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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 observe the effects of Cyanotype Triterpenoids on autophagy and apoptosis in STZ-injured INS-1 cells. Methods: The injury model was induced by streptozotocin (STZ) in INS-1 cells. MTT assay was used to determine the effects of different concentrations of Cyanatumientolide on the proliferation of INS-1 cells. Fluorescence microplate reader was used to detect the effect of Triterpene on intracellular ROS. Radioimmunoassay was used to measure the insulin secretion , The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) in the supernatant were measured by colorimetric method. The activity of Caspase-3 and Caspase-9 in cardiomyocytes was determined by colorimetric assay. The expressions of Bcl-2 and Bax mRNA were detected by real-time quantitative PCR. The ratio of Bcl-2 to Bax was calculated. Western blot was used to detect the expression of autophagy regulatory protein microtubules Protein light chain 3 (LC3) and apoptosis regulatory protein poly (ADP-ribose polymerase) (PARP) protein expression. Results: When Cyclocarya paliurus triterpenoids concentration was lower than 25μg / ml, both of them could promote the proliferation of INS-1 cells either at the concentration of 100μg / ml or with STZ combined with more than 50μg / ml could inhibit the growth of INS-1 cells, and all of them could inhibit the growth of INS-1 cells at 6.25,12.5,25μg / ml Increase the activity of SOD, CAT and GSH-Px in the supernatant, decrease the content of MDA, and decrease the protein expression of LC3-Ⅱ and Cleaved PARP, and up-regulate the expression of Bcl-2 mRNA Downregulate the expression of pro-apoptotic protein Bax and Bcl-2 / Bax, and decrease the activity of Caspase-9 and Caspase-3. CONCLUSION: Cyclotarbantol has good protective effect on STZ-injured INS-1 cells. It inhibits STZ-induced INS-1 cells over-production of ROS, enhances the activity of endogenous antioxidant enzymes GSH-Px, SOD, CAT, reduce the expression of autophagy regulatory protein LC3-Ⅱ and apoptosis regulatory protein Cleaved PARP, The anti-apoptotic protein Bcl-2 expression, the down-regulation of pro-apoptotic protein Bax, the decrease of Caspase-9 and Caspase-3 activity, and then the survival of INS-1 cells to play a protective effect on impaired INS-1 cells.