组胺H 1受体激动剂通过Akt/NF-κB通路抑制脂多糖诱导的星形胶质细胞炎症反应

Histamine 1 receptor agonist inhibits LPS-induced immune responses in astrocytes via Akt/NF-κB signaling pathway

目的探讨组胺H 1受体(histamine H 1 receptor,H 1R)对脂多糖(lipopolysaccharide,LPS)诱导的星形胶质细胞炎症反应的影响及其调控机制。方法采用LPS建立体外星形胶质细胞炎症模型,随机将大鼠原代星形胶质细胞分为对照组、LPS组、LPS+H 1R激动剂组(2-pyridylethlamine,Pyri)和H 1R激动剂组。对照组仅加入培养液,LPS+H 1R激动剂组提前在培养液中加入100μmol·L-1的Pyri,1 h后再加入终浓度为100μg·L-1的LPS。CCK-8法检测细胞活性;免疫荧光法检测活化标记物GFAP和H 1R的表达;倒置相差显微镜下观察星形胶质细胞的形态变化;ELISA法检测细胞上清中炎症因子TNF-α和IL-6水平;Western blot检测p-Akt、Akt、p-NF-κB p65、NF-κB p65的蛋白表达。结果与对照组比较,LPS组星形胶质细胞分支和辐射状突起减少,GFAP表达增加,细胞表面H 1R表达下调,上清液中TNF-α和IL-6含量增加,Akt和NF-κB p65的磷酸化水平明显增加;与LPS组相比,LPS+H 1R激动剂组激活态星形胶质细胞减少,GFAP表达降低,培养基上清中TNF-α和IL-6的含量明显下降,Akt和NF-κB p65的磷酸化表达明显降低。结论H 1R激动剂能够抑制LPS诱导的星形胶质细胞活化和炎症因子的表达,且Akt/NF-κB通路可能是H 1R参与星形胶质细胞免疫调节的重要途径。

Aim To investigate the effect of histamine H 1 receptor(H 1R)on the immune responses in astrocytes induced by lipopolysaccharide(LPS)and the regulatory mechanism of its signaling pathway.Methods LPS was used to establish an in vitro astrocyte inflammation model.Rat primary astrocytes were divided into the control group,LPS group,LPS+H 1R agonist group(2-pyridylethlamine,Pyri),and H 1R agonist group.Astrocytes were treated with Pyri 100μmol·L-1 for 1 h,then stimulated with LPS at 100μg·L-1 for 24 h.Cell viability was measured using the CCK-8 assay.The expression of GFAP and H 1R was detected by immunofluorescence.Glial morphological changes were observed under a microscope.The levels of proinflammatory mediators(TNF-αand IL-6)were detected by ELISA.The protein expressions of p-Akt,Akt,p-NF-κB p65,and NF-κB p65 were detected by Western blot.Results Compared with the control group,more activated astrocytes with fewer cell processes and branches were observed in the LPS group.Besides,LPS enhanced the GFAP expression level,reduced the H 1R expression level and stimulated the production of TNF-αand IL-6 from astrocytes.Pretreatment with Pyri for 1 h ameliorated the glial morphological changes stimulated by LPS,inhibited LPS-induced upregulation of GFAP level and the inflammatory factors secretion.In addition,LPS stimulated astrocytes showed a higher phosphorylation of Akt and NF-κB p65,which was also ameliorated by Pyri.Conclusions H 1R agonist can inhibit LPS-induced astrocyte activation and inflammatory factor secretion,and the Akt/NF-κB signaling pathway may be an important pathway for the involvement of H 1R in immune regulation.