| Non-alcoholic fatty liver disease(NAFLD)is considered to be the liver manifestation of metabolic syndrome(MS).It is one of the most common chronic liver diseases in the world.Type 2 diabetes mellitus(T2DM),as one of the most common chronic metabolic diseases in the world,is closely related to NAFLD.Studies have shown that there is a two-way relationship between the two diseases in China.The clinical relationship between the two diseases is closely related to their common pathogenesis.With the further study for the two diseases,the role of inflammatory factors,tumor necrosis factor-a(TNF-a),interleukin-6(IL-6)and C reactive protein(CRP),is obvious to all.Metformin,as a first-line drug for the treatment ofT2DM in"2013 Chinese guidelines for the Prevention and treatment of Type 2 Diabetes Mellitus",also has some therapeutic ef-fect on NAFLD.The aim of this study was to investigate the expression of inflammatory factors in T2DM combined with NAFLD rats and the changes after metformin treatment,and to investigate the mechanism of inflammatory factors in T2DM combined with NAFLD and the protective effect of metformin on T2DM combined with NAFLD rats.Objective1.To study the insulin resistance,liver function damage,dyslipidemia,hepatic lesions and the expression of TNF-??IL-6 and CRP in T2DM combined with NAFLD rats.2.To study the changes of the above indexes in T2DM combined with NAFLD rats after the intervention of metformin.3.To analyze the mechanism of TNF-??IL-6 and CRP in T2DM with NAFLD and the protective effect of metformin on T2DM with NAFLD rats.MethodsEstablishment of 4 groups of rat models:? Blank control group(A group);?T2DM group(B group);?T2DM+NAFLD group(C group);?T2DM+NAFLD+metformin group(D group).The weight of the rat was weighed,after modeling,the rat serum and liver samples were collected,and then the wet weight of the rat liver was weighed.The leves of glutenyl transpeptidase(GGT),low density lipoprotein cholesterol(LDL-C),aspertate aminotransferase(AST),high density lipoprotein cholesterol(HDL-C),total cholesterol(TC),alanine aminotransferase(ALT),fasting blood glucose,insulin,CRP and triglyceride(TG)in serum were measured by automatic biochemical analyzer.The liver index of rats was calculated,the homeostasis model of assessment for insulin resistance index(HOMA-IR)and the insulin sensitivity index(ISI)were evaluated.Oil red O staining,Masson staining,HE staining,and reticular fiber staining were respectively applied to liver tissue,and then according to the results of HE staining,the liver fat content score and NAFLD activity score(NAS)were carried out respectively to evaluate the degree of fat gathering and fatty inflammation in livers.And the degree of fat gathering is further judged according to the results of oil red O staining.The results of Masson staining and reticular fiber staining were used to evaluate the liver fibrosis and the degree of fibrosis in liver tissue.The expression of TNF-a,IL-6 and CRP protein in liver tissue were detected by immuneohistochemical method.Results1.The liver index of rats in T2DM group and T2DM+NAFLD group was significantly higher than that in control group,and the liver index in T2DM group and metformin intervention group was lower than that in T2DM+NAFLD group(P<0.01).2.Insulin resistance in T2DM group and T2DM+NAFLD group was significantly higher than that in control group and it in T2DM group and T2DM+NAFLD+metformin group was better than that in T2DM+NAFLD group.The levels of HOMA-IR in group B and C were higher than that in group A,and which in group B and group D is lower than that in C group(P<0.01).The ISI valued in group B and C were significantly lower than that in group A,and which in group C is higher than that in group B and group D(P<0.05).3.Compared with the control group,T2DM group and T2DM+NAFLD group rats had significant liver injury and dyslipidemia,and the degree of that in T2DM group and metformin intervention group was lighter than that in T2DM+NAFLD group.The levels of ALT,AST,GGT,TG,TC and LDL-C in group B and group C were significantly higher than those in group A,and those in group B and group D were lower than those in group C(P<0.01).The level of HDL-C in group B and C were significantly lower than that in group A(P<0.01),and group B and group D had no significant difference with C group(P>0.05).4.The levels of TNF-?,IL-6 and CRP inT2DM group were(3.28±1.10)ng/ml,(4.29±0.48)ng/l and(3.12± 1.04)mg/1,which were higher than those in control group(The level of CRP to group A(p<0.05)and the rest of the group(P<0.01));The levels of TNF-a,IL-6 and CRP inT2DM and NAFLD group were respectively(12.36±2.78)ng/ml,(10.34±2.1)ng/1 and(3.64±0.94)mg/l,which are obviously higher control group(P<0.01 =.The levels of above inflammatory factors in metformin intervention group were(2.11 ±0.98)ng/ml,(2.67±0.92)ng/l,and(2.36±0.67)mg/1,which were lower than T2DM and NAFLD group(P<0.01).TNF-a and IL-6 in simple T2DM group were lower than those in T2DM and NAFLD group(P<0.01),but there was no difference in CRP between T2DM group and T2DM and NAFLD group(P>0.05).The immunohistochemical results showed that the expression of TNF-a,IL-6 and CRP protein was high in T2DM group andT2DM and NAFLD group.The expression level of above inflammatory factor protein in metformin intervention group was lower than that in T2DM and NAFLD group(P<0.01),while the expression of TNF-a and IL-6 in simple T2DM group was lower than those in T2DM and NAFLD group(P<0.01).While,there was no significant difference in CRP between metformin intervention group and T2DM and NAFLD group(P>0.05).5.Analyzing the results of liver tissue HE staining,oil red O staining and NAS score,there was no significant difference in liver fatty degree and inflammatory activity between T2DM group and control group(P>0.05)and the degree of hepatic fatty degeneration and inflammatory activity in T2DM+NAFLD group were higher than those in control group and T2DM group(P<0.01).The degree of above pathological changes in metformin intervention group was lower than that in T2DM+NAFLD group(P<0.01).6.Analyzing the results of Masson staining and reticular fiber staining,there was no significant difference in liver fibrosis between T2DM group and control group(P>0.05)and the degree of hepatic fibrosis in T2DM+NAFLD group were higher than that in control group and T2DM group(P<0.01),and the results in metformin intervention group were lower than them in T2DM+NAFLD group(P<0.01).Conclusion1.TNF-?,IL-6 and CRP were over-expressed in T2DM combined with NAFLD rats.2.Metformin can improve insulin resistance,dyslipidemia,liver function abnormality,chronic inflammation and liver fibrosis in T2DM with NAFLD rats.The mechanism may be related to decreasing the expression of inflammatory factors and improving chronic inflammatory state. |
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