灰树花多糖对谷氨酸损伤PC12细胞的保护作用

Protective Effect of Grifola frondose Polysaccharide Against PC12 Cell Dam⁃age Induced by Glutamate Acid

通过谷氨酸诱导PC12细胞损伤,建立氧化应激损伤的体外模型,探讨灰树花多糖对细胞损伤的保护作用。试验分为模型组(谷氨酸15 mmol/L)、治疗组(灰树花多糖25、50、100、200 mg/L+谷氨酸15 mmol/L)、对照组(不添加谷氨酸和灰树花多糖);MTT法检测细胞活性;酶标仪及检测试剂盒检测抗氧化酶(过氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶)活性、抗超氧阴离子活力及丙二醛浓度;活性氧检测试剂盒(ROS Assay Kit)检测活性氧(reactive oxygen species,ROS)水平;流式细胞仪检测细胞凋亡率。结果表明:(1)治疗组细胞存活率、抗氧化酶的活性均高于模型组,且随着灰树花多糖质量浓度的增加呈现先上升后下降的趋势;丙二醛浓度则相反。(2)治疗组灰树花多糖质量浓度为100 mg/L时,细胞存活率最高(80.27%±0.82%),过氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶、抗超氧阴离子活性最大(分别为24.78μmol•min^(-1)•mL^(-1)、21.42μmol•min^(-1)•mL^(-1)、48.62μmol•min^(-1)•L^(-1)、137.65μmol•min^(-1)•L^(-1))、丙二醛浓度最小(0.92 nmol/mL),且与模型组相比,差异有统计学意义(P<0.05)。(3)治疗组灰树花多糖质量浓度为100 mg/L和200 mg/L时,随着多糖质量浓度的增加,细胞ROS水平升高、细胞凋亡率增大,但细胞ROS水平、凋亡率均显著低于模型组(P<0.05)。灰树花多糖能抑制细胞凋亡,提高抗氧化酶活性和抗超氧阴离子的活性,减少ROS和丙二醛的生成,对谷氨酸损伤具有一定的保护作用。

The in vitro model of oxidative stress injury was established by glutamate-induced PC12 cell injury,and the protective effect of Grifola frondosa polysaccharide on cell injury was investigated.The experiment was divided into a model group(glutamic acid 15 mmol/L),a treatment group(Grifola frondosa polysaccharide 25,50,100,200 mg/L+glu⁃tamic acid 15 mmol/L),a control group(without glutamic acid and Grifola frondosa polysaccharide).Cell activity was de⁃tected by MTT assay.The activity of antioxidant enzymes(catalase,superoxide dismutase,glutathione peroxidase),anti-superoxide anion activity and malondialdehyde concentration were detected by microplate reader and detection kit.The level of reactive oxygen species(ROS)was detected by ROS Assay Kit.The apoptosis rate was detected by flow cytometry.The results showed that:(1)The cell survival rate and antioxidant enzyme activity in the treatment group were higher than those in the model group,and increased first and then decreased with the increase of Grifola frondosa polysaccharide con⁃centration.The concentration of malondialdehyde was the opposite.(2)When the concentration of Grifola frondosa poly⁃saccharide in the treatment group was 100 mg/L,the cell survival rate was the highest(80.27%±0.82%),the activities of catalase,superoxide dismutase,glutathione peroxidase and anti-superoxide anion were the highest(24.78μmol·min^(-1)·mL^(-1),21.42μmol·min^(-1)·mL^(-1),48.62μmol·min^(-1)·L^(-1),137.65μmol·min^(-1)·L^(-1),respectively),and the concentration of malondialdehyde was the lowest(0.92 nmol/mL).Compared with the model group,the difference was statistically signifi⁃cant(P<0.05).(3)When the mass concentration of Grifola frondosa polysaccharide in the treatment group was 100 mg/L and 200 mg/L,with the increase of polysaccharide mass concentration,the level of ROS and apoptosis rate increased,but the level of ROS and apoptosis rate were significantly lower than those in the model group(P<0.05).Grifola frondosa polysaccharide can inhibit cell apoptosis,improve antioxidant enzyme activity and anti-superoxide anion activity,reduce the production of ROS and malondialdehyde,and has a certain protective effect on glutamate damage.