Loganin Alleviates CCl4-Induced Acute Liver Injury by Promoting Mitophagy and Inhibiting NLRP3 Inflammasome.

Abstract

Objective: To explore the protective effect and mechanism of loganin, a iridoid glycoside isolated from Corni Fructus, on carbon tetrachloride (CCl₄)-induced acute liver injury (ALI) mice model and L-02 cells.

Methods: ALI mice model was developed by an intraperitioneal injection of 0.3% CCl₄. Thirty 8-week-old male C57BL/6 mice were randomly divided into 5 groups using a random number table, including control, model, loganin (40 and 80 mg/kg), and silybin (100 mg/kg) groups (n=6). After 6 consecutive days of intragastric administration, serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using an automatic biochemical analyzer. Hepatic pathological changes were observed by hematoxylineosin (HE) staining. Levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) in liver tissue were detected by enzyme-linked immunosorbent assay (ELISA). Superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents were measured using biochemical kits. Mitochondrial ultrastructure and autophagosome formation were observed by transmission electron microscopy (TEM). Co-localization of mitophagy markers LC3 and TOMM20 were assessed by immunofluorescence (IF). Protein expressions of NOD-like receptor protein 3 (NLRP)3, LC3 II/LC3 I, p62, Beclin-1, Atg-7, Parkin, and PINK1 were determined by Western blot. In vitro, an injury model was established in L-02 hepatocytes stimulated with CCl4. Cell viability was assessed by CCK-8 assay, and related mechanisms were evaluated using the aforementioned methods. Besides, CCl₄-stimulated L-02 cells were intervened with NLRP3 shRNA or Mdivi-1.

Results: Compared with the model group, pre-treatment with loganin (40 and 80 mg/kg) significantly reduced serum AST and ALT levels (P<0.01), alleviated pathological injuries such as swelling, necrosis, inflammatory infiltration, and lipid vacuolation in liver tissue. Loganin also markedly decreased the levels of IL-6 and TNF-α, increased SOD activity, and reduced MDA content in liver tissues (P<0.05 or P<0.01). Mechanistically, loganin up-regulated the ratios of LC3 II/LC3 I and expressions of Beclin-1, Atg-7, Parkin, and PINK1, while down-regulated p62 and NLRP3 protein expressions (P<0.05). In vitro experiments further confirmed that loganin attenuated CCl₄-induced injury in L-02 cells by enhancing mitophagy and inhibiting NLRP3 inflammasome activation, which was reversed by the mitophagy inhibitor Mdivi-1.

Conclusion: Loganin protected against CCl₄-induced ALI both in vivo and in vitro by suppressing the NLRP3 inflammasome and enhancing mitophagy.