肝豆灵片通过调节TLR4/NF-κB/NLRP3信号通路减轻肝豆状核变性神经炎症的机制

doi:10.3976/j.issn.1002-4026.2023.04.006

摘要/Abstract

摘要：

基于TLR4/NF-κB/NLRP3信号通路,探讨肝豆灵片对TX小鼠肝豆状核变性神经炎症的影响以及对CuCl₂诱导的小胶质细胞炎性反应的作用机制。将TX小鼠分为对照组,模型组,肝豆灵片低、中、高剂量组,青霉胺组;BV2细胞分为对照组、模型组、肝豆灵片组、TAK-242组、肝豆灵片+TAK-242组,采用HE染色检测各组小鼠海马组织病理学改变,蛋白质免疫印迹检测各组小鼠海马组织及BV2细胞中TLR4、p65、NLRP3、IL-1β蛋白的表达,酶联免疫吸附测定法检测各组小鼠海马组织及BV2细胞中TNF-ɑ 、IL-1β、IL-6的含量,实时荧光定量聚合链式反应检测各组BV2细胞中TLR4、p65、NLRP3、TNF-ɑ 、IL-1β mRNA的表达。结果表明:与对照组相比,模型组海马组织出现明显炎性损伤,TLR4、p65、NLRP3、IL-1β蛋白表达均明显增高,TNF-ɑ、IL-1β、IL-6的含量明显升高(P<0.05);与模型组相比,肝豆灵片组和青霉胺组海马组织的病理损伤均得到改善,且肝豆灵片高剂量组效果更明显;肝豆灵片组和TAK-242组能抑制BV2细胞活化,TLR4、p65、NLRP3、IL-1β蛋白与mRNA表达较模型组均明显减少,TNF-ɑ、IL-1β、IL-6的含量明显降低(P<0.05)。肝豆灵片能减轻TX小鼠海马组织炎性损伤,抑制CuCl₂诱导的BV2细胞过度活化,其机制可能与下调 TLR4/NF-κB/NLRP3信号通路有关。

关键词: 肝豆灵片, 肝豆状核变性, TLR4/NF-κB/NLRP3, 神经炎症, 作用机制

Abstract:

This study aimed to investigate the effect of Gandouling tablet on neuroinflammation in hepatolenticular degeneration in TX mice and mechanism of generating CuCl₂-induced microglia inflammatory response based on TLR4/NF-κB/NLRP3 signaling pathway. TX mice were divided into control, model, Gandouling tablet low-dose, Gandouling tablet medium-dose, Gandouling tablet high-dose, and penicilamine groups. BV2 cells were divided into control, model, Gandouling tablet, TAK-242, and Gandouling tablet + TAK-242 groups. Hematoxylin and eosin staining was performed to detect histopathological changes in the hippocampus of mice in each group. Western blot was used to detect the expressions of TLR4, p65, NLRP3, and IL-1β in hippocampal tissue and BV2 cells of mice in each group. Enzyme-linked immunosorbent assay was performed to quantify TNF-α, IL-1β, and IL-6 levels in hippocampal tissue and BV2 cells of mice in each group. Real-time polymerase chain reaction was used to determine the expression of TLR4, p65, NLRP3, TNF-α, and IL-1β mRNA in all groups of BV2 cells. Compared with the control group, hippocampal tissue in the model group showed considerable inflammatory damage; increased protein expressions of TLR4, p65, NLRP3, and IL-1β; and significantly increased levels of TNF-α, IL-1β, and IL-6 (P<0.05). Compared with the model group, the pathological damage of hippocampal tissue improved in both Gandouling tablet and penicillamine groups, and the effect of Gandouling tablet in the Gandouling tablet high-dose group was more prominent than that in the other groups. The Gandouling tablet and TAK-242 groups inhibited the activation of BV2 cells. Additionally, the expression of TLR4, p65, NLRP3, and IL-1β protein and mRNA were significantly reduced in these two groups as compared with the model group, and TNF-α, IL-1β and IL-6 levels were significantly decreased (P < 0.05). Gandouling tablet can alleviate hippocampal inflammation and inhibit CuCl₂-induced hyperactivation of BV2 cells in TX mice probably by downregulating TLR4/NF-κB/NLRP3 signaling pathway.

Key words: Gandouling tablet, hepatolenticular degeneration, TLR4/NF-κB/NLRP3, neuroinflammation, action mechanism

中图分类号:

R285

闻雨雅, 董婷, 江张胜, 陈洁, 田丽伟, 赵晨玲, 唐露露. 肝豆灵片通过调节TLR4/NF-κB/NLRP3信号通路减轻肝豆状核变性神经炎症的机制[J]. 山东科学, 2023, 36(4): 42-51.

WEN Yuya, DONG Ting, JIANG Zhangsheng, CHEN Jie, TIAN Liwei, ZHAO Chenling, TANG Lulu. The mechanism of Gandouling tablet in alleviating hepatolenticular degeneration neuroinflammation via the regulation of the TLR4/NF-κB/NLRP3 signaling pathway[J]. Shandong Science, 2023, 36(4): 42-51.

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基因名称	引物序列	产物长度/bp
TLR4	上游5'- AATGAGGACTGGGTGAGAAATGAG-3' 下游5'-CTGTAGTGAAGGCAGAGGTGAAAG-3'	80
p65	上游5'-TCCTGTTCGAGTCTCCATGCAG-3' 下游5'-GGTCTCATAGGTCCTTTTGCGC-3'	133
NLRP3	上游5'-TCACAACTCGCCCAAGGAGGAA-3' 下游5'-AAGAGACCACGGCAGAAGCTAG-3'	147
TNF-α	上游5'- GGGTTGTACCTTGTCTACTCCCA-3' 下游5'-GAGATAGCAAATCGGCTGACG-3'	95
IL-1β	上游5'-TGGACCTTCCAGGATGAGGACA-3' 下游5'-GTTCATCTCGGAGCCTGTAGTG-3'	148
GAPDH	上游5'-GTGTTCCTACCCCCAATGTG-3' 下游5'-GTCATTGAGAGCAATGCCAG-3'	215