| Part1 Expression of Jagged2/Notch3 signaling molecules in pulmonary arterial hypertension rats induced by monocrotalineObjectives:To explore the expression of Jagged2/Notch3/Hes5 signaling molecule in pulmonary arterial hypertension(PAH)rats.Methods:1 Totally 45 Male Sprague-Dawley(SD) rats were randomly divided into normal control group(C group, n=15), solvent control group(S group, n =15) and monocrotaline(MCT) model group(M group, n =15). The model group were established by a single intraperitoneal injection of MCT(50mg/kg). The rats in S group were given a single intraperitoneal injection of the same dose of solvent. The untreated rats were used as normal control group.2 Four weeks later, the right ventricular systolic pressure(RVSP) and mean pulmonary artery pressure(m PAP) was determined by right heart catheterization. RVHI(right ventricle hypertrophy index) and the percentage of medial thickness(MT%) in each group were measured by histopathological analysis.3 When pulmonary arterial hypertension model was established successfully, all rats were killed directly through air embolism. Using dissection tools, pulmonary artery and pulmonary margin were removed to investigate the expression of Jagged2/Notch3/Hes5 in each group by real-time fluorescent quantitative polymerase chain reaction(RT-PCR) and immunohistochemical assays respectively. The correlation of Jagged2/Notch3 m RNA with m PAP in M group was analyzed statistically.4 Data were expressed as mean ± standard deviation(SD) and processed by SPSS18.0. One-way analysis of variance and linear correlation analysis were used to analyze the differences among all 3 groups.Results:1 Compared with S and C groups, we found that the blood vessel wall of M group were thickened and MT% of smaller arteries increased significantly(MT%: 0.71±0.04 vs 0.38±0.05 and 0.37±0.05, Mn=12, Sn=13, Cn=14, P<0.05). Besides, the levels of m PAP、RVSP and RVHI in M group were significantly higher than those in S and C groups(m PAP:29.92±5.03 mm Hg vs 16.82±2.17 mm Hg and 17.20±1.97 mm Hg;RVSP:56.09±18.89 mm Hg vs 25.47±1.56 mm Hg and 25.69±1.94 mm Hg;RVHI:0.42±0.04 vs 0.21±0.01 and 0.21±0.02;Mn=12,Sn=13,Cn=14,P<0.05). However, no difference was found between the S group and C group(P>0.05).2 Immunohistochemical staining and RT-PCR indicated that Notch3 and Hes5 mainly expressed in the medial smooth muscle cells, Jagged2 mainly expressed in the intima of small lung artery. Compared with S and C groups, the expression of Jagged2/Notch3/Hes5 was significantly increased in the lung small arteries of M group(Notch3 m RNA: 1.7080±0.1330 vs 0.9915±0.0378 and 0.9982±0.0007;Jagged2 m RNA: 10.2700±0.2543 vs 0.9917±0.0160 and 0.9982 ±0.0007; Hes5 m RNA: 1.8030±0.2783 vs 0.9825±0.0160 and 0.9982±0.0007;Mn=12,Sn=13,Cn=14,P<0.05). However, no difference was found between the S group and C group(P>0.05).3 There was a positive correlation between Notch3 m RNA and m PAP( r=0.587,p=0.045); However, no correlation was found between Jagged2 m RNA and m PAP( r=-0.353,p=0.260).Conclusion:Four weeks later,the expression of Jagged2/Notch3/Hes5 in the lung small arteries of M group was significantly increased after a single intraperitoneal injection of MCT. There was a positive correlation between Notch3 m RNA and m PAP. However, there was no correlation between Jagged2 m RNA and m PAP.Part2 Effect of simvastatin treatment on the expression of Jagged2/Notch3 signaling molecules in MCT induced PAH ratsObjectives:To explore the effect of simvastatin(2mg/kg/day) treatment on the expression of Jagged2/Notch3 signaling molecules in MCT induced PAH rats.Methods:1. 24 male Sprague-Dawley rats were treated as follows: intraperitoneal injection of monocrotaline(50 mg/kg)(n=24).After 3 weeks, MCT-treated rats were randomized to gavage with vehicle(0.9% saline) treatment group(C group,n =12)and simvastatin(2mg/kg/day) treatment group(S group,n =12). All drugs were administered by intragastric administration for 14 days.2. After treated with drugs for 2 weeks, and the hemodynamics were analyzed, all rats were killed directly through air embolism. Using dissection tools, pulmonary artery and pulmonary margin were removed to investigate the pathological shape and expression of Jagged2/Notch3 in each group by hematoxylin-eosin(HE) staining 、 RT-PCR 、immunofluorescence co-localization and western blotting(WB)assays respectively.3. Data were expressed as mean ± SD and processed by SPSS18.0. Independent-sample t test and linear correlation analysis were used to analyze the differences between 2 groups.Results:1.Compared with control group, we found that the blood vessel wall of simvastatin group became thin and various measures of vascular remodeling decreased significantly. The results of C and S groups are as follows(m PAP:31.55±3.63 mm Hg vs 21.11±3.09 mm Hg;RVSP: 56.09±18.89 mm Hg vs 30.06±3.08 mm Hg; RVHI:0.45±0.09 vs 0.34±0.04;MT%: 0.72±0.13 vs 0.51±0.05; Cn=12,Sn=12, P<0.05).2. Immunofluorescence co-localization indicated that Notch3 and Hes5 mainly expressed in the medial smooth muscle cells, Jagged2 mainly expressed on the intima of small lung artery. Compared with C group, the expression of Jagged2 was significantly increased in the lung small arteries of S group. Nevertheless, the level of Notch3 showed a downward trend in the S group. The results of C and S groups are as follows(Notch3:0.5165±0.0061 vs 0.4331±0.0074;Jagged2:0.4355±0.0140 vs 0.6160±0.0070;Notch3 m RNA: 2.1221±0.1766 vs 1.0821±0.1014; Jagged2 m RNA : 0.3874±0.0200 vs 0.7017±0.0850;Cn=12,Sn=12, P<0.05).Conclusion:Simvastatin relieved the severity of pulmonary vascular remodeling in MCT induced PAH rats, possibly by intervening on the Jagged2/Notch3 signaling. |
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