Calpain‐4 Knockdown Modulates Cholesterol Metabolism and LXRα Nuclear Localization in Experimental Alcohol‐Related Liver Disease

BACKGROUND: Ethanol affects lipid metabolism through multiple pathways, leading to fatty liver development in most alcohol-related liver disease (ALD) patients. Recent studies have highlighted the role of calpain, a calcium-dependent protease, in liver inflammation and fibrosis. Calpain activity is regulated by its essential subunit, Capns1 (calpain-4), which stabilizes and modulates the activity of its catalytic isoforms, calpain-1 and calpain-2. This study investigated calpain's impact on lipid metabolism in ALD. METHODS: Six-week-old C57Bl6/J mice were injected with rAAV8 vectors encoding Capns1 shRNA or control vectors. After 4 weeks, mice underwent a 10-day period of ad libitum ethanol consumption, followed by a single gavaged ethanol administration on day 11. RESULTS: Capns1 knockdown attenuated ethanol-induced microvesicular steatosis. Hepatic triglyceride and free fatty acid levels were not significantly altered, whereas cholesterol levels were significantly reduced in the ethanol group with Capns1 knockdown. Cpt1a expression increased significantly in the ethanol group with Capns1 knockdown. Western blot analysis revealed increased Cleaved-3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) to Pro-HMGCR ratio in Capns1-knockdown mice, suggesting reduced HMGCR activity and suppressed cholesterol biosynthesis. LXRα expression was mainly increased in the cytoplasm in the ethanol group, and following Capns1 knockdown, it was relocalized to the nucleus via its activation. In addition, RNA sequencing analysis indicated that Capns1 knockdown contributes to the reprogramming of ethanol-induced disruptions in metabolic pathways, primarily those involving cholesterol metabolism. CONCLUSION: Further investigation into the relationship between Capns1 and cholesterol biosynthesis proteins may provide insights into using calpain inhibitors as a therapeutic approach for alcohol-related liver disease.