木瓜三萜抗吲哚美辛诱导RGM-1细胞凋亡的作用

Anti-apoptotic effect of triterpene from Chaenomeles speciosa( sweet)Nakaion in indomethacin induced RGM-1 cells

目的:观察木瓜三萜对吲哚美辛诱导RGM-1细胞凋亡的影响,并探讨其可能的机制。方法:用吲哚美辛诱导RGM-1细胞建立细胞损伤模型,MTT法检测不同浓度木瓜三萜对RGM-1细胞增殖、流式细胞术和Hoechst33258染色分析木瓜三萜对吲哚美辛诱导细胞凋亡的影响,JC-1染色法测定细胞线粒体跨膜电位水平,Caspase试剂盒检测RGM-1细胞中Caspase-9和Caspase-3的活性,Real-time PCR检测RGM-1细胞中TFF1、p53、Bcl-2及Bax mRNA表达,Western blot检测RGM-1细胞中TFF1和p53蛋白表达。结果:当木瓜三萜浓度低于100μg/m L时,木瓜三萜对RGM-1细胞的生长无明显的影响;当木瓜三萜浓度低于50μg/m L时,木瓜三萜与吲哚美辛联用对RGM-1细胞的生长有促进作用;木瓜三萜可显著增加吲哚美辛诱导RGM-1细胞损伤的活性(P<0.05,P<0.01),升高线粒体膜电位,抑制细胞凋亡和改善细胞形态,上调TFF1蛋白表达及TFF1、Bcl-2 mRNA表达和Bcl-2与Bax的比值,下调p53蛋白表达及p53和Bax mRNA表达,降低Caspase-9及Caspase-3活性(P<0.05,P<0.01)。结论:木瓜三萜可有效抑制吲哚美辛诱导RGM-1细胞凋亡,其作用机制可能与上调TFF1、Bcl-2,下调p53、Bax、Caspase-3、Caspase-9表达有关。

AIM： To observe the effect of triterpene from Chaenomeles speciosa（ sweet） Nakaion in indomethacin induced RGM-1 cells,and to explore its possible mechanism. METHODS： The RGM-1 cell damage model induced by indomethacin was established. MTT assay was used to investigate the effect of triterpene from Chaenomeles speciosa（ sweet） Nakaion on proliferation of RGM-1 cells;flow cytometry and hoechst33258 staining were used to detect its effect on apoptosis,the caspase-3 and caspase-9 activities were detected by colorimetric method, the mitochondrial membrane potential of RGM-1 cells was measured by the JC-1 staining;Activities of caspase-9 and caspase-3 were measured by caspase assay kit, the mRNA expressions of TFF1,p53,Bcl-2 and Bax were analyzed by realtime quantitative PCR,the TFF1 and p53 protein expressions were detected by western blot. RESULTS： Triterpene from Chaenomeles speciosa（ sweet） Nakaion had no effect on RGM-1 cell grow when its concentration was less than 100 μg / m L;the combined application of indomethacin and triterpene from Chaenomeles speciosa（ sweet） Nakaion could promote RGM-1 cell growth when its concentration was less than 50 μg / m L. Triterpene from Chaenomeles speciosa（ sweet） Nakaion significantly increased RGM-1 cell activity（ P〈0. 05,P〈0. 01,respectively） and mitochondrial membrane potential,inhibited apoptosis,improved the cell morphology, upregulated the TFF1 protein expression,TFF1,Bcl-2 mRNA expressions and Bcl-2 / Bax ratio,down-regulated the p53 protein expression,p53 and Bax mRNA expressions and reduced caspase-9and caspase-3 activities compared with model group（ P〈0. 05,P〈0. 01,respectively）. CONCLUSION： Triterpene from Chaenomeles speciosa（ sweet） Nakaion could effectively inhibit the apoptosis of indomethacin induced RGM-1 cells and its mechanism may be related to the up-regulation of TFF1 and Bcl-2 expressions,accompanied with the down-regulation of p53,Bax,caspase-3 and caspase-9 expressions.