Rho/ROCK通路在野百合碱诱导大鼠肺动脉高压中的作用及法舒地尔的干预
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摘要
目的:建立野百合碱诱导的大鼠肺动脉高压模型,探讨Rho/ROCK信号转导通路在肺动脉高压发病机制中的作用,观察法舒地尔对该模型肺动脉高压的预防及治疗作用。
方法:采用一次性皮下注射1%野百合碱(MCT)50mg/Kg建立SD大鼠肺动脉高压模型;84只SD雄性大鼠随机分为:(1)预防组:正常4周组(N4)、模型4周组(M4)、地尔硫卓4周组(D4)、法舒地尔4周组(F4);(2)治疗组:正常8周组(N8)、模型8周组(M8)、地尔硫卓8周组(D8)、法舒地尔8周组(F8)。D4组、F4组于造模第3天开始分别予地尔硫卓(0.5mg/kg.d)、法舒地尔(15mg/kg.d)干预4周; D8组、F8组于造模第4周末开始分别予地尔硫卓(0.5mg/kg.d)、法舒地尔(15mg/kg.d)干预4周。各组干预结束后经心导管法测量平均右心室压力(mRVP)、平均肺动脉压力(mPAP)和右心室肥厚指数[RV/ (LV+S)];HE染色观察肺小动脉50-150μm血管厚度与血管外径之比(WT%)和管壁面积占血管总面积的百分比(WA%);免疫组化方法检测ROCK-1、内皮型一氧化氮合酶(eNOS)蛋白在肺组织的表达;逆转录聚合酶链式反应(RT-PCR)法检测ROCK-1、eNOS、前内皮素-1原(preproET-1)mRNA在肺组织的表达。
结果:(1)注射MCT4周后大鼠肺动脉高压模型建立成功, M4组mRVP、mPAP、RV/( LV+S)显著升高(P<0.01),HE染色显示肺小动脉内膜明显增生,中膜明显增厚,管腔狭窄,WT%、WA%显著升高(P<0.01),上述指标随时间进一步升高,M8组与M4组比较差异有统计学意义(P<0.05)。预防组中F4组、D4组mRVP、mPAP、RV/( LV+S)、WT%、WA%均较M4组下降(P<0.05),但F4组下降更明显,与D4组比较差异有统计学意义(P<0.01)。治疗组结果与预防组类似。
(2)预防组中: N4组ROCK-1 mRNA和蛋白仅少量表达,M4组表达显著升高(P<0.01);F4组ROCK-1 mRNA和蛋白表达较M4组下降(P<0.05),但仍高于N4组(P<0.05), D4组与M4组比较差异无统计学意义(P>0.05)。治疗组结果与预防组类似。
(3)预防组中:N4组preproET-1 mRNA少量表达,M4组表达显著升高(P<0.01);F4组较M4组表达显著下降(P<0.01),与N4组比较差异无统计学意义(P>0.05);D4组表达与M4组比较差异无统计学意义(P>0.05)。治疗组结果与预防组类似。
(4)预防组中:N4组eNOS mRNA和蛋白大量表达,M4组表达显著下降(P<0.01);F4组eNOS mRNA表达较M4组升高(P<0.05), D4组与M4组比较差异无统计学意义(P>0.05)。治疗组结果与预防组类似。
(5)ROCK-1表达与preproET-1表达、mPAP、WT%和WA%呈显著正相关(r=0.630、r=0.640、r=0.679、r=0.707,均P<0.01),ROCK-1表达与eNOS表达呈显著负相关(r=-0.815,P<0.01)。
结论:法舒地尔能有效地降低肺动脉压,抑制肺小动脉平滑肌的收缩、增生和肥厚,抑制肺小血管重构和右心室肥厚,阻止野百合碱诱导的肺动脉高压的形成或明显缓解已经形成的肺动脉高压,提示Rho/ ROCK信号通路在肺动脉高压的发病机制中起重要作用,这些作用可能通过激活ROCK-1直接影响肺动脉平滑肌收缩、上调preproET-1表达、下调eNOS表达及促进肺血管增生重构实现的。
Objectives: To examine whether the Rho/ROCK pathway is involved in the pathogenesis of monocrotaline-induced fatal pulmonary arterial hypertension (PAH) in rats,and the potential preventive of therapeutic effect of fasudil for PAH.
Methods: Sprague-Dawley rats received a subcutaneous injection of monocrotaline(50mg/Kg)once for all, which resulted in the development of severe PAH.A total of 84 adult male Sprague-Dawley rats were used, including 32 for prevention study, 52 for treatment study. Each part was randomly divided into 4 groups:(1)Prevention protocol:normal control group (4 weeks) (N4),model control group(4 weeks) (M4), dilthiazem group(4 weeks) (D4),fasudil group(4 weeks) (F4) ;(2)Treatment protocol: normal control group(8 weeks) (N8),model control group(8 weeks) (M8), dilthiazem group(8 weeks) (D8),fasudil group(8 weeks) (F8).Animals in D4 group and F4 group received intraperitoneal injection of dilthiazem(0.5mg/kg per day) and fasudil hydrochloride (15mg/kg per day) respectively from the 3th day to the end of the 4th week; Animals in D8 and F8 groups received intraperitoneal injection of dilthiazem (0.5mg/kg per day) and fasudil hydrochloride (15mg/kg per day) respectively from the end of the 4th week to the end of the 8th week.When the prevention and treatment ended,polyethylene catheters were inserted into the RV through the jugular vein for hemodynamic measurements. Mean right ventricular pressure(mRVP) and mean pulmonary artery pressure(mPAP) were measured with a polygraph system.RV hypertrophy index[RV/ (LV+S)] was also measured.Arteries of 50 to 150μm were evaluated for the median wall thickness and wall area by HE staining as follows: percent wall thickness(WT%)= [(medial thickness×2/external diameter)]×100 and percent wall area (WT%)= (wall area / total area)×100. The protein expressions of ROCK-1 and endothelial nitric oxide synthase (eNOS) in lungs were also analyzed by immunohistochemical analysis.The mRNA expression of ROCK-1,eNOS and preproET-1 in lungs was analyzed by reverse transcription-polymerase chain reaction(RT-PCR).
Results:
(1) The model groups developed severe PAH at the end of the 4th week after injected MCT with increased mRVP,mPAP and RV/( LV+S) (P<0.01) compared with the normal control groups, arteriole endomembrane ,medial thickness,WT% and WA% were also markedly increased(P<0.01),the parameters above increced over time.The differences of them between M8 group and M4 group were significant(P<0.05). In the prevention protocol,compared with M4 group, both fasudil and dilthiazem markedly suppressed the development of PAH in both D4 group and F4 group, but the effects in F4 group were better than those in D4 group, the differences between them were significant(P<0.01).The results in the treatment protocol were similar to those in the prevention protocol.
(2) In the prevention protocol, compared to N4 group,the expression of ROCK-1 mRNA and protein markedly increased in M4 group(P<0.01);they decreased in F4 group compared to M4 group(P<0.05),but they were still higher than in N4 group(P<0.05), the differences between D4 group and N4 group were not significant(P>0.05).The results in the treatment protocol were similar to those in the prevention protocol.
(3) In the prevention protocol, compared to N4 group,the expression of preproET-1 mRNA markedly increased in M4 group(P<0.01);it decreased in F4 group compared to M4 group(P<0.05),but the differences between F4 group and N4 group were not significant(P>0.05), the differences between D4 group and N4 group also were not significant(P>0.05).The results in the treatment protocol were similar to those in the prevention protocol.
(4) In the prevention protocol, compared to N4 group,the expression of eNOS mRNA and protein markedly decreased in M4 group(P<0.01);they increased in F4 group compared to M4 group(P<0.05);the differences between D4 group and N4 group were not significant(P>0.05).The results in the treatment protocol were similar to those in the prevention protocol.
(4)The expression of ROCK-1 was positively correlated with the expression of preproET-1 ,mPAP, WT% and WA%(r=0.630、r=0.640、r=0.679、r=0.707, P<0.01);The expression of ROCK-1 was negatively correlated with the expression of eNOS(r=-0.815,P<0.01).
Conclusion: Fasudil prevented or even caused a markedly improvement of the MCT-induced PAH through multiple mechanisms, including Fasudil markedly reduced PAP,suppressed proliferation and contraction of pulmonary VSMC, pulmonary remodeling, and RV hypertrophy.The result shows that Rho/ROCK pathway plays an important role in the pathogenesis of MCT-induced PAH. These may be contributed to improve contraction of pulmonary VSMC directly,up-regulate the expression of preproET-1,down-regulate the expression of eNOS and also enhance pulmonary remodeling.
方法:采用一次性皮下注射1%野百合碱(MCT)50mg/Kg建立SD大鼠肺动脉高压模型;84只SD雄性大鼠随机分为:(1)预防组:正常4周组(N4)、模型4周组(M4)、地尔硫卓4周组(D4)、法舒地尔4周组(F4);(2)治疗组:正常8周组(N8)、模型8周组(M8)、地尔硫卓8周组(D8)、法舒地尔8周组(F8)。D4组、F4组于造模第3天开始分别予地尔硫卓(0.5mg/kg.d)、法舒地尔(15mg/kg.d)干预4周; D8组、F8组于造模第4周末开始分别予地尔硫卓(0.5mg/kg.d)、法舒地尔(15mg/kg.d)干预4周。各组干预结束后经心导管法测量平均右心室压力(mRVP)、平均肺动脉压力(mPAP)和右心室肥厚指数[RV/ (LV+S)];HE染色观察肺小动脉50-150μm血管厚度与血管外径之比(WT%)和管壁面积占血管总面积的百分比(WA%);免疫组化方法检测ROCK-1、内皮型一氧化氮合酶(eNOS)蛋白在肺组织的表达;逆转录聚合酶链式反应(RT-PCR)法检测ROCK-1、eNOS、前内皮素-1原(preproET-1)mRNA在肺组织的表达。
结果:(1)注射MCT4周后大鼠肺动脉高压模型建立成功, M4组mRVP、mPAP、RV/( LV+S)显著升高(P<0.01),HE染色显示肺小动脉内膜明显增生,中膜明显增厚,管腔狭窄,WT%、WA%显著升高(P<0.01),上述指标随时间进一步升高,M8组与M4组比较差异有统计学意义(P<0.05)。预防组中F4组、D4组mRVP、mPAP、RV/( LV+S)、WT%、WA%均较M4组下降(P<0.05),但F4组下降更明显,与D4组比较差异有统计学意义(P<0.01)。治疗组结果与预防组类似。
(2)预防组中: N4组ROCK-1 mRNA和蛋白仅少量表达,M4组表达显著升高(P<0.01);F4组ROCK-1 mRNA和蛋白表达较M4组下降(P<0.05),但仍高于N4组(P<0.05), D4组与M4组比较差异无统计学意义(P>0.05)。治疗组结果与预防组类似。
(3)预防组中:N4组preproET-1 mRNA少量表达,M4组表达显著升高(P<0.01);F4组较M4组表达显著下降(P<0.01),与N4组比较差异无统计学意义(P>0.05);D4组表达与M4组比较差异无统计学意义(P>0.05)。治疗组结果与预防组类似。
(4)预防组中:N4组eNOS mRNA和蛋白大量表达,M4组表达显著下降(P<0.01);F4组eNOS mRNA表达较M4组升高(P<0.05), D4组与M4组比较差异无统计学意义(P>0.05)。治疗组结果与预防组类似。
(5)ROCK-1表达与preproET-1表达、mPAP、WT%和WA%呈显著正相关(r=0.630、r=0.640、r=0.679、r=0.707,均P<0.01),ROCK-1表达与eNOS表达呈显著负相关(r=-0.815,P<0.01)。
结论:法舒地尔能有效地降低肺动脉压,抑制肺小动脉平滑肌的收缩、增生和肥厚,抑制肺小血管重构和右心室肥厚,阻止野百合碱诱导的肺动脉高压的形成或明显缓解已经形成的肺动脉高压,提示Rho/ ROCK信号通路在肺动脉高压的发病机制中起重要作用,这些作用可能通过激活ROCK-1直接影响肺动脉平滑肌收缩、上调preproET-1表达、下调eNOS表达及促进肺血管增生重构实现的。
Objectives: To examine whether the Rho/ROCK pathway is involved in the pathogenesis of monocrotaline-induced fatal pulmonary arterial hypertension (PAH) in rats,and the potential preventive of therapeutic effect of fasudil for PAH.
Methods: Sprague-Dawley rats received a subcutaneous injection of monocrotaline(50mg/Kg)once for all, which resulted in the development of severe PAH.A total of 84 adult male Sprague-Dawley rats were used, including 32 for prevention study, 52 for treatment study. Each part was randomly divided into 4 groups:(1)Prevention protocol:normal control group (4 weeks) (N4),model control group(4 weeks) (M4), dilthiazem group(4 weeks) (D4),fasudil group(4 weeks) (F4) ;(2)Treatment protocol: normal control group(8 weeks) (N8),model control group(8 weeks) (M8), dilthiazem group(8 weeks) (D8),fasudil group(8 weeks) (F8).Animals in D4 group and F4 group received intraperitoneal injection of dilthiazem(0.5mg/kg per day) and fasudil hydrochloride (15mg/kg per day) respectively from the 3th day to the end of the 4th week; Animals in D8 and F8 groups received intraperitoneal injection of dilthiazem (0.5mg/kg per day) and fasudil hydrochloride (15mg/kg per day) respectively from the end of the 4th week to the end of the 8th week.When the prevention and treatment ended,polyethylene catheters were inserted into the RV through the jugular vein for hemodynamic measurements. Mean right ventricular pressure(mRVP) and mean pulmonary artery pressure(mPAP) were measured with a polygraph system.RV hypertrophy index[RV/ (LV+S)] was also measured.Arteries of 50 to 150μm were evaluated for the median wall thickness and wall area by HE staining as follows: percent wall thickness(WT%)= [(medial thickness×2/external diameter)]×100 and percent wall area (WT%)= (wall area / total area)×100. The protein expressions of ROCK-1 and endothelial nitric oxide synthase (eNOS) in lungs were also analyzed by immunohistochemical analysis.The mRNA expression of ROCK-1,eNOS and preproET-1 in lungs was analyzed by reverse transcription-polymerase chain reaction(RT-PCR).
Results:
(1) The model groups developed severe PAH at the end of the 4th week after injected MCT with increased mRVP,mPAP and RV/( LV+S) (P<0.01) compared with the normal control groups, arteriole endomembrane ,medial thickness,WT% and WA% were also markedly increased(P<0.01),the parameters above increced over time.The differences of them between M8 group and M4 group were significant(P<0.05). In the prevention protocol,compared with M4 group, both fasudil and dilthiazem markedly suppressed the development of PAH in both D4 group and F4 group, but the effects in F4 group were better than those in D4 group, the differences between them were significant(P<0.01).The results in the treatment protocol were similar to those in the prevention protocol.
(2) In the prevention protocol, compared to N4 group,the expression of ROCK-1 mRNA and protein markedly increased in M4 group(P<0.01);they decreased in F4 group compared to M4 group(P<0.05),but they were still higher than in N4 group(P<0.05), the differences between D4 group and N4 group were not significant(P>0.05).The results in the treatment protocol were similar to those in the prevention protocol.
(3) In the prevention protocol, compared to N4 group,the expression of preproET-1 mRNA markedly increased in M4 group(P<0.01);it decreased in F4 group compared to M4 group(P<0.05),but the differences between F4 group and N4 group were not significant(P>0.05), the differences between D4 group and N4 group also were not significant(P>0.05).The results in the treatment protocol were similar to those in the prevention protocol.
(4) In the prevention protocol, compared to N4 group,the expression of eNOS mRNA and protein markedly decreased in M4 group(P<0.01);they increased in F4 group compared to M4 group(P<0.05);the differences between D4 group and N4 group were not significant(P>0.05).The results in the treatment protocol were similar to those in the prevention protocol.
(4)The expression of ROCK-1 was positively correlated with the expression of preproET-1 ,mPAP, WT% and WA%(r=0.630、r=0.640、r=0.679、r=0.707, P<0.01);The expression of ROCK-1 was negatively correlated with the expression of eNOS(r=-0.815,P<0.01).
Conclusion: Fasudil prevented or even caused a markedly improvement of the MCT-induced PAH through multiple mechanisms, including Fasudil markedly reduced PAP,suppressed proliferation and contraction of pulmonary VSMC, pulmonary remodeling, and RV hypertrophy.The result shows that Rho/ROCK pathway plays an important role in the pathogenesis of MCT-induced PAH. These may be contributed to improve contraction of pulmonary VSMC directly,up-regulate the expression of preproET-1,down-regulate the expression of eNOS and also enhance pulmonary remodeling.
引文
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[32] Kphtaro Abe,Hiroaki S,Keiko M,et a1.Long-term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats[J].Circ Res,2004,94(3):385—393.
[33] Lee CG,Cho SJ,Kang MJ,et a1.Early growth response gene 1-mediated apoptosis is essential for transforming growth factor betal—induced pulmonary fibrosis[J].J Exp Med,2004,200(3):377—389.
[34] 陈慧,王师贞,白玉茹等.硝苯吡啶对先天性心脏病肺动脉高压的治疗作用[J] .中华心血管病杂志,1989,17(4):205.
[35] Ishikura K,Yamada,Ito M,et a1.Beneficial acute effects of Rho—Kinase inhibitor in patients with pulmonary arterial hypertension[J].Circ J,2006,70(2):174—178.
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[2] Etienne-M anneville S,Hall A.Rho GTPases in cell biology[J].Nature,2002,420(6916):629—635.
[3] Ishizaki T,Natio M,Fujisawa K,et a1.pl60ROCK,fl Rhoassociated coiled-coil forming protein kinase , works downstream of Rho and induces focal adhesions[J].FEBS Lett,1997,404(2-3):118--124.
[4] Etienne Manneville S,Hall A Nature,2002,420(6916):629—635.
[5] Somlyo AP,Somlyo AV.Ca2+ sensitivity of smooth m uscle and nonm uscle myosin II: modulated by G proteins kinases,and myosin phosphatase[J].Physiol Rev,2003,83(4):1325—1358.
[6] Wang Z,Jin N,Ganguli S,et a1.Rho-kinase activation is involved in hypoxia induced pulmonary vasoconstriction[J].Am J Respir Cell Mol Bio1,2001,25(5):628-635.
[7] Wang Z,Lanner MC,Jin N,et a1.Hypoxia inhibits myosin phosphatase in pulmonary arterial smooth muscle cells:role of Rho-kinase[J].Am J Respir Cell Mol Biol,2003,29(4):465—47l.
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[16] Palmer RMJ,Ferrige AG,Moncada SA,et a1.Nitric oxide release accounts for the biological activity of endoth elium derived relaxing factor[J].Nature,1987,327(5):524—526.
[17] Corone P.Nature history of ventricular septal defect .A study involing 790 cases[J].Circulation,1977,55(8):908.
[18] 师桃,耿希刚.先天性心脏病肺动脉高压患者肺组织内皮型一氧化氮合成酶的表达及肺血管重建[J] .西安交通大学学报(医学版),2003,24(1):37--39.
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[7] Wang Z,Lanner MC,Jin N,et a1.Hypoxia inhibits myosin phosphatase in pulmonary arterial smooth muscle cells:role of Rho-kinase[J].Am J Respir Cell Mol Biol,2003,29(4):465—47l.
[8] Robertson TP,Dipp M ,Ward JP,et a1.Inhibition of sustained hypoxic vasoconstriction by Y 27632 in isolated intrapulmonary arteries and perfused lung of the rat[J].Br J Pharmacol,2000.131(1):5—9.
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[1] Wettschureck N.offermanns S. Rho/Rho-kinase mediated signaling in physiology and pathophysiology[J].J Mol Med,2002,80(10):629—638.
[2] Etienne-M anneville S,Hall A.Rho GTPases in cell biology[J].Nature,2002,420(6916):629—635.
[3] Ishizaki T,Natio M,Fujisawa K,et a1.pl60ROCK,fl Rhoassociated coiled-coil forming protein kinase , works downstream of Rho and induces focal adhesions[J].FEBS Lett,1997,404(2-3):118--124.
[4] Etienne Manneville S,Hall A Nature,2002,420(6916):629—635.
[5] Somlyo AP,Somlyo AV.Ca2+ sensitivity of smooth m uscle and nonm uscle myosin II: modulated by G proteins kinases,and myosin phosphatase[J].Physiol Rev,2003,83(4):1325—1358.
[6] Wang Z,Jin N,Ganguli S,et a1.Rho-kinase activation is involved in hypoxia induced pulmonary vasoconstriction[J].Am J Respir Cell Mol Bio1,2001,25(5):628-635.
[7] Wang Z,Lanner MC,Jin N,et a1.Hypoxia inhibits myosin phosphatase in pulmonary arterial smooth muscle cells:role of Rho-kinase[J].Am J Respir Cell Mol Biol,2003,29(4):465—47l.
[8] Robertson TP,Dipp M ,Ward JP,et a1.Inhibition of sustained hypoxic vasoconstriction by Y 27632 in isolated intrapulmonary arteries and perfused lung of the rat[J].Br J Pharmacol,2000,131(1):5—9.
[9] Robertson TP,Aaronson PI,W ard JP.Hypoxic vasoconstriction and intracellular Cain pulmonary arteries : Evidence for PKC-independent Ca2+ sensitization[J].Am J Physiol,1 995,268(1-2):301—307.
[10] Uehata M ,Ishizaki T,Satoh H ,et a1.Nature,1997;389(6654):990--994
[11] Tadashi K,Iliroaki S,Yasushi M,et al.Involvement of Rho-kinasc in Agonists-induced contractions of arterioselerotic human arteries[J].Arterioscler Thromb Vasc Biol,2002,22:243-248.
[12] Feng J,Ito M,Iehikawa K,et al.Inhibitory phosphorylation site forRho-associated kinase on smooth muscle myosin phosphatase[J].J Biol Chem,1999,274:37385-37390.
[13] Kawano Y,Fukata Y,Oshiro N,et a1.Phosphorylation of myosin- binding suhunit(MBS) of myosin phusphatase by Rho-kinase in vivo[J].J Cell Biol,1999,147:1023一1038.
[14] Somlyo AP,Somlyo AV.Ca2+ sensitivity of smooth muscle and non-muscle myosin II : Modulated by G-proteins , kinases and myosin phosphatase[J]. Physiol Rev,2003,83 :1325—1358.
[15] 孙兴珍,王大为,李军等.Rock-1基因在缺氧性肺动脉高压大鼠肺组织中的表达及法舒地尔的预防作用[J].南京医科大学学报(自然科学版),2006,26(2):96—100.
[16] Palmer RMJ,Ferrige AG,Moncada SA,et a1.Nitric oxide release accounts for the biological activity of endoth elium derived relaxing factor[J].Nature,1987,327(5):524—526.
[17] Corone P.Nature history of ventricular septal defect .A study involing 790 cases[J].Circulation,1977,55(8):908.
[18] 师桃,耿希刚.先天性心脏病肺动脉高压患者肺组织内皮型一氧化氮合成酶的表达及肺血管重建[J] .西安交通大学学报(医学版),2003,24(1):37--39.
[19] Stendel W ,Inchinose F ,Huang PL ,et a1.Pulmonary vasoconstriction and hypertension in mice witll targeted disruption of the endothelial nitric oxide synthase (NOS3) gene[J].Circulation Research,1997,81(1):34—41.
[20] Takemoto M,Sun J,Hiroki J,et a1.Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase[J].Circulation,2002,106(1):57--62.
[21] 曾真,姜桢,王春生.肺循环血流增加造成肺高压模型大鼠 preproET-1mRNA和 eNOSmRNA 表达的改变[J].复旦学报(医学版),2006,33(2):157--160.
[22] Lee CG,Cho SJ,Kang MJ,et a1.Early growth response gene 1-mediated apoptosis is essential for transforming growth factor betal—induced pulmonaryfibrosis[J].J Exp Med,2004,200(3):377—389.
[23] 牟华明,祝之明,祝善俊等.NADPH 氧化酶与 Rho/Rho-激酶激活在外膜介导的血管重塑中的作用[J].中华心血管病杂志,2003,31(8):613—616.
[24] Kphtaro Abe,Hiroaki S,Keiko M,et a1.Long·term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats[J].Circ Res,2004,94(3):385—393.