苦豆碱对白细胞介素-1β诱导软骨细胞损伤的作用及其机制

Effect of aloperine on chondrocyte injury induced by interleukin-1β and its mechanism

目的 探讨苦豆碱(aloperine,ALO)对白细胞介素-1β(interleukin-1β,IL-1β)诱导软骨细胞损伤的作用及其机制。方法 将软骨细胞随机分为(1)空白对照(Con)、IL-1β、IL-1β+ALO-L(25 mg/L)、IL-1β+ALO-M(50 mg/L)、IL-1β+ALO-H(100 mg/L)组;(2)Con、IL-1β、IL-1β+miR-NC、IL-1β+miR-16-5p组;(3)Con、IL-1β、IL-1β+si-NC、IL-1β+si-SOX5组。IL-1β组细胞加入10 ng/mL的IL-1β,Con组不进行任何处理。qRT-PCR法检测软骨细胞中miR-16-5p和SOX5基因mRNA转录水平;ELISA法检测IL-6、TNF-α和IL-1β含量;Western blot法检测Bcl-2、Bax、SOX5蛋白表达水平;流式细胞术检测细胞凋亡情况。结果 与IL-1β组比较,IL-1β+ALO-L、IL-1β+ALO-M、IL-1β+ALO-H组软骨细胞中IL-6、TNF-α、IL-1β含量显著降低(t=5.002~20.653,P均<0.001),细胞凋亡率显著降低(t=5.473~17.371,P均<0.001),Bcl-2蛋白表达水平显著升高(t=7.800~16.100,P均<0.001),Bax蛋白表达水平显著降低(t=4.993~14.311,P均<0.001);miR-16-5p基因mRNA转录水平显著升高(t=6.688~16.545,P均<0.001),SOX5基因mRNA转录水平及其蛋白表达水平均明显降低(t=4.609~15.393,P均<0.001)。与IL-1β+miRNC组比较,IL-1β+miR-16-5p组min-16-5p基因mRNA转录水平明显升高(t=17.106,P <0.001),IL-6、TNF-α、IL-1β含量显著降低(t=15.030~20.013,P均<0.001),细胞凋亡率明显降低(t=12.273,P <0.001),Bcl-2蛋白表达水平显著升高(t=15.652,P <0.001),Bax蛋白表达水平显著降低(t=12.999,P <0.001)。与IL-1β+si-NC组比较,IL-1β+si-SOX5组SOX5蛋白表达水平显著降低(t=13.444,P <0.001),IL-6、TNF-α、IL-1β含量显著降低(t=14.087~17.103,P均<0.001),细胞凋亡率明显降低(t=11.991,P <0.001),Bcl-2蛋白表达水平显著升高(t=13.864,P <0.001),Bax蛋白表达水平显著降低(t=11.818,P <0.001)。结论 ALO可抑制IL-1β诱导的软骨细胞凋亡,从而降低软骨细胞损伤,其机制可能是通过调节软骨细胞中miR-16-5p和SOX5表达水平及炎症因子的分泌水平实现的。

Objective To investigate the effect of aloperine(ALO)on interleukin-1β(IL-1β)-induced chondrocyte injury and its mechanism.Methods Chondrocytes were randomly divided into control(Con)group,IL-1 β group,IL-1β + ALO-L(25 mg/L)group,IL-1β + ALO-M(50 mg/L)group and IL-1 β + ALO-H(100 mg/L)group;Con group,IL-1βgroup,IL-1β + miR-NC group and IL-1β + miR-16-5p group;Con group,IL-1β group,IL-1β + si-NC group and IL-1β + siSOX5 group.Cells in IL-1β group were treated with 10 ng/mL IL-1β,while no treatment was given in Con group.The transcription levels of miR-16-5p and SOX5 mRNA in chondrocytes were detected by qRT-PCR;The contents of IL-6,TNF-αand IL-1β were detected by ELISA;The expression levels of Bcl-2,Bax and SOX5 protein were detected by Western blot and the apoptosis was detected by flow cytometry.Results Compared with IL-1 β group,the contents of IL-6,TNF-α and IL-1βin IL-1β + ALO-L group,IL-1β + ALO-M group and IL-1β + ALO-H group decreased significantly(t = 5.002~20.653,each P<0.001),the apoptosis rate decreased significantly(t = 5.473~17.371,each P<0.001),the expression level of Bcl-2 protein increased significantly(t = 7.800~16.100,each P<0.001),and the expression level of Bax protein decreased significantly(t = 4.993~14.311,each P<0.001);The mRNA transcription level of miR-16-5p gene increased significantly(t = 6.688~16.545,each P<0.001),while the mRNA transcription level and protein expression level of SOX5 gene decreased significantly(t = 4.609~15.393,each P<0.001).Compared with the IL-1β + miR-NC group,the mRNA transcription level of miR-16-5p in the IL-1β + miR-16-5p group increased significantly(t = 17.106,P<0.001),the contents of IL-6,TNF-α and IL-1 β decreased significantly(t = 15.030~20.013,each P<0.001),the apoptosis rate decreased significantly(t = 12.273,P<0.001),the expression level of Bcl-2 protein increased significantly(t = 15.652,P<0.001),and the expression level of Bax protein decreased significantly(t = 12.999,P<0.001).Compared with IL-1β +si-NC group,the expression level of SOX5(t = 13.444,P<0.001),IL-6,TNF-α and IL-1β in IL-1β + si-SOX5 group decreased significantly(t = 14.087~17.103,each P<0.001),the apoptosis rate decreased significantly(t = 11.991,P<0.001),the expression level of Bcl-2 protein increased significantly(t = 13.864,P<0.001),and the expression level of Bax protein decreased significantly(t = 11.818,P<0.001).Conclusion Alo inhibited the apoptosis of chondrocytes induced by IL-1β,thus reducing the injury of chondrocytes,of which the mechanism might be regulating the expression of miR-16-5p and SOX5 and the secretion of inflammatory factors in chondrocytes.