Regulation Of Metformin On Hepatic Stellate Cell Epithelial Mesenchymal Transformation

Liver fibrosis is a chronic liver injury mainly caused by viral infection and alcoholism.Hepatic stellate cells（HSCs）participate in the process of liver fibrosis starting,progress and regression,the stillness of the Hepatic stellate cells（Q-HSCs）has the characteristics of epithelial cells and fat cells,its muscle to fibroblasts（MF-HSCs）transdifferentiation is a key link in the process of liver fibrosis,and the transformation of epithelial mesenchymal（epithelial to mesenchymal transition,EMT）involved in the link.EMT is the process by which cells lose their epithelial phenotype and gene expression characteristics and become mesenchymal cells.Most studies have shown that myofibroblasts are generated by EMT in various organs to cope with various chronic injuries,and this process can be reversed under certain conditions.Recent studies have shown that metformin（Met）has extensive pharmacological effects,and various pharmacological mechanisms include inhibition of the EMT process.At the same time,it has been proved that metformin can alleviate liver fibrosis to a certain extent,and it can reverse liver fibrosis by acting on HSCs.Therefore,metformin may inhibit liver fibrosis by reversing the EMT process,and further understanding of the mechanism of liver fibrosis reversal is expected to provide new theoretical basis for the treatment of liver fibrosis.ObjectiveTo investigate the effect of metformin on the expression of proteins related to hepatic fibrosis and EMT in mice.To investigate whether metformin inhibits the activation of hepatic stellate cells by regulating EMT.Methods1.4-week-old mice were randomly divided into 3 groups,15 mices in each group,named as the normal group,the model group（CCl₄ group）,and the CCl₄+metformin treatment group（CCl₄+Met group）.The model group was treated with a mixture of olive oil and CCl₄ at a ratio of 1:3 and intraperitoneal injection twice a week at a dose of 0.4ml/kg.The normal group was given the same volume of olive oil,intraperitoneal injection,2 times/week,the dose was 0.4ml/kg;The modeling method of metformin treatment group was the same as that of model group.Starting from 3 weeks,metformin was given by intragastric administration at 200mg/time and once/day.Normal group and model group were given normal saline by intragastric administration,200mg/time,1time/day,and mice were sacrificed until the 6th week of modeling,and liver tissue was taken.The liver index of mice was calculated,ALT and AST contents of liver function related indicators were detected,and HE and Sirius scarlet red staining were performed to observe the pathological changes of mice liver.By western blot and RT-PCR detection of liver fibrosis associated protein alpha smooth muscle excited protein（α-SMA）and typeⅠcollagen（collagenⅠ）content,EMT related protein epithelial markers such as E-cadherin and interstitial markers such as Vimentin,N-cadherin and Twist.2.LX-2 cells in logarithmic growth phase were digested with 0.25%trypsin to make cell suspension and inoculated in 12 petri dishes,which were divided into four groups on average,namely blank control group,TGF-β1 group,TGF-β1+Met low dose group,and TGF-β1+Met high dose group.The blank control group was not treated.TGF-1 group was given 10ng/ml cytokine TGF-β1 to induce cell activation.The low-dose TGF-β1+Met group was given 10ng/ml cytokine TGF-β1 and 5mmol/ml metformin.The high-dose TGF-β1+Met group was given 10ng/ml cytokine TGF-β1 and 10mmol/ml metformin.Cell status was determined by oil red O staining,cell activation,proliferation,and migration were detected by immunofluorescence,clonal formation,scratch,and Transwell invasion assay.By western blot and RT-PCR detection of liver fibrosis associated protein alpha smooth muscle excited protein（α-SMA）and typeⅠcollagen（collagenⅠ）content,EMT related protein epithelial markers such as E-cadherin and interstitial markers such as Vimentin,N-cadherin and Twist.Results（1）.Compared with the normal group,the liver index of CCl4 group was significantlyincreased,andthedifferencewasstatisticallysignificant（p<0.01）.Compared with the CCl4 group,the liver index of the CCl4+Met group decreased to a certain extent,but was higher than that of the normal group,with a significant difference and statistical significance（p<0.01）.Liver function indicators AST and ALT in CCl₄ group were significantly higher than those in the normal group（p<0.01）.Compared with the CCl₄ group,the AST and ALT levels in the CCl₄+Met group were significantly lower,with statistically significant differences（p<0.01）.The liver tissue of normal mice showed no obvious change.Compared with normal group,portal area of CCl₄ group mice article appear thick collagen fiber rope bag around,fiber interval obvious thickening,lobular structures apparently unusual,false flocculus to form,disorganized hepatocyte and interstitial inflammatory cell infiltration in clear,appear a large number of collagen deposition,widely distributed,the distribution between the portal area is more,α-SMA and CollagenⅠexpression significantly higher;Compared with CCl₄ group,CCl₄+Met in treatment group,the collagen fiber hyperplasia to reduce,the formation of false flocculus,inflammatory cells infiltration degree reducing,only part of the collagen hyperplasia,a small amount of interval is forming in the hepatic lobule,α-SMA and collagenⅠexpression decreased.WB and RT-PCR results showed that CCl₄ group in the liver of miceα-SMA and collagenⅠprotein expression significantly increased;Group compared with CCl₄ group,CCl₄+Met in mice liverα-SMA and CollagenⅠprotein expression was significantly reduced.In the CCl₄ group,the expression of epithelial markers E-cadherin was significantly decreased,while the expression of interstitial markers Vimentin,N-cadherin and Twist was significantly increased.Compared with the CCl₄ group,the expression of E-cadherin in the liver of mice in the CCl₄+Met group was significantly increased,while the expression of interstitial markers vimentin,N-cadherin and Twist was significantly decreased.（2）.Compared with the blank control group,the activation degree,proliferation ability and migration ability of cells in the TGF-β1 group were significantly increased;The ability of cell proliferation and migration decreased in a concentration dependent manner after the intervention of Met.WB and RT-PCR results showed that the expression ofα-SMA in TGF-β1 group was significantly higher than that in the blank control group.The expression ofα-SMA decreased in a concentration-dependent manner after Met intervention.Meanwhile,in the blank control group,the expression of intracellular epithelial markers was higher than that of interstitial markers.Compared with the blank control group,the expression of epithelial marker E-cadherin was significantly decreased in the TGF-β1 group,and the expression of interstitial markers vimentin,N-cadherin and Twist was significantly increased.After the intervention of Met,the expression of epithelial markers increased and the expression of interstitial markers decreased in a concentration-dependent manner.Conclusion1.After intraperitoneal injection of CCl4,elevated liver function index,liver fibrosis associatedα-SMA and collagenⅠprotein expression significantly increases,EMT related protein expression increased.2.Metformin can improve liver fibrosis,theα-SMA and collagenⅠexpress obviously lowered.3.Metformin not only improves the degree of hepatic fibrosis,but also inhibits the expression of liver EMT-related proteins.4.TGF-β1 induces the activation of HSCs and promotes the expression of EMT-related proteins in cells.5.Metformin inhibits the expression of EMT-related proteins in activated HSCs in a concentration-dependent manner.6.Under the action of metformin,the activation,proliferation and migration ability of HSCs decreased significantly,and showed a concentration dependence.