The effect and mechanism of Germacrone in ameliorating alcoholic fatty liver by inhibiting Nrf2/Rbp4.

Abstract

Background: Alcohol-related liver disease (ALD) is an important cause of the increase in liver disease-related morbidity and mortality worldwide. Its core pathological features are oxidative stress imbalance and lipid metabolism disorders. Nuclear factor E2-related factor 2 (Nrf2), a key regulator of oxidative stress, maintains cellular redox balance by activating antioxidant genes. However, over-activated Nrf2 may further exacerbate lipid accumulation. Retinol-binding protein 4 (Rbp4) is a key regulator of lipid metabolism, and its abnormal expression is closely related to hepatic steatosis. Therefore, regulating the balance between Nrf2 and Rbp4 may be an effective strategy to improve ALD. This study aims to explore the therapeutic effect of Germacrone on ALD and further reveal the molecular mechanism of Germacrone's improvement of oxidative stress and lipid metabolism disorder by regulating the Nrf2/Rbp4 signaling pathway.

Methods: An alcohol-induced ALD model was established in C57BL/6 mice. After continuous administration of Germacrone (21 days), the effect of Germacrone on liver lipid accumulation, oxidative stress, and pathological injury was evaluated. The core components and targets of JGST were screened by proteomics and network pharmacology, and the improvement effect of Germacrone on ALD was observed by H&E and oil red O staining, serum biochemical indices, and Western blot analysis. Subsequently, the binding of Nrf2 in the Rbp4 promoter region was analyzed by ChIP experiment. Finally, through in vivo and in vitro experiments, Nrf2 nuclear translocation and downstream target gene Rbp4 expression changes were detected, and Nrf2 knockdown or overexpression experiments were conducted to further verify its regulatory effect on Rbp4.

Results: Proteomic analysis showed that the expressions of HO-1, Gsta1 and Rbp4 in the ALD model were significantly increased, and Rbp4 expression was positively correlated with liver triglyceride (TG) level. Network pharmacological predictions found that Germacrone is the core component of JGST to improve ALD. Germacrone can significantly reduce alcohol-induced liver lipid deposition, oxidative stress, and histopathological damage and significantly reduce the abnormal expression of Nuclear Nrf2 and Rbp4. ChIP experiment results showed that Nrf2 could significantly bind the Rbp4 promoter region - 1534 to - 1473 bp and transcriptionally activate its expression. Meanwhile, In vitro and in vivo experiments further verified that overexpression or activation of Nrf2 could significantly up-regulate Rbp4 expression, while knockdown or inhibition of Nrf2 could significantly decrease Rbp4 expression.

Conclusion: Germacrone can protect the liver by inhibiting the Nrf2/Rbp4 signaling pathway, improving oxidative stress and lipid metabolism disorder in the ALD model. Rbp4 is a novel downstream target gene of Nrf2. As a potential drug candidate, Germacrone has great clinical application value.