The Role And Mechanism Of TLR4/RIAM In Osteogenic Differentiation Of Glycolipid Toxicity

Objective1.In vivo and in vitro studies were conducted to observe whether TLR4 and RIAM were involved in glycolipid-mediated bone metabolic damage.2.To investigate the effects of regulating TLR4 on the osteogenic differentiation and RIAM level under the condition of glycolipid toxicity.3.To analyze whether TLR4 mediates dysdifferentiation of osteoblasts induced by glycolipid toxicity through regulating RIAM-NF-?B nuclear transposition.Methods1.SPF grade SD rats were selected and TLR4 gene knockout rats were constructed,both induced by high-sugar and high-fat diet,then modeled T2 DM with a small dose of STZ treatment.The effects of T2 DM and TLR4 knockout on bone mineral density and RIAM expression in rats were observed after 16 weeks of high-glucose and high-fat intervention.2.The chronic glycolipid toxicity model was established by using the osteoblast precursor MC3T3-E1 cell line with high glucose(25m M)and high lipid(100 KB)induced differentiation for 14 days.Then to observe the effects of glycolipid toxicity on osteoblast differentiation and the expression of TLR4 and RIAM.3.MC3T3-E1 cells were transfected with lentiviral vector,and the TLR4 gene was knocked down or overexpressed,followed by intervention with high glucose and fat,to investigate the effect of regulating TLR4 on osteoblast differentiation and RIAM level induced by glycolipid toxicity.4.Stable transfection of RIAM gene knockdown and overexpression of MC3T3-E1 cell lines were prepared,respectively,treated with high glucose and fat,and further interfered with TLR4 agonist or inhibitor,to explore the effect of regulation of RIAM on osteogenic differentiation induced by glycolipid toxicity and its relationship with metabolic inflammation.The mutual regulation and interaction between NF-?B and RIAM were investigated by stimulating or inhibiting NF-?B and immunoprecipitation technique.5.Experimental techniques: Bone mineral density was determined by dual-energy X-ray absorptiometry.The expression of TLR4,RIAM,NF-?B and RUNX2 proteins were detected by Western Blot.ELISA was used to detect secreted proteins such as ALP and OCN.m RNA expression of RUNX2,ALP,OCN and other osteogenic differentiation indicators were detected by RT-PCR.The intracellular localization of RIAM protein under different treatment conditions was observed by immunofluorescence technique.The interaction between NF-?B and RIAM was verified by immunoprecipitation.Results1.The results of animal experiments showed that the expression of TLR4,RIAM and NF-?B in bone tissues of T2 DM rats were significantly increased and BMD was decreased(whole body,pelvis,spine,trunk and skull,P<0.05)compared with normal SD rats.Compared with SD rats with T2 DM,the expression of TLR4,RIAM and NF-?B in T2 DM rats with TLR4 knockout were all down-regulated,and BMD in the whole body,pelvis,spine,trunk and skull was increased,with statistically significant differences(P < 0.05).2.The results of in vitro cell experiments showed that the nuclear translocation of RIAM and NF-?B in MC3T3-E1 cells was caused by the intervention of high glucose and fat.The levels of TLR4,intracellular RIAM and NF-?B proteins increased,while matrix calcification nodules were fewer,and m RNA and protein expressions of RUNX2,ALP,and OCN were significantly decreased(P<0.05),which suggested that high glucose and fat inhibited the osteogenic ability of MC3T3-E1 cells.3.Western Blot,ELISA and RT-PCR results showed that,compared with the high-glucose and lipid groups,the intranuclear levels of RIAM and NF-?B in osteoblasts in the TLR4 knockdown+high-glucose and lipid groups were down-regulated,and the expressions of RUNX2,ALP and OCN were increased(P<0.05),which improved the disorder of osteoblast differentiation induced by high glucose and lipid.4.The interaction between RIAM and NF-?B increased with the intervention of high glucose and fat.Down-regulating the expression of RIAM reduces NF-?B activation and promotes osteogenic differentiation.Levels of NF-?B in the nucleus of osteoblasts in the RIAM knockdown + high-glucose and lipid groups were decreased,and m RNA and protein expressions of RUNX2,ALP and OCN were increased compared to high-sugar and lipid groups(P<0.05).Meanwhile,RIAM knockdown also improved the osteogenic differentiation disorder mediated by TLR4 activation.Compared with the TLR4 agonist group,the level of NF-?B in osteoblastic nuclei in the RIAM knockdown+ TLR4 agonist group was decreased,while the expression of RUNX2,ALP and OCN proteins was up-regulated(P < 0.05).On the contrary,high expression of RIAM increased nuclear translocation of NF-?B,promoted osteoblast damage induced by glycolipid toxicity,and weakened the improvement effect of TLR4 inhibition on osteogenic differentiation disorder induced by glycolipid toxicity.ConclusionTLR4 mediates osteoblast differentiation disorders induced by glycolipid toxicity by regulating RIAM activity,which may be related to the regulation of RIAM interactions with NF-?B and the further promotion of NF-?B nuclear transposition.