The Study Of PPAR-α Methylation And Inflammatory Factors In Type 2 Diabetes With Non-alcoholic Fatty Liver

Objective:Diabetes mellitus(DM)is a multi-etiological metabolic disease characterized by chronic hyperglycemia,accompanied by metabolic disorders of glucose,fat and protein due to defects in insulin secretion and/or actions.Nonalcoholic fatty liver disease(NAFLD)is a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility.Its main pathological manifestations are non-alcoholic,viral,and drug-induced hepatic fat content increase.Type 2 diabetes is a common disorder of lipid metabolism,and fatty liver and lipid metabolism are inseparable.Therefore,type 2 diabetes and fatty liver also have a certain relationship.The incidence of non-alcoholic fatty liver disease(NAFLD)is very common in people with type 2 diabetes.About 75%of patients with type 2 diabetes have clinical features of fatty liver.Diabetes is ranked third in the cause of fatty liver,second only to alcohol and obesity.Glucose and fatty acids in the body of diabetics are not well utilized,and lipoprotein synthesis is also impeded.As a result,most glucose and fatty acids are converted into fat in the liver and deposited in the liver,leading to fatty liver.NAFLD can also cause type 2 diabetes.The merger of the two forms a serious medical injury to the patient and a huge economic burden on society.Therefore,it is of far-reaching social significance to thoroughly analyze the occurrence and development mechanism of type 2 diabetes(T2DM)combined with non-alcoholic fatty liver disease(NAFLD).Studies have shown that peroxisome proliferators-activated receptors(PPARs)are widely involved in the regulation of energy metabolism,PPARs have three representative members:PPAR-α,PPAR-β/δ and PPAR-γ They play a decisive role in energy metabolism,but their active expression profiles are different.PPAR-steroids have roles in promoting adipogenesis and differentiation,preventing the formation of inflammation,maintaining homeostasis in glucose,and increasing insulin sensitivity.Therefore,there is a correlation between PPAR-steroids and the pathogenesis of fatty liver and T2DM.DNA methylation refers to a chemical modification method of adding an active methyl group to the 5-methylcytosine(5-mC)at the C5 position of cytosine under the catalysis of DNA methyltransferase.Methylation of the PPAR-a gene is an important factor affecting the development of metabolic diseases.However,it is unclear whether it affects the progression of fatty liver and T2DM.The results of animal experiments showed that after obesity was induced in rats fed with high-fat diet,the capillary density of the viscera became smaller and the blood flow was reduced.The adipocyte hyperplasia reduced the partial pressure of oxygen in the adipose tissue,resulting in hypoxia and apoptotic fat tissue.With inflammatory infiltration,macrophages infiltrating adipose tissue release excessive inflammatory mediators such as IL-6,IL-10,TNF-α,TGF-α1,and CRP,thereby triggering a chronic inflammatory response.The levels of these inflammatory factors in T2DM with NAFLD are not yet known.Until now,there is no literature to report a stable model of type 2 diabetes with NAFLD.In this study,we optimized the animal model of type 2 diabetes with NAFLD and explored the PPAR-a gene methylation in T2DM with NAFLD animal model in animal models.Whether the model changes during construction,whether it affects the expression of PPAR-a protein,and analyzes the methylation level and the expression levels of inflammatory factors IL-10,IL-6,TNF-a,TGF-β1,and hs-CRP Correlations and validation analysis in clinical specimensMethods:(1)Animal model of T2DM combined with NAFLD was established by feeding SD rats with high-fat diet and streptozotocin(STZ).The lipids were measured for total cholesterol(TC),triglyceride(TG),and high-density lipids.Protein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),liver function index alanine aminotransferase(ALT),aspartate aminotransferase(AST)and blood glucose(Glu),insulin(INS)levels Changes were evaluated by hematoxylin-eosin staining for hepatic steatosis.(2)Take tail vein whole blood of healthy control group and model group animal,extract whole blood DNA,and measure the methylation level of PPAR-α promoter region by DNA methylation kit.Total RNA was extracted from liver tissues of two groups and the expression of PPAR-a mRNA was analyzed by qPCR.Western blot analysis of PPAR-a protein expression was performed on liver tissue from two groups of animals.Serum inflammatory cytokines TNF-α,IL-6,IL-10,transforming growth factor were detected by enzyme-linked immunosorbent assay(ELISA).1(TGF-β1),C-reactive protein(hs-CRP)content.The protein expression of hs-CRP,TNF-a,IL-6 and TGF-PA1 in liver tissue of two groups was analyzed by Western blot.(3)Finally,in 32 cases of normal control(N group)and 35 cases of NAFLD patients(M group)whole blood samples,using commercially available kits the level of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were measured;the level of TNF-a,IL-β1,TGF-β1,IL-6 and IL10 were determined by qPCR and ELISA,the level of PPAR-a were measured by qPCR,according with verification of the relationship between expression of cytokines and the expression level of PPAR-a in clinical specimens of NAFLD patients with DM.These results were combined to verify the relationship between the expression of cytokines and the expression level of PPAR-a in clinical specimens of NAFLD patients in DM.(3)Thirty-two patients with normal(N group)and 35 T2DM patients with NAFLD(M group)were selected as subjects.Venous blood was taken from an empty stomach at 8:00 in the morning,and serum ALT,AST,TC,and TG levels were measured biochemically.The levels of hs-CRP,TNF-α,IL-6,IL-10,and TGF-β1 in the blood of the two groups were detected by fluorescent quantitative PCR.The levels of hs-CRP,TNF-α,IL-6,IL-10,and TGF-β1 protein in the blood of the two groups were analyzed by ELISA.The qPCR method was used to analyze the expression level of PPAR-a mRNA in both groups.The ELISA method was used to analyze the expression level of PPAR-α protein in the two groups of serum.The correlation between the expression levels of PPAR-a in the serum and the inflammatory factors hs-CRP,TNF-a,IL-6,IL-10 and TGF-β1 was analyzed.Results:(1)After 2 weeks of SD rats fed with high-fat diet and low-dose STZ(30mg/kg),HE staining of liver tissue sections showed that there were lipid droplets of different sizes in the hepatocytes of the model rats.A pathological lipolysis of vesicles occurred;abnormal glucose tolerance test results.The liver function indexes ALT,AST and blood lipids TC,TG levels increased with statistical significance(P<0.05 or 0.01);and the liver quality and liver index levels increased further and were higher than the control group(P<0.05 or 0.01).These findings indicate that the rat model of T2DM with NAFLD has been successfully constructed and can be used to study the mechanism of methylation of PPAR-a gene.(2)The methylation status of PPAR-α gene in healthy control and T2DM with NAFLD model rats was tested.The results showed that the methylation positive rate of PPAR-a promoter in healthy control and model groups was 10%and 65%(P<0.05).The mRNA and protein expression of PPAR-a in the model group was lower than that in the healthy control group.Compared with the healthy control group,the levels of hs-CRP,TNF-a,TGF-β1 and IL-6 in the model group increased,and the level of IL-10 decreased(all P<0.01).(3)Serum levels of ALT,AST,TC,TG and LDL in group M(T2DM patients with NAFLD)were significantly higher than those in group N(normal)(P<0.01),while HDL levels were significantly lower(P<0.01).P<0.01).The serum levels of hs-CRP,TNF-α,IL-6,and TGF-β1 in group M were significantly higher than those in group N(P<0.01),while the expression of IL-10 was significantly lower.N group subjects(P<0.01).The expression of hs-CRP,TNF-a,IL-6 and TGF-β1 protein in serum of group M was significantly higher than that of group N(P<0.01),while the expression of IL-10 was significantly lower than that of group N.The subjects(P<0.01)were consistent with the results of RNA level test.The mRNA expression of PPAR-a mRNA in serum of group M was significantly lower(P<0.01).The expression of PPAR-a protein in serum of group M was significantly lower than that of group M(P<0.01).Conclusion:1)Through the analysis of pathological and biochemical indicators,we first confirmed that a high-fat diet plus a small dose of STZ(30 mg/kg)can induce and successfully establish a stable rat animal model consistent with non-alcoholic fatty liver disease in clinical type 2 diabetes.It provides tools for the study of the mechanism and treatment of the disease.(2)The percentage of methylation of PPAR-a gene in T2DM patients with NAFLD increased significantly,and the expression of serum inflammatory factors increased,which may be one of the possible mechanisms of the increased incidence of fatty liver in type 2 diabetes.(3)The expression of PPAR-a protein was down-regulated in T2DM patients with NAFLD,and the abnormal expression of inflammatory factors in patients with T2DM was higher than that in the normal population.It was shown that PPAR-a is of great value in the study of NAFLD combined with DM,and severe lipid abnormalities and inflammatory reactions occur in T2DM patients with NAFLD.