Abstract
Pyrrolizidine alkaloids (PAs) are potent hepatotoxins that can cause liver damage. Hyperoside (Hyp), a natural flavonoid, can be extracted from medicinal plants. Hyp displays hepatoprotective activity in various liver diseases. However, the potential effect and mechanism of action of Hyp in ameliorating PA-induced liver injury remain obscure. This study aimed to explore the protective effect of Hyp against PA-induced hepatotoxicity and its underlying mechanism. We established an in vitro model of PAs in mouse primary hepatocytes and developed a mouse model of acute PA toxicity to investigate the protective effect of Hyp. We found that Hyp notably attenuated PA-induced hepatotoxicity. RNA-sequencing showed that the beneficial effect of Hyp against PA-induced hepatotoxicity was associated with the transcription factor EB (TFEB)-peroxisome proliferator-activated receptor-γ coactivator-1-α (PGC1α) pathway. Our results confirmed that both the autophagy-lysosomal pathway and mitochondrial biogenesis were induced by Hyp through TFEB nuclear translocation in PA-induced liver injury. Furthermore, we demonstrated that activation of the mechanistic target of rapamycin complex 1 (mTORC1) by MHY 1485 decreased TFEB nuclear translocation and abrogated the protective effect of Hyp against PA-induced liver injury in mice. In contrast, inhibition of mTORC1 activity increased the level of TFEB and reduced hepatotoxicity induced by PAs in mouse livers. Likewise, Hyp-induced TFEB activation was validated in vitro. In conclusion, Hyp can activate the TFEB-mediated autophagy-lysosomal pathway and mitochondrial biogenesis through inhibition of mTORC1 activity, alleviating the liver injury induced by PAs, thus suggesting the potential value of Hyp in the treatment of PA-induced hepatotoxicity.
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The data that support the findings of this study are not openly available due to resasons of sensitivity and are available from the corresponding author upon reasonable request.
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This work is financially supported by the National Natural Science Foundation of China (No. 81920108033; 82074011; 82130115) and the Program of Shanghai Municipal Health Commission (No. ZY (2021–2023)-0215).
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12272_2023_1460_MOESM2_ESM.tif
Supplementary file2 Supplementary Fig. 1 Hyp attenuates PA-induced liver injury by enhancing autophagy in primary hepatocytes. A Primary hepatocytes were treated with TA for 12 h or 24 h. Hyp was added to the cultures 1 h before TA treatment. The expression of the proteins LC3-II and SQSTM1/p62 were measured by western blotting in primary hepatocytes. Bar graphs show summary data. B Primary hepatocytes were treated with TA and Hyp in the presence or absence of Baf A1. The expression of the protein LC3-II was measured by western blotting in primary hepatocytes. C Primary hepatocytes were transfected with Ad-mRFP-GFP-LC3 for 24 h. After transfection, cells were treated with TA for 12 h or 24 h, Hyp was added to the cultures 1 h before TA treatment. Representative images of Ad-mRFP-GFP-LC3 in primary hepatocytes. Scale bars, 200 μm. B-C Hyp, 50 μM. Data were shown as the means ± SD from at least three independent experiments and analyzed by one-way ANOVA. ###P < 0.001 vs. Vehicle; *P < 0.05, **P < 0.01, ***P < 0.001 vs. TA. (TIF 4416 KB)
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Supplementary file3 Supplementary Fig. 2 Hyp attenuates PA-induced impairment of lysosomal function. A The expression of the protein LAMP1 was measured by western blotting in primary hepatocytes. Bar graphs show summary data. B The expression of the protein LAMP1 was measured by western blotting in mouse liver. Bar graphs show summary data (n = 3). B Hyp, 40 mg/kg. A Data are shown as the means ± SD from at least three independent experiments and analyzed by one-way ANOVA. B Data were shown as the means ± SEM and analyzed by one-way ANOVA. ###P < 0.001 vs. Vehicle; *P < 0.05, **P < 0.01, ***P < 0.001vs. TA. (TIF 1795 KB)
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Supplementary file4 Supplementary Fig. 3 Hyp induces mitochondrial biogenesis through the TFEB-PGC1α pathway in PA-treated primary hepatocytes. A Total intracellular ATP level. B ΔΨm determined by JC-1 red/green fluorescence ratio. C Representative images of the mitochondria of primary hepatocytes stained with MitoTracker Green. D Primary hepatocytes were fractionated into mitochondria and cytosol, and the level of Cyt C was analyzed by western blotting. Fraction quality was verified by immunoblotting with markers for the mitochondria (COX IV) and cytoplasm (α-Tubulin). E Representative TEM micrographs showing Hyp ameliorates mitochondria damage in PAs-treated primary hepatocytes. Scale bars, 2 μm (wireframe indicates the magnified image). F The expression of the protein PGC1α was measured by western blotting in primary hepatocytes. Bar graphs show summary data. G Primary hepatocytes were transfected with scRNA or siTFEB for 24 h and treated with TA in the presence or absence of Hyp for 12 h or 24 h. The expression of the proteins TFEB and PGC1α were measured by western blotting. H The expression of the proteins CPT1α and PPARα were measured by western blotting in primary hepatocytes. Bar graphs show summary data. A-E, G, Hyp, 50 μM. Data were shown as the means ± SD from at least three independent experiments and analyzed by one-way ANOVA. ###P < 0.001 vs. Vehicle; *P < 0.05, **P < 0.01, ***P < 0.001 vs. TA. (TIF 2214 KB)
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Supplementary file5 Supplementary Fig. 4 TFEB mediates the beneficial effects of Hyp in PA-induced liver injury. The mRNA levels of TFEB-associated genes (Map1lc3b, Uvrag, Wipi1, Lamp1, Pgc1α, Nrf1, Tfam) were evaluated by qRT-PCR. Data were shown as the means ± SEM and analyzed by one-way ANOVA or Student's t-test. ***P < 0.001 vs. TA; &&P < 0.01, &&&P < 0.001 vs. TA-Hyp (siRNA NC). (TIF 2275 KB)
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Supplementary file6 Supplementary Fig. 5 Primary hepatocytes were transfected with a plasmid carrying TFEB or empty vector and then treated to 50 μM Hyp for 12 h or 24 h. Effects of Hyp on TFEB nuclear translocation in GFP-TFEB primary hepatocytes. Scale bars, 200 μm. Data were representative of at least three independent experiments. (TIF 1608 KB)
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Supplementary file7 Supplementary Fig. 6 Inhibition of mTOR1 activity by Hyp attenuates PA-induced liver injury in primary hepatocytes. A The expression of the proteins p-mTORC and p-ERK1/2 were measured by western blotting in primary hepatocytes. Bar graphs show summary data. B The expression of the proteins p-S6 and p-eIF4E were measured by western blotting in primary hepatocytes. Bar graphs show summary data. C Primary hepatocytes treated with TA in the presence or absence of Torin1 for 12 h or 24 h. The expression of the nuclear and cytosolic TFEB proteins in primary hepatocytes were analyzed by western blotting. D Primary hepatocytes treated with TA and Hyp in the presence or absence of MHY 1485 for 12 h or 24 h. The expression of the nuclear and cytosolic TFEB proteins in primary hepatocytes were analyzed by western blotting. Data were shown as the means ± SD from at least three independent experiments and analyzed by one-way ANOVA. ##P < 0.01, ###P < 0.001 vs. Vehicle; *P < 0.05, **P < 0.01, ***P < 0.001 vs. TA. (TIF 4846 KB)
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Supplementary file8 Supplementary Fig. 7 Torin1 attenuates PA-induced liver injury in mice. The mice were orally administered TA, and then were intraperitoneally injected with Torin1 once at 6 h after TA administration. A Serum ALT activity (n = 6). B Serum AST activity (n = 6). C Serum TBA amount (n = 6). D Representative images of H&E-stained liver sections (scale bars, 50 μm). E Nuclear fractions of TFEB in mouse liver was analyzed by western blotting (n = 3). F The mRNA levels of TFEB target genes (Map1lc3b, Uvrag, Wipi1, Lamp1, Pgc1α) were evaluated by qRT-PCR (n = 6). G Paraffin sections of mouse liver were stained with TFEB (red) and DAPI (blue). Scale bars, 50 μm (wireframe indicates the magnified image). Data were shown as the means ± SEM and analyzed by one-way ANOVA. A-C ***P < 0.001 vs. TA. F ###P < 0.001 vs. Vehicle; **P < 0.01, ***P < 0.001 vs. TA. (TIF 6776 KB)
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Supplementary file9 Supplementary Fig. 8 Inhibition of mTORC1 activity by Hyp attenuates PA-induced liver injury in mice. Paraffin sections of mouse liver were stained with TFEB (red) and DAPI (blue). Scale bars, 50 μm (wireframe indicates the magnified image). (TIF 3939 KB)
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Xu, J., Xiong, A., Wang, X. et al. Hyperoside attenuates pyrrolizidine alkaloids-induced liver injury by ameliorating TFEB-mediated mitochondrial dysfunction. Arch. Pharm. Res. 46, 694–712 (2023). https://doi.org/10.1007/s12272-023-01460-3
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DOI: https://doi.org/10.1007/s12272-023-01460-3