清金化痰汤通过调节p62对AECOPD炎症因子的影响

Effect of Qingjin Huatantang on AECOPD Inflammatory Factors by Regulating p62

目的:通过清金化痰汤调节慢性阻塞性肺疾病急性加重期(AECOPD)大鼠气道上皮细胞的自噬蛋白p62,探讨其对白细胞介素(IL)-1β,肿瘤坏死因子-α(TNF-α),白三烯B(4LTB4)以及白三烯C(4LTC4)的影响。方法:原代培养气道上皮细胞和AECOPD气道上皮细胞,另将60只SPF级雄性SD大鼠随机分为6组清金化痰汤高、中、低剂量组、罗红霉素组、模型组以及正常组,各10只。除正常组外,其余各组采用脂多糖(LPS)气管滴入法+烟熏联合方法构建AECOPD模型。造模后清金化痰汤高、中、低剂量组分别灌胃给予30,15,7.5 g·kg^-1·d^-1剂量,罗红霉素组灌胃给予0.0175 g·kg^-1·d^-1剂量,模型组和正常组灌胃生理盐水,灌胃体积为20 mL·kg^-1·d^-1。给药2周后提取血清,用含药血清干预细胞,酶联免疫吸附测定法(ELISA)检测细胞上清液IL-1β,TNF-α,LTB4和LTC4的含量变化,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测肺气道上皮细胞中p62 mRNA及蛋白的表达。结果:ELISA结果显示,与正常组比较,模型组中的IL-1β,TNF-α,LTB4,LTC4显著升高(P<0.01),与模型组比较,各给药组细胞上清液中的IL-1β,TNF-α,LTB4,LTC4显著降低(P<0.01);p62mRNA和蛋白表达结果显示,与正常组比较,模型组p62 mRNA和蛋白表达显著降低(P<0.01),与模型组比较,各给药组P62中的mRNA和蛋白表达显著升高(P<0.01),清金化痰汤高剂量组与罗红霉素组自噬表达相当。结论:清金化痰汤能降低气道上皮细胞中炎症反应的机制可能与调节自噬蛋白p62有关。

Objective:To regulate autophagy protein p62 of airway epithelial cells in acute exacerbation of chronic obstructive pulmonary disease(AECOPD)rats with Qingjin Huatantang,in order to explore its effect on interleukin(IL)-1βand tumor necrosis,tumor necrosis factor-α(TNF-α),leukotriene B4(LTB4),and leukotriene C4(LTC4).Method:Airway epithelial cells and AECOPD airway epithelial cells were cultured.Sixty SPF male SD rats were randomly divided into 6 groups,namely high,medium and low-dose Qingjin Huatantang groups,western medicine group,model group and normal group.Except for the normal group,the remaining groups were included into the AECOPD model by lipopolysaccharide(LPS)tracheal instillation method+fumigation method.After modeling,the dosage of the high-dose traditional Chinese medicine group was 30 g·kg^-1·d^-1,that of the middle-dose group was 15 g·kg^-1·d^-1,that of the low-dose group was 7.5 g·kg^-1·d^-1,the positive control group was given luo erythromycin(0.0175 g·kg^-1·d^-1),the model group and the blank control group were orally given normal saline with the volume of 20 mL·kg^-1·d^-1.Serum was extracted two weeks after administration,and the cells were intervened with drug-containing serum.The content of interleukin IL-1β,TNF-α,and LTB4 in cell supernatants were detected by enzyme-linked immunosorbent assay(ELISA).And LTC4 content,p62 mRNA and protein expressions in lung airway epithelial cells were detected by quantitative real-time fluorescence polymerase chain reaction(Real-time PCR)and Western blot.Result:Compared with the normal group,ELISA results showed that IL-1β,TNF-α,LTB4,and LTC4 in the model group were significantly increased(P<0.01).Compared with the model group,IL-1β,TNF-α,LTB4,LTC4 in cell supernatants in each administration group were significantly reduced(P<0.01),mRNA and protein expressions in p62 showed that compared with the normal group,mRNA and protein expressions in p62 of model group significantly decreased(P<0.01).Compared with the model group,the mRNA and protein expressions of p62 in each administration group significantly increased to different degrees(P<0.01).The expression of autophagy in Qingjin Huatantang high-dose group and western medicine group was comparable.Conclusion:Qingjin Huatantang can reduce the inflammatory response in airway epithelial cells,which may be related to the regulation of autophagy protein p62.