五虎汤加味对RSV感染哮喘小鼠气道炎症及肺组织Muc5AC、STAT3、NF-κB、NLRP3蛋白表达的影响

Modified Wuhutang Alleviates Airway Inflammation and Down-regulates Expression of Muc5AC,STAT3,NF-κB,and NLRP3 in Lung Tissues of RSV-infected Asthmatic Mice

目的:探讨五虎汤加味对呼吸道合胞病毒(RSV)感染哮喘小鼠气道炎症及黏蛋白(Muc)5AC、转录激活因子3(STAT3)、核转录因子-κB(NF-κB)、NOD样受体蛋白3(NLRP3)蛋白表达的影响。方法:70只雄性6~8周龄BALB/c小鼠,每组10只,随机分为正常组、哮喘组(卵清蛋白,OVA)、RSV感染哮喘组(OVA+RSV)、五虎汤加味组(高、中、低剂量分别为4.08、2.04、1.02 g·kg^(-1)·d^(-1))、地塞米松组(0.1 g·kg^(-1)·d^(-1))。OVA致敏及雾化激发哮喘模型,经RSV滴鼻连续3次感染(1.0×10^(6) PFU·mL^(-1),50μL),构建RSV感染哮喘模型;五虎汤加味组予五虎汤加味灌胃;地塞米松组给予地塞米松注射液腹腔注射;正常组予等量生理盐水灌胃。末次激发24 h,在2.5%戊巴比妥钠麻醉下,留取肺组织进行苏木素-伊红(HE)、苦味酸-酸性品红(VG)染色观察气道炎症反应,免疫组织化学检测Muc5AC蛋白表达情况;蛋白免疫印迹法(Western blot)检测磷酸化(p)-STAT3、STAT3、p-NF-κB、NF-κB、NLRP3蛋白表达情况。结果:与正常组比较,OVA组小鼠气道炎性细胞浸润,组织充血水肿,胶原纤维沉积;OVA+RSV组小鼠气道炎性细胞浸润,组织充血水肿较OVA组更显著;与OVA+RSV组比较,五虎汤加味组能显著改善气道周围炎性细胞浸润,组织充血水肿,胶原纤维沉积明显减轻,其中高剂量组作用更加明显。免疫组化结果显示,与正常组比较,OVA组和OVA+RSV组Muc5AC蛋白表达水平均显著升高(P<0.01);与OVA+RSV组比较,五虎汤加味组可明显降低Muc5AC蛋白表达水平,其中高剂量组作用更加明显(P<0.05);与五虎汤加味高剂量组比较,地塞米松组可明显降低Muc5AC蛋白表达水平(P<0.05)。在蛋白表达水平,与正常组比较,OVA组、OVA+RSV组p-STAT3、p-NF-κB、NLRP3蛋白表达明显升高(P<0.05,P<0.01);与OVA+RSV组比较,五虎汤加味组可显著降低p-STAT3、p-NF-κB、NLRP3蛋白表达水平(P<0.01);与五虎汤加味高剂量组比较,地塞米松组可显著升高p-STAT3、p-NF-κB蛋白表达水平(P<0.01)。结论:五虎汤加味可以明显减轻RSV感染哮喘小鼠气道炎症,降低Muc5AC、p-STAT3、p-NF-κB、NLRP3蛋白的表达,提示五虎汤加味减轻RSV感染哮喘气道炎症可能与调控STAT3/NF-κB信号通路抑制NLRP3蛋白表达相关。

Objective:To explore effect of modified Wuhutang on airway inflammation and expression of mucin(Muc)5AC,signal transducer and activator of transcription 3(STAT3),nuclear factor kappa B(NF-κB),and NOD-like receptor thermal protein domain associated protein 3(NLRP3)in respiratory syncytial virus(RSV)-infected asthmatic mice.Method:Seventy male BALB/c mice of 6-8 weeks old were randomized into normal control(CON),asthma(ovalbumin,OVA),RSV infection-induced asthma(OVA+RSV),high-,medium-,and low-dose(4.08,2.04,1.02 g·kg^(-1)·d^(-1),respectively)modified Wuhutang,and dexamethasone(Dxms,0.1 g·kg^(-1) d^(-1))groups(n=10).The model of asthma was established by sensitization and atomization inhalation with OVA.The RSV infection-induced asthma model was established by three consecutive RSV nasal infusions(1.0×10^(6) PFU·mL^(-1),50μL).Wuhutang was administrated by gavage,and Dxms by intraperitoneal injection.The CON group was given the same amount of normal saline by gavage.The mice were anesthetized with 2.5%pentobarbital sodium 24 h after the last administration,and then the lung tissue was stained by hematoxylin-eosin(HE)and Van Gieson(VG)for observation of airway inflammation.The immunohistochemical assay was employed to detect the expression of Muc5AC.Western blot was employed to determine the protein levels of phosphorylated(p)-STAT3,STAT3,p-NF-κB,NF-κB,and NLRP3.Result:Compared with the CON group,the OVA group presented airway inflammatory cell infiltration,tissue hyperemia and edema,and collagen fiber deposition.The OVA+RSV group showed severer airway inflammatory cell infiltration and tissue hyperemia and edema than the OVA group.Compared with the OVA+RSV group,modified Wuhutang alleviated the airway inflammatory cell infiltration,tissue hyperemia and edema,and collagen fiber deposition,and the high-dose group had the best performance.Compared with the CON group,the OVA group and the OVA+RSV group showed increased expression level of Muc5AC(P<0.01).Compared with the OVA+RSV group,modified Wuhutang reduced the expression level of Muc5AC,and the reduction was significant in the high-dose group(P<0.05).Compared with the high-dose modified Wuhutang group,Dxms lowered the expression level of Muc5AC(P<0.05).Compared with the CON group,the OVA and OVA+RSV groups showed up-regulated protein levels of p-STAT3,p-NF-κB,and NLRP3(P<0.05,P<0.01).Compared with the OVA+RSV group,modified Wuhutang down-regulated the protein levels of p-STAT3,p-NF-κB,and NLRP3(P<0.01).Compared with the high-dose modified Wuhutang group,the Dxms group showed up-regulated levels of p-STAT3,p-NF-κB proteins(P<0.01).Conclusion:Modified Wuhutang can reduce airway inflammation and down-regulate the expression of Muc5AC,p-STAT3,p-NF-κB,and NLRP3 in RSV-infected asthmatic mice,which suggests that Wuhutang reduces airway inflammation in RSV-infected asthma by regulating the STAT3/NF-κB signaling pathway.