The Study On Obesity And Hepatic Fibrosis By Leptin Gene Edited Minipigs

Obesity and metabolic syndrome(MetS)have become serious diseases which threaten human health and life in recent years.Therefore,many animal models of obesity are studied and prepared for researching the mechanism of the disease and screening drug of diagnosis and treatment scheme in depth like ob/ob mouse.Leptin,ob gene encoded production,plays a key role in feeding and energy metabolism via signaling pathway like JAK-STAT to effect obesity.So rodent models based on Leptin gene editing like ob/ob mouse which has apparent obesity phenotype have been used widely in research of mechanism and clinic.However,the significant differences between rodents and human make them can't simulate human's disease phenotype very well.In contrast,minipig shows many similarities not only in the physiological structure and function,but also in the metabolic characteristics and genetic homology so that they have been used for diseases model popular.But in terms of obesity research,there are few genetically modified pig as obesity models,and most are mainly dietary induced.Our laboratory members had used ZFN technology to generate Leptin gene knockout minipigs in previous studies because of the advantages of minipig and the important role of Leptin gene.On the basis of this work,I had taken a series of molecular and phenotype identification to Lep?5bp/?2bp and WT-Lep+/+minipig.The results showed it appeared 3 new types of Leptin transcripts in adipose tissue of Lep?5bp/?2bp minipig.All of these would result in a shift mutation and premature translation termination of leptin.It also proved that the loss-function of leptin protein in Lep?5bp/?2bp minipigs by ELISA assay and specific Western Blot.The Lep?5bp/?2bp minipigs had significant peak weight gain(P<0.001)and high body fat rate(P<0.05)because of increased appetite caused by blocked JAK-STAT signaling pathway.Even more,Lep?/?2bp minipigs showed significantly increased obesity blood physiological and biochemical indexes(P<0.05)and type ? diabetes symptoms like insulin resistance and damaged glucose tolerance from the age of 12 months.These results suggested that the Lep?5bp/?2bp minipigs has been a good model of obesity and metabolic syndrome.Nonalcoholic fatty liver disease(NAFLD)with obesity is caused by excessive accumulation of lipid in liver,which is one of the most serious obesity complications of harming human health.The progression of NAFLD in human is characterized by progressive deterioration in pathology from the first stage called "non-alcoholic hepatic steatosis",then leading to non-alcoholic steatohepatitis(NASH),gradually developing into liver fibrosis,and deterioration to hepatic cirrhosis finally.But the rodent lack of leptin can only run to the first phase,the fatty liver and will not continue to deteriorate.So the current rodents NASH and liver fibrosis model were induced by drugs or surgery method.However,it was obviously different from human in the causes of NAFLD.Therefore,we explored the liver injury phenotype of Lep?5bp/?2bp minipigs in the second part of this research.The results of H&E and oil red O staining indicated that Lep?5bp/?2bp minipigs developed the phenotype with excessive accumulation of lipid in liver at the age of 12 months.The results of PAS staining and relative gene expression of inflammatory factor indicated that Lep?5bp/?2bp minipigs developed the phenotype of NASH at the age of 18 months.Fibrosis related specific staining,marker protein immunochemical staining and hematology indexes results showed that Lep?5bp/?2bp minipigs developed the phenotype of significant liver fibrosis at the age of 25 months.Furthermore,the scores showed that the fibrosis has developed to a moderate-severe stage(3.74±0.81).These results proved that the Lep?5bp/?2bp minipigs can simulate the progression of NAFLD in human perfectly and be used for research for mechanism of these diseases in depth.In order to explore the real reason of the phenotype of hepatic fibrosis of the Lep?5bp/?2bp minipigs which absolutely lacked in ob/ob mouse,we focused on the mechanism of hepatic fibrosis of the Lep?5bP/?2bp minipigs in the third part of this research.First,we took RNA-Seq assay with Lep?5bp/?2bp and WT-Lep+/+ minipigs and then got 148 differentially expressed genes(DEGs,Fold Change?2,FDR<0.05).The results of GO and KEGG enrichment with the DEGs showed it had 6 CYP2 genes'up-expression like CYP2E1(P<0.05)which exactly opposite of ob/ob mouse and it had been confirmed by Q-PCR and Western Blot.The further experiment that oxidative stress markers in liver and serum increased significantly proved that it had occurred severe oxidative stress in the liver of Lep?5bp/?2bp minipigs.However,combined with the prediction of protein cross-talk network and previous studies,we infered that leptin did not regulate the expression of CYP2 directly with signaling pathway network.The results of relative Q-PCR and Western Blot showed that Leptin knockout mainly affected the JAK-STAT and AMPK pathway.Through a series of further experiments with detecting relative protein factors in the two pathway we have confirmed that leptin deficiency enhanced the ?-oxidation and?-oxidation via down-regulated JAK-STAT pathway and enhanced the Parkin-mediated mitochondrial autophagy via down-regulatedAMPK pathway.They played common role in making liver injury secondary hit significantly enhanced via aggravated oxidative stress.This resulted in deterioration from adipose degeneration to fibrosis in liver of Lep?5bp/?2bp minipigs.The comparative verification experiment in Leptin-/-rats has further confirmed our judgment and conclusions.To sum up,we have established a genetic obesity model with Leptin-/-pig that can reflect the characterization of human diseases very well.This model compensated for the deficiedcies of the ob/ob mouse model which widely used in the kind of obesity research.We have delved into the molecular mechanism of development of liver fibrosis induced by obesity with Leptin-/-pig and found that the expression of factors in JAK-STAT and AMPK pathway regulated by Leptin was the important reason of deterioration from adipose degeneration to fibrosis.Our research provided not only a new model more closer to human disease for the current study of obesity and liver injury but also a powerful tool for research and development of effective treatment scheme and clinical drug screening.