Fluoxetine Protects Against MCT-induced Pulmonary Hypertension Accompanied By Increased Expression Of BMPR-â…¡ In Rats

ObjectivePulmonary hypertension (PHT) is a life - threatening disease, characterized by sustained elevation of small pulmonary arterial pressure, leading to right ventricular failure and death. PHT is pathologically characterized by vasocon-striction of the small pulmonary arteries, proliferation in all layers of the vessel wall, thrombosis, and inflammation. Many mediators were related with the development of PHT, such as 5 -HT, ET, ATâ…¡ and TXA₂.Serotonin (5 - HT) is one of the important vasomotor agents, and its importance in the pathogenesis of PHT is currently being investigated. Progression of PHT is associated with increased lung expression of the serotonin transporter (5 - HTT) , which leads to hyperplasia of PASMCs. 5 - HTT pathway is affected by BMPR - â…¡ mutation and BMPR - â…¡ gene played role in the pathophysiolo-gy of pulmonary hypertension. These mutations of BMPR - â…¡ are found in at least half of familial cases of pulmonary arterial hypertension and 10% - 25% of idiopathic pulmonary arterial hypertension patients, indicating that BMPR -â…¡ may contribute to the maintenance of normal pulmonary vascular structure and function. Reduced lung BMPR - â…¡ expression was found in pulmonary hypertension due to an elevated pulmonary vascular resistance in mice carrying a mutant BMPR - â…¡ gene, alluding that normal signaling from BMPR - â…¡ is associated with suppression of smooth muscle cell proliferation and hence may antagonize the effects of 5 - HT. BMPR - â…¡ and 5 - HTT may play independent roles inpathogenesis of the vascular lesion characteristic of PHT.Nevertheless , the cross - talk between 5 - HTT and BMPR - H is not yet to be clearly defined. The expression of BMPR - EmRNA level in PHT model induced by MCT in rats, and the effect of SSRI fluoxetine on BMPR - II mRNA level expression have not been fully elicited. So the objective of the present study includes the following aspects:1. To investigate the protection of fluoxetine against the pulmonary hypertension induced by monocrotaline in rats.2. To observe the expression changes of BMPR - II mRNA in PHT model induced by MCT.3. To investigate the relationship between 5 - HTT and BMPR - II through the effect of SSRI fluoxetine on the BMPR - II mRNA expression.MethodsExperimental animal Wistar rats 30 6 , Grade II , weighting 150 ± lOg, from Animal Resource Center, China Medical University, Certificate No: Liaon-ing 034.Drugs and reagents Monocrotaline was purchased from Sigma Co ( St Louis , USA) . Fluoxetine hydrochloride was from Eli Lilly Co ( USA) . TRIzol was purchased from Promega Co ( USA). RT - PCR kit was from Takara Co ( Japan ) . Primer of BMPR - II and p - actin were from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. Gel analyzer ( UVP, GDS8000, USA) , Uviospectrophotometer (UV -200, JAP) ,Metamorphy/BX41 (UIC/O-LYMPUS, US A/JAP).Monocrotaline ( MCT) model of PHT in rats Wistar rats were divided into three groups. MCT model group and fluoxetine - treated group rats were dealt with intraperitoneal injection of monocrotaline 60mg/kg, respectively, and then MCT model group rats were treated with vehicle, while the fluoxetine - treated group rats were treated with intragastric administration of fluoxetine hydrochloride 7mg/kg everyday. While the control rats were intraperitoneal injected with the solution of ethanol and saline, and intragastric administrated with an equalvolume of vehicle. And then these rats were fed with solid food and water ad lib in an alternating 12h light/dark cycle under controlled temperature (18 -22T! ) and humidity (50% -70% ) for 3 weeks.Hemodynamic measurement Rats in all groups were intraperitoneal anesthetized by 3% pentobarbital sodium ( 1. 5mg/kg) , and cervical arteries were dissected and intubated to estimate the systemic blood pressure, and then external jugular vein were dissected and catheterized into right ventricle to indirectly measure the pulmonary arterial pressure.Measurement of right ventricular index On the 21st day after administration of monocrotaline, fluoxetine or saline, these rats were killed and then taken out of hearts. The right ventricle (RV) , septum and the left ventricle ( S + LV) were dissected and weighed separately to evaluate the magnitude of the right ventricular hypertrophy which was expressed as the right ventricular index, RV/ ( S + LV).Morphological analysis of pulmonary arteries by HE staining The right lower lung tissues were cut into a size no larger than 3mm thick. After being fixed and paraffin embedded, lung sections (5|xm thick) were stained with he-matoxyline - eosine ( HE) , watched under light microscopy and analyzed by Metamorphy/BX41. The remodeling of pulmonary arteries was calculated as the ratio of PA medial wall thickness to PA external diameter.RT - PCR Pulmonary arteries ( PA) were dissected from peripheral tissues immediately and stored at — 70 °C. Total RNA was extracted with TRIzol Reagent as described in its directions. The concentration and purification of total RNA was estimated with ultra spectrophotometer. RT - PCR to identity mRNA expression of BMPR - H in rat PA was performed with RT - PCR kit. The gene of special primers of BMPR — II and p - actin were described as: BMPR — II a;ATA GGC GTG TGC CAA AAA TC;BMPR - II b:GCT AGG GAT TCG AGC TTG TG;3 - actin a: CAC CCT GTG CTG CTC ACC GAG GCC;p - actin b: CCA CAC AGA TGA CTT GCG CTC AGG. The reaction conditions were as follows;reserved transcription to synthesized cDNA at 50TI for 30 min, and then denature cDNA at 94V. for 3 min. Next 30 cycles ( 94^ for 45 sec, 57T: for 1 min, 12X1 for 1 min) were performed. The PCR products were checked with2% agarose gel electrophoresis stained with ethidium bromide (5mg/L) , and then scanned and semi - quantified with gel analyzer. Gene expression of BMPR - II was represented by the relative yield to the p - actin gene.Results1. Hemodynamic assessmentThe mean pulmonary artery pressures in control, MCT, MCT + Flu groups werell.7 ±2.5mmHg, 19.7 ±4.5mmHg and 13.9 ±2.8mmHg, respectively. In MCT - injected rats, the increased pulmonary arterial pressure (P<0.01 vs control) was reduced to control level by fluoxetine given during the entire experimental period (P <0. 01 vs MCT group). The systemic pressures in control, MCT, MCT + Flu groups were 116. 5 ± 15. 2 mmHg, 104. 3 ±9. 4 mmHg and 110.3 ±6.9 mmHg, but the pressures were similar in all groups.2. Right ventricular indexIn control, MCT, MCT + Flu groups, the right ventricle weights were 0. 163 ±0. 025g, 0. 218 ±0. 015g and 0. 186 ±0. 021 g;the weights of septum and the left ventricle were 0. 462 ± 0. 066g, 0. 458 ±0. 038g and 0. 502 ±0. 109g, respectively. Right ventricular index in control, MCT, MCT + Flu groups were 0. 355 ± 0. 052, 0. 478 ± 0. 042 and 0. 380 ± 0. 021, respectively. MCT can cause myocardial hypertrophy, and the right ventricular index was significantly increased in MCT group (P <0. 01 vs control) , and consistent with PA pressure data, but in fluoxetine administration group it was markedly decreased compared with the MCT group.3. Morphological analysis of pulmonary arteries by HE stainingIn control, MCT, MCT + Flu groups, the external diameters were 120.25 ±44.65 jxm, 141.66 ±42.25|xm and 151.47 ±12. 10|xm;the medial thickness were 40. 91 ±15. 79|xm, 59. 64 ±27. 46u,m and 57. 07 ± 12. 26jxm;the percentages of the medial wall thickness were 36. 33% ±6.31% , 47. 57% ±4. 91% and 38.33% ±3.27% , respectively. Medial hypertrophic and hyperplas-tic changes in pulmonary arteries were prominent in MCT?— injected group compared with normal control group;these features were markedly inhibited in flu-oxetine — treated group. Thus fluoxetine prevented the increase in the percent wall thickness.4. Expression of BMPR - H mRNA on the PA by RT - PCRIn control, MCT, MCT + Flu groups, the relative BMPR - II mRNA level in PA (BMPR - II mRNA /p - actin mRNA) were 0. 99 ±0. 09, 0. 77 ± 0. 07 and 0. 90 ±0. 08. BMPR - II mRNA level in PA from MCT group is significantly reduced (P <0.01 vs control);fluoxetine reversed the expression of BMPR - II mRNA level (P <0. 05 vs MCT group) .5. The relationship between the relative expression of BMPR - II mRNA and the mean pulmonary arterial pressureCorrelation analysis between the relative expression of BMPR - II mRNA and the mean pulmonary arterial pressure was identified significantly negative. Pearson R is 0. 649, P =0. 022 <0. 05. Regression equation;Y = 1. 125 -0. 01528X (Fig. 6).Conclusion1. Fluoxetine protects against pulmonary hypertension induced by monocro-taline in rats by decreasing the increased mPAP and RVI, and inhibiting the pulmonary vascular remodeling.2. MCT Reduced BMPR - II mRNA expression on PA compared with control groups.3. SSRI fluoxetine reversed BMPR - H mRNA expression on PA compared with MCT groups.4. There existed a significantly negative correlation between mean pulmonary artery pressure and the relative expression of BMPR - II mRNA.