青钱柳不同溶剂提取物对高糖诱导大鼠胰岛细胞损伤的保护作用

Protective Effects of Different Solvent Extracts of Cyclocarya Paliurus Against High Glucose Induced INS-1 Cell Injury

目的:观察青钱柳不同溶剂提取物对高糖诱导大鼠胰岛细胞瘤细胞(INS-1)损伤的保护作用。方法:用高糖诱导INS-1细胞损伤模型。MTT法检测青钱柳水提物、30%、60%和90%醇提物对INS-1细胞增殖的影响,荧光酶标仪检测青钱柳不同溶剂提取物对胞内活性氧(ROS)含量的影响,用ELISA法测定其胰岛素分泌能力,比色法测定上清液中乳酸脱氢酶(LDH)和一氧化氮(NO)及细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量;比色法测定INS-1中Caspase-3、Caspase-9活性,荧光定量PCR测定Bcl-2和Bax mRNA表达,并计算Bcl-2/Bax比率。结果:33 mmol/L葡萄糖处理INS-148 h后可见INS-1细胞数目明显减少、细胞边缘模糊、触角收缩聚集、形态变圆,LDH释放量增加,与正常对照组比较,培养液中的NO和细胞内的ROS、MDA含量、Caspase-3、Caspase-9活性和Bax mRNA表达明显升高,胰岛素分泌量和细胞内SOD、GSH-Px、CAT活性和Bcl-2 mRNA表达及Bcl-2/Bax比率明显降低(P<0.01)。与模型对照组比较,用青钱柳不同溶剂提取物干预后INS-1细胞凋亡被显著抑制,培养液或细胞中NO、ROS、MDA含量,Caspase-3、Caspase-9活性,LDH释放量及Bax mRNA表达明显降低,胰岛素分泌和细胞内SOD、GSH-Px、CAT活性、Bcl-2 mRNA表达及Bcl-2/Bax比率明显增加(P<0.05或者P<0.01)。结论:青钱柳不同溶剂提取物对高糖诱导INS-1细胞损伤有保护作用,其作用机制与增强内源性抗氧化系统功能、抑制线粒体依赖性凋亡密切相关;其作用效果由强到弱依次为青钱柳水提物、30%、60%和90%醇提物。

Objective:To observe the protective effects of different solvent extracts of Cyclocarya paliurus against high glucose-induced rat insulinoma(INS-1 cell)injury.Methods:INS-1 cell injury model was induced by high glucose.MTT assay was used to detect the effects of different solvent extracts of C.paliurus(water extract and 30%,60%,and 90%ethanol extracts)on the proliferation of INS-1 cells.The content of intracellular reactive oxygen species(ROS)was detected by fluorescence microplate assay.The secretion of insulin was measured by the enzyme-linked immunosorbent assay(ELISA).The content of lactate dehydrogenase(LDH),nitric oxide(NO),intracellular superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px),and malondialdehyde(MDA),and the activities of Caspase-3 and Caspase-9 in INS-1 cells were measured by colorimetry.The mRNA expression of Bcl-2 and Bax was measured by real-time polymerase chain reaction(PCR),and the ratio of Bcl-2/Bax was calculated.Results:After treatment with 33 mmol/L glucose for 48 h,INS-1 cells were reduced in number and blurred in cell edge with contracted and gathered antennae,round shape,and increased release of LDH.Compared with the normal control group,the model group showed increased NO content in the culture medium and the intracellular ROS and MDA,potentiated Caspase-3 and Caspase-9 activities,elevated Bax mRNA expression,declining insulin secretion,blunted SOD,GSH-Px,and CAT activities,and reduced Bcl-2 mRNA expression and Bcl-2/Bax ratio(P<0.01).Compared with the model group,the solvent extracts of C.paliurusgroups displayed inhibited apoptosis of INS-1 cells,declining content of NO,ROS,and MDA,diminished release of LDH,blunted Caspase-3 and Caspase-9 activities,decreased Bax mRNA expression,promoted insulin secretion,potentiated SOD,GSH-Px,and CAT activities,and increased Bcl-2 mRNA expression and Bcl-2/Bax ratio(P<0.05 or P<0.01).Conclusion:The different solvent extracts of C.paliuruspossess protective effects against high glucose-induced INS-1 cell injury.Its mechanism may be related to the enhancement of the function of the endogenous antioxidant system and the inhibition of mitochondrial-dependent apoptosis.The effects of different solvent extracts of C.paliurus are in the following descending order:water extract,30%ethanol extract,60%ethanol extract,and 90%ethanol extract.