摘要
目的探讨姜黄素对人椎间盘髓核细胞自噬和退变的影响。方法人椎间盘髓核组织来源于2018年3~11月四川省遂宁市中心医院骨科收治的7例椎间盘突出患者,并进行体外髓核细胞原代培养,将其分为三组:对照组、白细胞介素-1β(IL-1β)组和白细胞介素-1β+姜黄素组。四甲基偶氮唑盐比色法(MTT)检测姜黄素对人髓核细胞毒性情况,RT-PCR和Western blot分别检测各组细胞内基质金属蛋白酶-3(MMP-3)、MMP-13、聚集蛋白聚糖(aggrecan)和胶原蛋白(Collagen)Ⅱ的表达水平,同时检测各组细胞内自噬蛋白微管相关蛋白3(LC3)和Beclin 1的蛋白表达情况;采用GFP-LC3腺病毒转染观察各组细胞内自噬水平。结果 MTT结果显示,当姜黄素浓度<80μmol/L时对髓核细胞是没有细胞毒性的,考虑到对髓核细胞无毒性,同时对髓核细胞能发挥最大的药物作用,选择姜黄素浓度为80μmol/L进行后续的实验。姜黄素能显著增加髓核细胞内的自噬颗粒数,促进细胞自噬蛋白LC3的水平升高和P62蛋白水平下降(P<0.05);同时,姜黄素显著提高髓核细胞aggrecan和CollagenⅡ的水平,降低MMP-3和MMP-13的表达水平(P<0.05)。结论姜黄素能减缓椎间盘的退变,其机制可能是通过促进细胞的自噬水平。
Objective To investigate the effect of Curcumin on autophagy and degeneration of human intervertebral disc nucleus pulposus. Methods The human disc nucleus pulposus tissues were obtained from 7 patients with disc herniation admitted to the Department of Orthopedics, Suining Central Hospital of Sichuan Province from March to November2018. Primary culture of nucleus pulposus cells were performed in vitro and divided into three groups, control group,interleukin-1β(IL-1β) group and interleukin-1β+ Curcumin group. The toxicity of Curcumin to human nucleus pulposus cells was detected by tetramethylazoles salt colorimetry(MTT) assay;RT-PCR and Western blot were used respectively to detect each cell matrix metalloproteinases-3(MMP-3), MMP-13, gathered proteoglycans(aggrecan) and collagen(Collagen)Ⅱ expression level;meanwhile, protein expressions of autophagy protein microtubule-related protein 3(LC3) and Beclin 1 in each group were detected. The autophagy level in each group was observed by transfection with GFP-LC3 adenovirus. Results MTT results showed no cytotoxicity to nucleus pulposus cells when curcumin concentration was less than 80 μmol/L. Considering that it has no toxic effect on nucleus pulposus cells and can exert maximum drug effect on nucleus pulposus cells. Curcumin concentration of 80 μmol/L was selected for subsequent experiments. Curcumin can significantly increase the number of autophagy particles in nucleus pulposus cells, promote the increase of autophagy protein LC3 level and decrease of P62 protein level(P<0.05). Curcumin significantly improve aggrecan nucleus pulposus cells and Collagen Ⅱ level, reduce the MMP-3 and the expression of MMP-13 levels(P<0.05). Conclusion Curcumin can slow disc degeneration, possibly by promoting autophagy in cells.
作者
万俊锋
阳波
郑佳状
冉茂波
宋昭君
WAN Junfeng;YANG Bo;ZHENG Jiazhuang;RAN Maobo;SONG Zhaojun(Department of Orthopedics,Suining Central Hospital,Sichuan Province,Suining 629000,China)
出处
《中国医药导报》
CAS
2019年第25期11-15,I0003,共6页
China Medical Herald
基金
四川省卫生和计划生育委员会科研课题(16PJ523)
关键词
姜黄素
自噬
髓核细胞
退变
Curcumin
Autophagy
Nucleus pulposus cells
Degeneration
