The Effect And Mechanism Of Hepatocyte Nuclear Factor HNF1α On Non-alcoholic Fatty Liver Disease

【Background and Objective】 With the increasing number of obese people in the world,non-alcoholic fatty liver disease（NAFLD）has become a common disease,the trend of incidence of NAFLD increased year by year.In recent years,a number of studies have shown that NAFLD patients often associated with obesity,hyperglycemia,hyperlipidemia,hypertension and type 2 diabetes（T2DM）,metabolic syndrome and heart and cerebrovascular disease.NAFLD and T2 DM are metabolic-related diseases caused by glucose and lipid metabolism disorders,the two promote each other,mutual deterioration,has become a new challenge of clinical treatment in recent years.Hepatocyte nuclear factor（HNFs）is a transcription factor that is expressed in the liver,which including HNF1,HNF3,HNF4,HNF6 and CCAAT/enhancer binding protein（C/EBP）.HNFs play a key role in liver development,hepatocellular differentiation and functional maintenance by regulating the expression of a variety of important hepatocyte genes from downstream.HNF1α is one of the main members of the HNFs family and belongs to the POU-homologous domain family.HNF1 includes HNF1α and HNF1β,which have the highest expression level in the liver and also expressed in the kidney,intestine and pancreas.It can regulate many important functional genes of the liver and it is the necessary transcription factor for liver development.In the previous study,it was observed that HNF1α systemic knockout mice had hyperglycemia and hyperlipidemia.HNF1α gene mutation inhibited the function of liver-type of fatty acid binding protein（L-FABP）,causing a impairment of hepatocyte fatty acid transport function,eventually leading to hepatic steatosis.Above all,the study above suggesting that HNF1α may be an inhibitory factor in the process of glucose and lipid metabolism.However,it is also need to further confirm the regulation of HNF1α on glucose and lipid metabolism,clarify its role in the treatment of glucose and lipid metabolism disorders.Based on the above research background,the effect of HNF1α on glucose and lipid metabolism was analyzed by two aspects include NAFLD withT2 DM mouse model and HNF1α hepatocyte specific knockout mice,and its mechanism was further explored.【Methods】1.the expression of HNF1α in livers of NAFL mice model.To clarify the expression of HNF1α in lipid metabolism disorder,we established non-alcoholic fatty liver model mice with high fat diet.The peripheral blood glucose of mice was detected by IPGTT method.Paraffin sections were made by embedding part of tissues to observe whether the NAFL model was successful.The RNA and protein of liver were extracted.Real-time PCR,western blot and immunohistochemical method were used to detect HNF1α expression levels.2.The effect of specific regulation of HNF1α in hepatocyte on the development of non alcoholic fatty liver disease in mice.（1）To verify the expression of HNF1α in Hnf1a^H-KO mice liverHnf1af/f mice were crossed with Alb-Cre mice to obtain Hnf1a^H-KO mice,which was specifically knock out the HNF1 α in hepatocytes.All mice were sacrificed on the 15 th day after birth,and the tail DNA was extracted and the mouse genotype was detected by PCR.The expression of HNF1α mRNA level in liver tissues and hepatocytes of mice was detected by Real-time PCR and Western Blot.The expression of HNF1α level in mouse pancreas,intestine,kidney and liver was observed by immunohistochemistry.（2）The liver morphology of Hnf1a^H-KO mice and the detection of glucose and lipid metabolism genes in Hnf1a^H-KO mice.Mice were divided into two groups: HNF1α knockout group and control group(Hnf1a^H-KO and Hnf1af/f)according to the genotype.The mice were sacrificed at 10 weeks old and 30 weeks old.The liver phenotype,glucose and lipid metabolism related genes were observed.（3）The therapeutic effect of HNF1α on high fat diet mice model.The mice were randomly divided into two groups（n=5）,and then injected AAV-TBG and AAV-HNF1α（2×1011 Vg/per mice,once）via tail vein.The mice were sacrificed at the 20 th week after high fat diet and observed the body weight and liver weight.Liver specimens were collected and the liver phenotype was observed by HE and other histology methods.Real-time PCR,western Blot,immunohistochemistry and other methods to detect the expression of glucose and lipid related genes in the liver,to determine therapeutic effect of HNF1α on high fat diet mice model.3.The molecular mechanism of HNF1α inhibit the development of NAFLD1.Detecte the inflammatory factors and inflammation-related signal pathways in livers of Hnf1a^H-KO mice.（1）The expression of inflammatory factors mRNA level in liver tissues of Hnf1a^H-KO mice was detected by Real-time PCR and IHC.（2）Western Blot was used to detect the STAT3,NF-κB inflammation-related signal pathway in liver tissue of Hnf1a^H-KO mice.2.Detecte the inflammatory factors and inflammation-related signal pathways in hepatocyte of Hnf1a^H-KO mice.（1）The expression of inflammatory factors mRNA level in hepatocyte of Hnf1a^H-KO mice was detected by Real-time PCR.（2）Western Blot was used to detect the abnormal signal pathway in hepatocyte of Hnf1a^H-KO mice.3.Detecte the inflammatory factors and inflammation-related signal pathways in livers of NAFL mice model.（1）The expression of inflammatory factors mRNA level in liver tissues of NAFL mice model was detected by IHC.（2）Western Blot was used to detect the abnormal signal pathway in hepatocyte of NAFL mice model.4.Statistical analysisData were analyzed by SPSS 18.0 software.One-Way ANOVA analysis was used in the group.Student’s t-test analyze the data of experiments involving only two groups..Nonparametric test was used for non-homogeneity.The experimental data were expressed as X ± SD,P <0.05 was significant Differences,P <0.01 were very significant differences.【results】1.the expression of HNF1á in livers of NAFL mice model.Compared with the control group,the expression of HNF1α mRNA level after the high fat diet 10 wk has a transient increase,at 30 wk of high fat diet HNF1α mRNA level significantly reduced.Western blot and immunohistochemistry further confirmed that the expression of HNF1α in the liver of mice was significantly decreased at 30 wk in high fat diet.2.The effect of specific regulation of HNF1α in hepatocyte on the development of non-alcoholic fatty liver disease in mice.（1）To verify the expression of HNF1α in Hnf1a^H-KO mice liverTissue test results showed that HNF1α expression in the liver decreased significantly,while the expression of HNF1α in the intestine,kidney and pancreas did not change.the expression of HNF1α Hnf1a^H-KO mouse primary hepatocytes and non-hepatocyte,it was found that the expression of HNF1α in primary hepatocyte of mice was significantly lower than that in non-hepatocyte.（2）The liver morphology of Hnf1a^H-KO mice and the detection of glucose and lipid metabolism genes in Hnf1a^H-KO mice.Hnf1a^H-KO mice appeared hyperlipidemia,liver injury and blood glucose regulation damage,the occurrence of fatty liver and progress to NASH with hepatic fibrosis.Hnf1a^H-KO mice developed HCC enventually.The expression of ACC,SREBP-1c and FAS were significantly increased,which promoted the fatty acid synthesis.The expression of triglyceride in liver was increased and the expressions of GCK reduce.（3）The therapeutic effect of HNF1α on high fat diet mice model.The blood glucose of HFD AAV-HNF1α group was significantly lower than that of HFD AAV-TBG group,and hepatic steatosis was not found in liver.The hepatocytes were arranged neatly and there is no obvious inflammatory cell infiltration.The liver did not occurred NASH.collagen deposition were not found in liver,suggesting that there is no hepatic fibers appear.Compared with HFD AAV-TBG mice,the levels of SREBP-1c,FAS and ACC mRNA and liver TG in HFD AAV-HNF1α group were significantly decreased.On the other hand,in HFD AAV-HNF1α mice,the expression of GCK was significantly higher than that of HFD AAV-TBG group,and the expression of TNFα,TGFβ1 and IL-6 were significantly decreased.3.The molecular mechanism of HNF1α inhibit the development of NAFLD1.Detecte the inflammatory factors and inflammation-related signal pathways in livers of Hnf1a^H-KO mice.（1）the expression of TNFα,TGFβ1 and IL-6 was significantly increased in the liver of Hnf1a^H-KO mice.（2）HNF1α deletion lead to STAT3 and NF-κB signaling pathway activated in liver.2.Detecte the inflammatory factors and inflammation-related signal pathways in hepatocyte of Hnf1a^H-KO mice.（1）the expression of TNFα,TGFβ1 and IL-6 was significantly increased in hepatocyte of Hnf1a^H-KO mice.（2）HNF1α deletion lead to STAT3 and NF-κB signaling pathway activated in primary hepatocyte.3.Detecte the inflammatory factors and inflammation-related signal pathways in livers of NAFL mice model.（1）The expression of TNFα,TGFβ1 and IL-6 was significantly decreased in liver of NAFL mice model.（2）HNF1α overexpression leads STAT3 and NF-κB signaling pathway activated in liver of NAFL mice model.【in conclusion】1.HNF1α was significantly reduced in liver of non-alcoholic fatty liver disease,and it is closely related to heperglycemia and inflammatory.2.HNF1α hepatocyte-specific knockout mice developed fatty liver,NASH,hepatic fibrosis and HCC spontaneously.3.Hepatocyte-specific overexpression of HNF1α can alleviates fatty liver and NASH.4.Hepatocyte-specific HNF1α deficiency can cause STAT3 and NF-κB pathway activation in liver and hepatocytes.HNF1α can alleviates the fatty liver and liver insulin resistance through down-regulation STAT3 signaling pathway.