甘草查尔酮A通过调节TGF-β/Smad信号通路抑制小鼠肺纤维化
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摘要
目的:探索甘草查而酮A对博来霉素(bleomycin,BLM)诱导小鼠肺纤维化的治疗作用,并探讨其相关的作用机制。方法:本研究将30只小鼠随机平均分为5组,分别为正常组、模型组、甘草查尔酮A高、低剂量组、吡非尼酮组。气管滴注BLM 5 mg·kg~(-1)建立小鼠肺纤维化模型,次日治疗组灌胃给予甘草查尔酮A 15,30 mg·kg~(-1),吡非尼酮300 mg·kg~(-1),其余组给予生理盐水灌胃,28 d后处死小鼠。取肺组织称质量,进行肺组织苏木素-伊红(HE)染色和马松(Masson)染色、测定肺组织羟脯氨酸含量、通过蛋白免疫印迹法(Western blot)分析肺组织α-平滑肌肌动蛋白(α-SMA),I型胶原蛋白(Collagen I),纤连蛋白(FN)以及p-Smad2/3与Smad2/3表达水平。并考察甘草查尔酮A对转化生长因子-β1(TGF-β1)诱导成纤维细胞MRC-5细胞活化作用。结果:与正常组比较,模型组小鼠可见肺泡结构有不同程度的破坏,肺间质增生,有炎细胞浸润和胶原纤维增生形成,肺组织中羟脯胺酸,α-SMA,Collagen I蛋白表达水平明显增加(P<0.05,P<0.01);与模型组比较,各治疗组小鼠的肺指数明显下降(P<0.05,P<0.01),肺损伤与纤维化程度显著改善;肺组织的羟脯氨酸含量,α-SMA,Collagen I蛋白表达水平明显降低(P<0.05,P<0.01);肺组织的Smad2/3蛋白磷酸化水平显著降低(P<0.05,P<0.01);另外,甘草查尔酮A与吡非尼酮显著抑制TGF-β诱导MRC-5细胞转化,明显降低α-SMA,FN蛋白表达(P<0.05,P<0.01)。结论:甘草查而酮A可能通过阻断Smad相关信号通路,抑制成纤维细胞转化等发挥抗纤维化作用。
Objective:To explore the therapeutic effect of licochalcone A(Lico A) on pulmonary fibrosis(PF).Method:Thirty mice were divided into five group,namely sham,model,Lico A(15,30 mg·kg~(-1)) and pirfenidone(300 mg·kg~(-1)) groups.All of the groups except for sham group were intratracheally given bleomycin(BLM,5 mg·kg~(-1)).The sham group was given normal saline.On day 2,the mice were treated with Lico A and pirfenidone,respectively.On day 28,all of the mice were put to death.Then,lung tissues were collected and weighted.Pathological changes in lung tissue were measured by htoxylin eosin(HE) and Masson staining.Theα-smooth muscle actin(α-SMA),Collagen I,fibronectin p-Smad2/3 and Smad2/3 were analyzed by Western blot.Then,transforming growth factor-β1(TGF-β1)-induced MRC-5 cells were employed for evaluating the inhibitory activity of Lico A in vitro.Result:Compared with normal group,several pathological changes,including alveolar space collapse,emphysema,infiltration of inflammatory cells,and collagen deposition were observed in the BLM-treated mice,and these pathological changes were markedly attenuated by subsequent treatment with Lico A.Lico A could significantly inhibit BLM-induced up-regulation of α-SMA and Collagen I and phosphorylation of Smad2/3 in lung tissues of mice(P<0.05,P<0.01).In addition,Lico A could significantly suppressed TGF-β-induced α-SMA and fibronectin expression in MRC-5 cells(P<0.05,P<0.01).Conclusion:The preliminary mechanisms of the anti-fibrosis effect of Lico A may inhibit TGF-β/Smad pathway.
Objective:To explore the therapeutic effect of licochalcone A(Lico A) on pulmonary fibrosis(PF).Method:Thirty mice were divided into five group,namely sham,model,Lico A(15,30 mg·kg~(-1)) and pirfenidone(300 mg·kg~(-1)) groups.All of the groups except for sham group were intratracheally given bleomycin(BLM,5 mg·kg~(-1)).The sham group was given normal saline.On day 2,the mice were treated with Lico A and pirfenidone,respectively.On day 28,all of the mice were put to death.Then,lung tissues were collected and weighted.Pathological changes in lung tissue were measured by htoxylin eosin(HE) and Masson staining.Theα-smooth muscle actin(α-SMA),Collagen I,fibronectin p-Smad2/3 and Smad2/3 were analyzed by Western blot.Then,transforming growth factor-β1(TGF-β1)-induced MRC-5 cells were employed for evaluating the inhibitory activity of Lico A in vitro.Result:Compared with normal group,several pathological changes,including alveolar space collapse,emphysema,infiltration of inflammatory cells,and collagen deposition were observed in the BLM-treated mice,and these pathological changes were markedly attenuated by subsequent treatment with Lico A.Lico A could significantly inhibit BLM-induced up-regulation of α-SMA and Collagen I and phosphorylation of Smad2/3 in lung tissues of mice(P<0.05,P<0.01).In addition,Lico A could significantly suppressed TGF-β-induced α-SMA and fibronectin expression in MRC-5 cells(P<0.05,P<0.01).Conclusion:The preliminary mechanisms of the anti-fibrosis effect of Lico A may inhibit TGF-β/Smad pathway.
引文
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[23]唐磊,申萌萌,朱曼,等.右归饮对肺纤维化大鼠的疗效评价及作用机制[J].中国实验方剂学杂志,2018,24(13):93-99.
[24]王鹏飞,林昶,刘杨,等.黄芪甲苷对博来霉素诱导的小鼠特发性肺纤维化自噬和焦亡的影响[J].中国实验方剂学杂志,2018,24(10):144-149.
[25]HAN G,ZHOU Y F,ZHANG M S,et al.Angelica sinensis down-regulates hydroxyproline and Tgfb1 and provides protection in mice with radiation-induced pulmonary fibrosis[J].Radiat Res,2006,165(5):546-552.
[26]Willis B C,Borok Z.TGF-β-induced EMT:mechanisms and implications for fibrotic lung disease[J].Am J of Physiol Lung C,2007,293(3):L525-L534.
[27]Leask A,Abraham D J.TGF-βsignaling and the fibrotic response[J].FASEB J,2004,18(7):816-827.
[28]El Agha E,Kramann R,Schneider R K,et al.Mesenchymal stem cells in fibrotic disease[J].Cell Stem Cell,2017,21(2):166-177.
[29]El Agha E,Moiseenko A,Kheirollahi V,et al.Twoway conversion between lipogenic and myogenic fibroblastic phenotypes marks the progression and resolution of lung fibrosis[J].Cell Stem Cell,2017,20(2):261-273.
[30]庞立健,吕晓东,袁佺,等.益气养阴活血通络法干预肺成纤维细胞TGF-β1/Smads信号传导通路的机制[J].中国实验方剂学杂志,2017,23(22):125-131.
[2]Wollin L,Wex E,Pautsch A,et al.Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis[J].Eur Respir J,2015,45(5):1434-1445.
[3]Leask A,Abraham D J.TGF-βsignaling and the fibrotic response[J].FASEB J,2004,18(7):816-827.
[4]Kurita Y,Araya J,Minagawa S,et al.Pirfenidone inhibits myofibroblast differentiation and lung fibrosis development during insufficient mitophagy[J].Respir Res,2017,18(1):114.
[5]宋建平.特发性肺纤维化的中医论治[J].浙江中医学院学报,1997,21(2):4-5.
[6]吴传清.学习《金匮》肺痿证治的体会[J].泸州医学院学报,1984,6(3):13-16.
[7]康荻,李彧,朱曼,唐磊,等.肺痿论治干预肺纤维化大鼠肺功能的实验研究[J].世界中医药,2017,12(2):390-393,397.
[8]李健,卢朋,张瑞贤,信富荣,等.《中医方剂大辞典》中治疗肺痿方剂的用药规律分析[J].中国实验方剂学杂志,2012,18(10):1-5.
[9]陆国辉,李艳茹,高健美.甘草干姜汤对博莱霉素诱导的大鼠肺纤维化SIRT1和TGF-β1蛋白表达的影响[J].中药药理与临床,2014,30(6):25-28.
[10]黄云鉴,龚婕宁.中医治疗肺纤维化方药规律的文献分析[J].中国实验方剂学杂志,2016,22(15):206-210.
[11]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:86.
[12]李杰.《伤寒论》中甘草功效与配伍应用的规律研究[D].广州:广州中医药大学,2013.
[13]赵虹,蒋江涛,郑秋生.甘草查耳酮A药理作用研究进展[J].中国中药杂志,2013,38(22):3814-3818.
[14]Karkanis A,Martins N,Petropoulos S A,et al.Phytochemical composition,health effects,and crop management of liquorice(Glycyrrhiza glabra L.):a medicinal plant[J].Food Rev Int,2013,34(2):182-203.
[15]CHU X,CI X,WEI M,et al.Licochalcone a inhibits lipopolysaccharide-induced inflammatory response in vitro and in vivo[J].J Agric Food Chem,2012,60(15):3947-3954.
[16]CHU X,JIANG L,WEI M,et al.Attenuation of allergic airway inflammation in a murine model of asthma by Licochalcone A[J].Immunopharm Immunot,2013,35(6):653-661.
[17]任倩倩,汪丽佩,赵炜,等.甘草查尔酮A抑制ERK1/2/NF-κB途径减轻吸烟诱导的小鼠急性肺损伤[J].中国药理学通报,2016,32(5):643-651.
[18]JI Y,DOU Y,ZHAO Q,et al.Paeoniflorin suppresses TGF-βmediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway[J].Acta Pharmacol Sini,2016,37(6):794-804.
[19]LIU D,HUO X,GAO L,et al.NF-κB and Nrf2pathways contribute to the protective effect of Licochalcone A on dextran sulphate sodium-induced ulcerative colitis in mice[J].Biomed Pharmacother,2018,102:922-929.
[20]HU J,LIU J.Licochalcone A attenuates lipopolysaccharide-induced acute kidney injury by inhibiting NF-κB activation[J].Inflammation,2016,39(2):569-574.
[21]Szapiel S V,Elson N A,Fulmer J D,et al.Bleomycininduced interstitial pulmonary disease in the nude,athymic mouse[J].Am Rev Respir Dis,1979,120(4):893-899.
[22]Cho H Y,Reddy S P,Yamamoto M,et al.The transcription factor NRF2 protects against pulmonary fibrosis[J].FASEB J,2004,18(11):1258-1260.
[23]唐磊,申萌萌,朱曼,等.右归饮对肺纤维化大鼠的疗效评价及作用机制[J].中国实验方剂学杂志,2018,24(13):93-99.
[24]王鹏飞,林昶,刘杨,等.黄芪甲苷对博来霉素诱导的小鼠特发性肺纤维化自噬和焦亡的影响[J].中国实验方剂学杂志,2018,24(10):144-149.
[25]HAN G,ZHOU Y F,ZHANG M S,et al.Angelica sinensis down-regulates hydroxyproline and Tgfb1 and provides protection in mice with radiation-induced pulmonary fibrosis[J].Radiat Res,2006,165(5):546-552.
[26]Willis B C,Borok Z.TGF-β-induced EMT:mechanisms and implications for fibrotic lung disease[J].Am J of Physiol Lung C,2007,293(3):L525-L534.
[27]Leask A,Abraham D J.TGF-βsignaling and the fibrotic response[J].FASEB J,2004,18(7):816-827.
[28]El Agha E,Kramann R,Schneider R K,et al.Mesenchymal stem cells in fibrotic disease[J].Cell Stem Cell,2017,21(2):166-177.
[29]El Agha E,Moiseenko A,Kheirollahi V,et al.Twoway conversion between lipogenic and myogenic fibroblastic phenotypes marks the progression and resolution of lung fibrosis[J].Cell Stem Cell,2017,20(2):261-273.
[30]庞立健,吕晓东,袁佺,等.益气养阴活血通络法干预肺成纤维细胞TGF-β1/Smads信号传导通路的机制[J].中国实验方剂学杂志,2017,23(22):125-131.