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人参皂苷Rg1对LPS诱导的人鼻黏膜上皮细胞凋亡及黏蛋白分泌的影响
作者:王明香  张绍芳  陈薇  黄凌峰 
单位:郴州市第一人民医院 小儿耳鼻喉科, 湖南 郴州 423000
关键词:人参皂苷Rg1 脂多糖 人鼻黏膜上皮细胞 糖黏蛋白5AC 细胞凋亡 
分类号:R765.25
出版年·卷·期(页码):2022·50·第一期(15-22)
摘要:

目的:探讨人参皂苷Rg1(ginsenoside Rg1,GRg1)对脂多糖(lipopolysaccharide,LPS)诱导人的鼻黏膜上皮细胞中糖黏蛋白5AC(MUC5AC)表达的影响,并阐明其可能的作用机制。方法:采用不同浓度LPS或GRg1处理人鼻黏膜上皮细胞HNEpC,CCK-8法检测细胞增殖活性,筛选LPS和GRg1干预浓度。采用400 ng·ml-1 LPS或40 μmol·L-1 GRg1分别或同时处理HNEpC细胞,将细胞分为空白对照组(Blank组)、LPS组、LPS+GRg1组、LPS+3-MA和LPS+GRg1+3-MA组。采用ELISA法检测各组细胞上清液中MUC5AC含量;qRT-PCR检测各组细胞中MUC5AC mRNA表达水平;Annexin V/PI 双染色法检测各组细胞凋亡情况;单丹磺酰尸胺(monodansylcadaverin,MDC)染色检测细胞自噬囊泡聚集情况;透射电镜观察细胞自噬体形成情况;Western blot检测各组细胞中MUC5AC、Beclin 1、LC3Ⅰ和LC3Ⅱ蛋白表达水平。结果:与Blank组比较,LPS组细胞中MUC5AC mRNA和蛋白表达水平以及上清液中MUC5AC含量均显著上升(P<0.01),细胞凋亡率升高(P<0.01),细胞自噬水平增加,自噬相关蛋白Beclin 1表达水平和LC3 Ⅱ/Ⅰ值均显著升高(P<0.01);与LPS组比较,LPS+GRg1组和LPS+3-MA组细胞中MUC5AC mRNA和蛋白表达水平以及上清液中MUC5AC含量均显著下降(P<0.01),细胞凋亡率降低(P<0.01),细胞自噬水平降低,自噬相关蛋白Beclin 1表达水平和LC3Ⅱ/Ⅰ值均显著降低(P<0.01);与LPS+3-MA组比较,LPS+3-MA+GRg1组细胞以上各项指标均无显著性变化(P>0.05)。结论:人参皂苷Rg1可抑制LPS诱导的人鼻黏膜上皮细胞MUC5AC高表达,并同时抑制细胞凋亡,其机制可能与抑制细胞自噬有关。

Objective:To investigate the effect of Ginsenoside Rg1(GRg1) on the expression of mucin 5AC(MUC5AC) induced by lipopolysaccharide(LPS) in human nasal epithelial cells HNEpC, and to elucidate its possible mechanism. Methods: Human nasal mucosal epithelial cells HNEpC were treated with different concentrations of LPS or GRg1. The proliferation activity of cells was detected by CCK-8, and the intervention concentrations of LPS and GRg1 were confirmed. HNEpC cells were treated with 400 ng·ml-1 LPS or 40 μmol·L-1 GRg1 respectively or simultaneously, and the cells were divided into blank control(Blank) group, LPS group, LPS+GRg1 group, LPS+3-MA group and LPS+GRg1+3-MA group. The content of MUC5AC in the supernatant was detected by ELISA; the expression level of MUC5AC mRNA in cells was detected by qRT-PCR; the apoptosis rate was measured by Annexin V/PI staining method; the aggregation of autophagic vesicles was detected by Monodansyl cadaverine(MDC) staining; the formation of autophagosomes was observed under transmission electron microscope, and the expression levels of MUC5AC, Beclin 1, LC3 Ⅰ and LC3Ⅱ proteins were detected by Western blot. Results: Compared with the Blank group, the expression levels of MUC5AC mRNA and protein in the cells of the LPS group, and the MUC5AC content in the supernatant were significantly increased(P<0.01), the apoptosis rate was increased(P<0.01), the autophagy level increased, and the expression levels of autophagy-related proteins Beclin 1 and the LC3Ⅱ/Ⅰ ratio were markedly increased(P<0.01). Compared with the LPS group, the expression levels of MUC5AC mRNA and protein expression in the cells of the LPS+GRg1 group and LPS+3-MA group, and the content of MUC5AC in the supernatant were significantly reduced(P<0.01), the apoptosis rate was decreased(P<0.01), the autophagy level in the cells was decreased, and the expression level of autophagy-related proteins Beclin 1 and LC3Ⅱ/Ⅰ ratio were markedly decreased(P<0.01). Compared with the LPS+3-MA group, the LPS+3-MA+GRg1 group had no significant changes in the above indicators of cells(P>0.05). Conclusion: Ginsenoside Rg1 can inhibit the high expression of MUC5AC in human nasal epithelial cells induced by LPS, and at the same time inhibit cell apoptosis. The mechanism may be related to the inhibition of cell autophagy.

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