ATF4靶向AKT1对膝关节软骨细胞凋亡及软骨退变的影响

Effects of ATF4 on chondrocyte apoptosis and cartilage degeneration in knee joint via targeting AKT1

目的探讨活化转录因子4(ATF4)靶向蛋白激酶B(AKT1)对膝关节软骨细胞凋亡及软骨退变的影响。方法体外实验:①体外培养正常软骨细胞与骨关节炎(OA)软骨细胞,实时荧光定量PCR检测两组细胞内ATF4、AKT1的mRNA表达水平;②通过细胞转染法构建下调ATF4的OA软骨细胞;③MTT法及Annexin V/PI流式细胞术检测OA软骨细胞增殖及凋亡情况;④生物信息学预测ATF4靶向基因结合位点,双荧光素酶报告基因实验验证ATF4与AKT1的靶向关系;⑤共同转染构建ATF4下调及AKT1上调的OA软骨细胞,同样的方法检测其细胞增殖及凋亡情况。动物实验:取6个月月龄雄性Duncan豚鼠30只,随机均分为3组,A、B两组皮下均植入Alzet微量泵,ATF4组每天泵注含PBS稀释过的ATF4,PBS组泵注单纯PBS液,取豚鼠膝关节软骨,评估3组Mankin组织评分及膝关节软骨组织内IL⁃1及TNF⁃α水平。结果与正常软骨细胞相比,OA软骨细胞ATF4 mRNA表达量均显著上升(P<0.05),AKT1mRNA表达量均显著下降(P<0.05)。ATF4⁃mimics转染物可上调细胞ATF4的表达,且细胞增殖能力下降,凋亡能力上升,ATF⁃inhibitor能下调细胞ATF4表达,细胞则表现出相反的增殖及凋亡特点。靶基因预测ATF4与AKT13’UTR存在靶向结合位点,双荧光素酶报告基因验证了AKT1是ATF4的靶向基因。动物实验表明,ATF4组豚鼠膝关节软骨组织Mankin组织评分显著高于PBS组及空白组(P<0.05),膝关节软骨内IL⁃1及TNF⁃α表达水平明显高于阴性对照组及正常对照组(P<0.05)。结论ATF4在促进OA软骨细胞凋亡中发挥积极作用,其作用机制可能与靶向调控AKT1相关。

Objective To investigate the effects of activating transcription factor 4(ATF4)on osteoarthritic(OA)chondrocyte apoptosis and cartilage degeneration in knee joint via targeting protein kinase B(AKT1).Methods In vitro ex⁃periments:①Normal chondrocytes and OA chondrocytes were cultured in vitro,and the mRNA expression levels of ATF4 and AKT1 were detected by real⁃time fluorescence quantitative PCR.②OA chondrocytes with down⁃regulated ATF4 were constructed by cell transfection.③Proliferation and apoptosis of OA chondrocytes were detected by MTT assay and Annexin V/PI flow cytometry.④Bioinformatics was used to predict the binding sites of ATF4 targeted genes,and the targeting rela⁃tionship between ATF4 and AKT1 was verified by dual luciferase reporter experiment.⑤OA chondrocytes with DOWN⁃regu⁃lated ATF4 and up⁃regulated AKT1 were constructed by co⁃transfection,and cell proliferation and apoptosis were detected by the method mentioned above.Animal experiments:Thirty 6⁃month⁃old male Duncan guinea pigs were randomly divided into three groups.Alzet micro⁃osmotic pumps were implanted subcutaneously in both groups A and B,thereafter,group A re⁃ceived ATF4 diluted in PBS,and group B received PBS only.Group C was left untreated.The knee cartilage of guinea pigs was removed,then the Mankin scoring along with the levels of interleukin⁃1(IL⁃1)and tumor necrosis factor⁃α(TNF⁃α)in knee cartilage of three groups were assessed.Results Compared with normal chondrocytes,the mRNA expression of ATF4 was significantly increased(P<0.05),and AKT1 mRNA expression was significantly decreased in all OA chondrocytes(P<0.05).ATF4⁃mimics transfections up⁃regulated cellular ATF4 expression and decreased cellular proliferation and increased apoptosis,while ATF⁃inhibitor down⁃regulated cellular ATF4 expression and cells presented totally opposite proliferation and apoptosis characteristics.Target gene prediction showed that there was a targeted binding site between ATF4 and AKT13′UTR.The double luciferase reporter gene verified that AKT1 was the target gene of ATF4.Animal experiment showed that Mankin score of knee cartilage of guinea pigs in group A was significantly higher than that in group B and C(P<0.05),and the expression levels of IL⁃1 and TNF⁃αin knee cartilage of guinea pigs in group A were significantly higher than those in negative control group and normal control group(P<0.05).Conclusion ATF4 plays an active role in promoting the apopto⁃sis of OA chondrocytes.Its mechanism may be related to the targeted regulation of AKT1,which becomes a new direction for the targeted treatment of OA.