The Effects And Mechanism Of TLR4 Gene Knockout In Adipose Tissue Inflammation And Apoptosis In High-fat Diet-induced Obese Mice

ObjectiveThe study aims at investigating the effects of toll like receptor 4(TLR4)gene knock in adipose tissue inflammation and apoptosis in high-fat diet-induced obese mice,and its possible mechanism.MethodsFour-week-old male C57BL/6J(n=24)and TLR4 gene knockout mice(n=24)were randomly divided into normal control group(NC,n=8),obese group(OB,n=16),TLR4 gene knockout group(TK,n=8),TLR4 gene knockout obese group(TO,n=16).They were fed with normal or high-fat diets.After 16 weeks,mice whose weights surpassed the upper limit of normal-feeding mice and no diabetes were included into the obesity model and the following experiments were subsequently performed.1、Serum was collected to detect the level of a series of biochemical indexes including fasting blood glucose(FPG),total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL),high density lipoprotein(HDL)and free fatty acid(FFA)among groups.2、Hematoxylin-eosin staining(HE)was used to observe the morphological changes of adipose tissue cells among groups.3、Western blotting was performed to determine the expression of TLR4,myeloid differentiation factor 88(My D88),AMP-activated protein kinase α(AMPKα),phosphorylated AMP-activated protein kinase α(p-AMPKα),B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax).4、ELISA assay was used to detect the level of series of inflammatory chemokines including monocyte chemotactic protein 1(MCP-1),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-10(IL-10).Results1、Compared with the NC group,the serum level of FPG,TC,TG,LDL,and FFA were significantly increased(P < 0.01),the level of HDL was significantly decreased(P < 0.01);Compared with the OB group,the serum level of FPG,TC,TG,LDL,and FFA were significantly decreased(P < 0.01),the level of HDL level was significantly increased(P < 0.05).2、HE staining results showed that the adipose tissue cells were arranged closely,the size and morphology of adipocytes were relatively uniform,they were polygonal or circular in the NC group.Compared with the NC group,the size of the adipocytes was obvious increased in the OB group and TO group,exhibiting the irregular shapes,large vacuoles,thinner cell walls,and broader intercellular spaces;Compared with the OB group,the size of adipocytes was markedly reduced in the TK group and TO group,and the intercellular space is narrowed,showing more complete cell wall and smaller cell gap.3、Western blotting results showed that there were not significantly changes in the expression of the total AMPKα among groups.Compared with the NC group,the expression of TLR4,Myd88 and Bax were significantly increased(P < 0.01),and the expression of p-AMPKα and Bcl-2 were significantly reduced(P < 0.05)in epididymal adipose tissue in the OB group,however,the expression of Myd88 and Bax were significantly reduced(P < 0.01,P < 0.05),and the expression of p-AMPKα and Bcl-2 were significantly increased(P < 0.05)in the TK group;Compared with the OB group,the expression of TLR4,My D88 and Bax were significantly reduced(P < 0.01),and the expression of p-AMPKα and Bcl-2 protein were significantly increased(P <0.05)in the epididymal adipose tissue in the TO group.The immunohistochemistry results showed that p-AMPK was expressed in the epididymal adipose tissue of mice among groups.Compared with the NC group,the expression of p-AMPK in the OB group was significantly reduced,and the expression of p-AMPK was significantly increased in the TK group;Compared with the OB group,the expression of p-AMPK was significantly increased in the TK group,the result were consistent with the changes observed in western blotting.4、ELISA assay results revealed that compared with the NC group,the serum level of MCP-1,IL-6 and TNF-α were significantly increased(P < 0.01),and the level of IL-10 was significantly decreased(P < 0.05)in OB group,the serum level of MCP-1,IL-6 and TNF-α were significantly decreased(P < 0.01,P < 0.05),and the level of IL-10 was significantly increased(P < 0.01)in the TK group;Compared with the OB group,the serum level of MCP-1,IL-6 and TNF-αwere significantly decreased(P < 0.01),and the level of IL-10 was significantly increased(P < 0.01)in the TO group.ConclusionsTLR4 gene knockdown alleviates high-fat diet-induced adipose tissue chronic inflammation and apoptosis through My D88 dependent AMPK in obese mice.