| ObjectiveThis study aims to explore the regulatory relationship between miR-675 and TBK1 in OA,aiming at the mechanism of autophagy dysfunction in chondrocytes during the pathogenesis of osteoarthritis.Furthermore,the molecular mechanism of miR-675 overexpression or inhibition of TBK1 in chondrocyte autophagy was explored,and the molecular mechanism of the effect of TBK1 autophagy on chondrocyte function was further verified by Longbie capsule through miR-675 targeting TBK1.MethodsBased on other scholars and our previous studies,this study aims to explore the mechanism of autophagy dysregulation of chondrocytes in the pathogenesis of OA.Based on clinical tissue,cell and animal experiments,histochemical and pathological methods are adopted to explore the regulatory relationship between miR-675 and TBK1 in OA.The molecular mechanism of miR-675 overexpression or inhibition of TBK1 in chondrocyte autophagy was demonstrated by cellular and molecular biological techniques.Furthermore,Longbiie capsule was used to intervene chondrocytes in vitro to verify the biological effects and significance of Longbiie capsule on chondrocyte autophagy and other functions.Results1.Expression of miR-675 and TBK1 in articular cartilage of OA rats①OARSI score of articular cartilage pathology in SD rat OA model:Articular cavity injection collagen II proteinase to construct the SD rat model of OA by HE staining and his red-O-solid green OARSI rating after dyeing,the results showed that as the building time increasing,the model of OA articular cartilage pathological OARSI score significantly higher than the blank control group,difference has statistical significance(P<0.05).② Expression of miR-675 in articular cartilage of SD rat OA model:The expression level of miR-675 in articular cartilage of OA rats at 4,8 and 12 weeks after modeling was significantly reduced compared with the blank control group,with statistical significance(P<0.01),and with the extension of modeling time,the decrease of miR-675 expression was more obvious.③Expression of TBK1 in articular cartilage of SD rat OA model:The expression level of TBK1mRNA in articular cartilage tissue of OA rats at 4,8 and 12 weeks after modeling was significantly increased compared with that of blank control group(P<0.01),and the increase of TBK1mRNA was more obvious with the extension of modeling time.2.The mechanism of miR-675 targeting TBK1 affecting the function of OA chondrocytes①Effects of miR-675 mimics and miR-675 inhibitor on expression of miR-675 in chondrocytes:After transfection with miR-675 mimic and miR-675 inhibitor in human OA primary chondrocytes,the expression level of miR-675 in descending cells and miR-675 inhibitor could be up-regulated,respectively,with statistical significance compared with blank transfection control group(P<0.001).②Effects of miR-675 on proliferation of OA chondrocytes:After the chondrocytes were treated with miR-675mimic,the proliferation ability of chondrocytes was enhanced compared with the blank transfection group,and the difference was statistically significant(P<0.001).However,after the expression of miR-675 was inhibited,the proliferation ability of chondrocytes was significantly decreased compared with the blank transfection group,and the difference was statistically significant(P<0.01).③Effects of miR-675 on matrix metabolism of OA chondrocytes:Compared with the idling slow virus control group,miR-675 can increase Collagen in cartilage cells Ⅱ protein expression and the expression of MMP-9 protein.④ Effects of miR-675 on apoptosis of OA chondrocytes:After treated with miR-675mimic,the apoptosis rate of chondrocytes was lower than that of idle lentivirus control group,and the difference was statistically significant(P<0.001).However,after the expression of miR-675 was inhibited,the apoptosis rate of chondrocytes was significantly higher than that of idle lentivirus control group,with statistical significance(P<0.01).⑤Effects of miR-675 on the expression of autophagy-related proteins in OA chondrocytes:Compared with the idling slow virus control group,the use of miR-675 mimic processing cell,cartilage cells of Beclinl protein expression level,P62 protein expression is restrained,LC3A/B-Ⅱ/LC3A/B-Ⅰ ratio increases,the differences were statistically significant(P<0.05);With miR-675 inhibitor inhibit the expression of miR-675 in cartilage cells,cartilage cells Beclinl protein expression of cut,P62 protein expression level,LC3A/B-Ⅱ/LC3A/B-Ⅰ ratio decreased,the differences were statistically significant(P<0.05).⑥Effects of miR-675 on autophagy flow in OA chondrocytes:Compared with the idled lentivirus control group,the proportion of red fluorescence to green fluorescence in chondrocytes treated with miR-675mimic was significantly increased,and autophagy flow was enhanced.However,the ratio of red fluorescence to green fluorescence in chondrocytes treated with miR-675 inhibitor was significantly reduced,and autophagy flow was weakened.⑦Effects of siTBK1 and LvTBK1 on the expression of TBK1 protein in chondrocytes:Compared with the no-load lentivirus control group,LvTBK1 could significantly up-regulate the expression levels of TBK1 mRNA and protein in chondrocytes,and the difference was statistically significant(P<0.05).siTBK1 could significantly down-regulate the expression levels of TBK1mRNA and protein in chondrocytes,with statistical significance(P<0.05).⑧Effect of TBK1 on proliferation of OA chondrocytes:Compared with the no-load lentivirus control group,the proliferation rate of chondrocytes overexpressing TBK1 was significantly decreased,with statistical significance(P<0.01).⑨Effect of TBK1 on matrix metabolism of OA chondrocytes:Compared with light slow virus control group,siTBK1 processing after cartilage Collagen II expression,MMP-9 protein expression decreased;After the expression TBK1,MMP-9 protein,Collagen in cartilage cells Ⅱ protein expression.⑩Effect of TBK1 on apoptosis of OA chondrocytes:Compared with the no-load lentivirus control group,the apoptosis rate of chondrocytes was significantly increased after overexpression of TBK1,with statistical significance(P<0.05).(11)Effect of TBK1 on autophagy of OA chondrocytes:Compared with transfection blank control group,siTBK1 after transfection cartilage cells,cartilage cells of p-mTOR and P62 protein expression levels significantly cut,LC3A/B-Ⅱ/Ⅰ proportion is obviously higher,the differences were statistically significant(P<0.01).(12)MiR-675 targeted the expression of TBK1:The dual luciferase reporter gene assay showed that miR-675-5p significantly down-regulated the reporter fluorescence of TBK1-WT vector(P<0.05).The expression of miR-675-5p was interfered with miR-675-5p inhibitor,and the reported fluorescence of TBK1-WT vector was significantly up-regulated(P<0.05).(13)Effects of miR-675 on the proliferation of chondrocytes by regulating the expression of TBK1:After the overexpression of TBK1 in human OA primary chondrocytes,the proliferative activity of chondrocytes was inhibited.After the transfection of miR-675mimic,the proliferative activity of chondrocytes was significantly increased compared with the cells treated with only overexpression of TBK1,with statistical significance(P<0.01).(14)Effects of miR-675 on the apoptosis of chondrocytes by regulating the expression of TBK1:The overexpression of TBK1 promoted the apoptosis of chondrocytes,and the apoptosis rate of chondrocytes was significantly decreased after the use of miR-675mimic,with statistical significance(P<0.01).(15)Effects of miR-675 on autophagy of chondrocytes by regulating the expression of TBK1:Compared with the no-load lentivirus control group,after overexpression of TBK1,the mRNA expression levels of autophagy-related genes Atg5 and Atg7 in chondrocytes were down-regulated,the protein levels of mTOR were up-regulated,and the expressions of Beclinl and LC3 were inhibited,with statistical significance(P<0.01).After using miR-675mimic,mRNA expression levels of autophagy related genes Atg5 and Atg7 were up-regulated,mTOR protein level was down-regulated,and Beclinl and LC3 expression levels were up-regulated in chondrocytes,with statistical significance(P<0.01).3.Molecular mechanism by which Longbiie capsule regulates chondrocyte function through miR-675/TBK1 signaling axis①Effect of drug serum of Longbiie capsule on chondrocyte activity:The activity of chondrocytes increased with the increase of the working concentration of drug-containing serum in Longbiie capsule(0,0.625%,1.25%,2.5%,5%).There was statistical significance in the comparison of the cell viability of different concentrations of drug-containing serum after intervention(P<0.05).However,when the serum concentration rose to 10%,the activity of chondrocytes decreased.Under the action of drug-containing serum at the same concentration,the enhancement of cell viability was more and more obvious with the increase of intervention time,and the difference of cell viability at different intervention time was statistically significant(P<0.05).②The effect of Longbie capsule serum on chondrocyte proliferation:Compared with the blank group,the proliferation ability of chondrocytes in the intervention group(2.5%,5%)was significantly increased,and the difference was statistically significant(P<0.05).The proliferation ability of chondrocytes showed a trend of gradual enhancement with the increase of the intervention concentration of Longbiie capsules containing drug serum.③Effect of drug serum of Longbie capsule on matrix metabolism of Chondrocytes:Use the dragon turtle capsules containing medicine serum after the intervention of human original generation of OA chondrocytes II type collagen expression in obvious increase,as the dragon turtle capsules containing higher drug concentration of serum intervention,people in the original generation of OA chondrocytes II type collagen expression degree is becoming more and more obvious.④The effect of Longbiie capsule containing serum on the apoptosis of Chondrocytes:The apoptosis of chondrocytes was induced by IL-1(3,and the apoptosis rate of chondrocytes was significantly decreased after treatment with 5%LBJN(P<0.001).After treated with 2.5%and 5%Longbiie capsule serum for 48 hours,compared with the blank group,the expression of Bcl-2 protein in human OA primary chondrocytes was significantly increased,while the expression of CleavedCaspase-3 protein was decreased,with statistical significance(P<0.05).⑤Effect of drug serum of Longbie capsule on autophagy of chondrocytes:With 0%of the dragon turtle capsules containing medicine serum were examined,2.5%and 5%of the dragon turtle capsule medicated serum could significantly increase ATG5 and expression level of ATG7mRNA cartilage cells,inhibiting mTOR protein expression,raise Beclin 1 protein level,increase the LC3A/B-Ⅲ LC3A/B ratio,autophagy flow enhanced obviously,the differences were statistically significant(P<0.05).⑥Effects of drug containing serum of Longbiie capsule on expression of miR-675 and TBK1 in chondrocytes:The expression of miR-675 in chondrocytes was up-regulated by 2.5%and 5%Longbiie capsule drug serum,and the difference was statistically significant compared with the blank control group(P<0.05).Longbiie capsule drug serum could down-regulate the expression levels of TBK1mRNA and protein in chondrocytes,and the difference was statistically significant compared with the blank control group(P<0.05).⑦he effect of miR-675 on the function of chondrocytes in drug serum of Longbiie capsule:Compared with idle control group,the proliferation activity,apoptosis rate and autophagy of OA chondrocytes were significantly decreased after transfection with miR-675inhibitor,with statistical significance(P<0.05).When combined with 5%Longbiie capsule drug serum intervention,compared with cells treated with miR-675inhibitor alone,the proliferation activity of chondrocytes was significantly increased,the apoptosis rate was significantly decreased,and autophagy was significantly enhanced,with statistical significance(P<0.05).⑧The effect of TBK1 on the function of chondrocytes in Longbie capsule serum:Compared with light slow virus control group,the people in the original generation of OA chondrocytes after expressing TBK1,cartilage cell proliferation activity significantly lower rate increased significantly,autophagy and apoptosis related gene ATG5 and ATG7mRNA cut,mTOR protein expression level level increases,the expression level of Beclinl and LC3 is restrained,the differences were statistically significant(P<0.05);When used in combination with 5%dragon turtle capsules containing medicine serum intervention,compared with the simple expression of TBK1 cartilage cells,cartilage cell proliferation activity increased significantly,apoptosis rate significantly decreased,autophagy related gene ATG5 and ATG7mRNA expression level increases,mTOR cut,Beclinl protein levels and the expression of LC3 levels rise,the differences were statistically significant(P<0.05)ConclusionLongbiie capsule can enhance chondrocyte vitality,promote chondrocyte proliferation and autophagy,regulate chondrocyte matrix metabolism and inhibit chondrocyte apoptosis.MiR-675 can promote chondrocyte proliferation,inhibit chondrocyte apoptosis and cartilage matrix degradation,and enhance autophagy of chondrocytes,thus protecting articular cartilage.TBK1 can inhibit chondrocyte proliferation,inhibit chondrocyte autophagy,promote chondrocyte apoptosis,and promote cartilage matrix degradation.MiR-675 has a role in the targeted regulation of TBK1 expression.The drug-containing serum of Longbiie capsule could up-regulate the expression of miR-675 and down-regulate the expression of TBK1 protein in chondrocytes.The drug-containing serum of Longbiie capsule may inhibit the apoptosis of chondrocytes and promote autophagy and proliferation of chondrocytes by inhibiting the expression level of TBK1.The drug-containing serum of Longbiie capsule may inhibit the apoptosis of chondrocytes and promote autophagy and proliferation of chondrocytes by up-regulating the expression level of miR-675.The drug-containing serum of Longbie capsule may regulate autophagy through miR-675/TBK1 signal axis,inhibit the apoptosis of chondrocytes and enhance the proliferation activity of chondrocytes,so as to protect articular chondrocytes and delay the progression of OA. |
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