白芍总苷调控Nrf-2/HO-1信号通路对支气管哮喘小鼠气道重塑的影响

Effects of total glycosides of paeony on airway remodeling in mice with bronchial asthma by regulating Nrf-2/HO-1 signaling pathway

目的:探究白芍总苷(TGP)对支气管哮喘模型小鼠气道重塑的影响及潜在机制。方法:将50只小鼠随机分为对照组(Control组)、模型组(OVA组)、TGP低剂量组(TGP-L,460 mg/kg)、TGP高剂量组(TGP-H,920 mg/kg)、TGP+ML385组(TGP 920 mg/kg+ML385 30 mg/kg),每组10只。采用卵清白蛋白(OVA)致敏和激发两个阶段建立哮喘小鼠模型。在每次激发前1 h,TGP各剂量组灌胃相应剂量的TGP混悬液,TGP+ML385组给予30 mg/kg的ML385和920 mg/kg的TGP灌胃,连续给药8周,实验过程中观察各组小鼠的行为学变化,最后一次激发24 h后收集支气管肺泡灌洗液(BALF),ELISA检测BALF中TGF-β1、半胱氨酰白三烯1(CysLT1)、半胱氨酰白三烯受体1(CysLTR1)水平;检测肺匀浆中总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性;HE和AB-PAS染色观察肺组织病理学变化;RT-qPCR检测肺组织TGF-β1、基质金属蛋白酶9(MMP-9)和金属蛋白酶组织抑制因子1(TIMP-1)的mRNA表达;Western blot检测肺组织TGF-β1/Nrf-2/HO-1通路相关蛋白表达。结果:与Control组相比,OVA组小鼠出现典型的哮喘症状,如打喷嚏、烦躁不安、喘息,BALF中TGF-β1、CysLT1、CysLTR1水平、肺组织炎症和黏液分泌评分、TGF-β1 mRNA和蛋白、MMP-9 mRNA表达显著升高(P<0.05),肺组织中T-AOC、SOD、GSH-Px、CAT活性、TIMP-1 mRNA表达显著降低(P<0.05),肺组织Nrf-2和HO-1蛋白表达有所增加,但差异无统计学意义(P>0.05);与OVA组相比,TGP-L组和TGP-H组小鼠的哮喘症状明显减轻,BALF中TGF-β1、CysLT1、CysLTR1水平、肺组织炎症和黏液分泌评分、TGF-β1 mRNA和蛋白、MMP-9 mRNA表达显著降低(P<0.05),肺组织中T-AOC、SOD、GSH-Px、CAT活性、TIMP-1 mRNA表达、Nrf-2和HO-1蛋白表达显著升高(P<0.05);且使用Nrf2抑制剂ML385阻断Nrf-2/HO-1通路激活可明显减弱TGP对哮喘小鼠肺组织氧化应激和气道重塑的抑制作用。结论:TGP可调节氧化应激和炎症诱发的支气管哮喘小鼠的气道重塑,其作用机制可能与降低TGF-β1表达、激活Nrf-2/HO-1通路有关。

Objective:To explore the effect of total glucosides of paeony(TGP)on airway remodeling in mice with bronchial asthma and its underlying mechanism.Methods:Fifty mice were randomly divided into control group(Control group),model group(OVA group),TGP low-dose group(TGP-L,460 mg/kg),TGP high-dose group(TGP-H,920 mg/kg),TGP+ML385 group(TGP 920 mg/kg+ML38530 mg/kg),with 10 mice in each group.Two stages of ovalbumin(OVA)sensitization and stimulation were used to establish mice model of asthma.One hour before each stimulation,the TGP groups were given a corresponding dose of TGP suspension by gavage,and TGP+ML385 group was given 30 mg/kg of ML385 and 920 mg/kg TGP by gavage,administered continuously for 8 weeks.Behavioral changes of mice in each group were observed during the experiment,bronchoalveolar lavage fluid(BALF)was collected 24 hours after the last challenge,levels of TGF-β1,cysteyl leukotriene 1(CysLT1)and cysteyl leukotriene receptor 1(CysLTR1)in BALF were detected by ELISA;total antioxidant capacity(T-AOC),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT)activities in lung homogenate were detected;pathological changes of lung tissues were observed by HE and AB-PAS staining;expressions of lung tissue TGF-β1,matrix metalloproteinase 9(MMP-9)and tissue inhibitor of metalloproteinase 1(TIMP-1)mRNA were detected by RT-qPCR;expressions of TGF-β1/Nrf-2/HO-1 pathway related proteins in lung tissues were detected by Western blot.Results:Compared with control group,mice in OVA group showed typical asthma symptoms,such as sneezing,restlessness and wheezing,levels of TGF-β1,CysLT1,CysLTR1,lung inflammation and mucus secretion scores,expressions of TGF-β1 mRNA and protein,and MMP-9 mRNA in BALF were significantly increased(P<0.05),T-AOC,SOD,GSH-Px,CAT activity and expression of TIMP-1 mRNA in lung tissues were significantly reduced(P<0.05),expressions of Nrf-2 and HO-1 proteins in lung tissues were increased,while the difference was not statistically significant(P>0.05);compared with OVA group,mice in TGP-L and TGP-H groups had significantly reduced asthma symptoms,levels of TGF-β1,CysLT1,CysLTR1,lung inflammation and mucus secretion scores,expressions of TGF-β1 mRNA and protein,and MMP-9 mRNA in BALF were significantly reduced(P<0.05),T-AOC,SOD,GSH-Px,CAT activity and expressions of TIMP-1 mRNA,Nrf-2 and HO-1 proteins in lung tissues were significantly increased(P<0.05);using Nrf 2 inhibitor ML 385 to block the activation of Nrf-2/HO-1 pathway could significantly attenuate the inhibitory effect of TGP on lung tissue oxidative stress and airway remodeling in asthmatic mice.Conclusion:TGP can regulate airway remodeling in mice with bron‐chial asthma induced by oxidative stress and inflammation,and its mechanism may be related to reducing the expression of TGF-β1 and activating the Nrf-2/HO-1 pathway.