The Role Of TLR4/FTO In The Differentiation Of Osteoclasts Induced By High Glucose And Palmitate

Objective:1.To study the effect of TLR4 knockout on bone mass,body composition and m6A modifying enzymes in diabetic rats2.To explore whether TLR4 affects the differentiation of osteoclasts by regulating FTO-mediated high glucose and palmitate.Methods:1.Select female SPF SD rats,build a diabetic rat model through a high-sugar and high-fat diet with STZ injection,and construct a TLR4 knockout SD rat.Divided into NC group,DM group,TLR4^KONC group,TLR4^KODM group.the diabetic mice were modeled.The control group and the experimental group were observed for 6 weeks.Observe the effects of TLR4 gene knockout on bone density,body composition,blood lipids,bone metabolism indexes and m6A methylation modification enzymes in diabetic SD rats.2.Construct a model of osteoclast intervention with high glucose and palmitate.In vitro cell experiment uses osteoclast precursor cell RAW264.7 cell line.Observe the effect of inhibiting TLR4 activity on the intervention of high glucose and palmitate on osteoclast differentiation and the expression level of FTO.Western blot was used to detect the effect of TLR4 inhibitor CLI-095.TRAP staining was used to detect the number of osteoclasts;phalloidin fluorescent staining was used to detect the fluorescence intensity of the osteoclast skeleton F-actin ring;real-time quantitative PCR and Western blot were used to detect osteoclast differentiation-related genes（MMP-9,NFATc1,TRAP）and FTO protein expression.3.The effect of regulating FTO expression on the intervention of high glucose and palmitate on osteoclast differentiation and its relationship with TLR4.Construct a knockdown or overexpression FTO RAW264.7 cell line,and then intervene the cells with high glucose and palmitate+osteoclast inducer for 5 days.The differentiation of osteoclasts was detected by the same method as above;The expression of osteoclast differentiation genes m RNA、protein and the expression of TLR4 were detected by real-time quantitative PCR and Western blot.Results:1.TLR4 knockout regulates m6A modification enzyme to improve body composition disorder and bone mineral density in diabetic rats.We found that knockout of TLR4 did not have a significant effect on the weight and length of SD rats.The blood glucose and insulin levels of each group of rats confirmed that we successfully constructed a diabetic SD rat model.It was also found that knockout of TLR4 did not significantly affect blood glucose and insulin in SD rats.Detect the blood lipid levels of rats in each group.Diabetes will lead to increased blood lipids in rats.The bone metabolism indexes of rats in each group were tested,and it was found that diabetes would lead to the decrease of bone formation indexes in rats,the increase of bone resorption indexes,and the disorder of bone metabolism.Through dual-energy X-ray absorptiometry,it was found that the bone mineral density of the DM group decreased compared with the NC group.Compared with the DM group,BMD in the TLR4^KODM group was significantly increased.The body composition results indicated that compared with the NC group,the DM group had lower muscle and bone mineral salt content,while the fat mass increased.Compared with the DM group,the muscle and bone mineral salt content of the TLR4^KODM group increased,and the fat mass decreased.Lumbar 3（L3）Micro-CT results showed that compared with the DM group,the L3 volume bone density of the TLR4^KODM group increased.We detected m6A modified enzymes by real-time quantitative PCR.We found that compared with NC group,the FTO level of DM group decreased,while TLR4 gene knockout could up-regulate the FTO level of diabetic rats,while the other m6A modified enzymes did not have similar phenomena.The results suggest that knocking out TLR4 may regulate FTO to improve body composition disorder and bone mineral density in diabetic rats.2.Inhibition of TLR4 activity can attenuate osteoclast differentiation induced by high glucose and palmitate and up-regulate FTO expression.We found that compared with the NC group,the number of osteoclasts in the HG+PA group was significantly increased,and the fluorescence intensity of the osteoclast skeleton F-actin ring was stronger.Compared with the HG+PA group,the osteoclasts in the HG+PA+CLI-095 group the number decreases and the fluorescence intensity of the osteoclast skeleton F-actin ring decreases.Real-time quantitative PCR and Western blot results showed that high glucose and palmitate intervention promoted the m RNA and protein expression of osteoclast differentiation genes（MMP-9,NFATc1,TRAP）;compared with the HG+PA group,the HG+PA+CLI-095 group broke Osteocyte differentiation genes（MMP-9,NFATc1,TRAP）m RNA and protein expression decreased;the above results indicate that inhibiting the activity of TLR4 can weaken the osteoclast differentiation induced by high glucose and palmitate.Western blot found that high glucose and palmitate intervention can inhibit FTO protein expression;and inhibition of TLR4 activity can up-regulate FTO protein expression in RAW264.7 cells with high glucose and palmitate intervention;The results showed that inhibiting TLR4activity can attenuate the osteoclast differentiation induced by high glucose and palmitate and up-regulate FTO expression.3.Regulating FTO expression can affect osteoclast differentiation and TLR4expression induced by high glucose and palmitateWe found that compared with the HG+PA group,the number of osteoclasts increased significantly,the fluorescence intensity of the osteoclast skeleton F-actin ring was enhanced,and increased m RNA and protein expression of osteoclast differentiation genes（MMP-9,NFATc1,TRAP）in FTO^KD+HG+PA group;The number of osteoclasts in the FTO^OE+HG+PA group was significantly reduced,the fluorescence intensity of the F-actin ring of the osteoclast cytoskeleton decreased,and the m RNA and protein expression of osteoclast differentiation genes（MMP-9、NFATc1、TRAP）decreased.Compared with the HG+PA group,there was no significant difference in TLR4 protein expression between the FTO^KD+HG+PA group and the FTO^OE+HG+PA group.It is suggested that the expression level of FTO affects the differentiation of osteoclasts intervened by high glucose and palmitate.conclusions:High glucose and palmitate may promote osteoclast differentiation of RAW264.7 cells by activating TLR4 and inhibiting FTO.