柴胡皂甙D通过调控TGF-β1/Smads信号通路抑制人胚肺成纤维细胞增殖和胶原蛋白产生
|
摘要
目的研究柴胡皂甙D(SSD)对转化生长因子β1(TGF-β1)诱导的人胚肺成纤维细胞(HELF)增殖、转分化以及TGF-β1/果蝇抗生物皮肤生长因子蛋白家族(Smads)信号通路的影响。方法体外培养HELF,设立正常对照组、 1 ng/mL TGF-β1处理组、 1 ng/mL TGF-β1联合0.5μmol/L SSD处理组、 1 ng/mL TGF-β1联合1μmol/L SSD处理组、 1 ng/mL TGF-β1联合2μmol/L SSD处理组。采用CCK-8法检测HELF的增殖情况,荧光定量PCR法检测Smad2、 Smad3、 Smad7的mRNA水平, Western blot法检测α平滑肌肌动蛋白(α-SMA)、 1型胶原蛋白(Col1)、 Smad2、 Smad3、磷酸化的Smad2(p-Smad2)、 p-Smad3、 Smad7的蛋白水平。结果与正常对照组比较, TGF-β1处理组细胞增殖明显, Col1、α-SMA蛋白水平增加, Smad2和Smad3 mRNA和蛋白磷酸化水平显著增加, Smad7 mRNA和蛋白水平显著降低;与TGF-β1处理组比较,不同浓度SSD处理组细胞增殖降低,可逆转以上各指标的改变,呈一定的量效关系。结论 SSD通过调控TGF-β1/Smads信号通路,发挥抗肺纤维化的作用。
Objective To investigate the effect of saikosaponin D(SSD) on the proliferation and transformation of human embryonic lung fibroblasts(HELFs) induced by transforming growth factor-beta 1(TGF-β1) and the regulation of signal pathway of TGF-β1/Smads family. Methods HELFs were cultured in vitro and divided into 5 groups: a control group, 1 ng/mL TGF-β1-induced group, 1 ng/mL TGF-β1 combined with 0.5 μmol/L SSD treatment group, 1 ng/mL TGF-β1 combined with 1 μmol/L SSD treatment group, and 1 ng/mL TGF-β1 combined with 2 μmol/L SSD treatment group. Cell viability of HELFs was detected by CCK-8 assay. The expression of Smad2, Smad3 and Smad7 mRNA were detected by real-time fluorescence quantitative PCR. The protein levels of α-smooth muscle actin(α-SMA), type 1 collagen(Col1), Smad2, Smad3, phosphorylated Smad2(p-smad2), p-smad3 and Smad7 were assessed by Western blot analysis. Results Compared with the control group, TGF-β1-induced group showed the apparently increased proliferation ability, the increased protein levels of Col1 and α-SMA, the significantly increased mRNA and protein phosphorylation levels of Smad2 and Smad3, and the significantly decreased mRNA and protein expression of Smad7. Compared with the TGF-β1-induced group, the cell proliferation of HELFs in different concentrations of SSD treatment groups was reduced, which could reverse the changes of the above indicators in a dose-dependent manner. Conclusion SSD plays an important role in anti-pulmonary fibrosis by regulating TGF-β1/Smads signaling pathway.
Objective To investigate the effect of saikosaponin D(SSD) on the proliferation and transformation of human embryonic lung fibroblasts(HELFs) induced by transforming growth factor-beta 1(TGF-β1) and the regulation of signal pathway of TGF-β1/Smads family. Methods HELFs were cultured in vitro and divided into 5 groups: a control group, 1 ng/mL TGF-β1-induced group, 1 ng/mL TGF-β1 combined with 0.5 μmol/L SSD treatment group, 1 ng/mL TGF-β1 combined with 1 μmol/L SSD treatment group, and 1 ng/mL TGF-β1 combined with 2 μmol/L SSD treatment group. Cell viability of HELFs was detected by CCK-8 assay. The expression of Smad2, Smad3 and Smad7 mRNA were detected by real-time fluorescence quantitative PCR. The protein levels of α-smooth muscle actin(α-SMA), type 1 collagen(Col1), Smad2, Smad3, phosphorylated Smad2(p-smad2), p-smad3 and Smad7 were assessed by Western blot analysis. Results Compared with the control group, TGF-β1-induced group showed the apparently increased proliferation ability, the increased protein levels of Col1 and α-SMA, the significantly increased mRNA and protein phosphorylation levels of Smad2 and Smad3, and the significantly decreased mRNA and protein expression of Smad7. Compared with the TGF-β1-induced group, the cell proliferation of HELFs in different concentrations of SSD treatment groups was reduced, which could reverse the changes of the above indicators in a dose-dependent manner. Conclusion SSD plays an important role in anti-pulmonary fibrosis by regulating TGF-β1/Smads signaling pathway.
引文
[1] Barratt S L,Creamer A,Hayton C,et al.Idiopathic pulmonary fibrosis (IPF):an overview[J/OL].J Clin Med,2018,7(8):E201.DOI:10.3390/jcm7080201.
[2] Kim H Y,Kim M S,Kim S H,et al.Protective effects of nintedanib against polyhexamethylene guanidine phosphate-induced lung fibrosis in mice[J/OL].Molecules,2018,23(8):E1974.DOI:10.3390/molecules23081974.
[3] Ipatova A Y,Koerner P H,Miller R T,et al.Retrospective analysis of medication utilization and clinical outcomes in patients with idiopathic pulmonary fibrosis treated with nintedanib or pirfenidone[J/OL].Clin Med Insights Circ Respir Pulm Med,2019,13:1179548419834922.DOI:10.1177/1179548419834922.eCollection 2019.
[4] Millan-Billi P,Serra C,Alonso Leon A,et al.Comorbidities,complications and non-pharmacologic treatment in idiopathic pulmonary fibrosis[J/OL].Med Sci (Basel),2018,6(3):59.DOI:10.3390/medsci6030059.
[5] Hochhegger B,Marchiori E,Zanon M,et al.Imaging in idiopathic pulmonary fibrosis:diagnosis and mimics[J/OL].Clinics (Sao Paulo),2019,74:e225.DOI:10.6061/clinics/2019/e225.
[6] Trethewey S P,Walters G I.The role of occupational and environmental exposures in the pathogenesis of idiopathic pulmonary fibrosis:A narrative literature review[J/OL].Medicina (Kaunas),2018,54(6):108.DOI:10.3390/medicina54060108.
[7] Rangarajan S,Lee J S.The TAMing of the idiopathic pulmonary fibrosis myofibroblast.One step closer?[J].Am J Respir Crit Care Med,2018,197(11):1377-1378.
[8] Yu X,Zhang Y,Yang X,et al.The influence of BuqiHuoxueTongluo formula on histopathology and pulmonary function test in bleomycin-induced idiopathic pulmonary fibrosis in rats[J/OL].Evid Based Complement Alternat Med,2018,2018:8903021.DOI:10.1155/2018/8903021.eCollection 2018.
[9] Li X Q,Song Y N,Wang S J,et al.Saikosaponins:a review of pharmacological effects[J].J Asian Nat Prod Res,2018,20(5):399-411.
[10] 郑金旭,卢坤琴,夏德刚,等.柴胡皂甙D对博来霉素诱导肺纤维化小鼠的治疗作用及机制研究[J].中华医学杂志,2010,90(12):808-812.Zheng J,Lu K,Xia D,et al.The therapeutic effects and mechanism of saikosaponin-D on mice with bleomycin-induced pulmonary fibrosis [J].Zhonghua Yi Xue Za Zhi,2010,90(12):808-812.
[11] 郑金旭,许清,管淑红,等.柴胡皂甙D干预肺纤维化VEGF/PEDF表达的实验研究[J].天津医药,2012,40(8):796-799.Zheng J,Xu Q,Guan S,et al.Experimental study on the effect of saikosaponin on the expression of VEGF/PEDF in pulmonary fibrosis[J].Tian Jin Yi Yao,2012,40(8):796-799.
[12] Correll K A,Edeen K E,Redente E F,et al.TGF beta inhibits HGF,FGF7,and FGF10 expression in normal and IPF lung fibroblasts[J/OL].Physiol Rep,2018,6(16):e13794.DOI:10.14814/phy2.13794.
[13] Saito A,Horie M,Micke P,et al.The role of TGF-β signaling in lung cancer associated with idiopathic pulmonary fibrosis[J/OL].Int J Mol Sci,2018,19(11):3611.DOI:10.3390/ijms19113611.
[14] Na M,Hong X,Fuyu J,et al.Proteomic profile of TGF-β1 treated lung fibroblasts identifies novel markers of activated fibroblasts in the silia exposed rat lung[J].Exp Cell Res,2019,375(2):1-9.
[15] 刘理静,尹辉明,贺兼斌,等.大黄素抑制肺成纤维细胞增殖及转分化和胶原蛋白合成[J].细胞与分子免疫学杂志,2016,32(7):921-925.Liu L,Yin H,He J,et al.Emodin inhibits the proliferation,transdifferentiation and collagen synthesis of pulmonary fibroblas[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2016,32(7):921-925.
[16] Black M,Milewski D,Le T,et al.FOXF1 inhibits pulmonary fibrosis by preventing CDH2-CDH11 cadherin switch in myofibroblasts[J].Cell Rep,2018,23(2):442-458.
[17] Sgalla G,Iovene B,Calvello M,et al.Idiopathic pulmonary fibrosis:pathogenesis and management[J/OL].Respir Res,2018,19(1):32.DOI:10.1186/s12931-018-0730-2.
[18] Lederer D J,Martinez F J.Idiopathic pulmonary fibrosis[J].N Engl J Med,2018,378(19):1811-1823.
[19] Chen M F,Huang S J,Huang C C,et al.Saikosaponin d induces cell death through caspase-3-dependent,caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells[J/OL].BMC Cancer,2016,16(1):532.DOI:10.1186/s12885-016-2599-0.
[20] Liu Y,Gao L,Zhao X,et al.Saikosaponin A protects from pressure overload-induced cardiac fibrosis via inhibiting fibroblast activation or endothelial cell EndMT[J/OL].Int J Biol Sci,2018,14(13):1923-1934.
[21] 李敏,高凯,王华林,等.柴胡皂苷D-黄芩苷配伍对CCl4诱导大鼠HSC的TLR4-NF-κB通路作用研究[J].陕西中医,2018,39(1):3-5.Li M,Gao K,Wang H,et al.Inhibitory effects of saikosaponin D-baicalin on activation of rat HSC induced by CCl4 [J].Shan Xi Zhong Yi,2018,39(1):3-5.
[22] Morikawa M,Derynck R,Miyazono K.TGF-β and the TGF-β family:Context-dependent roles in cell and tissue physiology[J/OL].Cold Spring Harb Perspect Biol,2016,8(5):a021873.DOI:10.1101/cshperspect.a021873.
[23] Saito A,Nagase T.Hippo and TGF-β interplay in the lung field[J].Am J Physiol Lung Cell Mol Physiol,2015,309(8):L756-L767.
[24] Xu F,Liu C,Zhou D,et al.TGF/SMAD pathways and its regulation in hepatic fibrosis[J].J Histochem Cytochem,2016,64(3):157-167.
[25] Beppu H.Smad7-modified alleles by various gene-targeting strategies[J].J Biochem,2013,153(5):399-401.
[26] 管淑红,徐乾乾.柴胡皂苷D对人胚肺成纤维细胞增殖的影响及机制探讨[J].中国药物与临床,2015,15(12):1702-1704.Guan S,Xu Q.Effects and mechanism of saikosaponin D on proliferation of human embryonic lung fibroblasts[J].Zhong Guo Yao Wu Yu Lin Chuang,2015,15(12):1702-1704.
[2] Kim H Y,Kim M S,Kim S H,et al.Protective effects of nintedanib against polyhexamethylene guanidine phosphate-induced lung fibrosis in mice[J/OL].Molecules,2018,23(8):E1974.DOI:10.3390/molecules23081974.
[3] Ipatova A Y,Koerner P H,Miller R T,et al.Retrospective analysis of medication utilization and clinical outcomes in patients with idiopathic pulmonary fibrosis treated with nintedanib or pirfenidone[J/OL].Clin Med Insights Circ Respir Pulm Med,2019,13:1179548419834922.DOI:10.1177/1179548419834922.eCollection 2019.
[4] Millan-Billi P,Serra C,Alonso Leon A,et al.Comorbidities,complications and non-pharmacologic treatment in idiopathic pulmonary fibrosis[J/OL].Med Sci (Basel),2018,6(3):59.DOI:10.3390/medsci6030059.
[5] Hochhegger B,Marchiori E,Zanon M,et al.Imaging in idiopathic pulmonary fibrosis:diagnosis and mimics[J/OL].Clinics (Sao Paulo),2019,74:e225.DOI:10.6061/clinics/2019/e225.
[6] Trethewey S P,Walters G I.The role of occupational and environmental exposures in the pathogenesis of idiopathic pulmonary fibrosis:A narrative literature review[J/OL].Medicina (Kaunas),2018,54(6):108.DOI:10.3390/medicina54060108.
[7] Rangarajan S,Lee J S.The TAMing of the idiopathic pulmonary fibrosis myofibroblast.One step closer?[J].Am J Respir Crit Care Med,2018,197(11):1377-1378.
[8] Yu X,Zhang Y,Yang X,et al.The influence of BuqiHuoxueTongluo formula on histopathology and pulmonary function test in bleomycin-induced idiopathic pulmonary fibrosis in rats[J/OL].Evid Based Complement Alternat Med,2018,2018:8903021.DOI:10.1155/2018/8903021.eCollection 2018.
[9] Li X Q,Song Y N,Wang S J,et al.Saikosaponins:a review of pharmacological effects[J].J Asian Nat Prod Res,2018,20(5):399-411.
[10] 郑金旭,卢坤琴,夏德刚,等.柴胡皂甙D对博来霉素诱导肺纤维化小鼠的治疗作用及机制研究[J].中华医学杂志,2010,90(12):808-812.Zheng J,Lu K,Xia D,et al.The therapeutic effects and mechanism of saikosaponin-D on mice with bleomycin-induced pulmonary fibrosis [J].Zhonghua Yi Xue Za Zhi,2010,90(12):808-812.
[11] 郑金旭,许清,管淑红,等.柴胡皂甙D干预肺纤维化VEGF/PEDF表达的实验研究[J].天津医药,2012,40(8):796-799.Zheng J,Xu Q,Guan S,et al.Experimental study on the effect of saikosaponin on the expression of VEGF/PEDF in pulmonary fibrosis[J].Tian Jin Yi Yao,2012,40(8):796-799.
[12] Correll K A,Edeen K E,Redente E F,et al.TGF beta inhibits HGF,FGF7,and FGF10 expression in normal and IPF lung fibroblasts[J/OL].Physiol Rep,2018,6(16):e13794.DOI:10.14814/phy2.13794.
[13] Saito A,Horie M,Micke P,et al.The role of TGF-β signaling in lung cancer associated with idiopathic pulmonary fibrosis[J/OL].Int J Mol Sci,2018,19(11):3611.DOI:10.3390/ijms19113611.
[14] Na M,Hong X,Fuyu J,et al.Proteomic profile of TGF-β1 treated lung fibroblasts identifies novel markers of activated fibroblasts in the silia exposed rat lung[J].Exp Cell Res,2019,375(2):1-9.
[15] 刘理静,尹辉明,贺兼斌,等.大黄素抑制肺成纤维细胞增殖及转分化和胶原蛋白合成[J].细胞与分子免疫学杂志,2016,32(7):921-925.Liu L,Yin H,He J,et al.Emodin inhibits the proliferation,transdifferentiation and collagen synthesis of pulmonary fibroblas[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2016,32(7):921-925.
[16] Black M,Milewski D,Le T,et al.FOXF1 inhibits pulmonary fibrosis by preventing CDH2-CDH11 cadherin switch in myofibroblasts[J].Cell Rep,2018,23(2):442-458.
[17] Sgalla G,Iovene B,Calvello M,et al.Idiopathic pulmonary fibrosis:pathogenesis and management[J/OL].Respir Res,2018,19(1):32.DOI:10.1186/s12931-018-0730-2.
[18] Lederer D J,Martinez F J.Idiopathic pulmonary fibrosis[J].N Engl J Med,2018,378(19):1811-1823.
[19] Chen M F,Huang S J,Huang C C,et al.Saikosaponin d induces cell death through caspase-3-dependent,caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells[J/OL].BMC Cancer,2016,16(1):532.DOI:10.1186/s12885-016-2599-0.
[20] Liu Y,Gao L,Zhao X,et al.Saikosaponin A protects from pressure overload-induced cardiac fibrosis via inhibiting fibroblast activation or endothelial cell EndMT[J/OL].Int J Biol Sci,2018,14(13):1923-1934.
[21] 李敏,高凯,王华林,等.柴胡皂苷D-黄芩苷配伍对CCl4诱导大鼠HSC的TLR4-NF-κB通路作用研究[J].陕西中医,2018,39(1):3-5.Li M,Gao K,Wang H,et al.Inhibitory effects of saikosaponin D-baicalin on activation of rat HSC induced by CCl4 [J].Shan Xi Zhong Yi,2018,39(1):3-5.
[22] Morikawa M,Derynck R,Miyazono K.TGF-β and the TGF-β family:Context-dependent roles in cell and tissue physiology[J/OL].Cold Spring Harb Perspect Biol,2016,8(5):a021873.DOI:10.1101/cshperspect.a021873.
[23] Saito A,Nagase T.Hippo and TGF-β interplay in the lung field[J].Am J Physiol Lung Cell Mol Physiol,2015,309(8):L756-L767.
[24] Xu F,Liu C,Zhou D,et al.TGF/SMAD pathways and its regulation in hepatic fibrosis[J].J Histochem Cytochem,2016,64(3):157-167.
[25] Beppu H.Smad7-modified alleles by various gene-targeting strategies[J].J Biochem,2013,153(5):399-401.
[26] 管淑红,徐乾乾.柴胡皂苷D对人胚肺成纤维细胞增殖的影响及机制探讨[J].中国药物与临床,2015,15(12):1702-1704.Guan S,Xu Q.Effects and mechanism of saikosaponin D on proliferation of human embryonic lung fibroblasts[J].Zhong Guo Yao Wu Yu Lin Chuang,2015,15(12):1702-1704.