基于IRS-1/PI3K信号轴探究补肺健脾方对COPD大鼠骨骼肌线粒体损伤的影响

Role of IRS-1/PI3K signaling axis in the effects of Bufei Jianpi formula on mitochondrial damage in skeletal muscle of chronic obstructive pulmonary disease rats

目的探究补肺健脾方(Bufei Jianpi formula,BJF)通过调控IRS-1/PI3K信号轴对COPD大鼠骨骼肌线粒体损伤的影响。方法将60只SPF级SD大鼠随机分为空白(Control)组、COPD稳定期模型(Model)组、氨茶碱(Am)组、补肺健脾方(BJF)组、吡格列酮(PIO)组以及补肺健脾方+吡格列酮(BJF+PIO)组,10只/组。采用烟熏加鼻腔滴菌(肺炎克雷伯杆菌)的方法建立COPD稳定期大鼠模型,自第9周开始给药至20周结束后取材,每周给予大鼠体重测量。分别对肺组织和骨骼肌组织进行常规切片与HE染色,并于光镜下观察其相应的病理学改变。分别于第0、8、20周采用非束缚全身体积描记系统观察大鼠肺功能,包括VT、PEF、EF50。采用qPCR技术检测大鼠骨骼肌组织中IRS-1、PI3K、PGC-1α以及Leptin mRNA的表达。采用Western blot技术检测大鼠骨骼肌组织中IRS-1、PI3K、AKT、p-AKT、PGC-1α、TFAM和Leptin蛋白的表达。结果光镜观察显示与Control组比,Model组肺病理可见肺泡间质以及肺支气管存有大量的炎性细胞浸润,部分肺泡壁出现断裂并融合形成气腔、纤维网被破坏等;与Model组比,用药治疗后各组肺泡壁的断裂以及纤维网的破坏均得到改善,支气管中炎性细胞浸润减轻,其中以BJF组与Am组尤为明显。用药治疗后各组骨骼肌病理与Model组比,可不同程度改善肌纤维之间排列间隙、萎缩与断裂,肌细胞胞质染色不均一等,其中以BJF组疗效较为显著。与Control组比,Model组PEF、VT和EF50第8周起显著降低(P<0.01),BJF组、BJF+PIO组和Am组可以显著提高PEF、EF50(P<0.01)。与Control组比,Model组中IRS-1、PGC-1α和PI3K mRNA与蛋白表达水平显著降低(P<0.05,P<0.01),Leptin mRNA与蛋白表达水平显著增高(P<0.01);与Model组比,BJF组IRS-1、PGC-1α、PI3K mRNA与蛋白表达水平显著增高(P<0.05,P<0.01);PIO组IRS-1 mRNA表达水平显著增高(P<0.01);BJF+PIO组PGC-1αmRNA水平显著增高(P<0.01),IRS-1、PI3K mRNA与蛋白水平显著升高(P<0.05,P<0.01);Am组中PI3K mRNA与蛋白表达水平显著增高(P<0.01);4个用药组中Leptin mRNA的表达水平均显著降低(P<0.01),除Am组外,其余3个用药组Leptin蛋白表达显著降低(P<0.01);与Control组比,Model组股四头肌组织中TFAM、p-AKT蛋白表达有明显的下降趋势,各治疗组的TFAM、p-AKT蛋白表达均有升高趋势,但无显著性差异(P>0.05)。结论补肺健脾方可通过调控IRS-1/PI3K信号轴,改善骨骼肌线粒体的损伤,同时提高PGC-1α与线粒体转录因子TFAM的表达,增强线粒体的生物合成,从而减轻肺与骨骼肌组织的病理性损伤。

Objective To explore the action of Bufei Jianpi formula(BJF)on mitochondrial damage to skeletal muscle in chronic obstructive pulmonary disease(COPD)rats via its regulation of the IRS-1/PI3K signaling axis.Methods 60 SPF SD rats were randomly divided into Control group,Model group(COPD stable stage group),aminophylline(Am)group,BJF group,pioglitazone(PIO)group and BJF+PIO group,with 10 rats per group.A stable COPD rat model was established via forced smoking and Klebsiella pneumoniae nasal drip method.Samples were taken from the 9th week to the end of the 20th week,and the weight of the rats was measured every week.Routine sectioning and HE staining were performed on lung and skeletal muscle tissue,and corresponding pathological changes were observed under a light microscope.The lung function of the rats was observed by whole-body plethysmography in weeks 0,8,and 20,including tidal VT,PEF,and EF50.The mRNA expression of IRS-1,leptin,PGC1-α,and PI3K in rat skeletal muscle was detected by qPCR.The expression of PGC-1α,TFAM,IRS-1,PI3K,AKT,p-AKT,and leptin in rat skeletal muscle tissue was detected by Western blot.Results The Model group,but not the Control group,showed a large number of inflammatory cells infiltrating the alveolar interstitium and bronchus,indicative of lung disease;some alveolar walls had broken and fused to form air cavities,and fiber networks were destroyed.After drug treatment,the rats showed improved alveolar wall and fiber network integrity and reduced inflammatory cell infiltration in the bronchus,especially those in the BJF and Am groups.In the drug treatment groups,the skeletal muscle pathology of each group showed improved spatial arrangement,the atrophy and fracturing of muscle fibers were ameliorated to different degrees,and cytoplasmic staining of muscle cells was uneven,and the BJF group showed the most significant effects.Compared with the Control group,the Model group’s PEF,VT,and EF50 significantly decreased from week 8(P<0.01),while the BJF,BJF+PIO and Am groups had significantly increased PEF and EF50(P<0.01).Compared with Control group,the Model group’s mRNA and protein expression levels of IRS-1,PGC-1α,and PI3K were significantly decreased(P<0.05,P<0.01),the level of leptin was significantly increased(P<0.01).Compared with the Model group,the mRNA and protein expressions of IRS-1,PGC-1αand PI3K in the BJF group were significantly increased(P<0.05,P<0.01),and the mRNA expression of IRS-1 in the PIO group was significantly increased(P<0.01).The BJF+PIO group’s mRNA levels of PGC-1α(P<0.01)and mRNA and protein levels of IRS-1 and PI3K were significantly increased(P<0.05,P<0.01).The mRNA and protein expression levels of PI3K in the Am group were significantly increased(P<0.01).The expression levels of leptin mRNA were significantly decreased in the four treatment groups(P<0.01),and the expression of leptin protein was significantly decreased in all treatment groups except the Am group(P<0.01).Compared with the Control group,the Model group’s quadriceps femoris tissue showed a significant decrease in TFAM and p-AKT expression.TFAM and p-AKT expression in all the treatment groups showed an increasing trend,but the difference was not statistically significant(P>0.05).Conclusions By regulating the IRS-1/PI3K signaling axis,Bufei Jiempi reduces mitochondrial damage to skeletal muscle,increases the expression of PGC-1αand mitochondrial transcription factor TFAM,enhances mitochondrial biosynthesis,and reduces pathological damage to lung and skeletal muscle tissue.