丁苯酞对RSC96细胞糖尿病周围神经病变模型的保护作用及其分子机制研究

Protective effect of butylphthalein on RSC96 cell model of diabetic peripheral neuropathy and its molecular mechanism

目的研究丁苯酞(NBP)对大鼠施万细胞(RSC96)糖尿病周围神经病变(DPN)模型的保护作用及其分子机制。方法分别用不同浓度的H_(2)O_(2)作用于RSC96细胞,搭建DPN细胞模型;用不同浓度的NBP作用于RSC96细胞,确定干预组的药物浓度。DPN模型及干预组药物浓度确认后实验共分为四组(组1:空白对照组;组2:1.6 mmol/L H_(2)O_(2)DPN模型组;组3:1.6 mmol/L H_(2)O_(2)+2μmol/L丁苯酞处理组;组4:1.6 mmol/L H_(2)O_(2)+10μmol/L丁苯酞处理组)。用Cell Counting Kit-8测定各处理组RSC96细胞的增殖活性;2,7-二氯荧光素二乙酸酯荧光探针(DCFH-DA)染色后通过荧光倒置显微镜观察活性氧(ROS)荧光强度;采用蛋白印迹(Western blot)检测各处理组细胞核因子E2相关因子2(Nrf2)、单核细胞血红素氧合-1(HO-1)、醌氧化还原酶-1(NQO1)的分子表达情况;激光共聚焦观察各处理组表达的Nrf2分子由细胞质转移进入细胞核的情况。结果组1、组2、组3和组4细胞的增殖活性均值分别为100.0%、56.2%、73.1%、75.3%,组3与组4比较差异无统计学意义(P>0.05),其余各组间比较差异均有统计学意义(P<0.01);观察各组ROS荧光强度:与组1比较,组2、组3和组4的荧光强度增强;25μmol/L浓度以下的NBP没有明显的细胞毒性;与组1比较,组2、组3和组4的Total Nrf2、细胞核内Nrf2、HO-1、NQO1的表达量上调,差异有统计学意义(P<0.05);与组1比较,组2、组3、组4由细胞质转移至细胞核内的Nrf2增加,差异具有统计学意义(P<0.05)。结论丁苯酞通过上调Nrf2的表达对H_(2)O_(2)诱导的RSC96的糖尿病周围神经病变细胞模型发挥神经保护作用。

Objective To study the protective effect of butylphthalein on RSC96 cell model of diabetic peripheral neuropathy(DPN)and its molecular mechanism.Methods The cell models of DPN were established by treating RSC96 cells with different concentrations of H_(2)O_(2).Different concentrations of NBP were applied to RSC96 cells to determine the drug concentration in the intervention group.The DPN model and the intervention group were divided into 4 groups after the drug concentration was confirmed:group 1(blank control group),group 2(1.6 mmol/L H_(2)O_(2) DPN model group),group 3(1.6 mm H_(2)O_(2)+2μmol/L butylphthalein treatment group),group 4(1.6 mm H_(2)O_(2)+10μmol/L butylphthalein treatment group).Reproductive activity of RSC96 cells in each treatment group was measured by Cell Counting Kit-8.After staining with DCFH-DA probe,the fluorescence intensity of reactive oxygen species(ROS)was observed by inverted fluorescence microscope.The molecular expression of nuclear factor E2-related factor 2(Nrf2),monocyte heme oxygen-1(HO-1),and quinone oxidoreductase-1(NQO1)in each treatment group was detected by Western blot.The transfer of Nrf2 molecules from cytoplasm into nucleus was observed by confocal laser.Results The mean proliferative activity of cells in groups 1,2,3,and 4 was 100.0%,56.2%,73.1%,and 75.3%,respectively;there was no statistically significant difference between group 3 and group 4(P>0.05),while there was statistically significant difference between other groups(P<0.01).Compared with that of group 1,the fluorescence intensity of groups 2,3,and 4 was enhanced;NBP concentrations below 25μmol/L showed no significant cytotoxicity.Compared with those of group 1,the expression levels of Total Nrf2,nuclear Nrf2,HO-1,and NQO1 in groups 2,3,and 4 were significantly up-regulated(P<0.05).Compared with that of group 1,the Nrf2 transferred from cytoplasm to nucleus in groups 2,3,and 4 was significantly increased(P<0.05).Conclusion Butylphthalide plays a neuroprotective role in the H_(2)O_(2)-induced RSC96 cell model of DPN by up-regulating the expression of Nrf2.