钠钾泵Scr复合体调控幼年哮喘大鼠气道重塑的机制

Mechanisms of sodium-potassium pump Scr complex for regulating airway remodeling in juvenile asthmatic rats

目的探究钠钾泵琥珀酸-细胞色素c氧化还原酶(succinate-cytochrome c oxidoreductase,Scr)复合体调控幼年哮喘大鼠气道重塑的机制。方法筛选30只SD大鼠,随机分为对照组、卵白蛋白(ovalbumin,OVA)组和钠钾泵Scr复合体组,每组10只。对照组:经盐水处理并经雾化盐水激发的大鼠;OVA组:在OVA激发之前接受过盐水治疗的OVA致敏大鼠;钠钾泵Scr复合体组:将钠钾泵Scr复合体药物溶解在二甲亚砜中并用磷酸盐缓冲盐水(phosphate buffered saline,PBS)稀释,按照0.3 mg/kg体质量,腹膜给药。使用连接到呼吸机和雾化装置的气管插管,使用Buxco肺功能仪测量大鼠的气道反应性。肺组织石蜡切片用苏木精-伊红(HE)和高碘酸-席夫(PAS)染色以进行光学显微镜检查。通过蛋白质免疫印迹检测磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)和蛋白激酶B(protein kinase B,PKB)磷酸化。通过RT-qPCR检测哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA表达水平。通过细胞计数试剂8(cell counting kit-8,CCK-8)检测细胞增殖。结果与对照组相比,OVA组PC100值降低,气道反应性升高(P<0.05);与OVA组相比,钠钾泵Scr复合体组PC100值升高,气道反应性降低(P<0.05)。HE和PAS染色结果显示,与对照组相比,OVA组的肺组织显示出广泛的支气管周围和血管周围炎性细胞浸润,PAS阳性染色的上皮杯状细胞的百分比增加(P<0.05);与OVA组相比,钠钾泵Scr复合体组炎症细胞浸润减少,PAS阳性染色的上皮杯状细胞的百分比降低(P<0.05)。与对照组相比,OVA组PI3K和PKB磷酸化水平升高(P<0.05);与OVA组相比,钠钾泵Scr复合体组PI3K和PKB磷酸化水平降低(P<0.05)。与对照组相比,OVA组mTOR、HIF-1α和VEGF mRNA水平升高(P<0.05);与OVA组相比,钠钾泵Scr复合体组mTOR、HIF-1α和VEGF mRNA水平降低(P<0.05)。与对照组相比,OVA组48 h和72 h增殖速率增加(P<0.05);与OVA组相比,钠钾泵Scr复合体组48 h和72 h增殖速率增加(P<0.05)。与对照组相比,OVA组48 h和72 h增殖速率增加(P<0.05);与OVA组相比,钠钾泵Scr复合体组48 h和72 h增殖速率增加(P<0.05)。结论钠钾泵Scr复合体通过调节PI3K/PKB/mTOR/HIF-1α/VEGF,调控幼年哮喘大鼠气道重塑。钠钾泵Scr复合体可能是过敏性气道炎症的关键介质,也是治疗干预的有希望的靶标。

Objective To explore the mechanism by which the sodium potassium pump Scr complex regulates airway remodeling in juvenile asthmatic rats.Methods Thirty SD rats were screened and randomly divided into three groups:the control group(saline treated and atomized saline stimulated rats,n=10),OVA group[OVA-sensitized rats treated with saline prior to ovalbumin(OVA)challenge,n=10]and sodium-potassium pump Scr complex group(sodium-potassium pump Scr complex drug dissolved in dimethyl sulfoxide and diluted in PBS,0.3 mg/kg body weight,peritoneal administration,n=10).Airway responsiveness was measured in rats using an endotracheal tube connected to a ventilator and a nebulizer device using a Buxco pulmonary function meter.The paraffin sections of lung tissue were stained with hematoxylin-eosin(HE)and periodic acid-Schiff stain(PAS)for light microscopy.The phosphorylation of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(PKB)was detected by Western blotting.mRNA expression levels of mammalian target of rapamycin(mTOR),hypoxia inducible factor-1α(Hif-1α)and vascular endothelial growth factor(VEGF)were detected by RTqPCR.Cell proliferation was detected by Cell Counting KIT-8(CCK-8).Results Compared with the control group,PC100 value decreased and airway reactivity increased in the OVA group(P<0.05).Compared with the OVA group,PC100 value increased and airway reactivity decreased in the sodium potassium pump Scr complex group(P<0.05).The results of HE and PAS staining showed that compared with the control group,the lungs in the OVA group showed extensive infiltration of inflammatory cells around the bronchus and blood vessels,and that the percentage of PAS positive epithelial goblet cells increased(P<0.05).Compared with OVA group,the infiltration of inflammatory cells in the sodium potassium pump SCR complex group decreased,and the percentage of PAS positive epithelial goblet cells decreased(P<0.05).Compared with the control group,the phosphorylation levels of PI3K and PKB in the OVA group were increased(P<0.05).Compared with the OVA group,the phosphorylation levels of PI3K and PKB in the sodium potassium pump Scr complex group were decreased(P<0.05).Compared with the control group,the mRNA levels of mTOR,HIF-1αand VEGF in the OVA group were increased(P<0.05).Compared with the OVA group,the mRNA levels of mTOR,HIF-1αand VEGF in the sodium potassium pump Scr complex group were decreased(P<0.05).Compared with the control group,the proliferation rate at 48 h and 72 h in the OVA group increased(P<0.05).Compared with the OVA group,the proliferation rate at 48 h and 72 h in the sodium potassium pump Scr complex group increased(P<0.05).Compared with the control group,the proliferation rate at 48 h and 72 h in OVA group increased(P<0.05).Compared with the OVA group,the proliferation rate at 48 h and 72 h in the sodium potassium pump Scr complex group increased(P<0.05).Conclusion The sodium-potassium pump Scr complex can regulate airway remodeling in juvenile asthmatic rats by regulating levels of PI3K,PKB,mTOR,HIF-1αand VEGF,which may be a key mediator of allergic airway inflammation and a promising target for therapeutic intervention.