摘要
背景:椎间盘退变常会引发腰背痛、肩颈痛等,与椎间盘退变髓核细胞的凋亡、炎症及氧化应激反应联系密切,虎杖苷是虎杖的主要活性成分,具有抗炎、抗氧化等生物活性。目的:探讨虎杖苷对小鼠腰椎间盘退变髓核细胞凋亡及沉默信息调节因子1(silence information regulator 1,SIRT1)/哺乳动物雷帕毒素靶蛋白(mammalian target of rapamycin,mTOR)通路的影响。方法:采用胰蛋白酶分离培养原代小鼠髓核细胞,采用甲苯胺蓝染色及Ⅱ型胶原蛋白免疫细胞化学染色对髓核细胞进行鉴定并检测虎杖苷处理后的细胞毒性。将髓核细胞随机分为5组,空白组、模型组、虎杖苷低、中、高剂量组(分别为虎杖苷10,25,50 mg/L)。除空白组正常培养,其余各组均用白细胞介素1β诱导髓核细胞退变模型,并按照各组给药剂量进行培养;流式细胞仪检测各组髓核细胞凋亡情况,酶联免疫吸附(ELISA)法测定各组髓核细胞氧化应激因子及炎症因子表达水平;蛋白免疫印迹法检测髓核细胞凋亡相关蛋白及SIRT1/mTOR信号通路相关蛋白表达。研究经长江航运总医院伦理委员会批准同意,批准号:L20200059。结果与结论:①用胰蛋白酶成功分离较纯的小鼠腰椎间盘髓核细胞;②与空白组相比,模型组小鼠腰椎间盘髓核细胞凋亡率、半胱氨酸蛋白酶3、Bax蛋白、丙二醛、乳酸脱氢酶、白细胞介素6、诱导型一氧化氮合酶、肿瘤坏死因子α表达水平显著升高,虎杖苷各剂量组上述各指标较模型组显著降低;与空白组相比,模型组Bcl-2蛋白、超氧化物歧化酶、还原型谷胱甘肽及SIRT1、p-PI3K/PI3K、p-Akt/Akt、mTOR蛋白表达水平显著降低(P<0.05);虎杖苷各剂量组上述各指标较模型组显著升高(P<0.05);虎杖苷各剂量组之间呈现一定的剂量依赖效应,其中高剂量组效果较优;③结果表明,虎杖苷能显著减轻由白细胞介素1β引起的髓核细胞凋亡、炎症、氧化应激反应,可能是通过激活SIRT1/mTOR通路实现的。
BACKGROUND:Intervertebral disc degeneration often causes low back pain,shoulder and neck pain in the elderly,which is closely related to the apoptosis,inflammation and oxidative stress of nucleus pulposus cells in intervertebral disc degeneration.Polydatin is the main active component of Polygonum cuspidatum,which has anti-inflammatory,antioxidant and other biological activities.OBJECTIVE:To investigate the effects of polydatin on apoptosis of nucleus pulposus cells and silent information regulator 1(SIRT1)/mammalian target of rapamycin(mTOR)pathway in mice with intervertebral disc degeneration.METHODS:Primary mouse nucleus pulposus cells were isolated and cultured by trypsin.Cell identification and cytotoxicity measurement were conducted by toluidine blue staining and type II collagen immunocytochemistry staining.The nucleus pulposus cells were randomly divided into five groups:blank group,model group,low-,middle-,and high-dose polydatin groups(10,25,and 50 mg/L).The blank group was normally cultured;in addition,the other groups were induced by interleukin-1βto build the degenerative model of nucleus pulposus cells,followed by cultured with different doses of polydatin.Cell apoptosis in each group was detected by flow cytometry.The expression levels of oxidative stress factors and inflammatory factors were measured by enzyme-linked immunosorbent assay.The expression levels of apoptosis related proteins and SIRT1/mTOR signaling pathway related proteins in nucleus pulposus cells were detected by western blot method.The study protocol was ethically approved by the Ethics Committee of General Hospital of the Yangtze River Shipping with an approval No.L20200059.RESULTS AND CONCLUSION:The nucleus pulposus cells were successfully isolated and purified by trypsin.Compared with those in the blank group,the apoptotic rate of nucleus pulposus cells,the expression of Caspase-3 and Bax proteins,the expression levels of malondialdehyde,lactate dehydrogenase,interleukin-6,inducible nitric oxide synthase,and tumor necrosis factorαwere significantly higher in the model group.Compared with the model group,these indexes were significantly decreased in the polydatin groups.Compared with the blank group,the model group showed a significant reduction in the expression levels of Bcl-2 protein,superoxide dismutase,reduced glutathione,SIRT1,phosphorylated PI3K/phosphatidylinositol-3-kinase,phosphorylated Akt/protein kinase B and mTOR protein(P<0.05),and the polydatin groups had a significant increase in the above-mentioned indexes in a dose-dependent manner(P<0.05).To conclude,polydatin can significantly reduce the apoptosis,inflammation and oxidative stress of nucleus pulposus cells induced by interleukin-1β,which may be achieved by activating the SIRT1/mTOR pathway.
作者
左斌
夏晓枫
车彪
邹凯
唐家国
Zuo Bin;Xia Xiaofeng;Che Biao;Zou Kai;Tang Jiaguo(Department of Orthopedics,General Hospital of the Yangtze River Shipping,Wuhan 430010,Hubei Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2021年第35期5619-5625,共7页
Chinese Journal of Tissue Engineering Research
基金
湖北省卫生健康委员会联合基金立项项目(WJ2019H388),项目负责人:唐家国。
