The Effect And The Underlying Mechanisms Of Grape Seed Procyanidin Extract And Melatonin On Hypoxic Pulmonary Hypertension

BackgroundHypoxic pulmonary hypertension(HPH) is a serious disease characterized by hypoxia-induced increased pulmonary vascular resistance, pulmonary vascular remodeling and elevated pulmonary artery pressure. Although HPH is very common, there is no specific treatment for this serious disease, hunting for novel effective pharmacologic treatments for HPH is urgent. Current evidence suggests that reactive oxygen species(ROS) play an important role in HPH pathogenesis. In chronic hypoxiainduced PH, an increase in ROS production has been shown in the lung and pulmonary arteries. A variety of compounds with antioxidant properties have been shown to significantly attenuate pulmonary vasoconstriction due to hypoxia and have beneficial therapeutic effects in HPH. Thus, targeting of ROS appears as a potential approach in HPH treatment. Grape seed procyanidin extract(GSPE), a biologically active polyphenolic flavonoid combination that contains oligomeric proanthocyanidin, has been reported to exert biological, pharmacological, therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress. In addition, it has been reported GSPE has protective effects on various cardiac disorders. Although GSPE has been found to scavenge ROS and protect cardiovascular system, whether GSPE has effects on HPH has never been reported. The aim of this study was to investigate the possible effect and the underlying mechanisms of GSPE on HPH.ObjectiveTo investigate the possible effect and the underlying mechanisms of GSPE on HPH through observation on changes of pulmonary artery pressure, right ventricular hypertrophy, and vascular remodeling in a chronic hypoxia rat model.Methods1. Animal experimentsTwenty-eight male Sprague-Dawley rats(200-250 g) were randomly divided into 4 groups(7 rats per group):(1) normoxia group;(2) normoxia group treated with GSPE;(3) hypoxia group;(4) hypoxia group treated with GSPE. For group(2) and(4), rats were administered GSPE(250 mg/kg/day), intragastrically dissolved in normal saline for 1 week before and for the entire 4 weeks of nomoxia or hypoxia exposure.2. Hemodynamic experiments and morphological investigationAfter 4 weeks hypoxia exposure, the right ventricle systolic pressure(RVSP) was then recorded using Power Lab Software. After that, the right ventricle(RV) and left ventricle plus septum(LV+S) were collected, and the weight ratio of(RV/LV+S) was calculated as an index of RV hypertrophy. The lung slices were embedded in paraffin and cut into 4-μm thick sections and stained with hematoxylin and eosin. The outside diameter, inside diameter, medial wall area and total vessel area of pulmonary arteries were measured. The percent medial wall thickness(WT%) and percent medial wall area(WA%) were calculated to present pulmonary vascular structure remodeling. WT% =(outside diameter- inside diameter)/(outside diameter) × 100; WA% =(medial wall area)/(total vessel area) × 100.3. Immunohistochemical stainingSections were incubated overnight with anti-α-SMA mouse monoclonal antibody, anti-PCNA mouse monoclonal antibody at 4°C. Then, a biotinylated anti-mouse or rabbit IgG antibody and an avidin-biotinylated peroxidase complex were applied with 3,3-diaminobenzidine as a peroxidase substrate. Immunoreactivity was visualized using diaminobenzidine.4. Assay of MDA and SODThe content of malondialdehyde(MDA) and superoxide dismutase(SOD) were measured using commercial kits and analyzed with a spectrophotometer.5. Primary cells culture and in vitro hypoxiaPrimary PASMCs were cultured by tissue explant method and used for experiments between passages 3 and 6. For all experiments, cells were divided into six groups: normoxia, hypoxia, hypoxia + 20μg/ml GSPE, hypoxia + 40μg/ml GSPE, hypoxia + 80μg/ml GSPE and hypoxia + 120μg/ml GSPE. Cells were cultured either in 21% oxygen or 2% oxygen condition.6. Detection of intracellular ROSCells were cultured for 12 hours. Then the level of intracellular reactive oxygen species was measured using the Reactive Oxygen Species Assay Kit according to the manufacturer’s instructions.7. Cell proliferation analysisThe effect on proliferation of GSPE was analyzed using the Trypan Blue dyeexclusion assay. The cells were seeded at a density of 2 × 104 cells per well in a 24-well culture plate. After cultured for 48 hours, the cells were harvested with trypsinization. Trypan Blue(0.4 %) was added, and the number of viable cells that excluded the dye were counted with a hemocytometer.8. Western blotting analysisAfter cultured for 12 hours, cells were lysed. The primary antibodies were p-STAT3 antibody, total STAT3 antibody, cyclin D1 antibody and cyclin D3 antibody. The signals were detected by ECL kit.9. Quantitative real-time RT-PCR analysisCells were cultured for 12 hours. Then total RNAs of cells or lung tissues were extracted by using Trizol agent. Total RNAs were reverse-transcribed with oligo-dT primers. Quantitative real-time RT-PCR(qRT-PCR) was performed according to the manufacturer’s instructions.Results1. The average RVSP of hypoxia group was increased significantly compared with the normoxia group. However, the average RVSP of hypoxia treated with GSPE group was much lower than that of hypoxia alone group. In accordance with the RVSP, hypoxiainduced elevation of the ratio of RV/LV+S was inhibited by the application of GSPE. In addition, hypoxia markedly elevated WT% and WA%. While, WT% and WA% in hypoxia group treated with GSPE were much lower than in the hypoxia group.2. Integrated optical density value of α-SMA in hypoxia group was significantly higher than in normoxia group. With the treatment of GSPE, the integrated optical density value of α-SMA was reduced significantly.3. To determine GSPE’s anti-oxidative effect, SOD and MDA were analyzed both in lung tissue and in serum. The results showed that hypoxia decreased the levels of SOD, companied with an increased levels of MDA. GSPE treatment elevated the levels of SOD and reduced the levels of MDA both in lung tissue and in serum.4. Flow cytometry results presented that chronic hypoxia could increase the level of intracellular ROS in PASMCs significantly. However, GSPE obviously inhibited ROS production, and this inhibitory effect on ROS production was enhanced with increasing GSPE concentrations.5. Results confirmed that exposure to chronic hypoxia for 4 weeks increased the levels of Nox4 mRNA in rat lung, and treatment with GSPE prevented hypoxia-mediated Nox4 mRNA induction. Furthermore, hypoxia also increased the expression of Nox4 mRNA in PASMCs, and hypoxia-induced increases in Nox4 mRNA were reduced by treatment with GSPE.6. The percentage of PCNA-positive cells in hypoxia group was notably higher than in normoxia group. However, increased cell proliferation in hypoxia group was inhibited markedly by treatment of GSPE. In addition, the proliferation of PASMCs was significantly elevated in hypoxia compared with the cells under normoxic conditions. While, GSPE supplement exerted a significant antiproliferative effect.7. Western blotting results showed that hypoxia notably increased the p-STAT3, cyclin D1 and cyclin D3 protein levels in PASMCs. The expression of p-STAT3, cyclin D1 and cyclin D3 in the four dosages of GSPE treatment groups were all significantly lower compared with the hypoxia group.Conclusions1. GSPE attenuated hypoxia-induced pulmonary artery remodeling and pulmonary hypertension in rats.2. GSPE attenuated the hypoxia induced oxidative stress in rats.3. GSPE reversed the hypoxia-induced up-regulation of Nox4 mRNA levels both in PASMCs and in lung tissue.4. GSPE inhibited hypoxia-induced PASMCs proliferation.Background Hypoxic pulmonary hypertension(HPH) is a serious disease with poor prognosis. It is characterized by hypoxia-induced pulmonary vasoconstriction, pulmonary vascular remodeling and elevated pulmonary artery pressure. Hypoxia-induced inflammation and excessive proliferation of pulmonary artery smooth muscle cells(PASMCs) play important roles in the pathological process of HPH. Melatonin, an indolamine, is a small lipophilic molecule and ubiquitous physiological mediator that is synthesized in the pineal gland. Numerous studies have shown that melatonin plays crucial roles in several vital physiological and pathological processes, such as regulation of circadian rhythms, inhibition of tumor growth and metastasis, inhibition of inflammation and cell proliferation. In the cardiovascular system, melatonin has protective effects through the free radical scavenger activity and antioxidant properties. Although melatonin has been found to reduce inflammation and inhibit cell proliferation, whether melatonin has effects on HPH has never been reported. The aim of this study was to investigate the possible effect and the underlying mechanism of melatonin on HPH.Objective To investigate the possible effect and the underlying mechanisms of melatonin on HPH through observation on changes of pulmonary artery pressure, right ventricular hypertrophy, and vascular remodeling in a chronic hypoxia rat model.Methods 1. Animal experiments Twenty-eight male Sprague-Dawley rats(200-250 g) were randomly divided into 4 groups(7 rats per group):(1) normoxia group;(2) normoxia group treated with melatonin;(3) hypoxia group;(4) hypoxia group treated with melatonin. For group(2) and(4), each rat received melatonin(15 mg/kg/day) via intraperitoneal injection every morning prior to hypoxia exposure for 1 week before and for the entire 4 weeks of nomoxia or hypoxia exposure. 2. Hemodynamic experiments and morphological investigation After 4 weeks hypoxia exposure, the right ventricle systolic pressure(RVSP) was then recorded using Power Lab Software. After that, the right ventricle(RV) and left ventricle plus septum(LV+S) were collected, and the weight ratio of(RV/LV+S) was calculated as an index of RV hypertrophy. The lung slices were embedded in paraffin and cut into 4-μm thick sections and stained with hematoxylin and eosin. The outside diameter, inside diameter, medial wall area and total vessel area of pulmonary arteries were measured. The percent medial wall thickness(WT%) and percent medial wall area(WA%) were calculated to present pulmonary vascular structure remodeling. WT% =(outside diameter- inside diameter)/(outside diameter) × 100; WA% =(medial wall area)/(total vessel area) × 100. 3. Immunohistochemical staining Sections were incubated overnight with anti-α-SMA mouse monoclonal antibody, anti-PCNA mouse monoclonal antibody, anti-HIF-1α mouse monoclonal antibody, or anti-NF-κB p65 rabbit monoclonal antibody at 4°C. Then, a biotinylated anti-mouse or rabbit Ig G antibody and an avidin-biotinylated peroxidase complex were applied with 3,3-diaminobenzidine as a peroxidase substrate. Immunoreactivity was visualized using diaminobenzidine. 4. Primary cells culture and in vitro hypoxia Primary PASMCs were cultured by tissue explant method and used for experiments between passages 3 and 6. Cells were cultured either in 21% oxygen or 2% oxygen condition for 48 hours. 5. Cell proliferation analysis The cell viability of PASMCs under normoxia and hypoxia was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide(MTT) assay. 6. Western blotting analysis Cells lung homogenates were lysed. The primary antibodies were p-Akt antibody, p-ERK antibody, and NF-κB p65 antibody. The signals were detected by ECL kit. 7. Quantitative real-time RT-PCR analysis Total RNAs of lung tissues were extracted by using Trizol agent. Total RNAs were reverse-transcribed with oligo-d T primers. Quantitative real-time RT-PCR(q RT-PCR) was performed according to the manufacturer’s instructions.Results 1. The average RVSP of hypoxia group was increased significantly compared with the normoxia group. However, the average RVSP of hypoxia treated with melatonin group was much lower than that of hypoxia alone group. In accordance with the RVSP, hypoxiainduced elevation of the ratio of RV/LV+S was inhibited by the application of melatonin. In addition, hypoxia markedly elevated WT% and WA%. While, WT% and WA% in hypoxia group treated with melatonin were much lower than in the hypoxia group. 2. Integrated optical density value of α-SMA in hypoxia group was significantly higher than in normoxia group. With the treatment of melatonin, the integrated optical density value of α-SMA was reduced significantly. The percentage of PCNA-positive cells in hypoxia group was significantly higher than in normoxia group. However, increased cell proliferation in hypoxia group was inhibited markedly by treatment of melatonin. 3. The expression of HIF-1α in the medial wall of pulmonary arteries and the m RNA levels In the whole lungs were significantly elevated on hypoxic exposure compared with normoxic controls; However, HIF-1α levels were obviously down-regulated by the treatment of melatonin. 4. NF-κB p65 expression was up-regulated in the hypoxia group. However, melatonin suppressed NF-κB p65 expression in rat lungs markedly. 5. The proliferation of PASMCs was significantly elevated in hypoxia compared with the cells under normoxic conditions. While, GSPE supplement exerted a significant antiproliferative effect. 6. Western blotting results showed that hypoxia notably increased the p-AKT and pERK1/2 protein levels in PASMCs. However, melatonin repressed the protein expression of p-AKT and p-ERK1/2.Conclusions 1. Melatonin attenuated hypoxia-induced pulmonary artery remodeling and pulmonary hypertension in rats. 2. Melatonin attenuated the hypoxia induced inflammation in rats. 3. Melatonin inhibited hypoxia-induced PASMCs proliferation.