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

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*

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

This study was designed to observe the differences between main pulmonary arteries and the third-order branches of pulmonary arteries in the contractile response to phenylephrine (Phen), endothelin-1 (ET-1) and potassium chloride (KCl). The vascular tension changes of main and the third-order branches of pulmonary arteries induced by KCl, ET-1 and Phen were recorded by traditional vascular tone detection methods and microvascular ring technique, respectively. The results showed that Phen could cause a significant contraction in main pulmonary arteries, but did not induce apparent contraction in the third-order branches of pulmonary arteries. Compared with main pulmonary arteries, ET-1 contracted the third-order branches of pulmonary arteries with reduced maximal response value and PDvalue. In comparison with the main pulmonary arteries, contraction caused by KCl was enhanced in the third-order branches of pulmonary arteries. The results suggest that the vascular reactivity of main and the third-order branches of pulmonary arteries is different and it is important to study the vascular function of small branches of pulmonary arteries. This study could provide an important experimental basis for the further study on vascular function of small branches of pulmonary arteries and the functional changes in pulmonary hypertension.
Animals ; Endothelin-1 ; pharmacology ; Male ; Phenylephrine ; pharmacology ; Potassium Chloride ; pharmacology ; Pulmonary Artery ; drug effects ; Rats ; Vasoconstriction

OBJECTIVETo evaluate the effect of ulinastatin on hypoxia-induced phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) and explore the underlying mechanism.

METHODSCultured PASMCs from SD rats were exposed to normoxic condition, normoxia with ulinastatin treatment, hypoxia, or hypoxia with ulinastatin treatment. After 24 h of exposures, the cells were examined for SM-α-actin and caplonin expressions with immunofluorescence assay and for cell migration with CCK-8 andH-TdR assays. Western blotting was used for detecting the expressions of PPAR-γ in the cells, and PPAR-γ-responsive firefly luciferase reporter was employed for measuring the transcriptional activity of PPAR-γ. The PPAR-γ inhibitor GW9662 was used to explore the mechanism of the inhibitory effect of ulinastatin on hypoxia induced-phenotype modulation of PASMCs by measuring the changes in cell proliferation and migration.

RESULTSUlinastatin obviously enhanced the expressions of SM-α-actin and calponin (P<0.05), inhibited the proliferation and migration (P<0.05), and up-regulated the expression of PPAR-γ in PASMCs exposed to hypoxia (P<0.05). Pretreatment of the cells with GW9662 abolished the effect of ulinastatin on hypoxia-induced phenotype modulation of PASMCs and enhanced the cell proliferation and migration (P<0.05).

CONCLUSIONUlinastatin inhibits hypoxia-induced phenotype modulation of PASMCs from rats possibly by up-regulating the expression of PPAR-γ.

Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Glycoproteins ; pharmacology ; Microfilament Proteins ; metabolism ; Myocytes, Smooth Muscle ; cytology ; drug effects ; PPAR gamma ; metabolism ; Phenotype ; Pulmonary Artery ; cytology ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

To investigate the effects and the underlying molecular mechanisms of curcumin on pulmonary artery smooth muscle cells in rat model with chronic obstructive pulmonary disease (COPD).A total of 75 male Wistar rats were randomly divided into control group (group CN), model group (group M), low-dose curcumin group (group CL), medium-dose curcumin group (group CM) and high-dose curcumin group (group CH). HE staining was used to observe the morphology of pulmonary artery. Proliferating cell nuclear antigen (PCNA), apoptosis-related protein Bcl-2 and Bax were detected by immunohistochemical staining. TUNEL kit was used to analyze the effects of curcumin on apoptosis of smooth muscle cells, and the protein expressions of SOCS-3/JAK2/STAT pathway in lung tissues were determined by western blot.Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVMI) in group M were significantly higher than those in group CN, group CH and group CM (all<0.05). HE staining and TUNEL kit test showed that the number of pulmonary artery smooth muscle cells had a significant increase in group M, while the pulmonary artery tube became thin, and the smooth muscle cells shrinked in group CM and group CH. Immunohistochemistry showed that PCNA and Bcl-2 in group M were significantly higher than those in group CN (all<0.05), while Bax expression was significantly lower than that in group CN (<0.05). PCNA in group CM and group CH were significantly lower than that in group M (all<0.05), while Bax expression was significantly higher than that in group M (<0.05). Western blot showed that SOCS-3 protein was significantly decreased in group M, while the p-JAK2, p-STAT1, p-STAT3 were significantly increased (all<0.05). Compared with group M, SOCS-3 protein in group CM and group CH were significantly increased (all<0.05), while the p-JAK2, p-STAT3 were significantly reduced (all<0.05).Curcumin could promote the apoptosis of smooth muscle cells in rats with COPD, and improve the mean pulmonary artery pressure and RVMI through stimulating SOCS-3/JAK2/STAT signaling pathway.
Animals ; Apoptosis ; drug effects ; physiology ; Arterial Pressure ; drug effects ; physiology ; Curcumin ; pharmacology ; Hypertrophy, Right Ventricular ; pathology ; physiopathology ; Janus Kinase 2 ; drug effects ; physiology ; Lung ; chemistry ; drug effects ; Male ; Myocytes, Smooth Muscle ; drug effects ; pathology ; Proliferating Cell Nuclear Antigen ; drug effects ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; drug effects ; metabolism ; Pulmonary Artery ; drug effects ; pathology ; Pulmonary Disease, Chronic Obstructive ; pathology ; physiopathology ; Rats ; Rats, Wistar ; STAT Transcription Factors ; Suppressor of Cytokine Signaling 3 Protein ; drug effects ; physiology ; Ventricular Pressure ; drug effects ; bcl-2-Associated X Protein ; drug effects ; metabolism

OBJECTIVE: To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms.
 METHODS: Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR.
 RESULTS: RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression.
 CONCLUSION Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.
Animals ; Calpain ; genetics ; physiology ; Cell Proliferation ; Dipeptides ; physiology ; Hypertension, Pulmonary ; chemically induced ; genetics ; physiopathology ; Hypertrophy, Right Ventricular ; Hypoxia ; Ki-67 Antigen ; drug effects ; Myocytes, Smooth Muscle ; physiology ; Proliferating Cell Nuclear Antigen ; drug effects ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Up-Regulation ; Vascular Remodeling ; genetics ; physiology

OBJECTIVETo determine the effect of resveratrol on constrictions of isolated human intrapulmonary arteries and its mechanisms.

METHODSIntrapulmonary arteries (1-1.5 mm in diameter) were dissected and cut into rings (1.8-2.0 mm in length) under microscope, and were then mounted in a Multi Myograph system. The rings were stimulated with 100 nmol/L U46619, 30 nmol/L endothelin-1, or 60 mmol/L KCl to produce sustained contraction of the intrapulmonary arteries, after which resveratrol was applied cumulatively. Endothelium denudation, L-NAME and indomethecin were used to investigate the effect of resveratrol on constrictions of the isolated arteries, suing DMSO as the control.

RESULTSResveratrol induced concentration-dependent relaxations in endothelium-intact rings that contracted in response to stimulations with U46619, ET-1 and KCl, with pD2 of 3.82±0.20, 3.84±0.57, and 3.68±0.27, Emax of (99.58±0.83)%, 100%, and (99.65±0.98)%, respectively. Treatment of the arterial rings with the eNOS inhibitor L-NAME, but not with indomethecin or endothelium denudation, obviously affected the relaxant effects of resveratrol.

CONCLUSIONResveratrol can concentration-dependently produce relaxant effect on human intrapulmonary arteries independent of the endothelium possibly by promoting synthesis and release of NO.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; pharmacology ; Humans ; In Vitro Techniques ; Pulmonary Artery ; drug effects ; Stilbenes ; pharmacology ; Vasoconstriction ; drug effects

OBJECTIVETo explore the effects of NF-κB on proliferation of rat pulmonary artery smooth muscle cells (PASMC) inhibited by simvastatin.

METHODSPASMC isolated from rats and cultured in vitro were randomly divided into four groups (n=6 each): control, platelet-derived growth factor (PDGF) treatment, PDGF+simvastatin treatment, and PDGF+simvastatin+parthenolide (NF-κB inhibitor) treatment. MTT colorimetric assay and flow cytometry were performed to detect cell proliferation and cell cycle distribution. Immunohistochemistry was performed to detect the expression of NF-κB protein. Real-Time PCR was performed to detect NF-κB mRNA expression.

RESULTSCompared with the control group, MTT values of PASMC at all time points, cell proportion at the S phase and G2+M phase, NF-κB protein and mRNA expression increased significantly in the PDGF group (P<0.05). With the intervention of simvastatin, the levels of above indexes decreased compared with the PDGF group (P<0.05). With the intervention of simvastatin and parthenolide, the levels of above indexes decreased more obviously, but were not significantly different from those in the simvastatin intervention group.

CONCLUSIONSSimvastatin can inhibit proliferation of PASMC and cell cycle process. NF-κB may play an important role in the inhibitory effect of simvastatin on the proliferation of PASMC.

Animals ; Cell Proliferation ; drug effects ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; NF-kappa B ; analysis ; genetics ; physiology ; Pulmonary Artery ; cytology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Simvastatin ; pharmacology

This paper is to report the exploration of the activation of Rho/ROCK signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-Nis on this pathway. PASMCs were cultured with the explant technique. MTT assay was used to explore the proliferation of PASMCs after 5-HT treated for different time and the intervening effect of m-Nis. RT-PCR and Western blot were used respectively to explore the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 in 5-HT-treated PASMCs and intervening effect of m-Nis. The results of MTT assay suggested that 5-HT (1 µmol · L(-1)) treatment for 12-72 h significantly induced the proliferation of rat PASMCs (P<0.05 or P < 0.01), which were inhibited by m-Nis (1 x 10(-5), 1 x 10(-6), l x 10(-7), 1 x10(-8) mol · L(-1)) in dose-dependent manners (P < 0.05 or P < 0.01). Similarly, the mRNA expression of RhoA, ROCK1 and the protein expression of p-MYPT1 were also inhibited by m-Nis in different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Rho/ROCK pathway played an important role in 5-HT-induced proliferation of rat PASMCs, m-Nis inhibited 5-HT-induced proliferation obviously, which may be related to the blockage of Rho/ROCK signal pathway.
Animals ; Cell Proliferation ; drug effects ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Nisoldipine ; pharmacology ; Protein Phosphatase 1 ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Serotonin ; pharmacology ; Signal Transduction ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism