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

The mechanism of Gandouling tablet in alleviating hepatolenticular degeneration neuroinflammation via the regulation of the TLR4/NF-κB/NLRP3 signaling pathway

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CuCl 2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-1βmRNA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CuCl 2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。

This study aimed to investigate the effect of Gandouling tablet on neuroinflammation in hepatolenticular degeneration in TX mice and mechanism of generating CuCl 2-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-1βin 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-1βmRNA 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-1βprotein and mRNA were significantly reduced in these two groups as compared with the model group,and TNF-α,IL-1βand IL-6 levels were significantly decreased(P<0.05).Gandouling tablet can alleviate hippocampal inflammation and inhibit CuCl 2-induced hyperactivation of BV2 cells in TX mice probably by downregulating TLR4/NF-κB/NLRP3 signaling pathway.