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. 1A. As shown in Fig. 1B, 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. 1C). Observation of tissue appearance shows obvious necrotic spots and tissue hardening of the liver (Fig. 1D). In addition, APAP mice got high pathological scores which was significantly decreased in the high dose LOG-treated mice (p < 0.05) (Fig. 1E). 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. 1F-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. 1H).
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. 1I)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 APAP24. Our immunofluorescence data showed that CYP2E1 expression was strongly induced by APAP and apparently inhibited by LOG treatment (Fig. 1J). Emerging evidence demonstrated that CYP2E1 can induce oxidative stress and augment tissue injury25. Given rise to these aforementioned enzymes have important roles in resisting ROS and free radicals26, 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. 1K). 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. 2A-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 death27, was also potently induced by APAP, as indicated by the classic morphological abnormalities in mitochondria (Fig. 2C). 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. 2D-E). As to ferroptosis-related genes, Nrf2, a central transcription factor that regulates the antioxidant stress response28, 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. 2D and 2F). 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 H2O2-induced oxidative stress hepatocyte injury model was established in HepG2 cell line29, 30, 31. Considering ROS should be a central hub in the process of apoptosis and ferroptosis32, we firstly assessed the ROS levels in HepG2 cells. Our data demonstrated that ROS production was significantly increased in the H2O2-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 H2O2 (Fig. 3A and 3B). As depicted in Fig. 3C-D, exposure to H2O2 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 H2O2-induced group, LOG treatment led to an upregulation in the levels of SLC7A11, Nrf2, and GPX4 (Fig. 3E-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. 3G-H). As expected, elevated Fe2+/Fe3+ and LPO was detected in H2O2-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. 3I). 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. 3J). 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, H2O2-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. 4A-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 H2O2-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. 4C). 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. 4D-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. 4G-H), whereas the ferroptosis inhibitor Fer-1 potently recovered H2O2-suppresed Nrf2 (p < 0.01), GPX4 (p < 0.05), and SLC7A11 (p < 0.05) production, and the effects were comparable to those of LOG (Fig. 4I-J). Interestingly, Fer and LOG co-administration not show any bonus effect on these antioxidant proteins (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. 5A). 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. 5B). A click chemistry reaction between TAMRA-azide and the alkyne tag in LOG-P was performed to assess the subcellular localization of LOG33, and LOG is found to be mainly distributed in cytoplasm and nucleus (Fig. 5C). Subsequently, incubation of HepG2 cells with LOG-P exhibited that the labelled LOG can enrich numerous proteins in a dose- (Fig. 5D) and time-dependent (Fig. 5E) 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. 5F), 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. 5G). 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. 5H). Additionally, pathway analysis using KEGG orthology (KO) further elucidated the signaling pathways and molecular interactions involved in the hepatoprotective effects of LOG (Fig. 5I). Finally, 34 proteins were enriched in the LOG-P group compared to the “Compete” group (Fig. 5J). 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. 6A). As expected, TOP1MT level was significantly reduced upon H2O2 stimulation but greatly recovered by LOG treatment (Fig. 6B-C). A pull-down assay indicated direct interaction between TOP1MT and LOG (Fig. 6D). Additionally, the co-localization of LOG and TOP1MT in HepG2 cells provided further evidence that LOG can directly target TOP1MT (Fig. 6E).
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 Kd = 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. 6F). Then, a cellular thermal shift assay (CETSA) was conducted to further confirm the direct occupation of TOP1MT by LOG. As shown in Fig. 6G-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 H2O2-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 H2O2-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 H2O2, which was potently blocked by LOG addition. However, TOP1MT KD almost completely shielded this effect of LOG (Fig. 7A). Then, the mitochondrial membrane potential (MMP) was measured by the lipophilic cationic probe JC-1. As shown in Fig. 7B, H2O2 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 H2O2 promoted lipid peroxidation and LOG reversed this effect in a TOP1MT-dependent manner (Fig. 7C). Therefore, TOP1MT is extensively involved in LOG-regulated mitochondria function in H2O2-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 H2O2-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. 7D-E), as well as the substantial increase in the levels of lipid peroxidation (LPO) and Fe2+/Fe3+ (Fig. 7F-G). In addition, TEM results showed an obvious ferroptosis of TOP1MT knockdown HepG2 cells exposed to H2O2 which can not be saved by LOG pretreatment (Fig. 7H).
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. 8A). Two weeks later, AILI model was established by APAP administration and treated with LOG as aforementioned. As shown in Fig. 8B, 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. 8C). 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. 8D). 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. 8E-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. 8G).
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. 9A). 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. 9B) and TEM observation (Fig. 9C). 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. 9D-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.