West Femto Maximum Sensitivity Substrate in the ChemiDoc Imaging Systems (Bio-Rad, USA). Protein level was quantified by Image J, and normalized to β-actin loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCellular imaging\u003c/h2\u003e \u003cp\u003eHepG2 cells were cultured in 4-chamber glass bottom dishes and treated with LOG-P at different concentrations with or without LOG. After 4 h, cells were washed with PBS trice and fixed with 4% paraformaldehyde for 20 min at room temperature (RT), and then permeabilized with 0.5% Triton X-100. Freshly prepared clicked reaction cocktail (1 mM NaVc, 100 mM THPTA, 1 mM CuSO\u003csub\u003e4\u003c/sub\u003e and 50 \u0026micro;M TAMRA-N3) were added into cells and reacted with shake for 2 h at RT. Then, the cells were washed with PBS gently.\u003c/p\u003e \u003cp\u003eFor co-localization experiments, cells were fixed, permeabilized and blocked with 5% BSA. Then, the cells were incubated with primary antibodies (1:500) at 4℃ overnight, and gently washed with PBS, followed by incubation with secondary fluorescent antibody (Alexa Fluor 488, Beyotime, China) (1:500) for 2 h, and unbound antibodies were removed. Finally, the microscopic images were captured using a confocal microscope (Leica SP8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDetection of mitochondrial transmembrane potential (MTP)\u003c/h2\u003e \u003cp\u003eJC-1 dye (C2003, Beyotime) was used to measure the MTP of HepG2 cells. In brief, HepG2 cells were treated as indicated and JC-1 (10 \u0026micro;M) was added at 37\u0026deg;C for 20 min, followed by measurement of JC-1 aggregate fluorescence (red) (Ex/Em\u0026thinsp;=\u0026thinsp;488/583 nm) and JC-1 monomer fluorescence (green) (Ex/Em\u0026thinsp;=\u0026thinsp;488/ 525 nm) under a fluorescence microscope (Leica SP8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry determination\u003c/h2\u003e \u003cp\u003eHepG2 cell apoptosis was measured with the annexin V-FITC/PI apoptosis detection kit (Cat. No 556547, BD Biosciences) according to the manufacture's instruction. Briefly, the cells were treated with LOG and washed with PBS, resuspended in binding buffer gently, incubated with annexin-V-FITC and PI respectively in the dark for 10 min and detected by flow cytometry (BD Accuri C6). The data were analyzed using Flowjo Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn situ and in vitro fluorescence labeling\u003c/h2\u003e \u003cp\u003eFluorescence labeling experiments were performed according to standard protocol\u003csup\u003e36\u003c/sup\u003e. HepG2 cells were seeded in 6-well plates and allowed to proliferate up to 80% confluence. Then, LOG-P or DMSO was added for 2 h, followed by washing the cells twice with prechilled PBS. After collecting the cells with centrifugation, total proteins were extracted with RIPA buffer containing protease inhibitor and quantified with a BCA kit. Thereafter, equal amounts of cell lysates from different groups were incubated with the click chemistry reaction (1 mM NaVc, 100 mM THPTA, 1 mM CuSO\u003csub\u003e4\u003c/sub\u003e and 50 \u0026micro;M TAMRA-azide) at RT for 2 h with vigorous shaking, and labelled proteins were precipitated by prechilled acetone at -20℃ overnight. After that, the samples were separated by SDS-PAGE, and visualized in a laser scanner (Azure Sapphire RGBNIR, USA). Finally, the gel was stained with Instant Blue Coomassie Protein Stain (ab119211, Abcam).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLOG targets identification by pull-down and LC-MS/MS assay\u003c/h2\u003e \u003cp\u003eThe direct targets of LOG were screened by pull-down and LC-MS/MS strategy as previously described with optimization\u003csup\u003e36\u003c/sup\u003e. HepG2 cells were treated with competitors for 1 h, followed by incubation with the LOG-P (5 mM) or DMSO for 4 h further, the soluble proteins were then extracted to carry out the click reaction as aforementioned. After airdried and thoroughly dissolved with 1.5% SDS in PBS. The supernatant sample was added to 50 \u0026micro;L of streptavidin beads. The mixture was left to incubate for 4 h at RT before the beads were washed with 5 mL of PBS containing 1% SDS (thrice), 0.1% SDS (once), 6 M urea (thrice), and PBS (twice) sequentially.\u003c/p\u003e \u003cp\u003eTo validate targets via LC-MS/MS, the proteins enriched by streptavidin beads were separated using SDS-PAGE. The relevant bands were excised into small pieces and washed with 25 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e buffer and 50% acetonitrile (in 25 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e). Then, the samples were sequentially dehydrated via Speedvac, reduced with dithiothreitol (DTT), and alkylated by iodoacetamide (IAA) to eliminate free hydroxyl groups. Finally, peptide samples were obtained by digesting the samples with trypsin at 37℃ overnight, desalted with C18 column and analyzed with LC-MS/MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTarget protein analysis and gene ontology (GO) enrichment\u003c/h2\u003e \u003cp\u003eBased on the results of the DMSO (control) group, the LOG\u0026thinsp;+\u0026thinsp;LOG-P (compete) group and the LOG-P (treatment) group, GO enrichment analysis was performed on the targeted proteins utilizing the \u0026ldquo;clusterprofiler\u0026rdquo; package (version 3.18.1).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCellular thermal shift assay (CETSA)\u003c/h2\u003e \u003cp\u003eTo monitor LOG target engagement in HepG2 cell lysates, a cellular thermal shift assay was performed. In brief, lysates were taken from 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e HepG2 cells, diluted in PBS, and then separated into aliquots. Each aliquot was incubated with LOG (10 \u0026micro;M) or DMSO in PCR-tubes for 30 min at RT, and then heated to different temperatures individually as indicated using the Veriti thermal cycler from Applied Biosystems/Life Technologies. After removing the aggregated proteins by centrifugation, the supernatants were collected and subjected to Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eIsothermal titration calorimetry\u003c/h2\u003e \u003cp\u003eITC experiments were conducted using a MicroCal PEAQ-ITC Isothermal Titration Calorimeter (Malvern Panalytical) as described previously with minor modifications\u003csup\u003e16\u003c/sup\u003e. In summary, LOG and recombinant protein (TOP1MT) were immersed in ITC buffer (20 mM Bis-Tris, 150 mM NaCl, 2 mM DTT). LOG (200 \u0026micro;M) was then titrated against 20 \u0026micro;M of proteins over 13 injections of 2 \u0026micro;L of LOG solution at a rate of 2 s/\u0026micro;L at 150 s time intervals. The assay was executed at 25℃ with agitation at 750 rpm. The data generated was analyzed with the aid of the MicroCal PEAQ-ITC Analysis Software Setup. Additionally, the composite model was utilized to analyze three control titrations. These consisted of (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) titrating 1 into the buffer, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) titrating buffer into the proteins and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) titrating buffer into another buffer.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eLiver histopathology quantitively assay\u003c/h2\u003e \u003cp\u003eLiver morphology was scored by blind on 0 to 3 scale: 0, none; 1, few number of inflammatory and apoptosis cells; 2, tissue perivascular infiltration; 3, centrilobular necrosis and apoptosis. H\u0026amp;E staining was used to estimate the extent of inflammation, the specimen was observed under a light microscope, using ImageJ software to calculate the number of liver cell.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eAAV-shRNA-mediated TOP1MT knockdown in mice\u003c/h2\u003e \u003cp\u003eHBAAV2/8-TBG-mIR30-m-TOP1mt-LUC and HBAAV2/8-TBG-LUC (Control) viral plasmids were purchased from Hanheng Biology Company (Shanghai, China). Then, 6-8-week old male C57BL/6J mice were dosed with HBAAV2/8-TBG-mIR30-m-TOP1mt-LUC and HBAAV2/8-TBG-LUC (Control) at 10\u003csup\u003e12\u003c/sup\u003e viral particles/mouse. The mice were divided into 5 groups: Control, Control\u0026thinsp;+\u0026thinsp;HBAAV2/8-TBG-LUC, APAP (400mg/kg), APAP\u0026thinsp;+\u0026thinsp;HBAAV2/8-TBG-mIR30-m-TOP1mt-LUC, APAP\u0026thinsp;+\u0026thinsp;LOG and APAP\u0026thinsp;+\u0026thinsp;LOG\u0026thinsp;+\u0026thinsp;HBAAV2/8-TBG-mIR30-m-TOP1mt-LUC. After 7 days of adaptive feeding, each mouse was injected with corresponding 100 \u0026micro;L AAV-shRNA and the following treatment were as mentioned above.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed by using GraphPad Prism 9.0. Error bars indicate the standard deviations of the outcomes from three separate experiments, unless otherwise specified. For statistical analysis of significant differences between groups, we employed a One-way analysis of variance (ANOVA) unless otherwise noted. Throughout the study, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Technologies Research and Development Program for Modernization of Traditional Chinese Medicine (Grant No. 2017YFC1701300), the Key Research and Development Projects of Shaanxi Province, China (Grant No. 2020ZDLSF05-10 and 2021ZDLSF04-04), Special project of Shaanxi Administration of traditional Chinese Medicine (Grant No. 2021-QYZL-03), Open Fund Project of Key Science and Technology Innovation Platform of Central Universities (Grant No. GK202205001, GK202205010), Innovation Ability Improvement Plan Project of Hebei Province (Grant No. 225A2501D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiefang Kang and Xiaochang Xue designed the research, supervised the project and applied the grant proposals that supported this work. Dou Niu carried out most of the experiments. Jigang Wang designed and synthesized the LOP-P probe and guided the LOP targets screening. Yue Yang, Xiaobo Yu, Teng Hui and Meng Wang finished the knockdown animal model experiments. Dou Niu and Xiaochang Xue analyzed the data, drafted the manuscript; All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYan M, Huo Y, Yin S, Hu H. 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Small molecules in Chinese medicine have long been a treasured reservoir for drugs screening. Here, we reported that loganin (LOG), an active ingredient in Corni Fructus, exerts hepatoprotective effects as indicated by potently alleviated liver damages in APAP-induced liver injury (AILI) murine model. LOG reversed the decreased SOD, GSH and CAT levels, and reduced lipid peroxidation, ROS production, and iron overload and hence reduced apoptosis/ferroptosis of hepatocytes of AILI models, as apoptosis/ferroptosis inducers abolished, whereas their inhibitors enhanced the effect of LOG. Through the activity-based proteome profiling (ABPP) clickable alkyne-tagged LOG probe, mitochondrial topoisomerase I (TOP1MT) was captured as a direct target of LOG, which was further validated by CETSA and ITC assays. Deficiency of TOP1MT significantly compromised the effects of LOG on H2O2-induced oxidative stress cell model via regulating downstream apoptosis/ferroptosis regulators Bax, Bcl-2, NRF2, GSH, SLC7A11, and GPX4. Consistently, LOG effect was greatly eliminated in AILI mice once the endogenous hepatic TOP1MT was knocked-down by AAV-TOP1MT shRNA. Thus, TOP1MT might be a potential target for AILI treatment and LOG represents one of the most promising candidate drugs or lead compounds.\u003c/p\u003e","manuscriptTitle":"Loganin ameliorates acetaminophen-induced acute liver injury via targeting TOP1MT-mediated hepatocyte apoptosis and ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-14 10:33:16","doi":"10.21203/rs.3.rs-3994000/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d37392af-2d05-456d-b23b-65b16f1d4c25","owner":[],"postedDate":"March 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29388925,"name":"Biological sciences/Drug discovery/Target identification"},{"id":29388926,"name":"Biological sciences/Chemical biology/Mechanism of action"}],"tags":[],"updatedAt":"2024-04-18T11:40:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-14 10:33:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3994000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3994000","identity":"rs-3994000","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}