OBJECTIVES: To investigate the synergistic mechanism of the traditional Chinese medicine Rosa laevigata Michx. (RLM) for treatment of pulmonary arterial hypertension (PAH). METHODS: Network pharmacological analysis was carried out to screen the active ingredients of RLM and PAH disease targets and construct the "component-target-disease" interaction network, followed by gene enrichment analysis and molecular docking studies. In the cell experiments, primary cultures of rat pulmonary arterial smooth muscle cells were exposed to hypoxia for 24 h and treated with solvent or 100, 200 and 300 mg/mL RLM, and the changes in cell proliferation were detected using Western blotting for PCNA and immunofluorescence staining. In the animal experiment, male SD rats were randomized into 5 control group, monocrotaline (MCT) solvent group, and MCT with RLM (100, 200 and 300 mg/mL) treatment groups. HE staining and immunofluorescence staining were used to observe histopathological changes in the pulmonary blood vessels of the rats. RESULTS: Seven core active ingredients (including β-sitosterol and kaempferol) in RLM and 39 key disease targets were identified, and molecular docking showed that SRC was a high-affinity target. KEGG enrichment analysis showed that the differential genes were significantly enriched in calcium signaling and PI3K-AKT pathways. In rat pulmonary arterial smooth muscle cells, hypoxic exposure significantly up-regulated cellular expression of PCNA and phosphorylation levels of Src and AKT1, which were obviously lowered by RLM treatment. In RLM-treated rat models, the mean pulmonary artery pressure and right ventricular hypertrophy index (Fulton index) were significantly reduced, the tricuspid annular plane systolic excursion (TAPSE) was improved, and pulmonary vascular wall thickening and fibrosis were obviously ameliorated. CONCLUSIONS RLM inhibits pulmonary arterial smooth muscle cell proliferation in rat models of hypertension possibly by regulating the Src-AKT1 axis, suggesting the potential of RLM as a new natural drug for treatment of pulmonary hypertension.
Animals ; Cell Proliferation/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Pulmonary Artery/cytology* ; Male ; Rats ; Myocytes, Smooth Muscle/cytology* ; Hypertension, Pulmonary/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Muscle, Smooth, Vascular/cytology* ; src-Family Kinases/metabolism* ; Cells, Cultured

OBJECTIVES: To investigate the mechanism of DDX17 for regulating proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) during the development of pulmonary hypertension (PH). METHODS: In murine PASMCs cultured under normoxic or hypoxic conditions, the effects of transfection with si-Ddx17 and insulin treatment, alone or in combination, on cell proliferation and migration were evaluated using Ki-67 immunofluorescence staining, scratch assay and Transwell assay. Western Blotting was performed to detect the changes in protein expression levels of DDX17, 4EBP1, S6, p-4EBP1, and p-S6. In a mouse model of PH induced by intraperitoneal injection of monocrotaline (MCT), the changes in pulmonary vasculature were examined using HE staining following tail vein injection of AD-Ddx17i. RESULTS: The PASMCs in hypoxic culture exhibited significantly enhanced cell proliferation and migration and protein expressions of p-4EBP1 and p-S6, and these changes were obviously reversed by transfection with si-Ddx17. Treatment with insulin significantly attenuated the effect of si-Ddx17 against hypoxic exposure-induced changes in PASMCs. In the mouse model of MCT-induced PH, transfection with AD-Ddx17i obviously alleviated pulmonary vascular stenosis and intimal hyperplasia. CONCLUSIONS The expression of DDX17 is elevated in hypoxia-induced PASMCs and PH mice, and silencing DDX17 significantly inhibits PASMC proliferation and migration in vitro and pulmonary vascular remodeling in PH mice by reducing mTORC1 activity.
Animals ; Cell Proliferation ; Cell Movement ; DEAD-box RNA Helicases/metabolism* ; Myocytes, Smooth Muscle/metabolism* ; Mice ; Pulmonary Artery/cytology* ; Hypertension, Pulmonary/metabolism* ; Mechanistic Target of Rapamycin Complex 1 ; Cells, Cultured ; Muscle, Smooth, Vascular/cytology*

Humans ; Angioplasty, Balloon/adverse effects* ; Antithrombin III/metabolism* ; Chronic Disease ; Pulmonary Artery ; Pulmonary Embolism/etiology* ; Thromboembolism/therapy*

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.
Animals ; Male ; Rats ; Caspase 3/metabolism* ; Collagen ; Disease Models, Animal ; Hypertension, Pulmonary/drug therapy* ; Monocrotaline/adverse effects* ; Panax notoginseng/chemistry* ; Proliferating Cell Nuclear Antigen/pharmacology* ; Pulmonary Arterial Hypertension ; Pulmonary Artery/metabolism* ; Rats, Sprague-Dawley ; Receptor, Notch3/genetics* ; RNA, Messenger ; Saline Solution ; Signal Transduction ; Saponins/pharmacology*

OBJECTIVES: To study the effect of platelet-derived growth factor-BB (PDGF-BB) on pulmonary vascular remodeling in neonatal rats with hypoxic pulmonary hypertension (HPH). METHODS: A total of 128 neonatal rats were randomly divided into four groups: PDGF-BB+HPH, HPH, PDGF-BB+normal oxygen, and normal oxygen (n=32 each). The rats in the PDGF-BB+HPH and PDGF-BB+normal oxygen groups were given an injection of 13 μL 6×10¹⁰ PFU/mL adenovirus with PDGF-BB genevia the caudal vein. After 24 hours of adenovirus transfection, the rats in the HPH and PDGF-BB+HPH groups were used to establish a neonatal rat model of HPH. Right ventricular systolic pressure (RVSP) was measured on days 3, 7, 14, and 21 of hypoxia. Hematoxylin-eosin staining was used to observe pulmonary vascular morphological changes under an optical microscope, and vascular remodeling parameters (MA% and MT%) were also measured. Immunohistochemistry was used to measure the expression levels of PDGF-BB and proliferating cell nuclear antigen (PCNA) in lung tissue. RESULTS: The rats in the PDGF-BB+HPH and HPH groups had a significantly higher RVSP than those of the same age in the normal oxygen group at each time point (P<0.05). The rats in the PDGF-BB+HPH group showed vascular remodeling on day 3 of hypoxia, while those in the HPH showed vascular remodeling on day 7 of hypoxia. On day 3 of hypoxia, the PDGF-BB+HPH group had significantly higher MA% and MT% than the HPH, PDGF-BB+normal oxygen, and normal oxygen groups (P<0.05). On days 7, 14, and 21 of hypoxia, the PDGF-BB+HPH and HPH groups had significantly higher MA% and MT% than the PDGF-BB+normal oxygen and normal oxygen groups (P<0.05). The PDGF-BB+HPH and HPH groups had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group at all time points (P<0.05). On days 3, 7, and 14 of hypoxia, the PDGF-BB+HPH group had significantly higher expression levels of PDGF-BB and PCNA than the HPH group (P<0.05), while the PDGF-BB+normal oxygen group had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group (P<0.05). CONCLUSIONS Exogenous administration of PDGF-BB in neonatal rats with HPH may upregulate the expression of PCNA, promote pulmonary vascular remodeling, and increase pulmonary artery pressure.
Rats ; Animals ; Hypertension, Pulmonary ; Becaplermin ; Animals, Newborn ; Proliferating Cell Nuclear Antigen ; Vascular Remodeling ; Pulmonary Artery/metabolism* ; Hypoxia ; Oxygen ; Cell Proliferation ; Myocytes, Smooth Muscle/metabolism*

OBJECTIVE: To investigate the role of myelin and lymphocyte protein (MAL) in pulmonary hypertension (PAH). METHODS: Blood samples were collected from 50 patients with PAH (PAH group) and 50 healthy individuals for detection of plasma MAL expression using ELISA.According to the echocardiographic findings, the patients were divided into moderate/severe group (n=18) and mild group (n=32), and the correlation between MAL protein level and the severity of PAH was analyzed.In a pulmonary artery smooth muscle cell model of PAH with hypoxia-induced abnormal proliferation, the effects of mal gene knockdown and overexpression on cell growth, proliferation and starvation-induced apoptosis were observed; the changes in NK-κB signaling pathway in the transfected cells were detected to explore the molecular mechanism by which MAL regulates PAMSC proliferation and apoptosis. RESULTS: The plasma level of MAL was significantly higher in patients with PAH than in healthy individuals (P < 0.05), and the patients with moderate/severe PAH had significantly higher MAL level than those with mild PAH (P < 0.001).In PAMSCs, exposure to hypoxia significantly increased the mRNA and protein expression levels of MAL (P < 0.05), and MAL knockdown obviously inhibited hypoxia-induced proliferation and promoted starvation-induced apoptosis of the PAMSCs (P < 0.05).Knocking down mal significantly inhibited the activation of NK-κB signaling pathway that participated in regulation of PAMSC proliferation (P < 0.05). CONCLUSION The plasma level of MAL is elevated in PAH patients in positive correlation with the disease severity.MAL knockdown inhibits abnormal proliferation and promotes apoptosis of PAMSCs by targeted inhibition of the NF-κB signaling pathway to improve vascular remodeling in PAH.
Humans ; Pulmonary Artery ; Hypertension, Pulmonary ; Myelin Sheath/metabolism* ; Apoptosis ; Myocytes, Smooth Muscle ; Vascular Remodeling/genetics* ; Cell Proliferation ; Hypoxia/metabolism* ; Lymphocytes

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.
Animals ; Autophagy ; Beclin-1/pharmacology* ; Hypertension, Pulmonary/etiology* ; Hypoxia/complications* ; Myocytes, Smooth Muscle/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Umbelliferones/pharmacology* ; rho-Associated Kinases/pharmacology*

OBJECTIVE: To explore the therapeutic mechanism of tanshinone IIA in the treatment of pulmonary arterial hypertension (PAH) in rats. METHODS: A total of 100 male SD rats were randomized into 5 groups (n=20), and except for those in the control group with saline injection, all the rats were injected with monocrotaline (MCT) on the back of the neck to establish models of pulmonary hypertension. Two weeks after the injection, the rat models received intraperitoneal injections of tanshinone IIA (10 mg/kg), phosphatidylinositol 3 kinase (PI3K) inhibitor (1 mg/kg), both tanshinone IIA and PI3K inhibitor, or saline (model group) on a daily basis. After 2 weeks of treatment, HE staining and α-SMA immunofluorescence staining were used to evaluate the morphology of the pulmonary vessels of the rats. The phosphorylation levels of PI3K, protein kinase B (PKB/Akt) and endothelial nitric oxide synthase (eNOS) in the lung tissue were determined with Western blotting; the levels of eNOS and NO were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of HE staining and α-SMA immunofluorescence staining showed that tanshinone IIA effectively inhibited MCT-induced pulmonary artery intimamedia thickening and muscularization of the pulmonary arterioles (P < 0.01). The results of Western blotting showed that treatment with tanshinone IIA significantly increased the phosphorylation levels of PI3K, Akt and eNOS proteins in the lung tissue of PAH rats; ELISA results showed that the levels of eNOS and NO were significantly decreased in the rat models after tanshinone IIA treatment (P < 0.01). CONCLUSION Treatment with tanshinone IIA can improve MCT-induced pulmonary hypertension in rats through the PI3K/Akt-eNOS signaling pathway.
Abietanes ; Animals ; Hypertension, Pulmonary/drug therapy* ; Male ; Monocrotaline/toxicity* ; Nitric Oxide Synthase Type III/therapeutic use* ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

OBJECTIVE: To investigate the effects of apple polyphenols on pulmonary vascular remodeling in rats with pulmonary arterial hypertension and its mechanism. METHODS: Rats were randomly divided into 4 groups:control (Con) group, monocrotaline (MCT) group, apple polyphenol (APP) group,monocrotaline + apple polyphenol (MCT+APP) group. In Con group, rats received a subcutaneous injection of physical saline. In APP group, rats received intraperitoneal injection of 20 mg/kg APP, every other day. In MCT group, rats received a single subcutaneous injection of MCT(60 mg/kg). In MCT+APP group, rats received subcutaneous injection of 60 mg/kg MCT followed by an intraperitoneal injection of 20 mg/kg APP every other day. All the disposal lasted 3 weeks. Then the PAH-relevant indicators, such as mean pulmonary artery pressure(mPAP), pulmonary vascular resistance(PVR), right ventricular hypertrophy index (RVHI) ,wall thickness (WT%) and wall area (WA%) were tested. After that, the inflammatory pathway related indicators, such as interleukin1(IL-1),interleukin1(IL-6), tumor necrosis factor α(TNF-α), cyclooxygenase 2(COX-2) and myeloperoxidase(MPO) in pulmonary tissue and free intracellular Ca in pulmonary smooth muscle cell(PASMC), content of eNOS and NO in endothelial cells were determined. RESULTS: Compared with the control group, the levels of mPAP, PVR, RVHI, WA%, WT%, and IL-1, IL-6, TNF-α, COX-2, MPO in tissue and the expression of Ca in PASMC of MCT group were increased significantly, while the contents of eNOS and NO in endothelial cells were decreased significantly (P＜0.05). Compared with the MCT group, the apple polyphenol treatment could improve the above mentioned situation, and the COX-2 and Ca indicators of the apple polyphenol treatment group were decreased significantly (P＜0.05). CONCLUSION MCT can increase COX-2 expression and intracellular Ca in pulmonary artery smooth muscle cells, decrease the contents of eNOS and NO in endothelial cells, while apple polyphenols can significantly inhibit these effects.
Animals ; Calcium ; metabolism ; Cyclooxygenase 2 ; metabolism ; Cytokines ; metabolism ; Malus ; chemistry ; Monocrotaline ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Polyphenols ; pharmacology ; Pulmonary Artery ; drug effects ; pathology ; Random Allocation ; Rats ; Vascular Remodeling ; drug effects

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO, 21% O, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO, 5% O, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca]in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca]were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca].
Actins ; Animals ; Apoptosis ; Calcium ; metabolism ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Hypercapnia ; physiopathology ; Imidazoles ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Rats, Sprague-Dawley ; TRPC Cation Channels ; metabolism