白果内酯对脓毒症急性肺损伤大鼠TLR4NFκB信号通路及Th1Th2细胞的影响

Effects of Bilobalide on TLR4/NFκB signaling pathway and Th1/Th2 cells in rats with sepsis induced acute lung injury

目的探讨白果内酯(BB)对脓毒症致急性肺损伤(ALI)大鼠Toll样受体-4(TLR4)/核因子-κB(NF-κB)信号通路及辅助性T细胞1/2(Th1/Th2)的影响。方法50只SD大鼠随机分为假手术组(Sham组)、模型组(ALI组)、BB低(2.5 mg/kg)剂量组、BB高(10 mg/kg)剂量组、地塞米松阳性对照组(0.45 mg/kg),每组各10只,除Sham组外,其余各组采用盲肠结扎穿孔法复制脓毒症ALI模型,术后6 h各药物组经尾静脉注射相应剂量药物,Sham组、ALI组经尾静脉注射生理盐水,均3次/d,共3 d。末次给药1 h后处死大鼠,取肺组织,以苏木精-伊红染色(HE)检测各组大鼠肺组织病理变化;以酶联免疫吸附(ELISA)试剂盒检测大鼠肺组织氧化物酶(MPO)活性、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平;凝胶电泳迁移率转变分析(EMSA)法检测肺组织NF-κB活性;以蛋白免疫印迹法检测肺组织中TLR4通路蛋白表达;采用流式细胞仪(FACS)检测肺脏细胞中辅助性T细胞1/2(Th1/Th2)比值。结果与Sham组相比,ALI组大鼠肺组织可见水肿、炎性细胞浸润等病理损伤,肺组织MPO、IL-6、TNF-α含量、TLR4蛋白表达、NF-κB活性均明显升高(P<0.05),Th1/Th2、TNF-α/IL-6均明显降低(P<0.05)。与ALI组相比,BB低、高剂量组及地塞米松阳性组肺组织水肿、炎性细胞浸润等病理损伤减轻,肺组织MPO、IL-6、TNF-α含量、TLR4蛋白表达、NF-κB活性均明显降低(P<0.05),Th1/Th2、TNF-α/IL-6均明显升高(P<0.05)。与BB低剂量组相比,BB高剂量组及地塞米松阳性组肺组织水肿、炎性细胞浸润等病理损伤减轻,MPO、IL-6、TNF-α含量、TLR4蛋白表达、NF-κB活性均明显降低(P<0.05),Th1/Th2、TNF-α/IL-6均明显升高(P<0.05)。结论BB可抑制TLR4/NF-κB信号通路活化,调节Th1/Th2平衡,减轻炎性介质释放,改善脓毒症ALI大鼠肺组织损伤。

Objective To investigate the effects of Bilobalide(BB)on Toll like receptor-4(TLR4)/nuclear factor-kappa B(NF-κB)signaling pathway and helper T lymphocyte 1/2(Th1/Th2)in sepsis induced acute lung injury(ALI)rats.Methods Fifty SD rats were randomly divided into sham operation group(sham group),model group(ALI group),BB low(2.5 mg/kg)dose group,BB high dose(10 mg/kg)group and dexamethasone positive control group(0.45 mg/kg).Except for sham group,the other groups were made into Ali model by cecal ligation and puncture.6 hours after operation,the corresponding dose of drugs was injected into caudal vein in each drug group In group I,normal saline was injected through caudal vein for 3 days.After the last administration of 1H,the rats were sacrificed and the lung tissues were taken.Hematoxylin eosin staining(HE)was used to detect the pathological changes of lung tissue in each group.Enzyme linked immunosorbent assay(ELISA)kit was used to detect the activity of lung tissue MPO,interleukin 6(IL-6)and tumor necrosis factor alpha(TNF alpha)level,and the lung tissue was detected by electrophoretic mobility shift assay(EMSA).The expression of TLR4 pathway protein in lung tissue was detected by Western blot,and the ratio of helper T cell 1/2(Th1/Th2)in lung cells was detected by flow cytometry(FACS).Results Compared with sham group,ALI group showed edema,inflammatory cell infiltration and other pathological damage.The contents of MPO,IL-6,TNF-α,TLR4 Protein Expression and NF-κB activity were significantly increased in ALI group(P<0.05),while Th1/Th2 and TNF-α/IL-6 were significantly decreased in ALI group(P<0.05).Compared with ALI group,pulmonary edema and inflammatory cell infiltration were alleviated in BB low and high dose groups and dexamethasone positive group.MPO,IL-6,TNF-αcontent,TLR4 Protein Expression and NF-κB activity in lung tissue were significantly decreased(P<0.05),Th1/Th2 and TNF-α/IL-6 were significantly increased(P<0.05).Compared with low-dose BB group,pulmonary edema and inflammatory cell infiltration in high-dose BB group and dexamethasone positive group were significantly reduced,MPO,IL-6,TNF-αcontent,TLR4 Protein Expression and NF-κB activity were significantly decreased(P<0.05),Th1/Th2 and TNF-α/IL-6 were significantly increased(P<0.05).Conclusion BB can inhibit the activation of TLR4/NF-κB signal pathway,regulate the balance of Th1/Th2,reduce the release of inflammatory mediators,and improve the lung injury in sepsis ALI rats.