Necrostatin-1对压力诱导的髓核细胞自噬及凋亡的影响

Effect of necrostatin-1 on autophagy and apoptosis of nucleus pulposus cells induced by compression

目的观察Necrostatin-1对压力诱导的大鼠髓核细胞自噬及凋亡的影响。方法从3月龄大鼠腰段脊柱提取髓核细胞,选取第2代细胞进行实验,分为对照(生理盐水)组和Necrostatin-1组,1.0 Mpa压力条件下培养0、24、36 h。单丹黄酰尸胺(MDC)染色荧光显微镜观察自噬泡及细胞膜上MDC荧光表达情况,流式细胞仪检测MDC阳性率。膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙锭(PI)染色流式细胞仪定量Annexin V阳性率;Hoechst 33258染色荧光显微镜观测细胞凋亡。反转录-聚合酶链反应(RT-PCR)法检测自噬相关基因Beclin1、LC3B和凋亡相关基因半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、B细胞淋巴瘤/白血病-2相关X蛋白(bax)的表达水平。组间比较采用t检验。结果压力作用24、36 h,荧光显微镜观察显示,与对照组比较,Necrostatin-1可明显下调细胞MDC及Hoechst 33258的荧光强度。流式检测显示,与对照组24、36 h的MDC阳性率(13.97±0.85、23.52±1.92)%及Annexin V阳性率(12.72±0.89、22.13±1.46)%比较,Necrostatin-1可明显下调MDC阳性率(t=5.530、4.977,P<0.05)及Annexin V阳性率(t=4.702、4.970,P<0.05),差异有统计学意义。RT-PCR结果显示,与对照组24、36 h的Beclin1(2.76±0.13、2.65±0.14)、LC3B(3.62±0.18、4.54±0.65)及Caspase-3(2.04±0.15、3.28±0.39)、bax(3.56±0.42、4.82±0.66)基因表达比较,Necrostatin-1可明显抑制24、36 h压力诱导Beclin1(t=8.192、2.836,P<0.05)、LC3B(t=9.013、3.072,P<0.05)及Caspase-3(t=3.048、3.277,P<0.05)、bax(t=4.241、2.850,P<0.05)的基因表达,差异均有统计学意义。结论Necrostatin-1可明显抑制压力诱导的髓核细胞自噬及凋亡。

Objective To observe the effect of necrostatin-1 on autophagy and apoptosis of rat nucleus pulposus(NP)cells induced by compression.Methods The NP cells were extracted from the lumbar spine of 3-month-old rats,and the second generation cells were selected for experiment.They were divided into control(normal saline)group and Necrostatin-1 group,and cultured under 1.0 MPa compression for 0,24,and 36 h.Monodansylcadaverine(MDC)staining fluorescence microscope was used to observe the fluorescence expression of MDC on autophagic vesicles and cell membranes,and the positive rate of MDC was detected by flow cytometry.Annexin V-fluoresceine isothiocyanate(FITC)/propidium iodide(PI)staining flow cytometry was employed to quantify the positive rate of Annexin V.Hoechst 33258 staining fluorescence microscope was introduced to observe cell apoptosis.Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to detect the expression levels of autophagy-related genes Beclin1,LC3B and apoptosis-related genes Caspase-3,B cell lymphoma/leukemia-2 associated X protein(bax).The SPSS 18.0 statistical software was used for analysis.the measurement data were expressed as mean±standard deviation(±s),and t test was used for the comparison between two groups.Results Following 24 and 36 h compression,fluorescence microscope observation showed that as compared with the control group,Necrostatin-1 could significantly down-regulate the fluorescence intensity of MDC and Hoechst 33258 in NP cells.Flow cytometry demonstrated that as compared with the positive rate of MDC[(13.97±0.85)%,(23.52±1.92)%]and positive rate of Annexin V[(12.72±0.89)%,(22.13±1.46)%]of the control group at 24 and 36 h,Necrostatin-1 markedly down-regulated the positive rate of MDC(t=5.530,4.977,P<0.05)and the positive rate of Annexin V(t=4.702,4.970,P<0.05).RT-PCR results showed that as compared with the gene expression of Beclin1(2.76±0.13,2.65±0.14),LC3B(3.62±0.18,4.54±0.65),Caspase-3(2.04±0.15,3.28±0.39)and bax(3.56±0.42,4.82±0.66)of the control group at 24 and 36 h,Necrostatin-1 significantly inhibited the mRNA expression of Beclin1(t=8.192,2.836,P<0.05),LC3B(t=9.013,3.072,P<0.05)and Caspase-3(t=3.048,3.277,P<0.05),bax(t=4.241,2.850,P<0.05)in NP cells induced by compression.Conclusion Necrostatin-1 can efficiently downregulate the autophagy and apoptosis of NP cells induced by compression.