基于TLR4/NF-κB通路探讨阳和平喘颗粒对哮喘大鼠气道炎症和气道重塑的实验研究

Effect of Yanghe Pingchuan Granules(阳和平喘颗粒)on Airway Inflammation and Airway Remodeling in Asthmatic Rats Based on TLR4/NF-κB Pathway

目的:基于Toll样受体4/核转录因子-κB(TLR4/NF-κB)信号通路,研究Toll样受体4抑制剂干预后阳和平喘颗粒对哮喘模型大鼠肺组织气道炎症及气道重塑的影响,探讨阳和平喘颗粒治疗哮喘的作用机制。方法:随机数字法将SD大鼠分为正常对照组、模型对照组、阳和平喘颗粒7.74 g/kg组、Toll样受体4抑制剂0.001 g/kg组、阳和平喘颗粒7.74 g/kg+Toll样受体4抑制剂0.001 g/kg组,联合应用氢氧化铝和卵蛋白致敏诱发大鼠,建立哮喘动物模型,药物干预14 d后,采用HE染色观察肺组织病理改变,吉姆萨染色(Giemsa)观察肺泡灌洗液中炎性细胞的数目,酶联免疫吸附法检测大鼠血清中白介素-6(IL-6)、IL-1β、肿瘤坏死因子(TNF-α)含量;采用免疫荧光法检测TLR4、磷酸化核转录因子-κB(p-NF-κB)、α-平滑肌肌动蛋白(α-SMA)表达情况;qPCR法检测大鼠肺组织Tlr4、Nfkb、核转录因子-κB抑制蛋白(Ikb)mRNA的表达;Western blotting法检测大鼠肺组织TLR4、p-NF-κB、磷酸化核转录因子-κB抑制蛋白(p-IκB)、α-SMA蛋白的表达。结果:与正常对照组比较,模型对照组肺泡灌洗液中嗜酸性细胞(Eos)、中性粒细胞(Neu)、淋巴细胞(Lym)数目显著升高(P<0.01),血清中TNF-α、IL-6、IL-1β含量显著升高,肺组织Tlr4、Nfkb mRNA表达显著上调(P<0.01),Ikb mRNA显著下调,TLR4、p-NF-κB、α-SMA蛋白表达上调,p-IκB表达下调(P<0.01);与模型对照组比较,各给药组血清中TNF-α、IL-6、IL-1β含量显著下降(P<0.01),肺泡灌洗液中Eos、Neu、Lym数目明显减少,肺组织中Tlr4、Nfkb mRNA表达下调,Ikb mRNA表达上调,TLR4、p-NF-κB、α-SMA蛋白表达明显下调,p-IκB显著上调(P<0.05或P<0.01);与Toll样受体4抑制剂0.001 g/kg组相比,阳和平喘颗粒7.74 g/kg+Toll样受体4抑制剂0.001 g/kg组Eos、Lym数目及TNF-α、IL-6、IL-1β含量显著降低(P<0.01),TLR4、p-NF-κB、α-SMA蛋白表达明显下调,p-IκB表达明显上调,Tlr4、Nfkb mRNA下调,Ikb mRNA上调(P<0.01)。结论:阳和平喘颗粒可有效改善哮喘大鼠气道炎症和气道重塑,其作用机制可能与抑制哮喘大鼠肺组织TLR4/NF-κB信号通路和减少血清炎症因子TNF-α、IL-6、IL-1β的分泌相关。

Objective:Based on the Toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)signaling pathway,to investigate the effects of Yanghe Pingchuan Granules(阳和平喘颗粒)on airway inflammation and airway remodeling in the lung tissue of asthmatic rats after TLR4 inhibitor intervention,and to explore the mechanism of Yanghe Pingchuan Granules in the treatment of asthma.Methods:SD rats were randomly divided into a normal control group,a model control group,a 7.74 g/kg Yanghe Pingchuan Granule group,a 0.001 g/kg TLR4 inhibitor group,and a 7.74 g/kg Yanghe Pingchuan Granules+0.001 g/kg TLR4 inhibitor group by the random number table method.The rat model of asthma was induced by the combination of aluminium hydroxide and ovalbumin.After 14 days of drug intervention,hematoxylin-eosin(HE)staining was used to observe the pathological changes in the lung tissue,and Giemsa staining was used to observe the number of inflammatory cells in the alveolar lavage fluid.The content of interleukin-6(IL-6),tumor necrosis factor(TNF-α),and interleukin-1β(IL-1β)in the serum of rats was determined by the enzyme-linked immunosorbent assay(ELISA).The expression levels of TLR4,phosphorylated NF-κB(p-NF-κB),andα-smooth muscle actin(α-SMA)were determined by immunofluorescence.The mRNA expression levels of TLR4,NF-κB,and NF-κB inhibitor protein(IκB)in the lung tissue of rats in each group were determined by real-time quantitative polymerase chain reaction(real-time qPCR).The protein expression levels of TLR4,p-NF-κB,phosphorylated IκB(p-IκB),andα-SMA were determined by Western blot.Results:As compared with the normal control group,the numbers of eosinophils(Eos),neutrophils(Neu),and lymphocytes(Lym)in the model control group were significantly increased(P<0.01),the content of TNF-α,IL-6,and IL-1βin serum were significantly increased,the mRNA expression levels of Tlr4 and Nfκb in the lung tissue of rats were significantly up-regulated(P<0.01),whereas the mRNA expression level of Iκb was significantly down-regulated(P<0.01),the protein expression levels of TLR4,p-NF-κB,andα-SMA in the lung tissue were significantly up-regulated,and p-IκB was down-regulated(P<0.01).As compared with the model control group,the content of TNF-α,IL-6,and IL-1βin the serum of the 7.74 g/kg Yanghe Pingchuan Granule and 7.74 g/kg Yanghe Pingchuan Granule+0.001 g/kg TLR4 inhibitor group were significantly down-regulated(P<0.01),the numbers of Eos,Neu,and Lymin the alveolar lavage fluid were significantly down-regulated,the mRNA expression levels of Tlr4 and Nfkb in the lung tissue of rats were significantly down-regulated(P<0.01),whereas the mRNA expression level of Iκb was significantly up-regulated(P<0.01),the protein expression levels of TLR4,p-NF-κB,andα-SMA in the lung tissue were decreased.and the expression of p-IκB was increased(P<0.05,P<0.01).As compared with the 0.001 g/kg TLR4 inhibitor group,the numbers of Eos and Lym and the content of TNF-α,IL-6,and IL-1βwere significantly decreased in the 7.74 g/kg Yanghe Pingchuan Granule+0.001 g/kg TLR4 inhibitor group(P<0.01),the protein expression levels of TLR4,p-NF-κB,andα-SMA were significantly down-regulated,and the level of p-IκB was significantly up-regulated,the mRNA expression levels of Tlr4 and Nfkb in the lung tissue of rats were significantly down-regulated(P<0.01),whereas the mRNA expression level of Iκb was significantly up-regulated(P<0.01).Conclusion:Yanghe Pingchuan Granules can effectively improve airway inflammation and airway remodeling in asthmatic rats,and its mechanism may be related to the inhibition of the TLR4/NF-κB signaling pathway and the reduction of serum inflammatory factors TNF-α,IL-6,and IL-1βin asthmatic rats.