灯盏细辛与赤芍配伍组方对H2O2致PC12细胞损伤的保护作用

Protective Effects of Formula of Erigerontis Herba and Paeoniae Radix Rubra on Injury Induced by H2O2 of PC12 Cells

摘要:

目的：探讨灯盏细辛与赤芍配伍组方对H₂O₂诱导的PC12细胞氧化损伤的影响及其作用机制。方法：用H₂O₂（850 μmol·L^-1）处理PC12细胞6 h建立体外氧化应激模型，以不同质量浓度的灯盏细辛与赤芍配伍组方（6.25，12.5，25，50，100 mg·L^-1）预处理PC12细胞（3×10⁴~4×10⁴个/mL）24 h，采用MTT法检测细胞活力，比色法测定细胞培养液中乳酸脱氢酶（LDH）活性、细胞裂解液中超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平，利用荧光探针DCFH-DA和罗丹明123流式细胞术分别检测细胞内活性氧（ROS）和线粒体膜电位（Δψm）。结果：与正常对照组比较，模型组的细胞存活率下降至52.78%，细胞上清液中LDH释放量上升110.13 U·L^-1，细胞内MDA和ROS水平显著增高，细胞内SOD和Δψm水平显下降（P<0.01）；与模型组比较，灯盏细辛与赤芍配伍组方可明显增加细胞存活率，降低LDH活性，降低MDA水平，抑制细胞内活性氧的生成，增加SOD活性和使线粒体膜电位升高（P<0.05，P<0.01），且在一定范围呈剂量依赖性。结论：灯盏细辛与赤芍配伍组方对H₂O₂诱导的PC12细胞氧化损伤具有保护作用，其机制可能与其降低细胞脂质过氧化水平，提高抗氧化酶活力，抑制ROS生成和稳定线粒体膜电位有关。

Abstract:

Objective: To investigate the protective effect of the formula of Herba Erigerontis and Radix Paeoniae Rubra on H₂O₂-induced oxidative stress damage of PC12 cells and its possible mechanism. Method: The in vitro model of oxidative stress damage based on PC12 cells was established by treating with H₂O₂ (850 μmol·L^-1) for 6 h. The formula of Herba Erigerontis and Radix Paeoniae Rubra with different concentrations (6.25, 12.5, 25, 50, 100 mg·L^-1) pretreatment of PC12 cells (3×10⁴-4×10⁴ cells/mL)for 24 h. The MTT assay was used to determine the survival rate of PC12 cells. The activity of LDH in culture medium, the level of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in cell lysis solution were detected by colorimetric method. The fluorescent probe DCFH-DA and Rhodamine 123 reagent were employed to determine the level of intracellular reactive oxygen species (ROS) and the changes of mitochondrial membrane potential(Δψm) by flow cytometry, respectively. Result: Compared with the controls, the H₂O₂ model showed decreased to 52.78% in survival. The LDH release in culture supernatants increased 110.13 U·L^-1. In addition, the level of MDA and intracellular ROS were significantly increased, the activity of SOD and the Δψm were significantly decreased(P<0.01). Compared with model group, the survival rate of injured cells was increased, and the activity of LDH, the level of MDA and intracellular ROS were decreased in the formula group. In addition, the activity of SOD and the Δψm were significantly increased(P<0.05, P<0.01) in a certain range. Conclusion: The formula of Herba Erigerontis and Radix Paeoniae Rubra has the significant protective effects on the PC12 cells injured by H₂O₂, which may be associated with the inhibited of lipid peroxidation, the increased of antioxidase activity, the decrease of ROS and the increased stability of mitochondrial membrane potential.