Mechanism of TGF-β1/Smad2 pathway activated by curcumin to protect articular chondrocyte injury
Objective To study the protective effect of curcumin on articular chondrocyte injury and the effect on TGF-β1/Smad2 pathway.Methods The 6-week-old SD female rats were selected and sacrificed to iso-lated and obtained knee chondrocytes.And chondrocytes were identified by toluidine blue staining.The cells were divided into blank control group,model group and curcumin intervention group(low,medium and high dose).The model group and curcumin intervention group were pretreated with 5 μg/L lipopolysaccharide for 1h,the interven-tion group(low,medium and high dose)was added with medium containing 5,15 and 50 µmol/L curcumin,and the blank control group and model group were added with medium without curcumin.After culturing for 24 hours,Hoechst 33258 staining was used to observe the cell apoptosis,and Western blotting was used to detect cellular collagen(Bcl-2,Bax,caspase-3,caspase-9),protective extracellular matrix proteins(Aggrecan,COLL2)and the ex-pression levels of TGF-β1/Smad signaling pathway related proteins(TGF-β1,Smad2/3,Smad7),and real-time fluo-rescence quantitative PCR was used to detect the gene expression of TGF-β1/Smad2 signaling pathway.Results In the model group,the number of apoptotic bodies were decreased,and the cell nuclei were condensed and deeply stained.With the concentration of curcumin increased,the number of apoptotic bodies in the curcumin in-tervention group were gradually decreased compared with the model group.The expression levels of apoptotic pro-teins(Bcl-2,Bax,caspase-3,caspase-9)in the 5 groups were significantly differences(F=30.382,17.362,39.284,18.855;all P＜0.05).The expression levels of Bax,caspase-3,caspase-9 in the curcumin intervention group were higher than those in the blank control group,but lower than those in the model group.Bcl-2 in the intervention group was lower than that in the blank control group but higher than that in the model group.There were signifi-cant differences in the expression levels of chondrocyte extracellular matrix protein(Aggrecan and COLL2)in the 5 groups(F=26.605,40.699;all P＜0.05).The expression levels of Aggrecan and COLL2 in the curcumin interven-tion group were higher than those in the blank control group,and those in the model group were lower than those in the blank control group.The expression levels of extracellular matrix protein and apoptosis protein were dose-dependent(P＜0.05).There were statistically significant differences in the expression levels of TGF-β1/Smad signal-ing proteins and genes(TGF-β1,Smad2/3,Smad7)in the 5 groups(F=27.667,13.684,26.029,61.032,25.877,35.934;all P＜0.05),among which,the levels of TGF-β1 and Smad2/3 in curcumin intervention group were higher than those in blank control group and model group,while the levels of Smad7 were lower than those in blank con-trol group and model group.The protein and gene expression levels of TGF-β1/Smad signaling pathway were dose-dependent(P＜0.05).Conclusion Curcumin can improve the inflammatory injury of chondrocytes induced by lipopolysaccharide,inhibit cell apoptosis,and promote extracellular matrix synthesis,the mechanism of which may be related to the activation of TGF-β1/Smad2 pathway
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