竹叶总黄酮通过抑制Nox4减轻H2O2诱导的成骨细胞氧化损伤

摘要

目的 探究抑制烟酰胺腺嘌呤二核苷酸磷酸氧化酶4 (Nox4) 表达后竹叶总黄酮对H2O2诱导的成骨细胞氧化损伤的减轻作用.方法 M3T3-E1成骨细胞培养后分为5组, 正常组、损伤组、药物处理组、空白转染组、Nox4转染组.收集细胞上清液, 采用酶联免疫法测定超氧化物歧化酶 (SOD) 、乳酸脱氢酶 (LDH) 、丙二醛 (MDA) 含量, DCFH-DA荧光探针法检测活性氧 (ROS) 含量.钙结节染色、碱性磷酸酶 (ALP) 染色观察细胞成骨能力; MTT法检测M3T3-E1细胞活力;流式细胞仪检测M3T3-E1细胞凋亡情况; Western blot 检测Nox4蛋白的表达.结果 与正常组、药物处理组、NOX4转染组比较, 损伤组、空白转染组SOD含量降低, MDA、LDH、 ROS含量升高.ALP染色显示, 损伤组成骨细胞大量损伤, 数量明显减少, 药物处理组、NOX4转染组细胞损伤程度明显降低, 数量有所增加.茜红素染色显示, 损伤组细胞钙结节数量减少, 药物处理组、NOX4转染组细胞钙结节数量增多.与损伤组、空白转染组比较, 药物组、NOX4转染组ALP活性升高.MTT显示, 损伤组细胞活力明显降低, 药物处理组、NOX4转染组细胞活力升高.流式细胞仪结果显示, 损伤组、空白转染组细胞出现大量凋亡, 药物处理组、NOX4转染组凋亡程度较轻.Western blot 显示, 药物处理组、Nox4转染组M3T3-E1细胞Nox4蛋白相对表达量明显降低.结论 竹叶总黄酮对H2O2诱导的成骨细胞氧化损伤具有一定的保护作用, 其机制可能与抑制Nox4蛋白表达有关.%Objective To investigate the effect of total flavonoids of bamboo leaves on H2O2-induced oxidative damage in osteoblasts after inhibiting the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4). Methods M3T3-E1 osteoblasts were divided into 5 groups: normal group, injury group, drug treatment group, blank transfection group, and Nox4 transfection group. The supernatant of cells was collected and the contents of superoxide dismutase (SOD), lactic dehydrogenase (LDH) and malondialdehyde (MDA) were measured by enzyme linked immunosorbent assay, and the reactive oxygen species (ROS) content was detected by DCFH-DA. Calcium nodule staining and alkaline hosphatase (ALP) staining were used to observe the osteogenic ability of cells; M3T3-E1 cell viability was detected by MTTassay; the apoptosis of M3T3-E1 cells was detected by flow cytometry; the expression of Nox4 protein was detected by Western blot. Results Compared with the normal group, the drug treatment group and the NOX4 transfection group, the SODcontent in the injury group and the blank transfection group was decreased, and the contents of MDA, LDHand ROSwere increased. ALPstaining showed that the number of damaged bone cells was significantly damaged, and the number of cells in the drug treatment group and the NOX4 transfection group decreased significantly, and the number of the cells increased. Alizarin staining showed that the number of calcium nodules in the injury group decreased, and the number of calcium nodules increased in the drug treatment group and the NOX4 transfection group. Compared with the injury group and the blank transfection group, the activity of ALPin the drug treatment group and NOX4 transfection group increased. MTTshowed that the cell viability of the injury group was significantly lower, and the cell viability of the drug treatment group and the NOX4 transfection group increased. Flow cytometry showed that apoptotic cells in the injury group and the blank transfection group showed a great deal of apoptosis, and the apoptosis rate in the drug treatment group and the NOX4 transfection group was less. Western blot showed that the relative expression of Nox4 protein in M3T3-E1 treatment group and Nox4 transfection group was significantly decreased. Conclusion The total flavonoids of bamboo leaves can protect the osteoblasts from H2O2-induced oxidative damage, and its mechanism may be related to the inhibition of Nox4 protein expression.