基于Pkm2/Nrf2通路探讨补肺益肾方改善线粒体氧化损伤治疗慢性阻塞性肺疾病的机制

Mechanism of Bufei Yishen Formula on alleviating mitochondrial oxidative damage with chronic obstructive pulmonary disease via Pkm2/Nrf2 pathway

目的:探讨补肺益肾方调控Pkm2/Nrf2通路改善肺组织线粒体氧化损伤治疗慢性阻塞性肺疾病(COPD)机制。方法:将32只SD大鼠随机分为正常组、模型组、补肺益肾方组(简称BYF组)和乙酰半胱氨酸组(简称NAC组),每组8只。1~8周,采用香烟暴露联合肺炎克雷伯杆菌反复感染的方法制备COPD大鼠模型。9~16周,正常组和模型组给予0.9%氯化钠溶液灌胃(0.5 mL/100 g),BYF组和NAC组分别给予补肺益肾方灌胃(11.61 g·kg^(-1)·d^(-1))和乙酰半胱氨酸溶液灌胃(54 mg·kg^(-1)·d^(-1)),每日1次,16周结束后取材。检测肺功能、肺病理、线粒体超微结构、氧化应激相关指标、线粒体功能相关指标、Pkm2/Nrf2通路相关指标。结果:COPD大鼠肺功能降低(VT、PEF、EF50、FVC和FEV0.3降低,FRC升高),肺组织病理损伤(MLI、BWt升高,MAN降低),肺组织氧化损伤(T-SOD、T-AOC、GSH-Px、SOD2降低,MDA升高),线粒体功能受损(线粒体膜电位、线粒体呼吸链复合物Ⅰ和Ⅲ酶活性下降),Pkm2/Nrf2信号通路表达降低(Pkm2、Nrf2、GCLM、GCLC mRNA和核蛋白表达下降)(P<0.05,P<0.01)。BYF和NAC均可改善COPD大鼠肺功能(VT、PEF、EF50、FVC、FEV0.3升高,FRC降低),减轻肺组织病理损伤(MAN升高,MLI、BWt降低),提高肺组织抗氧化水平(T-SOD、GSH-Px、SOD2升高),保护线粒体功能(线粒体膜电位、线粒体呼吸链复合物Ⅰ、线粒体呼吸链复合物Ⅲ升高),激活Pkm2/Nrf2信号通路(Pkm2、Nrf2、GCLM、GCLC mRNA和核蛋白表达升高)(P<0.05,P<0.01);BYF组T-AOC显著升高,MDA显著降低(P<0.01,P<0.05);BYF在升高MAN、GSH-Px、T-AOC、Pkm2 mRNA表达及降低MLI的作用方面优于NAC组(P<0.05,P<0.01)。结论:补肺益肾方和乙酰半胱氨酸均可减轻COPD大鼠肺组织氧化应激,改善线粒体功能,提高抗氧化能力,其机制可能与调控Pkm2/Nrf2信号通路有关,补肺益肾方在改善肺泡结构及提高抗氧化酶活性方面优于乙酰半胱氨酸。

Objective:To investigate the mechanism of Bufei Yishen Formula(BYF)on improve mitochondrial oxidative damage with chronic obstructive pulmonary disease(COPD)via pyruvate kinase M2(Pkm2)/nuclear factor E2-related factor 2(Nrf2)signaling pathway.Methods:Totally 32 SD rats were randomly divided into normal group,model group,Bufei Yishen Formula(BYF)group and N-Acetylcysteine(NAC)group,8 rats in each group.The COPD rat model was established by cigarette smoke exposure combined with repeated infection of Klebsiella pneumoniae during 1 to 8 weeks.From week 9 to 16,the rats in the normal and model groups were given with saline solution by intragastric administration(O.5 mL/100 g),while the rats in BYF and NAC groups were given with Bufei Yishen Formula(11.61·kg^(-1)·d^(-1))and N-Acetylcysteine(54 mg·kg^(-1)·d^(-1)),respectively,once a day.The rats were euthanized at the end of week 16,and several indicators were measured as follows:Lung function,lung pathology,mitochondrial ultrastructure,oxidative stress indicators,mitochondrial function indicators and the indicator related to Pkm2/Nrf2 signal pathway.Results:In COPD rats,lung function(VT,PEF,EF50,FVC,and FEV0.3)decreased,while FRC increased.Lung tissue pathology showed MLI and BWt increased,while MAN decreased.Lung tissue oxidative damage was observed with decreased levels of T-SOD,T-AOC,GSH-Px and SOD2,and increased levels of MDA.Mitochondrial function was impaired with decreased mitochondrial membrane potential,as well as reduced enzyme activity of respiratory chain complexⅠandⅡ.The Pkm2/Nrf2 signaling pathway expression was suppressed with decreased mRNA and nuclear protein expression of Pkm2,Nrf2,GCLM,and GCLC(P<0.05,P<0.01).After treatment,BYF and NAC improved lung function(VT,PEF,EF50,FVC,and FEVO.3 increased,while FRC decreased)and alleviated lung tissue pathological injury(MAN increased,MLI and BWt decreased),increased antioxidant capacity(T-SOD,GSH-Px,SOD2 increased),protected mitochondrial function(increased mitochondrial membrane potential,respiratory chain complex I and Il enzyme activity),and activated the Pkm2/Nrf2 signaling pathway(Pkm2,Nrf2,GCLM,GCLC mRNA and nuclear protein expression increased)(P<0.05,P<0.01).The BYF group also showed a significant increase in T-AOC and decrease in MDA(P<0.01,P<0.05).Compared with NAC group,BYF group had a better effect in increasing MAN,GSH-Px,T-AOC and Pkm2 mRNA,as well as decreasing MLI(P<0.05,P<0.01).Conclusion:Bufei Yishen Formula and N-Acetylcysteine can ameliorate oxidative stress in lung tissue of COPD rats,improve mitochondrial function,the mechanism may be related to the activation of Pkm2/Nrf2 signal pathway.Bufei Yishen Formula had a better effect in alleviating lung pathology compared with N-Acetylcysteine.