Genistein Attenuates Monocrotaline-induced Pulmonary Arterial Hypertension In Rats By Activating PI3K/AKt-eNOS Signaling

Background:Pulmonary arterial hypertension (PAH) is a progressive and life-threateningdisease. Although the present therapeutic agent has been shown to attenuate PAH,drug resistance and the limited long-term effect continue to be major clinicalchallenges and PAH remains an incurable disease. Genistein is a kind ofphyto-oestrogen that has been shown to protect the vascular endothelium. Weestablished the monocrotaline (MCT)-induced PAH in rat and found that Genisteinprotected pulmonary artery effectively, which might be associated with PI3Ksignaling pathway. Based on the literature reports and our preliminary studies, wehypothesized that Genistein acted as an estrogen-like hormone that upregulated andbinded to the estrogen receptor β (ERβ), activated PI3K/AKT-eNOS signalingpathway, promoted nitric oxide (NO) synthesis, modulated vascular tone and finallyprotected right ventricle and prolonged survival rate. We expect to provide atheoretical and experimental basis both in vitro and in vivo for using Genstein to treatPAH in clinic.Objective:To study whether or not PI3K/AKT-eNOS signaling pathway could be involvedin the promotion of pulmonary hypertension in Monocrotaline-pretreated rats byGenistein.Methods:1. Thirty-two male SD rats were randomly divided into2groups (n=16) whichwere control group, monocrotaline group. One, two and three weeks after SD-ratswere injected Monocrotaline, we observed rat pulmonary artery diameter andtricuspid regurgitation velocity. Mean pulmonary arterial pressure （mpap) wasmeasured through the Right heart catheterization. We also got right ventricularhypertrophy index in rats by weighing method. Finally, the data described aboveshowed monocrotaline injection (50mg/kg) could induce pulmonary hypertension inrats. 2. Forty-eight male SD rats were randomly divided into six groups (n=8) whichwere control group, monocrotaline group,20μg/kg genistein treatment group,80μg/kg genistein treatment group,200μg/kg genistein treatment group, PI3Kinhibitor LY294002group (80μg/kg genistein treatment). Hemodynamics would bemonitored. We would weight right ventricleto detect hypertrophy index and observethe changes of pulmonary vascular structure by HE staining. The expression ofproteins would be measured by Western-Blot. A recording plotted curve of nitricoxide (NO) could be generated by nitrate reductase assay. Experimental data tomean±standard deviation, the experimental results of single factor analysis ofvariance or t-test, P<0.05was statistically significant with SPSS19.0statisticalsoftware.Result:1. Three weeks after injecting Monocrotaline, there is a significant differencebetween the two groups in tricuspid regurgitation velocity, pulmonary artery diameter,mPAP, RV/LV+S, so the model is successful.2. Compared to PAH group, tricuspid regurgitation velocity, pulmonary arterydiameter, mean pulmonary artery pressure and right ventricular hypertrophy indexsignificantly reduced in Genistein treatment group with increasing dose (p<0.05).However, in the PI3K inhibitor LY294002group tricuspid regurgitation velocity ratpulmonary artery, mean pulmonary artery pressure and right ventricular hypertrophyindex slightly reduced. There was no significant difference (p>0.05).3. The pulmonary vascular stenosis, smooth muscle proliferation, rightventricular hypertrophy and myocardial hypertrophy were more obvious in PAHgroup than that of control group, What’s more, Genistein could improve thepulmonary vascular stenosis, smooth muscle proliferation, the right ventriclehypertrophy and myocardial hypertrophy in a dose dependend manner. There was nosignificant difference between PI3K inhibitor LY294002group and PAH group.4. Genistein could promote the expression of rat lung tissue P-AKT, P-eNOS andNO with Genistein dose increasing (p<0.05)There was no significant differencecompared to PAH group, although the PI3K inhibitor LY294002could alsoupregulate the level of lung tissue P-AKT, P-eNOS and NO (p>0.05). 5. Each group rat survival rates were respectively100%,29%,63%,75%,100%and43%.Conclusion:Genistein can reduce pulmonary artery pressure and improve right ventricularfunction and survival rate of rat pretreated by Monocrotalin, which might beregulated by PI3K/AKT-eNOS signaling pathway.