人参根提取物对小鼠脾淋巴细胞自噬、增殖和细胞因子分泌的影响及机制研究

Study on the Effects of Ginseng Root Extract on Autophagy,Proliferation and Cytokine Secretion of Mice Splenic Lymphocytes and Its Mechanism

目的:研究人参根提取物对小鼠脾淋巴细胞自噬、增殖及细胞因子分泌的影响,并研究其作用机制。方法:将小鼠脾淋巴细胞分为空白对照组、阳性对照组(刀豆蛋白A,5μg/mL)和不同质量浓度人参根提取物组(32、125、500μg/mL,以冻干粉末计)。各组细胞加入相应药物培养48 h后,分别采用吖啶橙染色法检测细胞的自噬情况;采用CCK-8法检测细胞的增殖情况;采用酶联免疫吸附法测定细胞培养液中白细胞介素4(IL-4)、IL-6、肿瘤坏死因子α(TNF-α)的水平;采用Western blotting法检测细胞中微管相关蛋白轻链β3(LC3B)、酵母ATG6同源物(Beclin-1)的表达水平以及腺苷酸活化蛋白激酶(AMPK)、蛋白激酶B(AKT)、雷帕霉素靶蛋白(mTOR)的磷酸化水平。结果:与空白对照组比较,32、125、500μg/mL人参根提取物均能够增加小鼠脾淋巴细胞酸性自噬体的数量(P<0.05或P<0.01);125、500μg/mL人参根提取物均可显著提高细胞存活率,显著提高细胞中IL-4、IL-6和TNF-α的水平,显著促进细胞中LC3BⅠ向LC3BⅡ转化,显著上调细胞中Beclin-1蛋白表达水平,显著提高AMPK蛋白的磷酸化水平,显著降低AKT和mTOR蛋白的磷酸化水平,差异均有统计学意义(P<0.05或P<0.01)。结论:人参根提取物能通过激活AMPK、抑制AKT活化来抑制mTOR活性,从而诱导小鼠脾淋巴细胞发生自噬、激活脾淋巴细胞活性、调节细胞因子分泌以发挥免疫增强作用。

OBJECTIVE:To study the effects of ginseng root extract on autophagy,proliferation and cytokine of splenic lymphocyte of mice,and to study its mechanism.METHODS:The splenic lymphocyte of mice were divided into blank control group,positive control group(concanavalin A,5μg/m L),different concentration of ginseng root extract groups(32,125,500μg/m L,by lyophilized powder).After 48 h of culture with the corresponding medicine,acridine orange staining method was used to detect autophagy of splenic lymphocyte;CCK-8 assay was used to detect the cell proliferation;ELISA assay was used to determine the levels of IL-4,IL-6 and TNF-αin cell culture;Western blotting method was used to detect the expression of LC3B and Beclin-1,as well as the phosphorylation of AMPK,AKT and mTOR.RESULTS:Compared with blank control group,32,125,500μg/mL ginseng root extract could increase the number of acidic autophagosomes in splenic lymphocytes of mice(P<0.05 or P<0.01);125,500μg/m L Ginseng root extract could significantly enhance the survival rate of splenic lymphocytes,the levels of IL-4,IL-6 and TNF-αand the transformation of LC3BⅠto LC3BⅡ,significantly increased the protein expression of Beclin-1,the phosphorylation of AMPK protein,and significantly reduced the phosphorylation level of AKT and mTOR proteins,with statistical significance(P<0.05 or P<0.01).CONCLUSIONS:Ginseng root extract can induce autophagy of mice splenic lymphocytes,activate the activity of splenic lymphocytes,regulate the secretion of cytokine by activating AMPK and inhibiting the activation of AKT which can inhibit the activity of m TOR,so as to exert immune enhancement effect.