藁本内酯对白细胞介素-1β诱导大鼠软骨细胞凋亡的保护作用及其机制

Function and mechanism of ligustilide on apoptosis induced by interleukin-1βof cartilage cells

目的观察藁本内酯对白细胞介素(IL)-1β诱导大鼠软骨细胞凋亡的保护作用.方法选取健康2周龄无特定病原体(SPF)级SD大鼠,取膝关节软骨细胞进行分离、培养及鉴定.将终质量浓度为15、25、50、75μmol/L的藁本内酯加入二代软骨细胞培养板中,通过细胞计数试剂盒(CCK-8)检测细胞增殖;将藁本内酯加入合适浓度的IL-1β诱导的软骨细胞与对照组细胞内,流式细胞分析技术检测不同浓度藁本内酯对IL-1β诱导软骨细胞凋亡的影响,并检测细胞早期凋亡比例;通过Hoechst34580细胞核染色技术观察藁本内酯对凋亡软骨细胞核形态改变的影响;通过蛋白质印迹法(Western blot)法检测软骨细胞内半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、Caspase-9、B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)以及诱导型一氧化氮合酶(iNOS)等蛋白表达的变化.应用SPSS 20.0软件统计分析,计量资料如灰度值比值等采用t检验,计数资料采用Fish确切概率检验.结果CCK-8检测结果表明藁本内酯在15~70μmol/L浓度范围内对软骨细胞的增殖无明显促进作用(P>0.05).流式细胞检测结果表明25~75μmol/L终质量浓度的藁本内酯可降低IL-1β诱导的软骨细胞凋亡,凋亡比例分别为(20.12±2.07)%、(13.79±3.19)%和(7.86±2.14)%(t=17.260、13.140、6.350,P<O.05),Hoechst34580检测结果表明藁本内酯可降低IL-1β诱导软骨细胞凋亡引起的核改变.Western blot法检测结果表明藁本内酯能降低IL-1β诱导的软骨细胞中Caspase-3、Caspase-9、bax和iNOS的表达水平,以及增高bcl-2/bax比值(P<0.05).结论藁本内酯可抑制IL-1β诱导的软骨细胞凋亡,且呈剂量依赖性.

Objective To investigate the effect of different concentrations of the Z-ligustilide(LIG)on proliferation and apoptosis induced by interleukin(IL)-1βof cartilage cells in vitro.Methods Healthy specific pathogen free(SPF)Sprague Dawley rats aged 2 weeks were selected and chondrocytes from knee joints were isolated,cultured and identified.The final concentrations of 15,25,50,75μmol/L ligustilide were added to the second-generation chondrocyte culture plate,and cell proliferation was detected by cell counting kit-8(CCK-8).Ligustilide was added into chondrocytes induced by IL-1βof appropriate concentration and the cells in the control group.The effect of different concentrations of ligustilide on the apoptosis of chondrocytes induced by IL-1βwas detected by flow cytometry.The effect of ligustilide on nuclear morphological changes of apoptotic chondrocytes was observed by Hoechst34580 nuclear staining;Western blotting was used to detect the expression of cysteinyl aspartate-specific protease(Caspase)-3,Caspase-9,B cell lymphoma/leukemia-2(bcl-2),bcl-2 associated X protein(bax)and inducible nitric oxide synthase(iNOS)in chondrocytes.Results The results of CCK-8 showed that ligustilide had no significant effect on the proliferation of chondrocytes in the range of 15-70μmol/L(P>0.05).Flow cytometry results showed that ligustilide at a final concentration of 25-75μmol/L could reduce the apoptosis of chondrocytes induced by IL-1β[rate of apoptosis:(20.12±2.07)%,(13.79±3.19)%and(7.86±2.14)%,t=17.260,13.140,6.350,respectively,P<0.05].The results of Hoechst34580 showed that ligustilide can reduce the IL-1β-induced chondrocyte apoptosis.Western blotting analysis showed that ligustilide could decrease the expression of Caspase-3,Caspase-9,bax and iNOS and increase the bcl-2/bax ratio(P<0.05).Conclusion Ligustilide can inhibit the apoptosis of chondrocytes induced by IL-1βin a dose-dependent manner.