Expression And Implication Of TLR-4 And NF-KB In Rat Liver With Type 2 Diabetes

Objective:Toll like receptor 4(TLR4) is the key receptor of lipopolysaccharide (LPS).It links the innate immunity and the adaptive immunity as a member of the toll protein family. TLR4 mainly expressed in myeloid-derived cells. Its start-up of intracellular signal transduction plays an important role in the process of liver injury occurrence and development. Nuclear factor-kappa B (NF-KB) is widely present in mammalian cells as an important transcription factor. It can specific bind with a variety of cell gene promoter and enhancer sequence. Through the regulation of several important cytokines,adhesion molecules and chemokine expression,it plays an important role in the body's immune response,inflammation and cell growth regulation. Its activation is also involved in cell proliferation and apoptosis. This study was designed to understand the expression and implication of TLR4 and NF-KB in liver of type 2 diabetes rat model.Methods:40 healthy SD male rats,divided into four groups randomly:Group NC(normal control group,10);Group IR (insulin resistant group 10);Group DM4w (diabetes mellitus 4 weeks group,10);Group DM8w (diabetes mellitus 8 weeks group,10). All of them were fed with the regular diet. The rats of group IR,DM4w and DM8w were given intragastric administration with fat milk lml/100mg weight/ day for 7 weeks,the similar dosage of water was given to NC group rats. At the 7th weekend,the level of plasma glucose and insulin was detected,the results showed that the level of plasma insulin in the fat milk intragastric administration groups (IR, DM4w and DM8w group) were higher than normal control group. On the next morning,the weight of the rats were measured,and STZ was injected into the vena caudalis of the rats (30mg/kg) 3 days later,plasma glucose was measured. If the result of the plasma glucose was higher than 16.7mmol/L,it was succeed to establishing the type 2 diabetes mellitus rat model.After that,the rats were continuously given the regular diet and intragastric administration with fat milk (0.5ml/100mg weight/day).The serum insulin,lipid,FPG,GSP,FFA,ALT,AST were tested. The rats'liver thin slice was used to test the insulin receptor by ELISA. The expression of TLR4 mRNA on the liver of the rats was tested by RT-PCR. The expression of NF-KB protein was tested by immunohistochemistry. All the data were analyzed by using the SPSS 11.5 software.Result:After 7 weeks of the diets enriched with fat and sugar,contrast to control group,the serum insulin of groups which were given intragastric administration with fat milk were significant higher(P<0.05),the ISI of group IR,DM4w and DM8w were lower(P<0.05),the HOMA-IR of group IR,DM4w and DM8w were significant higher(P<0.05).Type 2 diabetes mellitus model in group DM4w and DM8w induced by STZ were all succeed. At the end of experiment,The serum ALT,AST,GSP of groups from NC and IR to groups DM4w and DM8w were progressively increased (P<0.05).The serum FPG,TG,TC of group DM4w and DM8w were significant higher than group NC and IR(P<0.05).Compared with the NC group,FINS,LDL-c,FFA of the other 3 groups were significantly increased while HDL-c was significantly decrease(P<0.05).Contrast to group NC,the insulin receptor level on rats liver cell membrane of group IR,DM4w,DM8w elevated significantly (P<0.05).With the progress of type 2 diabetes mellitus,the expression of TLR4 mRNA and NF-KB protein in liver tissue increased significantly(P<0.05).Pearson correlation analysis showed that the expression of TLR4 mRNA in hepatic tissue was correlate positively with NF-KB protein(r=0.706,P<0.01),ALT(r=0.974,P<0.01)and AST(r=0.950, P<0.01).And NF-KB protein was positively correlate with ALT(r=0.657,P<0.01) and AST(r=0.646,P<0.01)Conclusion:(1)Using intragastric administration with fat milk (high-glucose-high-fat diet) plus low dose STZ by vena caudalis,the type 2 diabetes mellitus SD rat model can be induced successfully, the method is reliable, and all the STZ injected SD rats feed with fat milk developed diabetes;(2)Because of the number of insulin receptor on insulin resistant and type 2 diabetes mellitus rats liver cell membrane is increased,possibly related to the decrease of the insulin sensitivity and effect;(3) With the progress of type 2 diabetes mellitus,the expression of TLR4 and NF-KB in liver tissue increased significantly,and they are correlate positively with liver damage. So the inflammatory reaction of TLR4/NF-κB signaling pathway plays an important role in the occurrence and development of diabetes mellitus and in the steatosis of hepatic tissue.