唑来膦酸对颞下颌关节骨关节炎软骨细胞增殖及凋亡的作用研究

Effects of zoledronate on the proliferation and apoptosis of temporomandibular joint osteoarthritic chondrocytes

目的探索唑来膦酸在颞下颌关节骨关节炎(TMJOA)软骨细胞增殖及凋亡的作用。方法用10 ng·mL^-1白细胞介素-1β(IL^-1β)诱导髁突软骨细胞构建TMJOA细胞模型,并随机分为对照组和低、高剂量实验组,分别用0,10和100 nmol·L^-1唑来膦酸干预培养。用Western Blot检测增殖细胞核抗原(PCNA)、胱天蛋白酶-3(Caspase-3)和Caspase-9蛋白的表达情况,用流式细胞技术检测细胞凋亡比例;最后加入Wnt/β-catenin通路抑制剂(XAV939)与激动剂(LiCl),在显微镜下观察软骨细胞状态变化。结果干预48 h后,对照组和低、高剂量实验组的PCNA蛋白相对表达量分别为0.36±0.01,0.21±0.01和0.20±0.01,Caspase-3蛋白相对表达量分别为0.75±0.02,1.11±0.03和1.03±0.03,低、高剂量实验组的上述指标与对照组相比,差异均有统计学意义(均P<0.001)。流式结果显示,高剂量实验组处理24和48 h后,软骨细胞凋亡比率分别为(29.05±3.43)%和(54.57±8.86)%,对照组的凋亡比率分别为(16.82±2.29)%和(17.23±1.91)%,高剂量实验组与对照组相比差异均有统计学意义(均P<0.05)。镜下观察显示:加入抑制剂XAV939后,软骨细胞多边形贴壁生长较好,一定程度上抑制唑来膦酸对软骨细胞的调控作用。结论唑来膦酸可抑制TMJOA软骨细胞增殖,促进软骨细胞凋亡,后者可能是通过Wnt/β-catenin通路实现。

Objective To evaluate the effects of zoledronate on the proliferation and apoptosis of condylar chondrocytes from temporomandibular joint osteoarthritis(TMJOA)cell model.Methods The cell model was constructed by 10 ng·mL^-1 interleukin-1β(IL^-1β)-induced condylar chondrocytes,and the cells were randomly divided into control group,experimental-L/-H groups,treated with 0,10,and 100 nmol·L^-1 zoledronate,respectively.Western blot was used to detect the protein levels of proliferation and apoptosis-related genes-proliferating cell nuclear antigen(PCNA),apoptosis-related cysteine peptidase-3(Caspase-3)and Caspase-9.The apoptosis rate was detected by flow cytometry.Finally,Wnt/β-catenin pathway inhibitor(XAV939)and activator(LiCl)were added to observe the morphological changes of chondrocytes under microscope.Results After intervention 48 h,the relative expressions of PCNA protein in control group,experimental-L/-H groups were 0.36±0.01,0.21±0.01 and 0.20±0.01,the relative expressions of Caspase-3 protein were 0.75±0.02,1.11±0.03 and 1.03±0.03,comparison between experimental-L/-H groups and control group showed significant differences(all P<0.001).Flow cytometry showed the apoptotic percentages of 24 and 48 hours action time in high concentration experimental groups were(29.05±3.43)%and(54.57±8.86)%,compared with control groups(16.82±2.29)%and(17.23±1.91)%,respectively,the differences were significant(all P<0.05).Furthermore,the effects of zoledronate were partly reversed by Wnt/β-catenin inhibitor-XAV-939.Conclusion Zoledronate could inhibit the proliferation and promote the apoptosis of TMJOA chondrocytes,which may be achieved by the Wnt/β-catenin pathway.