| Pulmonary artery hypertension (PAH) is a progressive and fatal disease characterized by an increase of vascular resistance, abnormal sustained pulmonary vasoconstriction, and progressive structural remodeling of pulmonary arteries, for which no clear pathogenesy has been developed. Recent studies suggest that constriction and remodeling of pulmonary arteries are the key point of PAH formation. The disequilibrium of proliferation of PASMCs and apoptosis results in pulmonary arteries remodeling. Therefore, the key points of PAH therapy include inhibiting the pulmonary artery constriction, improving the pulmonary circulation and preventing the proliferation of PASMCs.Calcium signals play an important role in the formation of PAH. Studies suggest that the elevation of [Ca2+]i is one of the most important initiation agents to trigger proliferation of PASMCs. In the progress of PAH, many cell factors including 5-HT can elevate the [Ca2+]i after they bind to their receptors. Therefore, the elevation of [Ca2+]i is important to PAH.Recently, calcium antagonists are frequently used in the treatment of PAH in clinic, but the clear mechanisms have not been elucidated. M-nisoldipine (m-Nis), a Ca2+ channel blocker of dihydropyridines (DHPs), is developed by Hebei Medical University. Early studies suggested that m-Nis had favourable stability and could inhibit vasoconstriction. Therefore, in the present study, monocrotaline (MCT) was use to induce PAH in rats, then the effect of m-Nis on PAH and pulmonary artery remodeling were studied. The methods such as immunhistochemistry, flow cytometry, confocal laser scanning microscope and molecular biology techniques were applied to explore the effect of m-Nis on 5-HT induced proliferation and migration of PASMCs and the mechanisms. Part 1 The effect of m-Nis on MCT-induced PAH in RatsObjective: To study the effect of m-Nis on MCT-induced PAH in rats.Methods: Rats were injected with a single dose (60mg/kg) of MCT subcutaneously to induce PAH. Pulmonary haemodynamic measurement and lung tissue morphological investigations were undertaken. The MDA production and SOD activity in the serum were tested. Expression of PCNA, ERK1 and p-ERK1/2 expression in pulmonary blood vessels was analyzed by western blot. The expression of 5-HT and PCNA in pulmonary tissues was observed by immunohistochemistry.Results:1 Effect of m-Nis on PAP and hemodynamic variables in MCT-induced PAH ratsCompared with the control group, mean PAP in the MCT group was significantly elevated (from 10.5±1.6 to 17.4±1.6mmHg, P<0.01). Compared with the MCT group, mean PAP in m-Nis 0.5, 1.0 and 2.0 mg/kg groups was decreased to 14.4±1.1,14.0±1.3 and 13.6±0.9 mmHg(P<0.01), respectively。Yet, SAP and heart rate did not differ significantly among the five groups.2 Effect of m-Nis on right ventricle hypertrophy in MCT-induced PAH ratsBody weight was significantly decreased by MCT injection (P<0.05). The ratio of right ventricular weight to body weight (RV/BW) was increased by MCT from 0.79±0.09 to 1.05±0.09 (P<0.01 vs control) and reduced by m-Nis 0.5, 1.0, 2.0mg/kg to 0.90±0.04 (P<0.05 vs MCT), 0.83±0.07 and 0.85±0.09 (P<0.01 vs MCT) respectively. Meanwhile, the right ventricular index RV/(LV+SP) was increased by MCT from 0.307±0.007 to 0.408±0.015 (P<0.01 vs control) and reduced obviously by m-Nis 0.5, 1.0, 2.0mg/kg to 0.365±0.013, 0.341±0.023 and 0.353±0.021 respectively (P<0.01 vs MCT).3 Effect of m-Nis on the morphological alterations of small pulmonary arteriesSmall pulmonary arteries in MCT group exhibited obvious medial wall thickening and lumen transverse area reducing, the MT% markedly increased, whereas the VA% decreased compared with those in control group (P<0.01). In m-Nis-treated groups, the pulmonary artery wall thickening was attenuated in different degrees, the MT% was decreased in m-Nis 0.5 mg/kg group (P<0.05) and m-Nis 1.0, 2.0 mg/kg groups (P<0.01) compared with that in MCT group. Meanwhile, the VA% was significantly increased in all the m-Nis groups compared with that in MCT group (P<0.01).4 Effect of m-Nis on the activity of SOD and the content of MDACompared with control group, the content of MDA in MCT group was significantly elevated from 6.32±0.27 to 7.62±0.63 (P<0.01), and the activity of SOD was decreased from 163±10 to 122±11 (P<0.01). Compared with the MCT group, a significant reduction in MDA production was observed in all three m-Nis 0.5, 1.0 and 2.0mg/kg groups (from 7.62±0.63 to 6.49±0.82, 6.76±0.38 and 6.40±0.60 respectively) (P<0.05), which was associated with the increase of SOD activity (from 122±11 to 142±12, 149±8 and 146±11 respectively) (P<0.05 or P<0.01).5 5-HT immunohistochemistry assessmentIn MCT group, the percentage of 5-HT-positive cells (14.2±3.0%) was higher than that in control group (4.2±2.1%) (P<0.01), which was reduced to 8.6±1.3%,7.1±1.9% and 7.2±1.0% by m-Nis 0.5, 1.0 and 2.0mg/kg treatment (P<0.01 vs MCT group).6 Effect of m-Nis on the expression of PCNAFrom the result of immunohistological staining, we found that in MCT group the percentage of PCNA-positive cells was remarkably increased compared with the control group (P<0.01), which was decreased by pretreatment with m-Nis (P<0.01). Western blot analysis indicated that the expression of PCNA was significantly higher in MCT group than that in control group (P<0.01), which was markedly decreased (P<0.05 vs MCT group) by m-Nis 0.5, 1.0 and 2.0mg/kg pretreatment.7 Effect of m-Nis on the activation of ERK1/2Western blot analysis showed that p-ERK1/2 expression in MCT group exhibited a significant increase compared with that in control group (P<0.01), which was obviously decreased by m-Nis 0.5, 1.0 and 2.0mg/kg (P<0.01). About the expression of ERK1/2, no statistical difference was found among the five groups. So, the ratio of p-ERK1/2/ERK1/2 increased significantly in MCT group compared with that in control group (P<0.01), which was decreased significantly by m-Nis (P<0.05).Conclusion: M-Nis protects against MCT-induced pulmonary artery hypertension, which may be related to the antioxidation effect, the reduction of 5-HT exression and the suppression of the ERK/MAPK signal pathway. Part 2 The effect of m-Nis on 5-HT-induced proliferation, migration of PASMCs and change of [Ca2+]iObjective: To study the effect of m-Nis on 5-HT-induced proliferation, migration of PASMCs and change of [Ca2+]i.Methods: PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Transwell chambers were used to detect the migration. Cell-cycle distribution was determined by flow cytometry assay. The expression of PCNA was evaluated by immunocytochemical stain and western blot. The change of [Ca2+]i was measured by confocal microscopy. The activity of CaN was measured using kits. The phosphorylation of ERK1/2 and JNK was detected by western blot. The mRNA expression of c-fos, c-jun, CaM and CaN was evaluated by RT-PCR.Results:1 Effect of m-Nis on 5-HT-induced proliferation of PASMCsThe results of MTT assay suggested that 5-HT significantly increased the A value from 0.48±0.04 to 0.74±0.09 (P<0.01), which was decreased to 0.54±0.05,0.57±0.07,0.59±0.06 (P<0.01) and 0.62±0.07 (P<0.05) by m-Nis 10-5,10-6,10-7,10-8 mol/L, respectively. Similarly, western blot analysis of PCNA indicated that the expression of PCNA was significantly higher in 5-HT group than that in control group (P <0.01). Whereas, in four m-Nis treated groups, the level of PCNA was markedly decreased (P<0.05 or P<0.01). Next, the results of immunocytochemical stain suggested that in 5-HT group the percentage of PCNA-positive cells was remarkably increased compared with that in control group (P < 0.01), which was decreased by pretreatment with m-Nis 10-5,10-6,10-7 and 10-8 mol/L (P < 0.01).2 Effect of m-Nis on 5-HT-induced PASMCs migrationCompared with control group, the number of migration cells increased from 34±7 to 95±13 (P<0.01) after 5-HT treatment, which was decreased to 43±9,44±10,58±9 and 62±9 (P <0.01) by m-Nis 10-5,10-6,10-7 and 10-8 mol/L pretreatment, respectively.3 Effect of m-Nis on cell-cycle progressionThe percentage of cells in S phase increased dramatically from 4% to 21% after 5-HT stimulation (P<0.01) and was accompanied by a reduction in the percentage of cells in G1 phase from 84% to 55% (P<0.01). M-Nis 10-5,10-6,10-7 mol/L markedly blocked 5-HT-induced cell-cycle progression, with the percentages of PASMCs entering the S phase decreasing to 6%, 10% and 11% (P<0.05 or P<0.01), and those arrested in the G0/G1 phase increasing to 78%, 71% and 64% respectively (P<0.05 or P<0.01). Meanwhile, the proliferation index increased significantly from 16% to 45% after 5-HT stimulation (P<0.01) and was decreased to 22%, 29% and 36% respectively by preincubation with m-Nis 10-5,10-6,10-7 mol/L (P<0.05 or P<0.01).4 Effect of 5-HT on fluorescence intensity of [Ca2+]i in rat PASMCsThe fluorescence intensity that represented the [Ca2+]i of PASMCs was significantly increased and reached the peak at 90 s after treated with 5-HT. After that, the [Ca2+]i decreased gradually and reached to a stable state at 200 s after 5-HT treatment.5 Effect of m-Nis on [Ca2+]i in rat PASMCsThe relative fluorescence intensity of intracellular Ca2+ was significantly increased from 3.0±1.0% to 132.4±4.3% after perfusion with 5-HT (P <0.01 vs control), which was decreased to 20.1±4.1%,33.6±9.0%,41.5±2.3% and 54.7±2.2% (P<0.01) respectively by preincubation with m-Nis 10-5,10-6,10-7 and 10-8 mol/L.6 Effect of m-Nis on the activity of CaN in rat PASMCsThe activity of CaN was significantly increased from 1.7±0.5 to 3.7±0.8 by 5-HT treatment (P <0.01 vs control), which was decreased to 2.4±0.6, 2.9±0.6 (P<0.01), 3.0±0.7 and 3.1±0.5 (P<0.05) respectively by preincubation with m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L.7 Effect of m-Nis on mRNA expression of CaM and CaN in rat PASMCsThe mRNA expression of CaM and CaN increased obviously after 1μmol/L 5 -HT treatment for 24h (P<0.01), which was significantly reduced (P<0.05 or P<0.01) by pretreatment with m-Nis 10-5, 10-6 and 10-7 mol/L. M-Nis 10-8 mol/L also significantly reduced the CaN mRNA expression, but have no effect on CaM mRNA expression.8 Effect of m-Nis on the phosphorylation of ERK1/2 and JNK in rat PASMCsTreatment of cells with 1μmol/L 5-HT caused phosphorylation of ERK1/2 and JNK with a peak at 15 min. M-Nis 10-5, 10-6 and 10-7 mol/L pretreatment reduced 5-HT-induced phosphorylation of ERK1/2 and JNK obviously (P<0.05 or P<0.01), and m-Nis 10-8 mol/L also significantly reduced the JNK phosphorylation, but have no significant effect on ERK1/2 phosphorylation.9 Effect of m-Nis on mRNA expression of c-fos and c-jun in rat PASMCsTreatment of cells with 1μmol/L 5-HT increased mRNA expression of c-fos and c-jun with a peak at 30 min, which was decreased in different degree by m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L pretreatment (P<0.05 or P<0.01).Conclusion: (1) M-Nis inhibited 5-HT-induced proliferation and migration of PASMCs, which may be related to the inhibition of PCNA expression and cell-cycle progression. (2) M-Nis inhibited 5-HT induced elevation of [Ca2+]i, mRNA expression of CaM, CaN, c-fos and c-jun, phosphorylation of ERK1/2 and JNK. So, the inhibition of m-Nis on proliferation of PASMCs may be related to the blockage of ERK1/2, JNK MAPK signal pathway through inhibiting the elevation of [Ca2+]i.Part 3 The activation of RhoA/ROCK signal pathway in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathwayObjective: To study the the activation of RhoA/ROCK signal pathway in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway.Methods: PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Cell-cycle distribution was determined by flow cytometry assay. The expression of PCNA, the phosphorylation of ERK1/2, JNK and MYPT1 was detected by western blot. The mRNA expression of Rho A, ROCK1, c-fos and c-jun was evaluated by RT-PCR.Results:1 Effect of fasudil on 5-HT-induced proliferation of PASMCsThe results of MTT assay suggested that 1μmol/L 5-HT significantly increased the A value from 0.37±0.05 to 0.61±0.031 (P<0.01), which was decreased to 0.48±0.05 , 0.46±0.03 and 0.43±0.04 by fasudil 25, 50, 100μmol/L pretreatment respectively (P<0.01). Similarly, western blot analysis of PCNA indicated that the expression of PCNA was higher in 5-HT group than that in control group (P <0.01), which was inhibited in different degree (P<0.05 or P<0.01) by pretreatment of PASMCs with fasudil.2 Effect of fasudil on cell-cycle progressionThe percentage of cells in S phase increased dramatically from 4% to 21% after 5-HT stimulation (P<0.01) and was accompanied by a reduction in the percentage of cells in G1 phase from 83% to 55% (P<0.01). Fasudil 25, 50, 100μmol/L markedly blocked 5-HT-induced cell-cycle progression, with the percentages of PASMCs entering the S phase decreasing to 12%, 11% and 9% (P<0.05 or P<0.01), and those arrested in the G0/G1 phase increasing to 65%, 70% and 78% respectively (P<0.05 or P<0.01). Meanwhile, the proliferation index increased dramatically from 17% to 45% after 5-HT stimulation (P < 0.01) and was decreased to 35%, 30% and 22% (P<0.05 or P<0.01) respectively by preincubation with fasudil 25, 50 and 100μmol/L.3 Fasudil inhibited 5-HT- mediated ROCK activation5-HT induced a significant upregulation of ROCK1 mRNA expression compared with that in quiescent PASMCs (P<0.01), and pretreatment with fasudil 25, 50, 100μmol/L resulted in a significant reduction in ROCK1 mRNA expression (P<0.05 or P<0.01). 5-HT caused phosphorylation of MYPT1, with a peak at 10 min (P<0.01), which was inhibited by fasudil in a concentration-dependent manner (P<0.05 or P<0.01).4 Effect of fasudil on ERK1/2 and JNK phosphorylation5-HT caused obvious phosphorylation of ERK1/2 and JNK with a peak at 15 min (P < 0.01). Pretreatment with fasudil 25, 50 and 100μmol/L did not block ERK1/2 phosphorylation, but reduced JNK phosphorylation obviously (P < 0.01). Next we examined the effect of fasudil on nuclear translocation of ERK1/2 induced by 5-HT. Result suggested that 5-HT increased nuclear translocation of ERK1/2 as determined by western blot analysis of nuclear-rich cellular fractions, with a peak at 15 min, consistent with its phosphorylation, which was reduced (P < 0.01) by pretreatment of cells with fasudil 25, 50 and 100μmol/L.5 Effect of fasudil on mRNA expression of c-fos and c-junTreatment of cells with 1μmol/L 5-HT for 30 min increased mRNA expression of c-fos and c-jun (P< 0.01), which was decreased in different degree by fasudil 25, 50, 100μmo/L pretreatment (P < 0.05 or P<0.01).6 Effect of m-Nis on 5-HT-mediated RhoA/ROCK signal pathway5-HT induced a significant upregulation of mRNA expression of RhoA and ROCK1 (P < 0.01), which was reduced significantly (P < 0.05 or P < 0.01) by pretreatment with m-Nis 10-5 mol/L, m-Nis 10-6 mol/L also reduced ROCK1 mRNA expression (P < 0.05), but had no effect on RhoA mRNA expression. Phosphorylation of MYPT1 was induced by 5-HT with a peak at 10 min (P < 0.01), which was inhibited by m-Nis 10-5,10-6 and 10-7 mol/L in different degree (P < 0.05 or P < 0.01). However, m-Nis 10-8 mol/L had no effect on p-MYPT1 expression.Conclusion: (1) 5-HT mediated ROCK activation, fasudil not only inhibited 5-HT-induced proliferation of PASMCs and cell-cycle progression, but also reduced JNK phosphorylation, nuclear translocation of ERK1/2 and c-fos, c-jun mRNA expression, which suggested that RhoA/ROCK signal pathway played an important role in 5-HT-induced proliferation of PASMCs. (2) M-Nis significantly inhibited 5-HT-induced RhoA, ROCK1 mRNA expression and the phosphorylation of MYPT1. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of RhoA/ROCK signal pathway.Part 4 Effect of ROS in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathwayObjective: To study the effect of ROS in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway.Methods: PASMCs were cultured with the explant technique. ROS was measured by confocal microscopy with DCFH-DA. The MDA production and SOD activity were measured using kits supplied by Nanjing Jiancheng Biotechnological Company. The proliferation of PASMCs was evaluated by MTT assay. The expression of PCNA, the phosphorylation of ERK1/2 and JNK were detected by western blot.Results:1 Effect of m-Nis on the production of ROS in rat PASMCsThe fluorescence intensity of ROS was significantly increased and reached the peak at 15 min after the cells were treated with 5-HT (P<0.01). After that, the fluorescence intensity decreased gradually and reached to a stable state at 120 min and 240 min after 5-HT treatment, but still stronger than that in control group (P<0.01). Compared with control group, the fluorescence intensity of ROS increased by 288% 15 min after 5-HT treatment (P<0.01). Pretreatment with m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L reduced the fluorescence intensity of ROS from 288±54% to 157±29%, 173±38%, 178±43% and 215±45%, respectively (P<0.01).2 Effect of m-Nis on the activity of SOD and the content of MDACompared with the control group, the activity of SOD decreased obviously (P<0.01), and the content of MDA increased significantly (P<0.01) after 5-HT treatment. Compared with the 5-HT group, a significant reduction in MDA production was observed in m-Nis 10-5,10-6,10-7 mol/L groups (P<0.05 or P<0.01), which was associated with an increase in the SOD activity (P<0.05 or P<0.01). Meanwhile, m-Nis 10-8 mol/L also reduced the content of MDA (P<0. 05), but had no effect on the SOD activity.3 Effect of m-Nis on H2O2-induced proliferation of PASMCsThe results of MTT assay suggested that 100μmol/L H2O2 significantly increased the A value (P<0.01), which was inhibited significantly (P <0.05 or P <0.01) by m-Nis 10-5,10-6,10-7 and 10-8 mol/L. Similarly, western blot analysis of PCNA indicated that the expression of PCNA was higher in H2O2 group than that in control group (P <0.01), which was also inhibited obviously by pretreatment of PASMCs with m-Nis (P<0.05 or P<0.01).4 Effect of m-Nis on H2O2-induced phosphorylation of ERK1/2 and JNKH2O2 caused obvious phosphorylation of ERK1/2 and JNK (P < 0.01), which was inhibited in different degree by m-Nis 10-5, 10-6 and 10-7 mol/L pretreatment (P < 0.05 or P < 0.01). Meanwhile, m-Nis 10-8 mol/L also inhibited the phosphorylation of ERK1/2 (P < 0.05), but had no effect on JNK activation.Conclusion: (1) 5-HT induced the production of ROS obviously. H2O2 not only mediated proliferation of PASMCs, but also induced ERK1/2 and JNK phosphorylation, which suggested that ROS played an important role in 5-HT-induced proliferation of PASMCs. (2) M-Nis not only inhibited 5-HT-induced production of ROS, but also reduced the proliferation of PASMCs and phosphorylation of ERK1/2 and JNK induced by H2O2. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of ROS production and the downstream ERK1/2,JNK MAPK signal pathway.CONCLUSIONS1 M-Nis protects against MCT-induced pulmonary artery hypertension, which may be related to the antioxidation effect, the reduction of 5-HT exression and the suppression of the ERK/MAPK signal pathway.2 M-Nis inhibited 5-HT-induced proliferation and migration of PASMCs, which may be related to the blockage of ERK1/2, JNK MAPK signal pathway through inhibiting the elevation of [Ca2+]i.3 Fasudil inhibited 5-HT-induced proliferation of PASMCs obviously, which suggested that RhoA/ROCK signal pathway played an important role in 5-HT-induced proliferation of PASMCs.4 M-Nis inhibited 5-HT-induced RhoA, ROCK1 mRNA expression and the phosphorylation of MYPT1 in different degree. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of RhoA/ROCK signal pathway.5 ROS played an important role in 5-HT-induced proliferation of PASMCs. The inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of ROS production and the downstream ERK1/2,JNK MAPK signal pathway. |
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