百令胶囊上调miR-145-5p逆转TGF-β_(1)/Smad3通路机制初探

Preliminary Study on the Mechanism of Bailing Capsules Reversing TGF-β_(1)/Smad3 Pathway by Upregulation of miR-145-5p

目的探讨百令胶囊(BLC)对慢性阻塞性肺疾病(COPD)模型大鼠肺损伤的改善作用,以及对香烟烟雾提取物(CSE)作用下人支气管上皮细胞BEAS-2B生长的影响。方法将60只SD大鼠随机分为正常对照组(A组,灌胃生理盐水2 mL),模型组(B组,灌胃生理盐水2 mL),BLC组(C组,灌胃BLC 5.5 mg/kg),miR-145-5p inhibitorNC组(D组,灌胃BLC 5.5 mg/kg+尾静脉注射miR-145-5p inhibitorNC 6μL),miR-145-5p inhibitor组(E组,灌胃BLC 5.5 mg/kg+尾静脉注射miR-145-5p inhibitor 6μL),各12只。以香烟烟雾吸入法复制大鼠COPD模型。建模成功后灌胃相应药物或生理盐水(每日1次),尾静脉注射相应药物(每周1次),连续8周。以0(空白对照),3%,5%,10%,20%CSE,以及加5%,10%,20%,40%的BLC药物血清(BLC-S)孵育细胞48 h。检测大鼠潮气量(TV)、呼气峰流量(PEF)、分钟通气量(MV)、用力肺活量(FVC)和第0.3秒用力呼气容积(FEV_(0.3));观察肺组织病理形态;免疫组化法检测Smad3表达水平,用酶联免疫吸附试验(ELISA)法测定转化生长因子(TGF)-β_(1)表达水平,采用实时荧光定量聚合酶链反应(RT-qPCR)法检测miR-145-5p mRNA表达水平。采用CCK-8法检测细胞活力。结果与B组比较,C组及D组大鼠TV,PEF,MV,FVC,FEV0.3均显著升高;与D组比较,E组大鼠上述指标均显著降低(P<0.05)。与B组比较,C组及D组大鼠肺组织病理形态显著改善,平均肺泡数显著增加,肺泡直径显著减小;与D组比较,E组大鼠肺组织病理形态未改善,且平均肺泡数显著减少,肺泡直径显著增长(P<0.05)。与5%CSE条件比较,5%CSE+20%BLC-S条件下的细胞活力显著增强(P<0.05)。与B组比较,C组及D组大鼠肺组织中Smad3,TGF-β_(1)的表达水平均显著降低,miR-145-5p mRNA表达水平显著升高(P<0.05);与D组比较,E组大鼠肺组织中Smad3,TGF-β_(1)的表达水平均显著升高,miR-145-5p mRNA表达水平显著降低(P<0.05)。与5%CSE条件比较,5%CSE+20%BLC-S条件下细胞Smad3和TGF-β_(1)的表达水平均显著降低,miR-145-5p mRNA表达水平显著升高(P<0.05);与5%CSE+20%BLC-S+inhibitor NC条件比较,同水平inhibitor条件下细胞Smad3及TGF-β_(1)表达水平均显著升高(P<0.05)。结论BLC在体内和体外均显示出对香烟诱导COPD的保护作用,其机制可能与上调miR-145-5p水平逆转香烟暴露激活的TGF-β_(1)/Smad3信号通路有关。

Objective To investigate the improvement effect of Bailing Capsules(BLC)on the lung injury in model rats with chronic obstructive pulmonary disease(COPD),and its effect on the growth of human bronchial epithelial cells BEAS-2B exposed to cigarette smoke extract(CSE).Methods Sixty SD rats were randomly divided into the normal control group(group A,2 mL physiological saline by gavage),the model group(group B,2 mL physiological saline by gavage),the BLC group(group C,5.5 mg/kg BLC by gavage),the miR-145-5p inhibitor NC group(group D,5.5 mg/kg BLC by gavage+6µL miR-145-5p inhibitor NC by tail vein injection),the miR-145-5p inhibitor group(group E,5.5 mg/kg BLC by gavage+6µL miR-145-5p inhibitor by tail vein injection),with 12 rats in each group.Rat models with COPD were replicated by the cigarette smoke inhalation method.After successful modeling,corresponding drugs or physiological saline were given by gavage(once a day),and corresponding drugs were injected into the tail vein(once a week).All rats were administered continuously for eight weeks.Cells were exposed to 0,3%,5%,10%,20%CSE(with 0 as the blank control)and incubated with the addition of 5%,10%,20%,40%BLC serum(BLC-S)for 48 h.Tidal volume(TV),peak expiratory flow(PEF),minute ventilation(MV),forced vital capacity(FVC)and forced expiratory volume in 0.3 second(FEV_(0.3))of rats were measured.The pathological morphology of lung tissue were observed.The expression level of Smad3 was detected by the immunohistochemical assay,the expression level of transforming growth factor-β_(1)(TGF-β_(1))was detected by the enzyme-linked immunosorbent assay(ELISA),and the expression level of miR-145-5p mRNA was detected by the real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Cell viability was detected by the CCK-8 method.Results Compared with those in the group B,the TV,PEF,MV,FVC and FEV_(0.3) in the groups C and D significantly increased,and compared with those in the group D,the above indicators in the group E significantly decreased(P<0.05).Compared with those in the group B,the pathological morphology of lung tissue in the groups C and D significantly improved,the average alveolus count significantly increased,and the alveolus diameter was significantly shorter(P<0.05).Compared with those in the group D,the pathological morphology of lung tissue in the group E did not improve,the average alveolus count significantly decreased,and the alveolus diameter was significantly longer(P<0.05).Compared with that at 5%CSE,the cell viability at 5%CSE+20%BLC-S significantly increased(P<0.05).Compared with those in the group B,the Smad3 and TGF-β_(1)expression levels of lung tissue in the groups C and D significantly decreased,and the miR-145-5p mRNA expression level significantly increased(P<0.05).Compared with those in the group D,the Smad3 and TGF-β_(1)expression levels of lung tissue in the group E significantly increased,and the miR-145-5p mRNA expression level significantly decreased(P<0.05).Compared with those at 5%CSE,the Smad3 and TGF-β_(1)expression levels at 5%CSE+20%BLC-S significantly decreased,and the miR-145-5p mRNA expression level significantly increased(P<0.05).Compared with those at 5%CSE+20%BLC-S+inhibitor NC,Smad3 and TGF-β_(1)expression levels at 5%CSE+20%BLC-S+inhibitor significantly increased(P<0.05).Conclusion BLC shows protective effect on the cigarette-induced COPD in vivo and in vitro,and its mechanism may be related to reversing cigarette-activated TGF-β_(1)/Smad3 signaling pathway by upregulating the level of miR-145-5p.