Effect Of TLR4 On Glycolipid Toxicity Induced Osteoblast Mitochondrial Dysfunction And Its Relationship With NLRP3-MAVS

Objective:1.To observe the effects of high glucose and palmitic acid on mitochondrial function of osteoblasts and the effect of regulating TLR4 expression on it.2.To explore the effect of TLR4 on expression of NLRP3 and MAVS proteins in glycolipid toxic osteoblasts.Methods:1.Experimental design:In the experiment,MC3T3-E1 subclone 14 was selected as the resesrch objiect.The experiment would be divided into two parts:The first part is the effects of high glucose and palmitic acid on mitochondrial function of osteoblasts and the effect of regulating TLR4 expression on it.The second part explores the effect of TLR4 on expression of NLRP3 and MAVS proteins in glycolipid toxic osteoblasts.2.Evaluation of osteogenesis induction models:After osteoblasts were cultured in osteogenic induction medium for 14 days,they were stained with alizarin red staining solution,and the mineralized nodules secreted by osteoblasts can be seen under the microscope.3.Establishment of lentiviral transfection model:Dilute the lentiviral containing vector,TLR4-si RNA and TLR4-OE to 1×10~8TU/ml with the enhanced infection solution,the osteoblasts were infected with the complete medium containing the lentiviral vector.,incubate for 12h,replaced with the complete medium,and analyze TLR4 expression by Western Blot.4.Osteoblast mitochondrial function test:JC-1 staining method and ATP detection kit were used to detect the effects of high glucose and palmitic acid intervention and regulation of TLR4 expression on mitochondrial membrane potential and ATP content of osteoblasts.5.Detection of osteoblast differentiation proteins and apoptotic proteins:The expressions of osteoblasts differentiation proteins ALP and OCN were detected by ELISA,and the expressions of RUNX2,Bcl-2,and Bax proteins were detected by Western Blot.6.Signal protein detection:Western Blot was used to detect the effects of high glucose and palmitic acid intervention on the expression of TLR4,NLRP3,and MAVS proteins and the effect of regulating TLR4 expression on the expression of NLRP3,MAVS proteins.7.Proteins interaction:Immunofluorescencec and co-immunoprecipitation to explore the effect of inhibiting TLR4 activity on the regulation of NLRP3-MAVS expression by high glucose and palmitic acid.Results:1.Effect of high glucose and palmitic acid intervention on mitochondrial function and differentiation function of osteoblasts.JC-1 staining results showed that compared with the Control group,HG+PA group osteoblastic mitochondrial membrane potentials and ATP content decreased.Western Blot results showed that the expression of proapoptotic protein Bax increased and the expression of anti-apoptotic protein Bc-2 decreased,the expression of osteogenic differentiation proteins(ALP,OCN,RUNX2)decreased.The results suggest that high glucose and palmitic acid can lead to mitochondrial dysfunction of osteoblasts and promotes osteoblasts apoptosis,and inhibits osteogenic differentiation.2.Effects of regulating TLR4 expression on mitochondrial function and osteogenic differentiation of glycolipid toxic osteoblasts.Compared with the HG+PA group,HG+PA+TLR4-si RNA group increased the expression of osteoblasts differentiation proteins(ALP,OCN,RUNX2),osteoblast mitochondrial membrane potential and ATP content and anti-apoptotic protein Bcl-2 and the expression of pro-apoptotic protein Bax expression decreased.While HG+PA+TLR4-OE group decreased the expression of osteoblasts differentiation proteins and osteoblast mitochondrial membrane potential and ATP content and anti-apoptotic protein Bcl-2,the expression of pro-apoptotic protein Bax expression increased.The results suggest that regulating the expression level of TLR4 can affects the mitochondrial function of osteoblasts and the influence of osteogenic differentiation caused by glycolipid toxicity.3.Effects of regulating the expression of TLR4 on the expression of NLRP3 and MAVS proteins in glycolipid toxic osteoblasts.Intervention of high glucose and palmitic acid level can increase the expression of osteoblast chronic inflammation signaling proteins(TLR4,NLRP3,MAVS).Silencing TLR4 can inhibit the expression of NLRP3 and MAVS protein induced by high glucose and palmitic acid.Overexpression of TLR4 can cooperate with the increase of NLRP3and MAVS protein expression caused by high glucose and palmitic acid.The results suggest that regulation of TLR4 expression level can affect the regulation of NLRP3and MAVS proteins of osteoblasts by glycolipid toxicity.4.Effect of inhibiting TLR4 activity on the regulation of NLRP3-MAVS expression by high glucose and palmitic acid.Confocal laser scanning showed that compared with the Control group,the fluorescence intensity of NLRP3 and MAVS in the bone cytoplasm increased by HG+PA group,Compared with HG+PA group,the fluorescence intensity of NLRP3 and MAVS in the bone cytoplasm of TLR4 inhibitor(CLI-095)is weakened.Further immunoprecipitation showed that MAVS protein could not be detected in the experimental group without NLRP3 antibody,while MAVS protein was detected in the experimental group with NLRP3 antibody,Compared with the Control group,MAVS protein increased in the HG+PA group,and decreased in the TLR4 inhibitor(CLI-095)group compared with the HG+PA group.The results suggest that there is an interaction between NLRP3 and MAVS,and inhibition of TLR4 activity affects the expression of NLRP3-MAVS in glycolipid toxicity osteoblasts.Conclusions:TLR4 can mediate the osteogenic differentiation and mitochondrial dysfunction of osteoblasts caused by high glucose and palmitic acid,the above-mentioned mediated effect of TLR4 may be related to the regulation of the interaction between NLRP3-MAVS.