[Effects and mechanisms of hesperetin in attenuating alcoholic liver injury by targeting mitochondria to regulate hepatocyte pyroptosis].

This study investigated the effects and potential mechanisms of hesperetin(HST) in targeting mitochondria to regulate hepatocyte pyroptosis and alleviate alcoholic liver injury through in vitro experiments. Human immortalized hepatocytes(THLE-2) were used to establish an in vitro alcoholic liver injury model induced by 200 mmol·L~(-1) ethanol. The effective concentrations of HST(40, 80, and 160 μmol·L~(-1)) were determined using the MTT assay. The effects of HST on hepatocyte pyroptosis were evaluated by optical microscopy, lactate dehydrogenase(LDH) release assay, and flow cytometry, and its effects on lipid accumulation were assessed using Nile red staining and cholesterol and triglyceride assay kits. Further intervention with a high dose of HST was performed, and the expression levels of pyroptosis-related molecules, including caspase-1, gasdermin D(GSDMD), interleukin-18(IL-18), and interleukin-1β(IL-1β), were detected by Western blot and PCR. In addition, a caspase-1 inhibitor(Z-YVAD-FMK) was used to reversely validate the regulatory effect of HST on the classical pyroptosis pathway. The morphology of the endoplasmic reticulum, lysosomes, and mitochondria was observed using confocal laser scanning microscopy. Superoxide dismutase(SOD) levels were measured using a commercial assay kit, mitochondrial membrane potential was analyzed by flow cytometry, and the gene expression levels of mitochondrial membrane transport proteins, including voltage-dependent anion channel 1(VDAC1), voltage-dependent anion channel 2(VDAC2), translocase of the outer mitochondrial membrane 20(TOM20), and translocase of the outer mitochondrial membrane 34(TOM34), were determined by PCR. Meanwhile, the mitochondrial reactive oxygen species(mtROS) inducer rotenone was employed to validate the mitochondria-targeted regulatory effects of HST on mitochondrial function. The experimental results showed that HST concentration-dependently increased hepatocyte viability, reduced LDH levels, significantly alleviated hepatocyte pyroptosis, and improved lipid accumulation, while downregulating the expression of caspase-1, GSDMD, IL-18, and IL-1β. After the addition of a caspase-1 inhibitor, the inhibitory effect of HST on hepatocyte pyroptosis was further confirmed. Mechanistic investigations revealed that HST targeted mitochondria by scavenging mtROS, upregulating SOD expression, restoring mitochondrial membrane potential, and downregulating the expression of mitochondrial membrane-associated transport proteins VDAC1, VDAC2, TOM20, and TOM34. Furthermore, the addition of the mtROS inducer rotenone reversely validated the role of HST in alleviating hepatocyte pyroptosis through mitochondrial targeting. In summary, HST alleviates hepatocyte pyroptosis and improves lipid metabolism disorders by targeting mitochondria and regulating the classical pyroptosis pathway, thereby mitigating alcoholic liver injury. These findings provide a theoretical basis for the potential clinical application of HST in the treatment of alcoholic liver injury.