没药甾酮下调TLR4/NF-κB通路减轻脓毒症相关性脑病神经炎症反应机制探究

Guggulsterone Reduced the Neuroinflammatory Response Through Downregulating the TLR4/NF-κB Pathway in Sepsis-associated Encephalopathy

目的探讨没药甾酮(GS)下调Toll样受体4(TLR4)/核转录因子-κB(NF-κB)通路减轻脓毒症相关性脑病(SAE)神经炎症反应的机制。方法采用脂多糖(LPS)诱导小鼠小胶质细胞系(BV2细胞)制备脓毒症相关性脑病细胞模型。在此细胞模型中,通过CCK-8法检测BV2细胞的增殖凋亡情况,确定LPS和GS的最佳给药浓度,在此给药浓度基础上分为空白对照组(NC组)、LPS组及LPS+GS组,采用Western blotting法检测给药6、12、24 h后TLR4/NF-κB通路关键蛋白的表达情况,最后,采用ELISA法检测细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、转化生长因子-β(TGF-β)、白细胞介素-10(IL-10)的表达情况。结果当LPS浓度≥3μg/mL和GS浓度>30μg/mL时细胞增殖被显著抑制(P<0.01),选择LPS(3μg/mL)和GS(30μg/mL)作为联合给药浓度,在此浓度基础上联合给药最长至168 h,BV2细胞增殖仍稳定,故选择此浓度作为最佳给药浓度。与NC组相比,LPS诱导后各时间点TLR4/NF-κB通路蛋白表达均有不同程度的增加,差异有统计学意义(P<0.05)。LPS诱导后,促炎细胞因子TNF-α、IL-1β表达较NC组显著增加(P<0.05),与LPS组相比,GS干预后能在一定程度上下调TNF-α、IL-1β表达,并上调TGF-β、IL-10的表达,差异有统计学意义(P<0.05)。结论在LPS诱导的SAE细胞模型中,炎症通路TLR4/NF-κB被激活,没药甾酮能够下调此通路关键蛋白的表达,减轻脓毒症相关性脑病神经炎症反应。

Objective:To investigate the mechanisms of guggulsterone(GS)downregulated the TLR4/NF-κB pathway to reduce the neuroinflammatory response in sepsis-associated encephalopathy(SAE).Methods:A sepsis-associated encephalopathy cell model was prepared by using Lipopolysaccharide(LPS)-induced mouse microglial cell line(BV2 cells).In this cell model,the optimal apoptotic concentrations of LPS and GS was determined by CCK-8 cell proliferation and apoptosis.On the basis of this administration concentration,it was divided into non-specific control(NC group),LPS group,LPS+GS group.Western blotting was used to determine the expression of key proteins of TLR4/NF-κB pathway at 6,12 and 24 h.The expression of the cytokines TNF-α,IL-1,TGF-β,and IL-10 was determined by the ELISA assay.Results:When the LPS concentration of 3 u g/mL and GS concentration>30μg/mL was significantly inhibited(P<0.01),LPS(3μg/mL)and GS(30μg/mL)were selected as the combined administration concentration,on the combination dose as long as 168 h,BV2 cell proliferation was still stable,so this concentration was chosen as the optimal administration concentration.Compared with the NC group,the protein expression of TLR4/NF-κB pathway increased to different degrees after LPS induction,suggesting that the TLR4/NF-κB pathway was activated during SAE,and the expression level of each protein could be reduced after GS intervention,which was statistically significant(P<0.05).After LPS induction,the expression of TNF-αand IL-1βincreased significantly compared with the NC group(P<0.05);compared with the LPS group,GS intervention could downregulated TNF-αand IL-1βexpression and upregulated TGF-βand IL-10 expression,and the difference was statistically significant was significant(P<0.05).Conclusion:In the LPS-induced SAE cell model,the inflammatory pathway TLR4/NF-κB is activated,and GS is able to downregulate the expression of key proteins of this pathway and alleviate the neuroinflammatory response in SAE.