It is used in the study of the influence of imperatorin on HNF-4α regulating the process of liver fibrosis
Using Thioacetamide (Thioacetamide, TAA) to induce liver fibrosis in mice, explore the effect of imperatorin on HNF-4α on the process of liver fibrosis, and provide certain evidence for imperatorin as a potential drug for the treatment of liver fibrosis Theoretical basis.
Methods: (1) C57BL/6 mice were selected for in vivo experiments and randomly divided into six groups: normal group, model group, IM low-dose administration group (20 mg/kg), IM high-dose administration group (50 mg/kg ), curcumin positive control group (20 mg/kg), IM single administration group (50 mg/kg). Liver fibrosis model was established by intraperitoneal injection of TAA (100 mg/kg or 200 mg/kg) for 5 weeks. Serum was collected to detect liver transaminase levels in mice; liver histopathological changes were observed by H&E staining and Sirius red staining; liver fibrosis indicators (α-SMA, Collagen-I, TIMP-1, MMP -13), inflammatory cytokines (F4/80, MPO, caspase-1, IL1R1, IL-1β); use western blot, RT-PCR, immunofluorescence and immunohistochemistry to detect HNF-4α in total protein, plasma protein and regulatory roles in nucleoproteins.
(2) For in vitro experiments, rat hepatic stellate cells (HSCs) were activated with TGF-β (10 ng/m L) for 1 h, and incubated with different concentrations of IM (3.125, 6.25, 12.5 μM) for 6 h, Western blot and RT-PCR were used to detect the protein and mRNA expression levels of HNF-4α, liver fibrosis indicators and inflammatory cytokines; and the different expression of HNF-4α in nucleoprotein and plasma protein.
(3) The siHNF-4α experiment was performed on human hepatic stellate cells (LX-2). Under the condition that the expression of HNF-4α was reduced, the indicators of liver fibrosis (α-SMA, Collagen-I, TIMP-1, MMP -13) and the expression changes of inflammatory cytokines (caspase-1, IL1R1, IL-1β). Results: In TAA-induced liver fibrosis experiments, the levels of ALT and AST in serum increased, and the levels of ALT and AST could be reduced after IM administration. H&E staining showed that IM alleviated the pathological damage of mouse liver tissue.
Sirius red staining showed that collagen deposition in mice was improved after IM administration. The results of Western blot and RT-PCR showed that IM significantly inhibited the expression levels of liver fibrosis indicators α-SMA, Collagen-I, TIMP-1, and MMP-13, and decreased the expression levels of MPO, F4/80, IL1R1, caspase-1, and IL The release of inflammatory cytokines such as -1β can reduce liver inflammation. In addition, IM can up-regulate the expression of HNF-4α in total protein and plasma protein, inhibit its expression in nucleus, and improve liver fibrosis through the activation of HNF-4α.
In cell experiments, IM can reduce the protein and mRNA levels of α-SMA, Collagen-I, and TIMP-1 in activated HSCs, down-regulate the ratio of TIMP-1/MMP-13, and inhibit IL1R1, caspase-1, and IL-1. Release of inflammatory cytokines such as 1β. And IM can inhibit the activation of hepatic stellate cells by up-regulating the expression level of HNF-4α in total cell protein and plasma protein. After silencing HNF-4α siRNA, the expression of HNF-4α decreased, HNF-4α was activated after IM administration, and the activation of HNF-4α inhibited the expression of α-SMA, Collagen-I, caspase-1, IL-1β, etc. However, the inhibitory effect of IM on inflammatory cytokines and liver fibrosis markers was weakened under the condition of HNF-4α inhibition.
Conclusion: Imeratorin reduces ECM deposition and inhibits the activation of hepatic stellate cells by up-regulating the expression of HNF-4α; imperatorin inhibits the release of inflammatory cytokines such as F4/80, IL1R1, caspase-1, and reduces liver inflammatory response. Imimperatorin improved TAA-induced liver fibrosis by activating the expression of HNF-4α in the liver, reducing hepatic collagen deposition and secretion of inflammatory cytokines.
