The Protective Effect Of PKG-1 On Nonalcoholic Fatty Liver

Background:Nonalcoholic fatty liver disease(NAFLD)is the most common liver disease at present.It is the main cause of other chronic liver diseases.The further development of NAFLD will lead to the occurrence of nonalcoholic steatohepatitis(NASH),liver cirrhosis and hepatocellular carcinoma.Worldwide,the prevalence of NAFLD is about 25%,of which South America(31%)and the Middle East(32%)have the highest incidence,followed by Asia(27%),the United States(24%)and Europe(23%),while NAFLD is rare in Africa(14%).It is a chronic liver disease closely related to obesity and diabetes.The incidence rate in China is as high as 15-20%,which has brought heavy burden to the national economy.At present,there is no drug for the treatment of NAFLD.Therefore,it is an important topic to study the pathogenesis of NAFLD and develop related drugs.Protein kinase G(PKG)is serine / tyrosine specific kinase,and is the main role of cGMP signal.At the transcriptional level,a series of factors have been found to regulate the expression of PKG1,such as Sp1 / Sp3,KLF4,p53,FoxO1,USF1/2 and RhoA,and most of them are associated with the NO/ cyclic nucleotide.Our previous studies have confirmed that overexpressed protein kinase G-(PKG-1)can regulate lipid metabolism in obese mice induced by high fat diet,reduce the accumulation of lipid in mice,and obesity often accompanied by the occurrence of NAFLD,but the mechanism of PKG-1 on NAFLD is not clear.Therefore,this study used simple obesity in mice to be late in the study.As an animal model,the effects and molecular mechanisms of PKG1 gene regulation on nonalcoholic fatty liver were studied by biochemical and molecular biological techniques,which provide experimental support for the research and development of PKG1 related drugs for the treatment of NAFLD.Objective:To verify the protective effect of PKG-1 on non-alcoholic fatty liver disease and its mechanismMethod:1.Establishment and experimental grouping of mouse fatty liver modelThe mice were divided into four groups: ob-/-CD+ group,ob+/-CD+ group,ob-/-CD-group and ob-/-CD-group.Group ob-/-is a obese gene stealth homozygous mouse.It is an animal model commonly used in type two diabetes research.Ob+/-mice were hybridized and bred by ob-/-mice and wild type mice.The mice in group CD+,which were overexpressed by PKG-1 gene,were selected from the mice in Professor Wang Shuxia's laboratory in Kentucky,USA,and through the genotype identification of CD+ mice and ob-/-mice,four groups of ob-/-CD+,ob+/-CD+,ob-/-CD-and ob+/-CD-were selected,and 6 female mice in each group were tested.2.Calculation of liver weight and liver index in miceThe weight of the mice was weighed at 20 weeks of age.The mice were dissected and the weight of the liver was weighed.The liver index was obtained by dividing the weight of the liver with the weight of the mice.3.Observation on histomorphology of mice liverAfter immobilization of 10% formalin in mice,the liver tissue was embedded in paraffin.After making paraffin section,the liver tissue was stained by HE.The structure of liver tissue was observed under microscope.The fibrosis degree of liver tissue was determined by the distribution of collagen fibers under microscope after staining with Masson and Sirius red.4.Detection of transcription level of obesity and fibrosis related genes in miceThe Q-pcr method was used to detect the genes related to lipid synthesis and degradation,such as CPT1 A,PGC1 alpha,PPAR GAMA,SREBP and FASN,and the transcription of fibrosis related genes,such as CollagenI/III/IV,TGF-b and Fibronectin.Result:1.PKG-1 overexpression can significantly improve the fatty liver in mice In the pure and obese ob-/-models,the overexpressed PKG-1 mice were significantly lower in weight and liver index than in the knockout PKG-1 mice,and the percentage of triglyceride and total protein decreased significantly HE staining showed that compared with PKG-1 knockout mice,overexpression of PKG-1 vacuoles significantly reduced fat vacuoles and liver tissue was intact These results indicate that overexpression of PKG-1 can significantly improve the fatty liver in obese mice2.PKG-1 overexpression reduced the expression of genes related to fat degradation,fat synthesis and free fatty acid absorption.In the obesity model,the overexpressed PKG-1 mice were compared with the non overexpressed PKG-1 mice.The related genes related to the oxidative decomposition of fat,CPT1 a and PGC1 a,were related genes of PPAR GAMA,SREBP and FASN,and the genes related to the absorption of free fatty acids,CD36,FATP2 and FATP5,were significantly down.It indicates that PKG-1overexpression regulates the transformation process of downstream fat degradation,synthesis and free fatty acid absorption.3.PKG-1 overexpression weakens the inflammatory response in liver cells In obese mice,the overexpression of PKG-1 mice was significantly lower than that of PKG-1 mice The decrease in inflammatory response may be due to a decrease in fat4.PKG-1 overexpression can attenuate fibrosis in liver tissue In obese mice,after PKG-1overexpression,the color fraction of fiber staining was significantly less than that of PKG-1 deletion In addition,the liver cell fibrosis related genes CollagenI/III/IV,TGF-b and Fibronectin were significantly lower in the obese model mice with PKG-1 overexpression than in the obese model mice with the PKG-1deletion These results indicate that PKG-1 can inhibit fibrosis in liver tissueConclusion:1.In the absence of obese gene,the over expression of PKG-1 can reduce the adipose accumulation of nonalcoholic fatty acids.2.Over expression of PKG-1 can improve the degree of liver fibrosis.3.Over expression of PKG-1 may protect non-alcoholic fatty liver by reducing the inflammatory reaction of liver.