Effect Of Inhibiting Mitochondrial Pyruvate Carrier Activity On The Progression Of Nonalcoholic Fatty Liver Disease And Its Underlying Mechanisms

Background and aims:In the development of non-alcoholic fatty liver disease(NAFLD),the expression of mitochondrial pyruvate carrier(MPC)is increased.But the related molecular mechanism is still unclear.Therefore,we established a NAFLD mouse model,and by the means of in vitro or in vivo inhibiting MPC,we explored the effect of MPC inhibition on NAFLD pathology and the underlying mechanisms.These preliminary results hopefully could provide new clues for the development of drugs for treatment of NAFLD.Methods:Seven-weeks male C57BL/6 mice were randomly divided into two groups(n=6 per group).Group#1 were considered as control group and were given standard chow and normal drinking water.Group#2 were fed with high-fat and highglucose diet to construct a mouse NAFLD model.We used HE staining to observe the liver tissue morphology change,oil red O staining to observe lipid accumulation,Sirius red staining to observe collagen deposition in liver tissue,immunohistochemical staining to observe MPC1 expression and distribution in liver.We also used RT-PCR to detect mRNA expression of MPC1,MPC2,AMPK,ACC,carnitine palmitoyltransferase 1 A(CPT1A),Sterol regulatory element-binding protein 1(SREBP1),ACTA2,CTGF,TGF-β,TIMP1 mRNA.And we used automatic protein analyze to detect MPC1,AMPK,p-AMPK,ACC,p-ACC,SREBP1 protein e level.2.We investigated the effect of inhibiting MPC on the progression of hepatic steatosis,We started with the in vivo study.Mice were divided into a control group,a model group and treatment group.The treatment groups were administrated with MPC1 inhibitor UK5099.In the in vitro study,the steatosis modeling of HepG2 cells were induced by PA.HepG2 cells were divided into a control group,a model group and treatment groups,treatment groups were administrated with MPC1 inhibitor UK5099.Significant lipid accumulation was documented by oil red O staining.RTPCR were used to detect the expression level of AMPK,ACC,CPT1A,SREBP1 mRNA.Automatic protein analyze were used to detect the expression of AMPK,pAMPK,ACC,p-ACC,SREBP1 protein level.3.We then investigated the effect of inhibiting MPC on the progression of fibrosis in NAFLD.For the in vivo observation,mice were divided into a control group,a model group and treatment groups,the treatment groups were administrated with MPC1 inhibitor UK5099.For the in vitro study,LX2 cell lines and LX2 cellHepG2 cell co-culture model were used to investigate the mechanism of MPC1 and liver fibrosis.RT-PCR were used to detect the expression level of ACTA2,CTGF,TGF-β,TIMP1 mRNA.Automatic protein analyze were used to detect a-SMA,CTGF,TGF-β protein level.Results:1.Compared with the normal diet group,steatosis appeared in the liver portal region of HFG model group.As the molding time goes on,the steatosis of the portal region was aggravated,with bullae mixed vesicular steatosis of hepatocytes appeared.The steatosis region spread from the portal region to the central venous region,and filamentous fibers appeared in the portal region in the HFG model group.suggesting a stable mice model of NAFLD was established.MPC1 immunohistochemical,automatic protein analysis experiment and RT-PCR results suggest that compared to the diet group,MPC1 gene and protein expression levels were elevated in HFG model group mice liver.AMPK-ACC pathway is restrained,with decreased p-AMPK and p-ACC protein levels,increased SREBP1 protein levels.The expression level of fibrotic related molecule ACTA2,CTGF,TGF-β mRNA were increased.2.In the animal model,the AMPK-ACC pathways were activated,with increased p-AMPK and p-ACC protein levels,decreased SREBP1 protein levels,and increased CPT1A mRNA expression level in MPC inhibitor treatment group mice liver.In cell culture model,PA significantly induced lipid accumulated,increased the ACC,and reduced the p-AMPK,p-ACC protein level.MPC inhibitor treatment decreased the number of cells containing lipid droplets induced by PA,and increased the expression ofp-AMPK and p-ACC protein level.3.In the animal model,MPC inhibitor treatment decreased the HFG diet induced expression of fibrosis related factor a-SMA、CTGF、TGF-β protein levels.In cell culture model,when LX2 cell co-cultured with HepG2 cells by Transwell,the mRNA level of CTGF,TGF-β,TIMP1 were significantly decreased in LX2 cell treated with MPC inhibitor.The automatic protein quantitative analyzer result showed that,compared with the normal control group,the a-SMA、CTGF、TGF-β protein level was decreased in LX2 cells.Conclusions:We found that HFG treated mice,the hepatic steatosis and fibrosis progression accompanied with high expression levels of MPC gene and protein,and the AMPK-ACC pathways was inhibited.Inhibition of MPC by UK5099 can improve lipid accumulation through activation of the AMPK-ACC pathways.Inhibition of MPC may indirectly inhibit the activation of hepatic stellate cells by inhibiting the secretion of pro-fibrosis factors by hepatocytes,thereby preventing the progression of liver fibrosis.