青钱柳三萜对链脲佐菌素损伤的INS-1细胞自噬和凋亡的影响

Protective effects of total triterpenoids from Cyclocarya paliurus(Batal.) Iljinskaja on-INS1 cells stimulated by STZ through regulation of autophagy and apoptosis

目的:观察青钱柳三萜对STZ损伤的INS-1细胞自噬和凋亡的影响。方法:用链脲佐菌素(STZ)诱导INS-1细胞建立损伤模型。MTT法检测不同浓度青钱柳三萜对INS-1细胞增殖的影响,荧光酶标仪检测青钱柳三萜对胞内活性氧(ROS)含量的影响,用放射免疫法测定其胰岛素分泌能力,比色法测定上清液中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)含量;比色法测定心肌细胞中Caspase-3、Caspase-9的活性,荧光定量PCR测定Bcl-2和Bax mRNA表达,并计算Bcl-2与Bax的比值,Western blot检测细胞中自噬调控蛋白微管相关蛋白轻链3(LC3)和凋亡调控蛋白聚腺苷二磷酸核糖聚合酶(PARP)蛋白表达。结果:当青钱柳三萜浓度低于25μg/ml时,无论单用青钱柳三萜还是与STZ联用均对INS-1细胞增殖具有促进作用,但是当其浓度大于100μg/ml或与STZ联用大于50μg/ml时,均对其细胞生长具有抑制作用;青钱柳三萜(6.25、12.5、25μg/ml)可抑制INS-1细胞凋亡,促进INS-1细胞分泌胰岛素,降低细胞内ROS含量;升高上清液中SOD、CAT和GSH-Px活性,降低MDA含量,降低自噬调控蛋白LC3-Ⅱ和凋亡调控蛋白Cleaved PARP的蛋白表达,上抗凋亡蛋白Bcl-2 mRNA表达,下调促凋亡蛋白Bax mRNA表达及Bcl-2/Bax,降低Caspase-9及Caspase-3的活性。结论:青钱柳三萜对STZ损伤的INS-1细胞具有较好的保护作用。它通过抑制STZ损伤的INS-1细胞过量产生ROS,增强内源性抗氧化酶GSH-Px、SOD、CAT的活性,降低自噬调控蛋白LC3-Ⅱ和凋亡调控蛋白Cleaved PARP的表达,上抗凋亡蛋白Bcl-2表达,下调促凋亡蛋白Bax表达及降低Caspase-9及Caspase-3的活性,进而提高INS-1细胞存活率,来发挥对受损INS-1细胞的保护作用。

Objective： To explore the protective effects of total triterpenoids from Cyclocarya paliurus（ Batal.） Iljinskaja on-INS-1 cells stimulated by STZ through regulating of autophagy and apoptosis.Methods： INS-1 cells were cultured in media containing 3n M STZ and different doses of total triterpenoids from Cyclocarya paliurus（ Batal.） Iljinskaja.The proliferation of cells was examined by MTT assay,ROS content were detected by fluorescence enzyme label; The levels of superoxide dismutase（ SOD）,glutathione peroxidase（ GSH-Px）,hydrogen peroxidase（ CAT）,malondialdehyde（ MDA） were also measured by colorimetry; The activities of Caspase-3,9 were also observed by Caspase colorimetric assay kit in each group; The expressions of Bcl-2 mRNA and Bax mRNA were detected by Real time PCR,protein expression of LC3-Ⅱand PARP were detected by western blotting.Results： Compared with the control group,total triterpenoids from Cyclocarya paliurus（ Batal.） Iljinskaja could promote the proliferation of INS-1 cells no matter with STZ or not when its concentration was lower than 25μg/ml;But when its concentration was higher than 100μg/ml（ individually use） or 50μg/ml（ combined use）,total triterpenoids from Cyclocarya paliurus（ Batal.） Iljinskaja can significantly inhibit the growth of the cells whether STZ existed or not.Total triterpenoids from Cyclocarya paliurus（ Batal.） Iljinskaja（ 6.25,12.5,25μg/ml） can inhibit INS-1 cell apoptosis,decrease the contents of intra-cellular ROS,improve activities of SOD,GSH-PX,CAT in the supernatant liquid,decrease MDA level,promote INS-1 cell secreting insulin,decrease the protein expressions of autophagy protein LC3 II and apoptosis regulating protein cleaved PARP protein,up-regulate the anti-apoptotic protein Bcl-2 mRNA expression,down-regulate the pro-apoptotic protein Bax mRNA expression and Bcl-2 and Bax,reduce the activity of caspase-9 and caspase-3（ P 〈 0.05 or P 〈 0.01）.Conclusion： Total triterpenoids from Cyclocarya paliurus（ Batal.） Iljinskaja has good protective effect on-d INS-1 cells stimulated by STZ.It can inhibit STZ to injure INS-1 cells to overproduce ROS,enhance endogenous antioxidant enzymes（ GSH-Px,OD,CAT activities）,reduce the expression of autophagy protein LC3 II and apoptosis regulating protein cleaved PARP,upreglute the anti-apoptosis protein Bcl-2 expression,down-regulate the pro-apoptotic protein Bax expression,decrease activities of the Caspase-9 and Caspase-3,and improve the survival rate of INS-1 cell,then played a protective effect on damaged INS-1 cells.