Study On The Relationship Between NAFLD And The Expression Of Transcription Factors TWIST And PPARγ

Objective:With the improvement of people’s living standard,unhealthy lifestyles such as increased fat intake and reduced physical activities have led to an increasing proportion of people suffering from obesity and other metabolic disorders,and the prevalence of the non-alcoholic fatty liver disease(NAFLD)has increased year by year.Through the study of serum specimens of patients with nonalcoholic fatty liver disease,the mice model with a fatty liver induced by high-fat diet and hepatocellular model induced by oleic acid,the subject aims to explore the transcription factors TWIST1,TWIST2 and PPARy expression changes in the nonalcoholic fatty liver disease,providing new clues for the diagnosis or treatment of non-alcoholic fatty liver disease.Methods:1.Collection and detection of clinical specimens:A total of 406 serum specimens were collected from the physical examination center of Shandong Provincial Qianfoshan Hospital from December,2017 to April,2018.According to the imaging results,they were divided into healthy group,mild NAFLD group,moderate NAFLD group and severe NAFLD group.The concentrations of TWIST1,TWIST2 and PPAR’y in serum samples were detected by ELISA.2.The animal model’s establishment and experiments:We established nonalcoholic fatty liver disease model of C57/BL6 mice by high fat diet.The mice were sacrificed after feeding for 4,8,12,16 weeks respectively.We took the mice’s liver tissue for H E staining,oil red O staining,and Western Blot experiments to detect transcription factors TWIST],TWIST2 and PPARly expression changes.The intraperitoneal glucose tolerance test(IPGTT)and the intraperitoneal insulin tolerance test(IPITT)were performed before the mice’s sacrifice at the end of the 16th week.3.Establishment and experiments of cell model:We established the adipose model of hepatocyte induced by oleic acid(50μg/mL).The formation of lipid droplets was observed by oil red O staining as well as semi-quantitative analysis was conducted4.LcRNA/mRNA expression profiling of L02 cells:completed by Shanghai Kangcheng Bioengineering Co.,Ltd.Results:1.Human serum samples:As the severity of the non-alcoholic fatty liver disease increased,the body mass index(BMI),fasting blood glucose level,fasting insulin level and insulin resistance index HOMA-IR of the patients were all increased,with statistically significant differences(P<0.05).TWIST1 level in serum of mild NAFLD patients was lower than that of other groups(P<0.05).TWIST1 concentration in serum of patients with moderate NAFLD was higher than that of the other three groups(P<0.05).The concentration of TWIST2 in serum of patients with non-alcoholic fatty liver disease was lower than that in the healthy group(P<0.05).There was no difference in serum TWIST2 concentration among each group patients with non-alcoholic fatty liver disease.There was no significant difference in the serum PPAR,y concentration among the three NAFLD groups and healthy controls.The correlation analysis of TWIST]and TWIST2 in serum showed that the Pearson correlation coefficient was 0.810.The correlation analysis of TWIST1 and PPARγ in serum showed that the Pearson correlation coefficient was 0.797.The correlation analysis of TWIST2 and PPARy in serum showed that the Pearson correlation coefficient was 0.812.2.The animal experiments:After feeding for 5 weeks,the weight of C57/BL6 mice in the high-fat diet(HFD)group was higher than that in the control group(P<0.05).Compared with the control group,the liver’s surface of mice in the HFD group was greasy and white.The frozen section of liver tissue were dyed by Haematoxylin and eosin,which showed that in the hepatic acinar area were mainly steatotic hepatocytes,accompanied by hepatocytes balloon-like changes,and there were various inflammatory cells in the hepatic lobules infiltrate.At the same time,the oil red O staining were conducted and showed obvious lipid droplets in the liver tissue of HFD group.At the end of week 16,mice were subjected to intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT).The blood glucose concentrations of the HFD group mice were higher than those of the control group at different time points.The blood glucose concentration of the HFD group decreased slowly after injection of glucose,and the reactivity to insulin was worse than that of the control group,suggesting that the mice of HFD group existed insulin resistance.Western Blot analysis of liver tissue proteins were conducted.There was no differences between the expression of TWIST1 or PPARy in the mice’s liver tissue of HFD group and control group.The transcription factor TWIST2 is down-regulated in the HFD group mice fed for 3 months by 45.30%(P<0.05).3.Experiments of hepatocytes:Oleic acid-induced hepatocyte adiposis model was dyed with oil red O,as well as the normal hepatocytes.We observed lipid droplets in the hepatocytes of model group obviously.Semi-quantitative analysis of oil red O after decolorization with isopropanol,the absorbance of the model group was significantly increased.The total protein of hepatocytes was extracted for Western Blot analysis,and the expression levels of TWIST1 and PPAR,y were not significantly changed in the model group and control group at the same time.But the expression of transcription factors TWIST1 and PPARy in L02 cells increased with the prolongation of cell culture time.The expression of TWIST2 in the model group decreased by 23.49%and 25.68%at the molding time of 48h and 72h,respectively(P<0.05).4.LcRNA/mRNA expression profiling of L02 cells:We conducted the mRNA expression profile differences in L02 cells induced by oleic acid(50 μg/mL)and control cells after 48 hours.13823 gene mRNAs signals were detected by microarray,among which 230 genes mRNAs were up-regulated and 377 genes mRNAs were down-regulated.The enrichment analysis of biological function prediction of differential expressed mRNAs showed that a total of 507 differential expressed mRNAs were involved in the biological process.Compared with the control group,the differential expressed mRNAs in the model group participated in 1095 biological processes(P<0.05),among which the up-regulated difference mRNAs participated in 586 biological processes,while the down-regulated difference mRNAs participated in 509 biological processes.The biological processes of insulin signal transduction and glucose transport in the model group were significantly different from those in the control group.Compared with the control group,the differential expressed mRNAs were involved in 41 signaling pathways,among which the up-regulated mRNAs were mainly involved in 36 signaling pathways,while the down-regulated mRNAs were mainly involved in 5 signaling pathways.The differential expressed mRNAs were significantly enriched in the PPAR signaling pathway.Conclusion:1.The TWIST2 concentration in the serum of patients with non-alcoholic fatty liver disease decreased than that of healthy controls,which was consistent with that of non-alcoholic fatty liver disease model mice fed for 3 months and hepatocyte steatosis model in vitro.2.There was a certain positive correlation between the transcription factors TWISTI,TWIST2 and PPARy in human serum.3.During the formation of non-alcoholic fatty liver disease,there is a series of differential expressed mRNAs,as well as insulin resistance exists.The Differentially expressed mRNAs were significantly enriched in the PPAR pathway.