补肾通络方对肺纤维化大鼠肺组织TGF-β_1/Smads通路的影响
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摘要
目的:观察补肾通络方对博莱霉素所致肺纤维化大鼠肺组织病理及转化生长因子β_1(TGF-β_1)/Smads通路蛋白的影响,揭示该方抑制肺纤维化的作用机制。方法:48只清洁级雄性SD大鼠随机分为正常组8只,模型组、补肾通络低、高剂量组、氢化可的松琥珀酸钠组各10只。除正常组外,各组均采用博莱霉素法诱导肺纤维化制备模型。从术后第3天开始,补肾通络高、低剂量组分别给予14. 2,7. 1 g·kg~(-1)补肾通络方药灌胃,氢化可的松琥珀酸钠组给予4. 58 g·kg-1氢化可的松琥珀酸钠溶液腹腔注射,正常组和模型组给予等体积生理盐水灌胃,共治疗28 d。大鼠处死后测定血清羟脯氨酸(HYP),透明质酸(HA),层粘连蛋白(LN)的含量,取肺组织,采用苏木精-伊红染色法(HE)及马松(Masson)三色染色法观察肺组织病理形态变化,采用实时荧光定量PCR法(Real-time PCR)和蛋白免疫印迹法(Western blot)观察各组大鼠肺组织TGF-β_1,Smad2,Smad3,Smad7等mRNA和蛋白表达变化。结果:与正常组相比,模型组大鼠肺纤维化明显,HYP,HA,LN含量升高(P <0. 05,P <0. 01),肺组织TGF-β_1,Smad2,Smad3蛋白和mRNA表达升高(P <0. 05,P <0. 01),Smad7蛋白和mRNA表达降低(P <0. 01);与模型组相比,补肾通络高剂量组血清HYP,HA,LN的含量下降(P <0. 05,P <0. 01)肺组织TGF-β_1,Smad2,Smad3蛋白和mRNA表达降低(P <0. 05),Smad7蛋白和mRNA表达升高(P <0. 01)。结论:补肾通络方可有效改善肺纤维化的病变进程,其机制可能与调节TGF-β_1/Smads通路有关。
Objective: To observe the effect of Bushen Tongluo prescription on the pathogenesis of lung fibrosis induced by bleomycin and the transforming growth factor β_1( TGF-β_1)/Smads pathway protein in rats,and reveal the mechanism of this formula in inhibiting pulmonary fibrosis. Method: Totally 48 male SD rats were randomly divided into five groups: normal group( 8 rats),model group( 10 rats),high and low-dose Bushen Tongluo group( 10 rats) and hydrocortisone sodium succinate group( 10 rats). The model of pulmonary fibrosis was made with bleomycin except for normal group. Since day 3 after surgery,14. 2,7. 1 g·kg~(-1) Bushen Tongluo prescription were given by ig in high and low-dose Bushen Tongluo groups,and 4. 58 g·kg~(-1) hydrocortisone sodium succinate was given by gavage( ig) in normal group and model group. All of the rats were put to death after 28 days. The contents of hydroxyproline( HYP),hyaluronic acid( HA) and laminin( LN) were measured after the death of the rats. Hematoxylin-eosin staining( HE) and Masson tricolor staining were used to observe the pathological changes of lung tissue. Real-time PCR and Western blot were used to observe the changes of mRNA and protein expressions of lung tissues TGF-β_1,Smad2,Smad3,Smad7 in each group. Result: Compared with normal group,the level of pulmonary fibrosis was more significant in model group,and the serum HYP,HA and LN were increased obviously( P < 0. 05,P < 0. 01). The expressions of TGF-β_1,Smad2,Smad3 proteins and mRNA in lung tissues were increased obviously( P < 0. 05,P < 0. 01). The expressions of Smad7 protein and mRNA were decreased significantly( P < 0. 01). Compared with model group,the contents of HYP,HA and LN in high-dose Bushen Tongluo group were decreased obviously( P < 0. 05,P < 0. 01),the expressions of lung tissues TGF-β_1,Smad2,Smad3 protein and mRNA were decreased obviously( P < 0. 05),and the expressions of Smad7 protein and mRNA was increased significantly( P < 0. 01). Conclusion: Bushen Tongluo prescription can effectively improve the pathological process of pulmonary fibrosis,and the mechanism may be related to the regulation of TGF-β_1/Smads pathway.
Objective: To observe the effect of Bushen Tongluo prescription on the pathogenesis of lung fibrosis induced by bleomycin and the transforming growth factor β_1( TGF-β_1)/Smads pathway protein in rats,and reveal the mechanism of this formula in inhibiting pulmonary fibrosis. Method: Totally 48 male SD rats were randomly divided into five groups: normal group( 8 rats),model group( 10 rats),high and low-dose Bushen Tongluo group( 10 rats) and hydrocortisone sodium succinate group( 10 rats). The model of pulmonary fibrosis was made with bleomycin except for normal group. Since day 3 after surgery,14. 2,7. 1 g·kg~(-1) Bushen Tongluo prescription were given by ig in high and low-dose Bushen Tongluo groups,and 4. 58 g·kg~(-1) hydrocortisone sodium succinate was given by gavage( ig) in normal group and model group. All of the rats were put to death after 28 days. The contents of hydroxyproline( HYP),hyaluronic acid( HA) and laminin( LN) were measured after the death of the rats. Hematoxylin-eosin staining( HE) and Masson tricolor staining were used to observe the pathological changes of lung tissue. Real-time PCR and Western blot were used to observe the changes of mRNA and protein expressions of lung tissues TGF-β_1,Smad2,Smad3,Smad7 in each group. Result: Compared with normal group,the level of pulmonary fibrosis was more significant in model group,and the serum HYP,HA and LN were increased obviously( P < 0. 05,P < 0. 01). The expressions of TGF-β_1,Smad2,Smad3 proteins and mRNA in lung tissues were increased obviously( P < 0. 05,P < 0. 01). The expressions of Smad7 protein and mRNA were decreased significantly( P < 0. 01). Compared with model group,the contents of HYP,HA and LN in high-dose Bushen Tongluo group were decreased obviously( P < 0. 05,P < 0. 01),the expressions of lung tissues TGF-β_1,Smad2,Smad3 protein and mRNA were decreased obviously( P < 0. 05),and the expressions of Smad7 protein and mRNA was increased significantly( P < 0. 01). Conclusion: Bushen Tongluo prescription can effectively improve the pathological process of pulmonary fibrosis,and the mechanism may be related to the regulation of TGF-β_1/Smads pathway.
引文
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[5]姜岩,于功昌,张兴国.转化生长因子-β1及其介导的信号通路在百草枯致肺上皮间质转化中的作用[J].环境与职业医学,2017,34(8):729-733.
[6]苏凤哲,赵昕,周梦佳.补肾通络治疗老年肺纤维化临床研究[J].中医临床研究,2014,6(3):107-109.
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[9]金晓光,代华平,庞宝森,等.博莱霉素致大鼠肺纤维化模型肺组织的动态病理变化及其发生机制[J].中国病理生理杂志,2009,25(4):708-714.
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[12]张丽萍,樊香,郝莉,等.瓜蒌薤白汤对肺纤维化早期阶段肺组织中TGF-β、Smad3、Smad7表达的影响[J].中医学报,2017,32(11):2064-2069.
[13]丛鲁红,吴依娜,段军,等.可吸入颗粒物2. 5对大鼠肺纤维化的影响及糖皮质激素的干预作用[J].心肺血管病杂志,2017,36(6):482-487.
[14]孙莉,荀平.西洋参茎叶皂苷抗大鼠心肌缺血再灌注损伤的作用及机制[J].中国实验方剂学杂志,2014,20(24):176-179.
[15]李启欢,阳如春,阮志忠,等.虫草素的全合成研究[J].有机化学,2013,33(6):1340-1344.
[16]李晶峰,孙佳明,张辉.僵蚕的化学成分及药理活性研究[J].吉林中医药,2015,35(2):175-177.
[17]史磊,张天锡,杜聪颖,等.中药全蝎活性成分、药理作用及临床应用研究进展[J].辽宁中医药大学学报,2015,17(4):89-91.
[18]王仁芳,范令刚,高文远,等.桃仁化学成分与药理活性研究进展[J].现代药物与临床,2010,25(6):426-429.
[19]潘有禄,黄文海,沈正荣,等.肺纤维化发生机制及治疗研究进展[J].中国药学杂志,2012,47(23):1873-1876.
[20]郭敬文,张振玲,邵华.肺纤维化药物治疗机制研究进展[J].中国职业医学,2017,44(5):633-637.
[21]张会超,韩丽华,索红亮,等.基于TGF-β1/Smad3信号通路调控的温阳益气方对心梗后结构重塑的干预作用[J].中国实验方剂学杂志,2018,24(6):114-120.
[22]潘志强,方肇勤,卢文丽,等. Smad信号转导通路在不同证候H22荷瘤小鼠肾上腺的基因表达[J].北京中医药大学学报,2009,32(5):336-340.
[23]王芳芹,刘涛,张攀,等.虾青素通过TGF-β1介导的Smads/ERK通路抑制肺纤维化[J].第三军医大学学报,2017,39(5):442-447.
[24]高向朋,刘清,刘涛,等. TGF-β1/Smad7信号通路在哈萨克族食管癌上皮间质转化中的作用[J].中山大学学报:医学科学版,2015,36(1):78-87.
[25]肖洁,张惠丽,徐米清,等.冬虫夏草菌粉对梗阻性肾病大鼠肾组织羟脯氨酸含量及p21表达的影响[J].中华生物志,2015(4):319-324.
[26]苏奕亮,翁东,周瑛,等.血清LN、1V C、PⅢNP、HA与特发性肺纤维化严重度和预后的相关性[J].中华急诊医学杂志,2018,27(2):188-193.
[27]宋占帅,张娟,张蓉,等.补阳还五汤联合汉防己甲素片改善矽肺肺纤维化效果观察[J].山东医药,2017,57(42):51-53.
[28]柳湘洁,姚弘毅,涂艳,等.呼吸康复训练联合百令胶囊对矽肺合并COPD患者炎症反应及肺纤维化的影响[J].海南医学院学报,2018,24(4):480-483.
[2]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.
[3] HOU J,MA T,CAO H H,et al. TNF-α-induced NF-κB activation promotes myofibroblast differentiation of LRMSCs and exacerbates bleomycin-induced pulmonary fibrosis[J]. J Cell Physiol,2018,233(3):2409-2419.
[4]赵存刚,黄文海,王尊元,等.转化生长因子-β1在特发性肺纤维化中的研究新进展[J].中国药学杂志,2016,51(5):341-345.
[5]姜岩,于功昌,张兴国.转化生长因子-β1及其介导的信号通路在百草枯致肺上皮间质转化中的作用[J].环境与职业医学,2017,34(8):729-733.
[6]苏凤哲,赵昕,周梦佳.补肾通络治疗老年肺纤维化临床研究[J].中医临床研究,2014,6(3):107-109.
[7]刘晓艳,苏凤哲,刘真.补肾通络方治疗老年特发性肺纤维化的临床体会[J].环球中医药,2017,10(9):1112-1113.
[8]刘真,苏凤哲,刘晓艳.自拟方补肾通络汤治疗老年特发性肺纤维化的临床研究[J].世界中西医结合杂志,2017,12(12):1715-1718,1722.
[9]金晓光,代华平,庞宝森,等.博莱霉素致大鼠肺纤维化模型肺组织的动态病理变化及其发生机制[J].中国病理生理杂志,2009,25(4):708-714.
[10]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993:33.
[11] Respir G,Richeldi L. Current approaches to the management of idiopathic pulmonary fibrosis[J]. Respir Med,2017,8(129):24-30.
[12]张丽萍,樊香,郝莉,等.瓜蒌薤白汤对肺纤维化早期阶段肺组织中TGF-β、Smad3、Smad7表达的影响[J].中医学报,2017,32(11):2064-2069.
[13]丛鲁红,吴依娜,段军,等.可吸入颗粒物2. 5对大鼠肺纤维化的影响及糖皮质激素的干预作用[J].心肺血管病杂志,2017,36(6):482-487.
[14]孙莉,荀平.西洋参茎叶皂苷抗大鼠心肌缺血再灌注损伤的作用及机制[J].中国实验方剂学杂志,2014,20(24):176-179.
[15]李启欢,阳如春,阮志忠,等.虫草素的全合成研究[J].有机化学,2013,33(6):1340-1344.
[16]李晶峰,孙佳明,张辉.僵蚕的化学成分及药理活性研究[J].吉林中医药,2015,35(2):175-177.
[17]史磊,张天锡,杜聪颖,等.中药全蝎活性成分、药理作用及临床应用研究进展[J].辽宁中医药大学学报,2015,17(4):89-91.
[18]王仁芳,范令刚,高文远,等.桃仁化学成分与药理活性研究进展[J].现代药物与临床,2010,25(6):426-429.
[19]潘有禄,黄文海,沈正荣,等.肺纤维化发生机制及治疗研究进展[J].中国药学杂志,2012,47(23):1873-1876.
[20]郭敬文,张振玲,邵华.肺纤维化药物治疗机制研究进展[J].中国职业医学,2017,44(5):633-637.
[21]张会超,韩丽华,索红亮,等.基于TGF-β1/Smad3信号通路调控的温阳益气方对心梗后结构重塑的干预作用[J].中国实验方剂学杂志,2018,24(6):114-120.
[22]潘志强,方肇勤,卢文丽,等. Smad信号转导通路在不同证候H22荷瘤小鼠肾上腺的基因表达[J].北京中医药大学学报,2009,32(5):336-340.
[23]王芳芹,刘涛,张攀,等.虾青素通过TGF-β1介导的Smads/ERK通路抑制肺纤维化[J].第三军医大学学报,2017,39(5):442-447.
[24]高向朋,刘清,刘涛,等. TGF-β1/Smad7信号通路在哈萨克族食管癌上皮间质转化中的作用[J].中山大学学报:医学科学版,2015,36(1):78-87.
[25]肖洁,张惠丽,徐米清,等.冬虫夏草菌粉对梗阻性肾病大鼠肾组织羟脯氨酸含量及p21表达的影响[J].中华生物志,2015(4):319-324.
[26]苏奕亮,翁东,周瑛,等.血清LN、1V C、PⅢNP、HA与特发性肺纤维化严重度和预后的相关性[J].中华急诊医学杂志,2018,27(2):188-193.
[27]宋占帅,张娟,张蓉,等.补阳还五汤联合汉防己甲素片改善矽肺肺纤维化效果观察[J].山东医药,2017,57(42):51-53.
[28]柳湘洁,姚弘毅,涂艳,等.呼吸康复训练联合百令胶囊对矽肺合并COPD患者炎症反应及肺纤维化的影响[J].海南医学院学报,2018,24(4):480-483.