The Effect Of Metformin To TLR4/MYD88/IL-23 Signaling Pathway And Its Mechanism On Skeletal Muscle In Diabetic Rats With STZ

1 Background and Objectives In recent years,the process of urbanization and aging has accelerated.At the same time,with the change of lifestyle,the number of obese and overweight people has increased,and the incidence show that type 2 diabetes has been increasing year by year.As the largest country in the world in diabetes,China has a total of 109.6million people in 2015 and 1.3 million people died of diabetes and its complications.Therefore,prevention and treatment of type 2 diabetes is an important problem to be solved in human health.As a chronic metabolic disease,type 2 diabetes mellitus(T2DM)has complicated pathogenesis involving many factors such as inheritance,immune inflammation,oxidative stress and microorganism.Eventually,they all develop into insulin resistance and decreased islet?cell function,cause metabolism disorders of glucose and lipid,which result in a series of complications.Insulin resistance is an early pathological change in type 2 diabetes.,obesity is closely related to the occurrence of insulin resistance,dyslipidemia,atherosclerosis and other metabolic syndrome,and chronic low-grade inflammatory is considered to be the key intermediate links connected with obesity and type 2 diabetes.Up to 40% of the cells in adipose tissue are macrophages,and highly activated inflammatory pathways are observed in adipose tissue-derived macrophages(ATM),leading to over-secretion of various inflammatory factors such as TNF-a and IL-1?,those proinflammatory cytokines can act locally through a paracrine manner,but also enter the circulation to act on other insulin target organs to reduce insulin sensitivity,such as liver,muscle,pancreas,hypothalamus,etc.The activation of inflammatory signaling pathway and the secretion of inflammatory cytokines are the key joints that lead to insulin resistance.TLR4/My D88 signaling pathway is a key pathway regulating innate immunity and adaptive immune response in vivo,which plays an important role in inflammatory diseases and autoimmune diseases.In recent years,it has been proved that the signaling pathway can affect insulin signaling and plays an important role in the development of insulin resistance.In addition,it has been demonstrated that the TLR4 / My D88 signaling pathway partially synthesizes IL-23 through the activation of NF?B,thus IL-23 can be considered as a downstream inflammatory factor in the TLR4/My D88 pathway.The current study is more about the relationship between IL-23 and type 1 diabetes.It has been shown that IL-23 is involved in the islet immune response,leading to the development of type 1 diabetes.However the relationship between IL-23 and type 2 diabetes is poorly understood.Therefore,this study will explore the relationship between the type 2 diabetes mellitus and TLR4 /My D88/IL-23 signaling pathway.Metformin is the first-line treatment of type 2 diabetes,its hypoglycemic mechanism mainly lies in the inhibition of hepatic glycogen output and improve the sensitivity of surrounding organs and tissues to insulin utilization,while the molecular mechanism of metformin is divided into AMP-dependent protein kinase(AMPK)-dependent pathway and AMPK-independent pathway.This experiment will focus on TLR4/My D88/IL-23 signaling pathway,exploring the anti-inflammatory mechanism of metformin.2 Materials and Methods2.1 Grouping and feeding methods40 SPF SD male rats(89.62±7.42g)were randomly divided into 4 groups after7 days of adaptive feeding: normal diet group(NC group),model group(DM group),modeling and injected with insulin(DM+INS group),modeling and fed with metformin(DM+MET group).The model was built with high-fat high-sugar diet and intraperitoneal injection of STZ(30mg/kg).MET was intragastrically administered at 200mg/kg.Rats without MET were given intragastric administration of normal saline,insulin was administered with insulin glargine,and the dose was used according to the experimental admiration.Rats were sacrificed at the end of the12 th week.The abdominal aorta was used to take blood in a fasting state.The upper serum was collected by centrifugation,and the skeletal muscle tissue was isolated and stored in a refrigerator at-80?.2.2 Observation indicators Fasting blood glucose(FBG,unit mmol/L)was measured by automatic biochemical analyzer.The fasting blood insulin(FINS,unit m IU/L)was measured by electrochemiluminescence.The insulin resistance index HOMA-IR was calculated as FBG × FINS / 22.5.The level of IL-23 in serum was measured by ELISA.Western bloting detect the levels of AMPK,TLR4,My D88 and NF?B in muscle tissue.RT-PCR detect levels of TLR4-m RNA and My D88 m RNA in muscle Tissue.Immunofluorescence observe the expression of IL-23 in muscle tissue.3 Results3.1 HOMA-IR Serum HOMA-IR level between different groups was statistically significant(F=586.168,P<0.05).Compared with NC group,the HOMA-IR of DM group increased(P<0.05).Compared with DM group,the HOMA-IR of DM+MET group decreased(P<0.05).There was no significant difference between DM+INS group and DM group(P<0.05).Compared with DM+INS group,the HOMA-IR of DM+MET group decreased(P<0.05). 3.2 Serum IL-23 level The mean serum level of IL-23 in each group were statistically significant(F=37.941,P<0.05).Compared with NC group,the expression of IL-23 in DM group increased(P<0.05).Compared with DM group,the expression of IL-23 in DM+INS group decreased(P<0.05),the expression of IL-23 in DM+MET group decreased obviously(P<0.05).Compared with DM+INS group,IL-23 in DM+MET group was lower(P<0.05).3.3 Correlation analysis between HOMA-IR and IL-23 in rats In DM group,HOMA-IR and serum IL-23 was positively correlated,r=0.716,P<0.05;In DM+MET group,HOMA-IR and serum IL-23 was positively correlated,r =0.725,P<0.05.3.4 Expression of My D88-m RNA and TLR4-m RNA in muscle tissue of rats The mean level of TLR4-m RNA in each group were statistically significant(F=37.458,P<0.05).Compared with NC group,the expression of TLR4-m RNA in DM group increased(P<0.05).Compared with DM group,the expression of TLR4-m RNA in DM+INS group decreased(P<0.05),the expression of TLR4-m RNA in DM+MET group decreased(P<0.05).Compared with DM+INS group,TLR4-m RNA in DM+MET group was lower(P<0.05).The My D88-m RNA expression in muscle in each group had statistical significance(F=45.599,P<0.05).Compared with NC group,the expression of My D88-m RNA in DM group increased(P<0.05).Compared with DM group,the expression of My D88-m RNA in DM+INS group decreased(P<0.05),the expression of My D88-m RNA in DM+MET group decreased(P<0.05).Compared with DM+INS group,My D88-m RNA in DM+MET group was lower(P<0.05).3.5AMPK expression in muscle tissue.The expression of AMPK in muscle tissue of each group was statistically significant(F=69.920,P<0.05).AMPK expression in DM+MET group was higher than that in NC group,DM group and DM+INS group(P<0.05).There was no significant difference in AMPK expression between NC group,DM group and DM+INS group(P>0.05). 3.6TLR4?Myd88 and NFk B level in muscle tissue.TLR4 in muscle tissue in each group differed significantly(F=94.836,P<0.05).Compared with NC group,the expression of TLR4 in DM group increased(P<0.05).Compared with DM group,the expression of TLR4 in DM+INS group decreased(P<0.05),TLR4 in DM+MET group decreased(P<0.05).Compared with DM+INS group,TLR4 in DM+MET group was lower(P<0.05).My D88 expression in muscle tissue differed statistically(F=131.266,P<0.05).Compared with NC group,My D88 in DM group increased(P<0.05).Compared with DM group,My D88 in DM+INS group decreased(P<0.05),the expression of My D88 in DM+MET group decreased(P<0.05).Compared with DM+INS group,My D88 in DM+MET group was lower(P<0.05).NFk B in muscle tissue was significantly different(F=176.829,P<0.05);Compared with NC group,NFk B in DM group increased(P<0.05).Compared with DM group,NFk B in DM+INS group decreased(P<0.05),the expression of NFk B in DM+MET group decreased(P<0.05).Compared with DM+INS group,NFk B in DM+MET group was lower(P<0.05).3.7 Immunofluorescence observed IL-23 expression in muscle tissue of rats There was a significant difference in the expression of IL-23 between the muscles of rats in each group(F=37.274,P<0.05).Compared with NC group,IL-23 in skeletal muscle of DM group increased(P<0.05).Compared with DM group,the expression of IL-23 in skeletal muscle of DM+INS group decreased(P<0.05),IL-23 in skeletal muscle of DM+MET group decreased(P<0.05).Compared with DM+INS group,IL-23 in skeletal muscle of DM+MET group was lower(P<0.05).4 Conclusion4.1 The expression of IL-23 in serum and skeletal muscle of type 2 diabetic rats increased.4.2 Inflammatory factor IL-23 is associated with insulin resistance in type 2 diabetic rats.4.3 TLR4/My D88 signaling pathway in skeletal muscle of type 2 diabetic rats was activated.4.4 Metformin inhibits the TLR4/My D88 signaling pathway in skeletal muscle by activating AMPK and further inhibits the expression of the downstream inflammatory factor IL-23,directly or indirectly improving insulin resistance.