补肺健脾方对COPD大鼠骨骼肌TNF-α、Caspase-8及Caspase-3的影响
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摘要
目的:观察补肺健脾方对慢性阻塞性肺疾病(COPD)模型大鼠骨骼肌肿瘤坏死因子-α(TNF-α)、Caspase-8及Caspase-3的影响。方法:48只SD大鼠随机分为对照组、模型组、补肺健脾组和氨茶碱组,采用香烟烟雾暴露联合肺炎克雷伯杆菌感染的方法建立COPD稳定期大鼠模型,从第9周起,对照组和模型组分别给予0.9%氯化钠溶液(2m L/只)灌胃,补肺健脾组和氨茶碱组分别给予补肺健脾方(5g·kg-1·d-1)、氨茶碱(2.3mg·kg-1·d-1)灌胃,至第20周结束后取材。光镜下观察大鼠股四头肌、肋间肌、肱二头肌及比目鱼肌组织病理变化,采用荧光定量PCR和Western Blot技术检测4种骨骼肌组织TNF-α、Caspase-8和Caspase-3 m RNA和蛋白表达。结果:模型组4种骨骼肌TNF-α、Caspase-8和Caspase-3基因和蛋白表达均较对照组显著升高(P<0.01)。补肺健脾组、氨茶碱组上述基因和蛋白表达较模型组显著降低(P<0.01),且补肺健脾组疗效优于氨茶碱组。结论:补肺健脾方能够降低骨骼肌组织中TNF-α、Caspase-8和Caspase-3基因和蛋白表达,这可能是其调控炎性反应和细胞凋亡改善COPD骨骼肌功能的重要机制。
Objective: To observe the effects of Bufei Jianpi Formula on TNF-α, Caspase-8 and Caspase-3 in skeletal muscles in rats with chronic obstructive pulmonary disease(COPD). Methods: Sprague-Dawley rats were randomized into the control, the model, the Bufei Jianpi, and the aminophylline group. The stable COPD rat model was duplicated by the combination of cigarette smoke inhalations and bacterial infections. From the 9 th week, rats were administrated intragastrically by normal saline(2 m L per rat), Bufei Jianpi Formula(5 g·kg-1·d-1) and aminophylline(2.3 mg·kg-1·d-1), respectively. Animals were sacrificed at the end of 20 th week. The pathological changes of quadriceps, intercostal, biceps, soleus muscles were observed by light microscope. The rates of cell apoptosis in the four skeletal muscles were determined by terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling(TUNEL) method. The expressions of TNF-α, Caspase-8 and Caspase-3 m RNA and proteins were detected by real-time fluorescence quantitative polymerase chain reaction(q PCR) and Western blotting. Results: The expressions of TNF-α, Caspase-8, and Caspase-3 were significantly higher in the four skeletal muscles in model rats than the control group(P<0.01). Bufei Jianpi Formula and aminophylline down-regulated all above-mentioned genes and proteins(P<0.01). The effect of Bufei Jianpi Formula was better than aminophylline. Conclusion: Bufei Jianpi Granules can down-regulate the expressions of TNF-α, Caspase-8, and Caspase-3, which may be the mechanisms of improving skeletal muscle via regulating inflammation and cell apoptosis.
Objective: To observe the effects of Bufei Jianpi Formula on TNF-α, Caspase-8 and Caspase-3 in skeletal muscles in rats with chronic obstructive pulmonary disease(COPD). Methods: Sprague-Dawley rats were randomized into the control, the model, the Bufei Jianpi, and the aminophylline group. The stable COPD rat model was duplicated by the combination of cigarette smoke inhalations and bacterial infections. From the 9 th week, rats were administrated intragastrically by normal saline(2 m L per rat), Bufei Jianpi Formula(5 g·kg-1·d-1) and aminophylline(2.3 mg·kg-1·d-1), respectively. Animals were sacrificed at the end of 20 th week. The pathological changes of quadriceps, intercostal, biceps, soleus muscles were observed by light microscope. The rates of cell apoptosis in the four skeletal muscles were determined by terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling(TUNEL) method. The expressions of TNF-α, Caspase-8 and Caspase-3 m RNA and proteins were detected by real-time fluorescence quantitative polymerase chain reaction(q PCR) and Western blotting. Results: The expressions of TNF-α, Caspase-8, and Caspase-3 were significantly higher in the four skeletal muscles in model rats than the control group(P<0.01). Bufei Jianpi Formula and aminophylline down-regulated all above-mentioned genes and proteins(P<0.01). The effect of Bufei Jianpi Formula was better than aminophylline. Conclusion: Bufei Jianpi Granules can down-regulate the expressions of TNF-α, Caspase-8, and Caspase-3, which may be the mechanisms of improving skeletal muscle via regulating inflammation and cell apoptosis.
引文
[1]Global Initiative for Chronic Obstructive Lung Disease.Global strategy for the diagnosis,management,and prevention of chronic obstructive pulmonary disease,updated 2014.http://www.goldcopd.org.Accessed at Feb.20,2014
[2]Choudhury G,Rabinovich R,Mac Nee W.Comorbidities and systemic effects of chronic obstructive pulmonary disease.Clin Chest Med,2014,35(1):101-130
[3]孙圣华,唐文祥,刘纯,等.TNF-α对COPD大鼠模型呼吸肌蛋白质分解代谢的影响.中华结核和呼吸杂志,2007,30(3):55-56
[4]Liao J,Zhang L,Zheng J,et al.Electroacupuncture inhibits annulus fibrosis cell apoptosis in vivo via TNF-α-TNFR1-Caspase-8and integrinβ1/Akt signaling pathways.J Tradit Chin Med,2014,34(6):685
[5]赵世飞.肌肉运动和骨骼肌细胞的凋亡.中国组织工程研究和临床康复,2010,14(11):2030-2033
[6]Liu Q S,Wang H F,Sun A K,et al.A comparative study on inhibition of total astragalus saponins and astragalosideⅣon TNFR1-mediated signaling pathways in arterial endothelial cells.PLo S One,2014,9(7):e101504
[7]李亚,毛静,董玉琼,等.补肺健脾方对慢性阻塞性肺疾病大鼠骨骼肌Fas/Fas L/Caspase-3的影响.中华中医药杂志,2017,32(3):1238-1241
[8]Dong Y Q,Li Ya,Sun Y F,et al.Bufei Jianpi granules improve skeletal muscle and mitochondrial dysfunction in rats with chronic obstructive pulmonary disease.BMC Complementary and Alternative Medicine,2015,15:51
[9]Li Y,Li S Y,Li J S,et al.A rat model for stable chronic obstructive pulmonary disease induced by cigarette smoke inhalation and repetitive bacterial infection.Biol Pharm Bull,2012,35(10):1752-1760
[10]李素云,李亚,李建生,等.补肺健脾方对慢性阻塞性肺疾病大鼠膈神经放电和膈肌功能的影响.中国中西医结合杂志,2012,32(6):812-816
[11]Sakuma K,Yamaguchi A.Sarcopenia and cachexia:The adaptations of negative regulators of skeletal muscle mass.J Cachexia Sarcopenia Muscle,2012,3(2):77-94
[12]Oudijk E J,Lammers J W,Koenderman L.Systemic inflammation in chronic obstructive pulmonary disease.Eur Respir J Suppl,2003,22(46):5-13
[13]Caron M A,Debigare R,Dekhuijzen P N,et al.Comparative assessment of the quadriceps and the diaphragm in patients with COPD.Appl Physiol,2009,107(3):952-961
[14]Zuo L,Hallman A H,Roberts W J,et al.Superoxide release from contracting skeletal muscle in pulmonary TNF-αover expression mice.Am J Physiol Regul Inteqr Comp Physiol,2014,306(1):75-81
[15]Casadevall C,Coronell C,Ramirez-Sarmiento AL,et al.Upregulation of pro-inflammatory cytokines in the intercostal muscles of COPD patients.Eur Respir J,2007,30(4):701-707
[16]Vescovo G,Dalla Libera L.Skeletal apoptosis in experimental heart failure,the only link between inflammation and skeletal muscle wastage?Curr Opin Clin Nutr Metab Care,2006,9(4):416-422
[17]Guicciardi M E,Mott J L,Bronk S F,et al.Cellular inhibitor of apoptosis 1(c IAP-1)degradation by caspase 8 during TNF-related apoptosis-inducing ligand(TRAIL)-induced apoptosis.Exp Cell Res,2011,317(1):107-116
[18]De Martino L,Marfe G,Longo M,et al.Bid cleavage,cytochromec release and caspase activation in canine coronavirus-induced apoptosis.Vet Microbiol,2010,141(2):36-45
[19]Fulda S.Caspase-8 in cancer biology and therapy.Cancer Lett,2009,281(2):128-133
[2]Choudhury G,Rabinovich R,Mac Nee W.Comorbidities and systemic effects of chronic obstructive pulmonary disease.Clin Chest Med,2014,35(1):101-130
[3]孙圣华,唐文祥,刘纯,等.TNF-α对COPD大鼠模型呼吸肌蛋白质分解代谢的影响.中华结核和呼吸杂志,2007,30(3):55-56
[4]Liao J,Zhang L,Zheng J,et al.Electroacupuncture inhibits annulus fibrosis cell apoptosis in vivo via TNF-α-TNFR1-Caspase-8and integrinβ1/Akt signaling pathways.J Tradit Chin Med,2014,34(6):685
[5]赵世飞.肌肉运动和骨骼肌细胞的凋亡.中国组织工程研究和临床康复,2010,14(11):2030-2033
[6]Liu Q S,Wang H F,Sun A K,et al.A comparative study on inhibition of total astragalus saponins and astragalosideⅣon TNFR1-mediated signaling pathways in arterial endothelial cells.PLo S One,2014,9(7):e101504
[7]李亚,毛静,董玉琼,等.补肺健脾方对慢性阻塞性肺疾病大鼠骨骼肌Fas/Fas L/Caspase-3的影响.中华中医药杂志,2017,32(3):1238-1241
[8]Dong Y Q,Li Ya,Sun Y F,et al.Bufei Jianpi granules improve skeletal muscle and mitochondrial dysfunction in rats with chronic obstructive pulmonary disease.BMC Complementary and Alternative Medicine,2015,15:51
[9]Li Y,Li S Y,Li J S,et al.A rat model for stable chronic obstructive pulmonary disease induced by cigarette smoke inhalation and repetitive bacterial infection.Biol Pharm Bull,2012,35(10):1752-1760
[10]李素云,李亚,李建生,等.补肺健脾方对慢性阻塞性肺疾病大鼠膈神经放电和膈肌功能的影响.中国中西医结合杂志,2012,32(6):812-816
[11]Sakuma K,Yamaguchi A.Sarcopenia and cachexia:The adaptations of negative regulators of skeletal muscle mass.J Cachexia Sarcopenia Muscle,2012,3(2):77-94
[12]Oudijk E J,Lammers J W,Koenderman L.Systemic inflammation in chronic obstructive pulmonary disease.Eur Respir J Suppl,2003,22(46):5-13
[13]Caron M A,Debigare R,Dekhuijzen P N,et al.Comparative assessment of the quadriceps and the diaphragm in patients with COPD.Appl Physiol,2009,107(3):952-961
[14]Zuo L,Hallman A H,Roberts W J,et al.Superoxide release from contracting skeletal muscle in pulmonary TNF-αover expression mice.Am J Physiol Regul Inteqr Comp Physiol,2014,306(1):75-81
[15]Casadevall C,Coronell C,Ramirez-Sarmiento AL,et al.Upregulation of pro-inflammatory cytokines in the intercostal muscles of COPD patients.Eur Respir J,2007,30(4):701-707
[16]Vescovo G,Dalla Libera L.Skeletal apoptosis in experimental heart failure,the only link between inflammation and skeletal muscle wastage?Curr Opin Clin Nutr Metab Care,2006,9(4):416-422
[17]Guicciardi M E,Mott J L,Bronk S F,et al.Cellular inhibitor of apoptosis 1(c IAP-1)degradation by caspase 8 during TNF-related apoptosis-inducing ligand(TRAIL)-induced apoptosis.Exp Cell Res,2011,317(1):107-116
[18]De Martino L,Marfe G,Longo M,et al.Bid cleavage,cytochromec release and caspase activation in canine coronavirus-induced apoptosis.Vet Microbiol,2010,141(2):36-45
[19]Fulda S.Caspase-8 in cancer biology and therapy.Cancer Lett,2009,281(2):128-133