标题：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

      作者：张金桂 1，王超 2    张金桂 1，王超 21.海南省人民医院Ⅰ期临床试验病房，海南 海口 570311；2.琼海市人民医院药剂科，海南 琼海 571400

      卷次： 2024年35卷14期

      【摘要】 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H2O2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 H2O2 DPN模型组；组3：1.6 mmol/L H2O2+2 μmol/L丁苯酞处理组；组4：1.6 mmol/L H2O2+10 μmol/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；25 μmol/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H2O2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。
      【关键词】 糖尿病周围神经病变；丁苯酞；核因子E2相关因子2；单核细胞血红素氧合酶-1；醌氧化还原酶-1；作用机制
      【中图分类号】 R-332 【文献标识码】 A 【文章编号】 1003—6350（2024）14—1977—05

Protective effect of butylphthalein on RSC96 cell model of diabetic peripheral neuropathy and its molecularmechanism.
ZHANG Jin-gui 1, WANG Chao 2. 1. Phase Ⅰ Clinical Trial Ward, Hainan General Hospital, Haikou 570311,Hainan, CHINA; 2. Department of Pharmacy, Qionghai People's Hospital, Qionghai 571400, Hainan, CHINA
【Abstract】 Objective To study the protective effect of butylphthalein on RSC96 cell model of diabetic periph-eral neuropathy (DPN) and its molecular mechanism. Methods The cell models of DPN were established by treatingRSC96 cells with different concentrations of H2O2. Different concentrations of NBP were applied to RSC96 cells to deter-mine the drug concentration in the intervention group. The DPN model and the intervention group were divided into4 groups after the drug concentration was confirmed: group 1 (blank control group), group 2 (1.6 mmol/L H2O2 DPN mod-el group), group 3 (1.6 mm H2O2+2 μmol/L butylphthalein treatment group), group 4 (1.6 mm H2O2+10 μmol/L butylphtha-lein treatment group). Reproductive activity of RSC96 cells in each treatment group was measured by Cell CountingKit-8. After staining with DCFH-DA probe, the fluorescence intensity of reactive oxygen species (ROS) was observedby inverted fluorescence microscope. The molecular expression of nuclear factor E2-related factor 2 (Nrf2), monocyteheme 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 prolif-erative activity of cells in groups 1, 2, 3, and 4 was 100.0%, 56.2%, 73.1%, and 75.3%, respectively; there was no statisti-cally significant difference between group 3 and group 4 (P>0.05), while there was statistically significant difference be-tween other groups (P<0.01). Compared with that of group 1, the fluorescence intensity of groups 2, 3, and 4 was en-hanced; NBP concentrations below 25 μmol/L showed no significant cytotoxicity. Compared with those of group 1, theexpression 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 signifi-cantly increased (P<0.05). Conclusion Butylphthalide plays a neuroprotective role in the H2O2-induced RSC96 cellmodel of DPN by up-regulating the expression of Nrf2.
      【Key words】 Diabetes peripheral neuropathy; Butylphthalein; Nuclear factor E2 related factor 2 (Nrf2); Mono-cyte heme oxygenase-1 (HO-1); Quinone oxidoreductase-1 (NQO1); Mechanism