锌指蛋白440在人膝关节OA软骨细胞损伤中的促进作用及机制

Promoting effect and mechanism of zinc finger protein 440 on chondrocyte injury in human knee osteoarthritis

目的探讨锌指蛋白440(ZNF440)在膝关节(Knee)骨性关节炎(OA)软骨细胞损伤和退化变性病理生理学中的作用。方法Knee软骨中分离人软骨细胞,分为对照组、Knee OA组、ZNF440-GFP(绿色荧光蛋白)组、ZNF440-siRNA组和Scriptaid组。通过免疫组化和Western blotting测定ZNF440在Knee OA软骨中的表达。用ZNF440-GFP过表达和ZNF440-siRNA转染细胞,并给予白细胞介素-1β(IL-1β)刺激。分别用qPCR和WB检测分解代谢、合成代谢和凋亡相关标志物的mRNA和蛋白表达水平。生物信息学分析有可能抑制ZNF440表达的化合物。结果与对照组相比,ZNF440在Knee OA软骨中的表达有所增加(P<0.05);ZNF440过表达明显增加基质金属蛋白酶(MMP)13和PARP p85的表达,并降低COL2A1表达(P<0.05);siRNA敲除ZNF440可部分逆转IL-1β刺激诱导的人Knee OA软骨细胞的分解代谢和细胞凋亡(P<0.05)。通过生物信息学分析和验证实验,确定Scriptaid是一种有可能下调ZNF440表达的化合物。Scriptaid处理可降低OA软骨细胞中ZNF440的表达,同时降低过表达ZNF440的人Knee OA软骨细胞中MMP13和PARP p85的表达(P<0.05)。结论ZNF440在人Knee OA软骨中的表达明显增加,可能通过调节细胞中炎症、分解代谢和凋亡标志物的表达参与软骨退行性机制。此外,Scriptaid可降低ZNF440的表达,并抑制其在OA软骨细胞中的破坏作用。

Objective To study the role of zinc finger protein 440(znf440)in the pathophysiology of chondrocyte injury and degeneration in knee osteoarthritis(OA).Methods Human chondrocytes were isolated from knee cartilage and divided into control group,knee OA group,ZNF440-GFP(green fluorescent protein)group,ZNF440-siRNA group and Scriptaid group.The expression of ZNF440 in Knee OA cartilage was determined by immunohistochemistry and Western blotting.Cells were transfected with ZNF440-GFP overexpression and ZNF440-siRNA and given interleukin-1β(IL-1β)stimulated.The mRNA and protein expression levels of catabolism,anabolism and apoptosis related markers were detected by qPCR and WB,respectively.Bioinformatics was used to analyze the compounds that may inhibit the expression of ZNF440.Results Compared with the control group,the expression of ZNF440 in Knee OA cartilage increased.ZNF440 overexpression significantly increased the expression of MMP13 and PARP p85,and decreased the expression of COL2A1.siRNA knockout ZNF440 can partially reverse IL-1βstimulation induced catabolism and apoptosis of human knee OA chondrocytes.Through bioinformatics analysis and validation experiments,Scriptaid was determined to be a compound that may down regulate the expression of ZNF440.Scriptaid treatment could reduce the expression of ZNF440 in OA chondrocytes and reduce the expression of MMP13 and PARP p85 in ZNF440-overexpressing human Knee OA chondrocytes.Conclusion The expression of ZNF440 in human knee OA cartilage is significantly increased,which may participate in the mechanism of cartilage degeneration by regulating the expression of inflammatory,catabolic and apoptotic markers in cells.In addition,Scriptaid can reduce the expression of ZNF440 and inhibit its destructive effect in OA chondrocytes.