樟芝多糖通过ROS-NOX2-NLRP1信号减轻皮层神经细胞炎症损伤的作用研究  

Antrodia camphorata polysaccharide alleviates inflammatory damage of cortical neurons through ROS-NOX2-NLRP1 signal pathway

作　　者：沈和平 官俏兵 郝亚南 俞晓翔 王云 翟丽萍 韩晨阳 SHEN Heping;GUAN Qiaobing;HAO Yanan;YU Xiaoxiang;WANG Yun;ZHAI Liping;HAN Chenyang(Department of Infection and Immunology,Jiaxing Second Hospital,Jiaxing 314001,China)

机构地区：[1]嘉兴市第二医院感染与免疫专科,314001

出　　处：《浙江医学》2021年第3期244-249,I0004,共7页Zhejiang Medical Journal

基　　金：浙江省基础公益研究计划项目(LYY19H280003、LGF19H090012)。

摘　　要：目的探讨樟芝多糖(ACP)通过抑制活性氧(ROS)-NADPH氧化酶2(NOX2)-NLRP1信号减轻皮层神经细胞(CNC)炎症损伤的作用和机制。方法在研究ACP抗炎症反应作用的实验中,将CNC分为对照组、脂多糖(LPS)组、ACP 5 mg/L组、ACP 10 mg/L组和ACP 20 mg/L组。对照组为常规培养的细胞,无LPS和ACP干预;LPS组用0.1 mg/L LPS诱导细胞炎症反应;ACP各组分别使用终浓度为5、10、20 mg/L的ACP进行预处理6 h后,再用0.1 mg/L LPS诱导细胞炎症反应。在研究ACP通过抑制ROS发挥作用的机制研究中,将CNC分为LPS组、N-乙酰半胱氨酸(NAC)组和NAC+ACP组。LPS组用0.1 mg/L LPS诱导细胞炎症反应;NAC组用10 mM NAC预处理4 h后,再用0.1 mg/L LPS诱导细胞炎症反应;NAC+ACP组同时用10 mM NAC和20 mg/L ACP预处理4 h后,再用0.1 mg/L LPS诱导细胞炎症反应。采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡水平,探针检测ROS的表达,Western blot法检测NOX2、NLRP1蛋白表达水平,ELISA法检测培养基中IL-1β、IL-6及TNF-α表达水平。结果ACP预处理后,与LPS组比较,ACP 5 mg/L组、ACP 10 mg/L组和ACP 20 mg/L组细胞活力均明显上调,细胞凋亡率均下调,细胞中ROS的荧光光度值均降低,NOX2和NLRP1蛋白表达水平均降低,IL-1β、IL-6和TNF-α水平均降低(均P<0.05)。ROS抑制剂NAC预处理后,与LPS组比较,NAC组和NAC+ACP组细胞凋亡率均下调,细胞中ROS的荧光光度值均降低,NOX2和NLRP1蛋白表达水平均降低,IL-1β、IL-6和TNF-α表达水平均降低(均P<0.05);而NAC组和NAC+ACP组细胞凋亡率、细胞中ROS的荧光光度值、NOX2和NLRP1蛋白表达水平及L-1β、IL-6和TNF-α表达水平比较差异均无统计学意义(均P>0.05)。结论ACP可以通过抑制ROS的表达来抑制NOX2-NLRP1的激活,从而减轻CNC的炎症反应。Objective To explore the effect of antrodia camphorata polysaccharide(ACP)on inflammatory damage of cortical neurons and its mechanism.Methods Mouse cortical neuron cell(CNC)was divided into control group,lipopolysac-charide(LPS)group,5,10,20 mg/L ACP treatment groups.LPS group was treated with 0.1 mg/L LPS to induce cellular inflammation,ACP treatment groups was pretreated with ACP at final concentrations of 5,10,and 20 mg/L for 6 h,respectively,then treated with 0.1 mg/L LPS,control group were cultured routinely.In studying the mechanism of ACP,CNC were treated with 10 mM ROS inhibitor N-acetylcysteine(NAC group)or 10 mM NAC and 20 mg/L ACP(NAC+ACP group)for 4 h before LPS treatment.CCK-8 method was used to detect cell viability,flow cytometry was used to detect cell apoptosis,probes were used to detect ROS expression,Western blot was used to detect NOX2 and NLRP1 protein expression levels,and ELISA method was used to detect IL-1β,IL-6 and TNF-αexpression level.Results After ACP pretreatment,compared with the LPS group,the cell viability of three ACP treatment groups were significantly up-regulated,the apoptosis rate was down-regulated,and the fluorescence value of ROS in the cells was decreased,the expression of NOX2 and NLRP1 protein were decreased,the expression of IL-1β,IL-6 and TNF-αlevels were decreased(all P<0.05).After pretreatment with NAC,compared with the LPS group,the apoptosis rate of the NAC group and the NAC+ACP group was down-regulated,the fluorescence value of ROS in the cells was decreased,the expression of NOX2 and NLRP1 protein were decreased,the expression levels of IL-1β,IL-6 and TNF-αwere decreased(all P<0.05).There were no significant differences in the apoptotic rate,the fluorescence value of ROS,the expression of NOX2 and NLRP1 protein,and the expression levels of IL-1β,IL-6 and TNF-αbetween NAC group and NAC+ACP group(all P>0.05).Conclusion ACP can inhibit the activation of NOX2-NLRP1 pathway and regulate the inflammatory response of cortical neurons by inhibiting the expressi

关 键 词：樟芝多糖 皮层神经细胞 活性氧 炎症反应 

分 类 号：R285.5[医药卫生—中药学]