当归芍药散含药血清对ET-1诱导的HSC-T6细胞内p-MLCⅡ,MLCⅡ蛋白表达的影响
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摘要
目的:探究内皮素-1(endothelin-1,ET-1)对大鼠肝星状细胞HSC-T6内磷酸化肌球蛋白轻链Ⅱ(phosphorylated myosin light chainⅡ,p-MLCⅡ),肌球蛋白轻链Ⅱ(myosin light chainⅡ,MLCⅡ)蛋白表达的影响及当归芍药散(Danggui Shaoyao San)含药血清对其的干预作用。方法:将HSC-T6细胞种板后,各组每孔加入DMEM和终体积分数分别为2. 5%,5%,10%,15%,20%的空白大鼠血清,采用噻唑蓝(MTT)比色法测定HSC-T6细胞的活力,筛选出适合的大鼠血清浓度范围;将细胞分为空白血清组(5%,10%,15%)和当归芍药散含药血清组(5%,10%,15%),酶联免疫吸附测定(ELISA)检测基础状态下细胞培养上清液中ET-1的含量;细胞分为空白血清组(10%),当归芍药散含药血清低、中、高剂量组(5%,10%,15%),实时荧光定量聚合酶链式反应(Real-time PCR)检测基础状态下细胞培养上清液中ET-1 mRNA的水平;将细胞分为空白血清组(10%),模型组(10%),当归芍药散含药血清低、中、高剂量组(5%,10%,15%),Y-27632抑制剂组(100μmol·L~(-1)),除空白血清组外,其余各组均加入10 nmol·L~(-1)ET-1诱导HSC-T6细胞,蛋白免疫印迹法(Western blot)检测ET-1诱导的HSC-T6细胞中p-MLCⅡ,MLCⅡ蛋白表达。结果:选用血清浓度为5%,10%,15%作为含药血清浓度。与空白血清组比较,当归芍药散含药血清组明显降低基础状态下ET-1含量,ET-1 mRNA相对含量(P <0. 05,P <0. 01)。与空白血清组比较,模型组细胞内p-MLCⅡ,MLCⅡ蛋白表达水平均显著升高(P <0. 01);与模型组比较,当归芍药散含药血清各剂量组及Y-27632抑制剂组均可明显下调p-MLCⅡ,MLCⅡ蛋白表达(P <0. 05,P <0. 01)。结论:当归芍药散含药血清可能通过下调ET-1的含量,抑制ET-1的自分泌,从而下调p-MLCⅡ,MLCⅡ蛋白表达。
Objective: To investigate the effect of endothelin-1( ET-1) on the expression of phosphorylated myosin light chain Ⅱ( p-MLC Ⅱ) and myosin light chain Ⅱ( MLC Ⅱ) protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San( DSS) drug-containing serum.Method: After HSC-T6 cells were seeded,DMEM and blank rat serum with final concentrations of 2. 5%,5%,10%,15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium( MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group( 5%,10%,15%) and DSS drug-containing serum group( 5%,10%,15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group( 10%),DSS drug-containing serum low( 5%),medium( 10%) and high dose( 15%) groups.Real-time fluorescent quantitative polymerase chain reaction( Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group( 10%),model group( 10%),DSS drug-containing serum low( 5%),medium( 10%),high dose( 15%)groups and Y-27632 inhibitor group( 100 μmol·L~(-1)). Except the blank serum control group,the other groups all received 10 nmol·L~(-1) ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡand MLCⅡ in HSC-T6 cells induced by ET-1. Result: Serum concentrations of 5%,10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group,the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state( P < 0. 05,P <0. 01). As compared with the blank serum control group,the expression of p-MLCⅡ and MLCⅡ protein in the model group was significantly increased( P < 0. 01); DSS drug-containing serum groups and Y-27632 inhibitor group can significantly down-regulate p-MLC Ⅱ and MLC Ⅱ protein expression( P < 0. 05, P < 0. 01).Conclusion: DSS drug-containing serum may down-regulate the expression of p-MLC Ⅱ and MLC Ⅱ by downregulating the content of ET-1 and inhibiting the autocrine of ET-1.
Objective: To investigate the effect of endothelin-1( ET-1) on the expression of phosphorylated myosin light chain Ⅱ( p-MLC Ⅱ) and myosin light chain Ⅱ( MLC Ⅱ) protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San( DSS) drug-containing serum.Method: After HSC-T6 cells were seeded,DMEM and blank rat serum with final concentrations of 2. 5%,5%,10%,15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium( MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group( 5%,10%,15%) and DSS drug-containing serum group( 5%,10%,15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group( 10%),DSS drug-containing serum low( 5%),medium( 10%) and high dose( 15%) groups.Real-time fluorescent quantitative polymerase chain reaction( Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group( 10%),model group( 10%),DSS drug-containing serum low( 5%),medium( 10%),high dose( 15%)groups and Y-27632 inhibitor group( 100 μmol·L~(-1)). Except the blank serum control group,the other groups all received 10 nmol·L~(-1) ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡand MLCⅡ in HSC-T6 cells induced by ET-1. Result: Serum concentrations of 5%,10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group,the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state( P < 0. 05,P <0. 01). As compared with the blank serum control group,the expression of p-MLCⅡ and MLCⅡ protein in the model group was significantly increased( P < 0. 01); DSS drug-containing serum groups and Y-27632 inhibitor group can significantly down-regulate p-MLC Ⅱ and MLC Ⅱ protein expression( P < 0. 05, P < 0. 01).Conclusion: DSS drug-containing serum may down-regulate the expression of p-MLC Ⅱ and MLC Ⅱ by downregulating the content of ET-1 and inhibiting the autocrine of ET-1.
引文
[1]李文静,兴桂华,刘军,等.狼毒大戟配伍大枣对诱导肝硬化大鼠的抵抗作用及机制[J].中国实验方剂学杂志,2017,23(21):117-123.
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[4] Rodríguez-Pascual F, Busnadiego O, GonzálezSantamaría J. The profibrotic role of endothelin-1:is the door still open for the treatment of fibrotic diseases[J].Life Sci,2014,118(2):156-164.
[5]熊莎,高建蓉,胡祖良,等.鳖甲提取物对抑制TGF-β诱导的大鼠肝星状细胞活化的影响[J].中国实验方剂学杂志,2017,23(19):155-159.
[6]甘霞,赵新芳,林红,等.加味赤石脂禹余粮汤对脾肾阳虚证肝硬化腹水患者的影响及疗效分析[J].中国实验方剂学杂志,2016,22(6):172-176.
[7] JI H,MENG Y,ZHANG X,et al. Aldosterone induction of hepatic stellate cell contraction through activation of Rho A/ROCK-2 signaling pathway[J]. Regul Pept,2011,169(1/3):13-20.
[8] SHI M,WEI J,MENG W Y,et al. Effects of phased joint intervention on Rho/ROCK expression levels in patients with portal hypertension[J]. Exp Ther Med,2016,12(3):1618-1624.
[9]路燕,宋育林,季巍巍,等.磷酸化肌球蛋白轻链在大鼠非酒精性脂肪性肝纤维化中的表达[J].安徽医科大学学报,2012,47(4):370-374.
[10] ZHANG C G,ZHANG B,DENG W S,et al. Role of estrogen receptorβselective agonist in ameliorating portal hypertension in rats with CCl4-induced liver cirrhosis[J]. World J Gastroenterol,2016,22(18):4484-4500.
[11]许钒,宋欣,陶春蕾,等.当归芍药散对慢性应激抑郁模型大鼠行为及中枢单胺类神经递质的影响[J].中国中药杂志,2011,36(13):1824-1826.
[12]王成业,许钒,王满媛,等.当归芍药散对肝硬化腹水大鼠的干预作用研究[J].中国中药杂志,2013,38(6):871-874.
[13]潘永福,许钒,王成业,等.基于水通道蛋白研究当归芍药散对肝硬化腹水大鼠的干预作用[J].中国实验方剂学杂志,2015,21(8):111-115.
[14] Sakamoto Y,Sakai M,Watari T. Hepatic and plasma endothelin-1 in dogs with chronic hepatitis[J]. J Vet Intern Med,2017,31(3):764-769.
[15] Klein S,Rick J,Lehmann J,et al. Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis[J]. Gut,2017,66(1):145-155.
[16]吴惠春,张斌.肝星状细胞与抗肝纤维化治疗的研究进展[J].实用肝脏病杂志,2012,15(2):172-173.
[17] Thabut D,Shah V. Intrahepatic angiogenesis and sinusoidal remodeling in chronic liver disease:new targets for the treatment of portal hypertension[J]. J Hepatol,2010,53(5):976-980.
[18] Kisseleva T,Brenner D A. Hepatic stellate cells and the reversal of fibrosis[J]. J Gastroenterol Hepatol,2006,21(3):S84-S87.
[19] Rockey D. The cellular pathogenesis of portal hypertension:stellate cell contractility,endothelin,and nitric oxide[J]. Hepatology,1997,25(1):2-5.
[20] FAN M,SU C,LU L,et al. Ultrasonic diagnosis and vasoactive substances examination in patients with cirrhosis[J]. Asian Pac J Trop Med,2014,7(4):329-332.
[21]张军,张忠涛.内皮素对肝星状细胞作用的研究进展(文献综述)[J].国外医学:外科学分册,2004,31(2):100-102.
[22] Rockey D C,Fouassier L,Chung J J,et al. Cellular localization of endothelin-1 and increased production in liver injury in the rat:potential for autocrine and paracrine effects on stellate cells[J]. Hepatology,1998,27(2):472-480.
[23] Sward K,Dreja K,Susnjar M,et al. Inhibition of Rhoassociated kinase blocks agonist-induced Ca2+sensitization of myosin phosphorlation and force. in guinea pig ileum[J]. J Physiol,2000,522(1):33-49.
[24] Bi D,Nishimura J,Niiro N,et al. Contraetile properties of the cultured vaseular smooth musele cells:the crueial role played by rho A in the regulation of contraetility[J].Cire Res,2005,96(8):890-897.
[25] Hersch E,HUANG J,Grider J R,et al. Gq/G13signaling by ET-1 in Smooth muscle:MYPT1phosphorylation via ETA and CPI-17 dephosphorylation via ETB[J]. Am J Physiol Cell Physiol,2004,287(5):C1209-C1218.
[26] SONG J,LU Y P,LUO G H,et al. Effects of mycophenolate mofetil on chronic allograft nephropathy by affecting RHO/ROCK signal pathways[J].Transplant Proc,2008,40(8):2790-2794.
[27]毛萧萧,周正翔,夏珂,等. Y-27632通过抑制ROCK通路降低内皮细胞中基质金属蛋白酶2和9表达[J].中南大学学报:医学版,2016,41(6):566-570.
[28] LIU C,ZUO J,Janssen L J. Regulation of airway smooth muscle Rho/ROCK activities by cholinergic and bronchodilator stimuli[J]. Eur Respir J,2006,10(28):703-711.
[2] Schepis F,Turco L,Bianchini M,et al. Prevention and management of bleeding risk related to invasive procedures in cirrhosis[J]. Semin Liver Dis,2018,38(3):215-229.
[3] Khimji A K,Rockey D C. Endothelin and hepatic wound healing[J]. Pharmacol Res,2011,63(6):512-518.
[4] Rodríguez-Pascual F, Busnadiego O, GonzálezSantamaría J. The profibrotic role of endothelin-1:is the door still open for the treatment of fibrotic diseases[J].Life Sci,2014,118(2):156-164.
[5]熊莎,高建蓉,胡祖良,等.鳖甲提取物对抑制TGF-β诱导的大鼠肝星状细胞活化的影响[J].中国实验方剂学杂志,2017,23(19):155-159.
[6]甘霞,赵新芳,林红,等.加味赤石脂禹余粮汤对脾肾阳虚证肝硬化腹水患者的影响及疗效分析[J].中国实验方剂学杂志,2016,22(6):172-176.
[7] JI H,MENG Y,ZHANG X,et al. Aldosterone induction of hepatic stellate cell contraction through activation of Rho A/ROCK-2 signaling pathway[J]. Regul Pept,2011,169(1/3):13-20.
[8] SHI M,WEI J,MENG W Y,et al. Effects of phased joint intervention on Rho/ROCK expression levels in patients with portal hypertension[J]. Exp Ther Med,2016,12(3):1618-1624.
[9]路燕,宋育林,季巍巍,等.磷酸化肌球蛋白轻链在大鼠非酒精性脂肪性肝纤维化中的表达[J].安徽医科大学学报,2012,47(4):370-374.
[10] ZHANG C G,ZHANG B,DENG W S,et al. Role of estrogen receptorβselective agonist in ameliorating portal hypertension in rats with CCl4-induced liver cirrhosis[J]. World J Gastroenterol,2016,22(18):4484-4500.
[11]许钒,宋欣,陶春蕾,等.当归芍药散对慢性应激抑郁模型大鼠行为及中枢单胺类神经递质的影响[J].中国中药杂志,2011,36(13):1824-1826.
[12]王成业,许钒,王满媛,等.当归芍药散对肝硬化腹水大鼠的干预作用研究[J].中国中药杂志,2013,38(6):871-874.
[13]潘永福,许钒,王成业,等.基于水通道蛋白研究当归芍药散对肝硬化腹水大鼠的干预作用[J].中国实验方剂学杂志,2015,21(8):111-115.
[14] Sakamoto Y,Sakai M,Watari T. Hepatic and plasma endothelin-1 in dogs with chronic hepatitis[J]. J Vet Intern Med,2017,31(3):764-769.
[15] Klein S,Rick J,Lehmann J,et al. Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis[J]. Gut,2017,66(1):145-155.
[16]吴惠春,张斌.肝星状细胞与抗肝纤维化治疗的研究进展[J].实用肝脏病杂志,2012,15(2):172-173.
[17] Thabut D,Shah V. Intrahepatic angiogenesis and sinusoidal remodeling in chronic liver disease:new targets for the treatment of portal hypertension[J]. J Hepatol,2010,53(5):976-980.
[18] Kisseleva T,Brenner D A. Hepatic stellate cells and the reversal of fibrosis[J]. J Gastroenterol Hepatol,2006,21(3):S84-S87.
[19] Rockey D. The cellular pathogenesis of portal hypertension:stellate cell contractility,endothelin,and nitric oxide[J]. Hepatology,1997,25(1):2-5.
[20] FAN M,SU C,LU L,et al. Ultrasonic diagnosis and vasoactive substances examination in patients with cirrhosis[J]. Asian Pac J Trop Med,2014,7(4):329-332.
[21]张军,张忠涛.内皮素对肝星状细胞作用的研究进展(文献综述)[J].国外医学:外科学分册,2004,31(2):100-102.
[22] Rockey D C,Fouassier L,Chung J J,et al. Cellular localization of endothelin-1 and increased production in liver injury in the rat:potential for autocrine and paracrine effects on stellate cells[J]. Hepatology,1998,27(2):472-480.
[23] Sward K,Dreja K,Susnjar M,et al. Inhibition of Rhoassociated kinase blocks agonist-induced Ca2+sensitization of myosin phosphorlation and force. in guinea pig ileum[J]. J Physiol,2000,522(1):33-49.
[24] Bi D,Nishimura J,Niiro N,et al. Contraetile properties of the cultured vaseular smooth musele cells:the crueial role played by rho A in the regulation of contraetility[J].Cire Res,2005,96(8):890-897.
[25] Hersch E,HUANG J,Grider J R,et al. Gq/G13signaling by ET-1 in Smooth muscle:MYPT1phosphorylation via ETA and CPI-17 dephosphorylation via ETB[J]. Am J Physiol Cell Physiol,2004,287(5):C1209-C1218.
[26] SONG J,LU Y P,LUO G H,et al. Effects of mycophenolate mofetil on chronic allograft nephropathy by affecting RHO/ROCK signal pathways[J].Transplant Proc,2008,40(8):2790-2794.
[27]毛萧萧,周正翔,夏珂,等. Y-27632通过抑制ROCK通路降低内皮细胞中基质金属蛋白酶2和9表达[J].中南大学学报:医学版,2016,41(6):566-570.
[28] LIU C,ZUO J,Janssen L J. Regulation of airway smooth muscle Rho/ROCK activities by cholinergic and bronchodilator stimuli[J]. Eur Respir J,2006,10(28):703-711.