The Study Of The Effects Of Fasudil On Monocrotaline-Induced Pulmonary Hypertension In Rats

Objectives: To discuss the therapeutic effect of fasudil for monocrotaline-induced pulmonary arterial hypertension (PAH) in rats, further study the pathogenesis of the Rho/ROCK pathway which is involved in 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 56 healthy male Sprague-Dawley rats were used, were randomly divided into 5 groups: normal control group (4 weeks) (N4),model control group(4 weeks) (M4), normal control group(8 weeks) (N8),model control group(8 weeks) (M8), fasudil group(8 weeks) (F8).Animals in F8 group received intraperitoneal injection of fasudil hydrochloride (15mg/kg per day) respectively from the end of the 4th week to the end of the 8th week. Normal control group and PAH group just received vehicle by the same way and dose. When the ended, polyethylene catheters were inserted into the RV through the jugular vein for hemodynamic measurements. Right ventricular systolic pressure (RVSP) 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 MYPT-1 in lungs were also analyzed by western blot, as well as p-MYPT-1.Results:(1) Survival analysis: At the end of the prevention protocol, all rats were survived except 41rats which were involved in group N4,M4 and N8, survived rates of group M8,F8 were 32.25%and 75.00%. (2)Hemodynamic: At the end of the 4th week after injected MCT with increased RVSP and mPAP(P<0.01) compared with the normal control groups, what confirmed that the model of PAH was success established. At the end of 8th week, the parameters above increased over time. The differences of them between F8 group and M8 group were significant(P<0.05), although still higher than in N8group(P<0.05).(3)Right ventricular hypertrophy assessment: Compared to N4 group, the RV/(LV+S) was markedly increased in M4 group(P<0.05);At the end of 8th week, it was more increased in M8 group(P<0.01);The cured group F8 was markedly decreased the RV/(LV+S), but the differences between F8 group and N8groupwere not significant(P>0.05).(4)Morphology: The M4 group the arteriole endomembrane ,medial thickness, WT% and WA% were also markedly increased(P<0.05),the parameters above increased over time. The differences of them between M8 group and M4 group were significant(P<0.05).WT% and WA% were also markedly increased(P<0.01), The fasudil markedly suppressed the development of PAH in F8 group, the differences between them were significant(P<0.01).(5) Protein expression:①Immunohistochemistry:Compare with N4 group, the expression of ROCK-1 were markedly inceased(P<0.01), M8 and N8 were the same(P<0.01), The differences of them between M8 group and M4 group were significan(tP<0.05); It decreased in F8 group compared to M8 group(P<0.01),but also significant with N8 group(P<0.05);②Western blot: The protein expression of ROCK-1 markedly increased in M4 and M8 group(P<0.01), the differences between M4 group and M8 group were not significant(P>0.05). It decreased in F8 group compared to M8 group(P<0.01),but also significant with N8 group(P<0.05). The protein expression of ROCK-1 was positively correlated with mPAP, RV/(LV+S)(r=0.897,r=0.747, P<0.01). The extent of MYPT-1 phosphorylation was markedly increased in M4 and M8 group(P<0.01). Although still higher than in N8group(P<0.05), it was markedly decreased in F8 group(P<0.01)after the therapy. The extent of MYPT-1 phosphorylation was positively correlated with the protein expression of ROCK-1,as well as the mPAP and the RV/(LV+S)(r=0.724, r=0.727, r=0.689, P<0.01).Conclusions: Fasudil can reverse the MCT-induced PAH effectually in rats, which could induce medial thickness, arteriole endomembrane, myocardium remolding, and the sustained contraction of PVSMC and the increase of mPAP. The activation of ROCK-1 directly contributed to the contraction of PVSMC, also regulated the expression of the endothelia1 cell factor, and further shows that Rho/ROCK pathway plays a key role in the pathogenesis of MCT-induced PAH.