化浊解毒活血通络方对脑缺血再灌注损伤大鼠LPS及TLR4/NF-κB信号通路的影响

Effect of Huazhuo Jiedu Huoxue Tongluo Decoction on LPS and TLR4/NF-κB signaling pathway of cerebral ischemia-reperfusion injury rats

目的:观察化浊解毒活血通络方对大鼠脑缺血再灌注损伤(CIRI)的保护作用及对LPS及TLR4/NF-κB信号通路的影响。方法:60只SPF级雄性SD大鼠随机分为假手术组(Sham)、模型组(MCAO)、化浊解毒活血通络方低(TCM-L)、中(TCM-M)、高(TCM-H)剂量组,每组12只。Longa法结合既往研究经验制备永久性大脑中动脉梗塞大鼠模型。化浊解毒活血通络方低、中、高剂量组大鼠灌胃给予6.25、12.5、25 g/kg化浊解毒活血通络方,假手术与模型组灌胃给予2 ml生理盐水,给药后72 h检测大鼠神经功能缺失情况;HE染色观察脑、肠组织病理改变;TUNEL检测脑组织凋亡;TTC染色观察脑梗死面积;鲎试剂检测血清LPS浓度;ELISA检测血清DAO、脑组织IL-1β、TNF-α表达;Western blot检测脑组织TLR4、NF-κB、肠组织ZO-1、Occludin蛋白表达;RT-PCR检测脑组织MyD88、IRAK、TRAF6 mRNA表达。结果:与假手术组相比,模型组大鼠神经功能缺失加重,脑梗死面积增大,脑组织细胞凋亡增加,血清DAO、脑组织IL-1β、TNF-α表达升高(P<0.05),血清LPS表达升高(P<0.05),肠组织ZO-1、Occludin蛋白表达降低,脑组织TLR4、NF-κB、MyD88、IRAK、TRAF6蛋白及mRNA表达明显升高(P<0.05);与模型组相比,化浊解毒活血通络方各组大鼠神经功能缺失减轻,脑梗死面积减小,脑组织细胞凋亡减少,血清DAO、脑组织IL-1β、TNF-α表达降低(P<0.05),血清LPS表达降低(P<0.05),肠组织ZO-1、Occludin蛋白表达升高,脑组织TLR4、NF-κB、MyD88、IRAK、TRAF6蛋白及mRNA表达明显降低(P<0.05),TCM-H组最为显著,血清LPS表达下降(P<0.05)。结论:化浊解毒活血通络方可改善CIRI,其机制可能与降低LPS含量、保护肠道屏障、调控TLR4/NF-κB信号通路减轻炎症反应有关。

Objective:To observe protective effect of Huazhuo Jiedu Huoxue Tongluo Decoction on cerebral ischemia-reperfusion injury(CIRI)of rats and its effect on LPS and TLR4/NF-κB signaling pathway.Methods:Sixty SPF grade male SD rats were randomly divided into sham-operation group(Sham),model group(MCAO),Huazhuo Jiedu Huoxue Tongluo Decoction low(TCM-L),medium(TCM-M),high(TCM-H)doses groups,with 12 rats in each group.Permanent middle cerebral artery infarction rat model was established by Longa method and previous research experience.TCM-L,TCM-M and TCM-H groups were gastricly given 6.25,12.5,25 g/kg Huazhuo Jiedu Huoxue Tongluo Decoction.Sham group and MCAO group were gastricly given 2 ml saline.Neurological function defects of rats were detected at 72 h after administration.Pathological changes of brain and intestinal tissue were observed by HE staining.TUNEL to detect brain tissue apoptosis.TTC staining was used to was used to observe area of cerebral infarction.Serum LPS concentrations were detected by limulus reagent.Serum DAO expression,IL-1βand TNF-αexpressions in brain tissue were detected by ELISA.Protein expressions of TLR4 and NF-κB in brain tissue and ZO-1 and Occludin in intestinal tissues were detected by Western blot.mRNA expressions of MyD88,IRAK and TRAF6 in brain tissues were detected by RT-PCR.Results:Compared with Sham group,neural function impairment in MCAO group was aggravated,cerebral infarction area was increased,apoptosis of brain tissue cells was increased,expressions of serum DAO,IL-1βand TNF-αin brain tissue were increased(P<0.05),expression of serum LPS was increased(P<0.05),and expressions of ZO-1 and Occludin in intestinal tissue were decreased,protein and mRNA expressions of TLR4,NF-κB,MyD88,IRAK and TRAF6 in brain tissues were significantly increased(P<0.05).Compared with MCAO group,neurological function impairment was reduced in Huazhuo Jiedu Huoxue Tongluo Decoction groups,cerebral infarction area was decreased,brain tissue cell apoptosis was decreased,serum DAO,IL-1β,TNF-αexpressions were decreased(P<0.05),serum LPS expression was decreased(P<0.05),expressions of ZO-1 and Occludin in intestinal tissue were increased,protein and mRNA expressions of TLR4,NF-κB,MyD88,IRAK and TRAF6 in brain tissue were significantly decreased(P<0.05),and the most significant was found in TCM-H group,serum LPS expression was decreased(P<0.05).Conclusion:Huazhuo Jiedu Huoxue Tongluo Decoction can improve CIRI,whose mechanism may be related to reducing LPS content,protecting intestinal barrier,regulating TLR4/NF-κB signaling pathway and alleviating inflammatory response.