基于TLR4/MyD88/NF-κB信号通路探讨复方石斛汤对气道炎症模型大鼠的影响

Effect of Compound Shihu Decoction on Airway Inflammation Model Rats Based on TLR4/MyD88/NF-κB Signal Pathway

目的:基于Toll样受体4(toll-like receptor 4,TLR4)/髓样分化因子88(myeloid differentiation factor 88,MyD88)/核转录因子-κB(nuclear transcription factor-κB,NF-κB)信号通路探讨复方石斛汤干预气道炎症的作用与机制。方法:84只Wistar大鼠随机分为空白组、模型组、甲强龙组(2 mg·kg^(-1))、复方石斛汤预防组(5.46 g·kg^(-1))及复方石斛汤高(10.92 g·kg^(-1))、中(5.46 g·kg^(-1))、低(2.73 g·kg^(-1))剂量组。除空白组外,其余组大鼠于实验第1、14天气道内滴注200μL脂多糖(1 g·L^(-1)),并于第2—13天及第15—28天置于烟熏箱内烟熏40 min,复方石斛汤预防组从实验第1天起灌胃复方石斛汤,复方石斛汤高、中、低剂量组及甲强龙组于实验第29—42天给予相应药物,空白组和模型组灌胃生理盐水。观察大鼠一般情况;HE染色观察肺组织病变;ELISA检测支气管肺泡灌洗液肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6水平;Western Blot及免疫组化检测肺组织TLR4、MyD88、NF-κB p65蛋白表达水平;RT-PCR检测肺组织TLR4 mRNA、MyD88 mRNA、NF-κB p65 mRNA表达水平。结果:与空白组比较,模型组大鼠精神、身体状态欠佳,咳嗽气促,肺组织炎性细胞浸润严重,TNF-α、IL-1β、IL-6水平显著升高(P<0.01),TLR4、MyD88、NF-κB p65蛋白与mRNA表达水平显著升高(P<0.05);与模型组比较,复方石斛汤各组大鼠精神、身体状态尚可,肺组织炎性细胞浸润减少,TNF-α、IL-1β、IL-6水平显著降低(P<0.01),TLR4、MyD88、NF-κB p65蛋白与mRNA表达水平显著降低(P<0.05);免疫组化显示TLR4、MyD88、NF-κB p65阳性表达降低(P<0.05)。结论:复方石斛汤在减轻呼吸道症状、抑制气道炎症方面起到了治疗及预防作用,常规剂量下,越早用药肺组织病变越轻微,抗炎效果越显著,且其机制可能与下调TLR4/MyD88/NF-κB信号通路表达有关。

Objective:Based on Toll like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear transcription factor-κB(NF-κB)to explore the effect and mechanism of compound Shihu Decoction on airway inflammation through signal pathways.Method:84 Wistar rats were randomly divided into a blank group,a model group,a methylprednisolone group(2 mg·kg^(-1)),a compound Dendrobium decoction prevention group(5.46 g·kg^(-1)),and a compound Dendrobium decoction high(10.92 g·kg^(-1)),medium(5.46 g·kg^(-1)),and low(2.73 g·kg^(-1))dose group.Except the blank group,rats in other groups were given Lipopolysaccharide(1 g·L^(-1).200μL).On days between 2-13 and 15-28,smoke in a fumigation box for 40 minutes.The prevention group of Compound Dendrobium Decoction was orally administered from the first day of the experiment.The high,medium,and low dose groups of Compound Dendrobium Decoction and the methylprednisolone group were given corresponding drugs on days 29-42 of the experiment.The blank group and model group were orally administered with physiological saline.The general situation of rats was observed;HE staining was used to observe lung tissue lesions;TNF-α,IL-1βand IL-6 level were detected with ELISA;immune-histochemical detection of TLR4,MyD88,NF-κB p65 protein expression level was made with Western Blot;TLR4,MyD88,NF-κB p65 mRNA expression levels were detected with RT-PCR.Results:Compared with the blank group,the model group rats showed poor mental and physical condition,cough and shortness of breath,severe infiltration of inflammatory cells in lung tissue,and levels of TNF-α,IL-1βand IL-6 significantly increased(P<0.01);the expression of TLR4,MyD88,NF-κβand Bp65 protein and mRNA significantly increased(P<0.05);Compared with the model group,the mental and physical states of the rats in each group of compound Shihu Decoction were still good,and the infiltration of inflammatory cells in lung tissue was reduced,and TNF-α,IL-1βand IL-6 levels in each group significantly decreased(P<0.01),The expression of TLR4,MyD88,NF-κB p65 protein and mRNA was significantly reduced(P<0.05).The positive expression of TLR4,MyD88,NF-κB p65 decreased in immuno-histochemical test(P<0.05).Conclusion:Compound Shihu Decoction has played a therapeutic and preventive role in reducing respiratory symptoms and inhibiting airway inflammation.Under conventional doses,the earlier the medication is used,the milder the lung tissue lesion,and the more significant the anti-inflammatory effect.Its mechanism may be related to downregulating the expression of the TLR4/MyD88/NF-κB signaling pathway.