Inhibition Of The Glycolytic Active Hepatic Stellate Cells And The BDL-induced Liver Fibrosis In Mice By Anti-HIF1 Drugs

Objective:Hepatic fibrosis is a pathological repair reaction caused by chronic liver injury,characterized by the transformation of hepatic stellate cells（HSCs）from a resting to an activated,enhanced proliferation and migration,and the secretion of large amounts of extracellular matrix（ECM）,resulting in the abnormal deposition of type I collagen-based ECM proteins in the liver parenchyma.Previous studies in the laboratory have found that abnormally high glycolytic metabolic activity in activated HSCs,inhibition of cellular glycolysis,or expression of HIF1αprotein in cells,can reverse the activated phenotype of myofibroblast-like hepatic stellate cells.This thesis focuses on the effects of various anti-HIF1 drugs on HIF1 in activated state HSCs and the effects of anti-HIF1 drugs on the glycolytic process and MF phenotype of HSCs,and evaluates the effects of anti-HIF1 drugs on the improvement of liver fibrosis in mice in order to find effective drugs for the treatment of liver fibrosis.Method:We first investigated the effects of five anti-HIF1 drugs:Camptochecin（CPT）,SN-38,Formononetin（FMN）,Acriflavine and Bortezomib on the cell viability,HIF1αprotein expression and HIF1transcriptional activity of rat activated hepatic stellate cells（HSC-T6）,and then selected the effective anti-HIF1 drugs.We further investigated the effects of the camptothecin representative drug CPT and its hydroxyl derivative hydroxy camptothecin（HCPT）on the role of activated state HSC.We first compared the effects of CPT and HCPT drugs on HSC-T6,human activated stellate cells LX-2 and then examine whether they alter proliferation,migration,and apoptosis in two types of HSC cells and further examine their effects on the expression of HIF1α,the glycolytic enzyme PKM2 and the MF phenotypic signature proteinαSMA in two cells.To further investigate the in vivo effects of CPT and HCPT on the basis of pre-laboratory studies we encapsulate CPT and HCPT with VA-PEG-PCL nanomicelles ligated with retinol targets to achieve their targeting effects on HSCs in gallbladder ligation（BDL）-induced hepatic fibrosis mice,and then carried out biochemical,molecular biology and pathological histological investigations to analyze the in vivo anti-HSC activation and anti-fibrosis activities of the two drugs.Results:The results showed that,except FMN had no significant inhibitory effect on HSC-T6 cell viability,CPT,SN-38,Acriflavine and Bortezomib all showed dose-dependent inhibition of HSC-T6 cell viability.Among them,CPT can inhibit HIF1αprotein expression in HSC-T6 at low doses of Bortezomib and Acriflavine.It had no effect on HIF1αprotein expression at its IC₅₀ concentration,but inhibited the expression of the glycolysis-limiting enzyme PKM2 protein.However,luciferase reporter gene analysis failed to show an inhibitory effect of the drug on the transcriptional activity of HIF1;The results revealed that both CPT and HCPT dose-dependently inhibited all three cell viabilities;both at 1/2 IC₅₀ and IC₅₀treatment concentrations significantly inhibited the proliferation and migration of HSC-T6 and LX-2,while promoting HSC-T6 apoptosis.More interestingly,we found that at the same concentrations,CPT but not HCPT significantly inhibited the expression of HIF1αprotein and glycolysis-restricted enzyme PKM2 in HSC-T6 and LX-2,and thus inhibited the expression of the MF phenotype proteinαSMA;We found that the serum aspartate aminotransferase（AST）,alanine aminotransferase（ALT）and bilirubin（BIL）as well as the ratio of mouse liver weight to body weight in BDL mice increased significantly,the necrotic mass of liver tissue increased significantly,pseudobullet appeared,and the deposition of collagen in liver parenchyma increased significantly;meanwhile,the amount ofαSMA and PKM2 positive cells in liver tissue increased significantly,and the expression of HIF1αand PKM2 proteins in the liver increased significantly.In the drug-treated group,CPT-containing micelles more significantly reduced AST,ALT and BIL and the ratio of liver weight to body weight more significantly than HCPT-containing micelles.It also attenuated the increase of HIF1αand PKM2 protein expression and the expansion of activated HSC cells in liver after BDL injury;it also eliminated the necrotic mass and collagen deposition in liver tissue and effectively repaired the structure and function of the liver.Conclusions:Our experimental results indicate that activated HSCs are selective for anti-HIF1 drugs.CPT can be more effective than HCPT Inhibit HIF1αprotein expression and cytosolic glycolysis in activated HSCs,thereby reversing their MF phenotypes and suppressing their mass production,ultimately significantly improving the degree of liver fibrosis and restoring the outcome and function of the damaged liver in mice.Therefore,CPT is expected to be an effective drug in the treatment of liver fibrosis,and CPT-containing micelles are also expected to be an effective target drug in the treatment of liver fibrosis.