| 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. |
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