TLR4 Knockout Improves The Obesity Induced By High-sugar High-fat Diet Without Increasing Risk Of Bone Loss

Objective:1.To observe the effect of TLR4 knockout on body weight and body composition of obese rats with high-sugar high-fat diet.2.To observe the effect of TLR4 knockout on femur proteomics in obese rats with high-sugar high fat diet.Methods:1.30 Male SD rats and 30 TLR4 knockout SD rats were randomly divided into control group?NC group,n=15?and high-sugar high-fat group?HSFD group,n=15?.The NC group was given normal diet for 16 weeks,and the HSFD group was fed with high-sugar high-fat diet for 16 weeks.2.Under the anesthetic condition,the bone mineral density and body composition of each group were determined by DEXA method.3.Rats abdominal aortic blood were collected from each group and the biochemical parameters?FPG,TCH,TG,LDL,ALT,AST,INS?and inflammatory factors?TNF-?,IL-1,IL-6?were measured.4.Femur was separated,the 10%femur homogenates were used to detect the levels of ALP,OCN and TRAP by ELISA.5.Femur was separated and used for protein quantification,GO enrichment,KEGG pathway,and PPI analysis by iTRAQ methods.Results:1.Body weight and body composition:In the SD-HSFD group,the body weight increased significantly compared with the SD-NC group.The weight gain of TLR4^KOO rats was significantly lower than that of control SD rats regardless of the normal diet or high-sugar high-fat diet,and the difference in body weight was statistically significant after the 4th week of feeding.Compared with the normal diet,the fat content was mainly increased whereas the bone mineral content and muscle content in the torso and whole body were decreased in high-sugar high-fat diet rats(including SD rats and TLR4^KO rats).However in the TLR4^KO-HSFD group,the bone mineral content and muscle content of the torso and whole body were increased whereas the fat content was decreased compared with the SD-HSFD group.2.Biochemical parameters:in the TLR4^KO-HSFD group,the insulin level was elevated,while the HOMA-IR was decreased compared with the SD-HSFD group.Comparing with the SD-NC group,the inflammatory factor levels were significantly increased in the SD-HSFD group,while the result was opposite in the TLR4^KOO rats.And there was no significant difference in FPG,TCH,TG,LDL,ALT and AST between groups.3.Bone metabolism:compared with SD rats,the BMD of torso,spine and whole body were decreased in TLR4^KO rats?including normal diet and high-sugar high-fat diet?,however,there was no significant difference in BMD after adjusting for body weight.4.Quantitative analysis of the proteins:TLR4 knockout reduced the increased expression level of 10 proteins and upregulated the expression level of 11 proteins in Femur of rats by high-sugar high-fat diet.5.GO analysis:the main biological processes of TLR4^KO in HSFD rats were localization,establishment of localization,transport,immune response,and gene expression.6.KEGG analysis:TLR4^KO-HSFD vs SD-HSFD groups,the pathways involved in differential proteins mainly include Ferroptosis,Necroptosis,Mineral absorption,Oxidative phosphorylation,Thermogenesis,Parkinson's disease,Huntington's disease,Alzheimer's disease,and Non-alcoholic fatty disease?NAFLD?.7.PPI analysis:the interaction between proteins were ferritin and ferritin light chain 1,which were involved in the pathways of mineral absorption and ferroptosis.Conclusions:TLR4 knockout significantly improved the obesity and body composition disorder induced by high-sugar high-fat diet in rats and did not cause bone loss,and the underlying mechanism might be involved in regulating the interaction of ferritin and ferritin light chain 1 which were involved in the pathways of mineral absorption and ferroptosis.