樟芝多糖抑制6-OHDA诱导的多巴胺能神经元细胞炎症反应的作用机制研究

Study on Mechanism of Antrodia Camphorata Polysaccharide Inhibits Inflammatory Response of Dopaminergic Neurons Cells Induced by 6-OHDA

关键词:  樟芝多糖  多巴胺能神经元  6-羟基多巴胺  炎症反应

Key words:  Antrodia camphorata polysaccharide  dopaminergic neurons  6-hydroxydopamine  inflammatory response

摘要:

目的 探究樟芝多糖通过抑制ROS-NLRP3-caspase-1途径调节6-羟基多巴胺（6-hydroxydopamine，6-OHDA）诱导的多巴胺能神经元（dopaminergic neurons，DAN）细胞炎症反应的作用。方法 分离小鼠中脑DAN细胞，采用6-OHDA体外构建帕金森病细胞模型，将细胞分为正常组、模型组、对照组、实验组。正常组为常规培养的DAN细胞，模型组为6-OHDA处理的DAN细胞，对照组为ROS抑制剂乙酰半胱氨酸（NAC）+6-OHDA处理的DAN细胞，实验组为6-OHDA+樟芝多糖处理的DAN细胞。采用CCK-8法检测细胞活力，流式细胞术和免疫荧光染色法检测ROS的水平，流式细胞术检测细胞凋亡水平，Hoechst 33342染色活细胞，蛋白免疫印迹（Western-bolt）法检测细胞中NLRP3、caspase-1、pro-caspase-1的表达水平，酶联免疫吸附（Elisa）法检测上清中IL-1β、IL-6和IL-18的分泌水平。结果 模型组中6-OHDA可以诱导DAN细胞炎症反应，ROS表达增高，NLRP3-caspase-1炎性小体水平增高，细胞凋亡率增高，相比正常组具有显著性差异（P<0.05）。樟芝多糖干预后，ROS的水平下调，NLRP3-caspase-1炎性小体水平降低，细胞凋亡率下调，相比模型组具有显著性差异（P<0.05）。结论 樟芝多糖可以通过抑制ROS-NLRP3-caspase-1途径调节6-OHDA诱导DAN炎症反应，这可能是樟芝多糖在帕金森病炎症反应中的作用机制之一。

Abstract:

OBJECTIVE To explore the effect of Antrodia camphorata polysaccharide on the inflammatory response of dopaminergic neurons (DAN) cells induced by 6-OHDA through inhibiting ROS-NLRP3-caspase-1 pathway. METHODS To isolate the DAN from midbrain. Parkinson cell model was constructed in vitro by 6-OHDA. The cells were divided into normal group, model group, control group and experimental group. In the normal group, the DAN cells were cultured; in model group the DAN cells treated by 6-OHDA; in control group the DAN cells treated by ROS inhibitor NAC+6-OHDA; in experimental group, the DAN cells treated by 6-OHDA+Antrodia camphorata polysaccharide. Cell viability was detected by CCK-8 assay, ROS level was detected by flow cytometry and immunofluorescence staining, cell apoptosis was detected by flow cytometry. Hoechst 33342 staining of living cells, the expression of NLRP3, caspase-1 and pro-caspase-1 in cells was detected by protein immunoblotting (Western-bolt), and the secretion level of IL-1β, IL-6 and IL-18 in the supernatant was detected by enzyme linked immunosorbent assay(Elisa). RESULTS In the model group, 6-OHDA could induce the inflammatory response of DAN cells, the expression of ROS increased, the level of NLRP3-caspase-1 inflammatory corpuscle increased and the rate of apoptosis increased, which was significantly different from that of the normal group(P<0.05). After the Antrodia camphorata polysaccharide intervention, the level of ROS was down regulated, the level of NLRP3-caspase-1 inflammatory corpuscle was decreased, and the rate of apoptosis was down regulated, which was significantly different from that of the model group(P<0.05). CONCLUSION Antrodia camphorata polysaccharide can regulate the inflammatory response of DAN cells by inhibiting the regulation of 6-OHDA in ROS-NLRP3-caspase-1 pathway, which is one of the mechanisms of Antrodia camphorata polysaccharide in the inflammatory response of Parkinson disease.