|Year : 2019 | Volume
| Issue : 4 | Page : 157-165
Aloperine suppresses human pulmonary vascular smooth muscle cell proliferation via inhibiting inflammatory response
Zhi Chang1, Peng Zhang2, Min Zhang1, Feng Jun1, Zhiqiang Hu2, Jiamei Yang1, Yuhua Wu2, Ru Zhou3
1 Department of Pharmacology, College of Pharmacy, Ningxia Medical University, Yinchuan 750004, PR China
2 General Hospital of Ningxia Medical University, Yinchuan 750004, PR China
3 Department of Pharmacology, College of Pharmacy; Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education; Ningxia Hui Medicine Modern Engineering Research Center and Collaborative Innovation Center, Ningxia Medical University, Yinchuan 750004, PR China
|Date of Submission||27-Mar-2019|
|Date of Decision||14-Jun-2019|
|Date of Acceptance||01-Aug-2019|
|Date of Web Publication||29-Aug-2019|
Prof. Ru Zhou
Department of Pharmacology, Ningxia Medical University, 1160 Shengli Street, Yinchuan 750004
Prof. Yuhua Wu
General Hospital of Ningxia Medical University, 804 Shengli Street, Yinchuan 750004
Source of Support: None, Conflict of Interest: None
Abnormal pulmonary arterial vascular smooth muscle cells (PASMCs) proliferation is critical pathological feature of pulmonary vascular remodeling that acts as driving force in the initiation and development of pulmonary arterial hypertension (PAH), ultimately leading to pulmonary hypertension. Aloperine is a main active alkaloid extracted from the traditional Chinese herbal Sophora alopecuroides and possesses outstanding antioxidation and anti-inflammatory effects. Our group found Aloperine has protective effects on monocroline-induced pulmonary hypertension in rats by inhibiting oxidative stress in previous researches. However, the anti-inflammation effects of Aloperine on PAH remain unclear. Therefore, to further explore whether the beneficial role of Aloperine on PAH was connected with its anti-inflammatory effects, we performed experiments in vitro. Aloperine significantly inhibited the proliferation and DNA synthesis of human pulmonary artery smooth muscle cells (HPASMCs) induced by platelet-derived growth factor-BB, blocked progression through G0/G1to S phase of the cell cycle and promoted total ratio of apoptosis. In summary, these results suggested that Aloperine negatively regulated nuclear factor-κB signaling pathway activity to exert protective effects on PAH and suppressed HPASMCs proliferation therefore has a potential value in the treatment of pulmonary hypertension by negatively modulating pulmonary vascular remodeling.
Keywords: Aloperine, inflammatory response, pulmonary arterial hypertension, pulmonary arterial vascular smooth muscle cell
|How to cite this article:|
Chang Z, Zhang P, Zhang M, Jun F, Hu Z, Yang J, Wu Y, Zhou R. Aloperine suppresses human pulmonary vascular smooth muscle cell proliferation via inhibiting inflammatory response. Chin J Physiol 2019;62:157-65
|How to cite this URL:|
Chang Z, Zhang P, Zhang M, Jun F, Hu Z, Yang J, Wu Y, Zhou R. Aloperine suppresses human pulmonary vascular smooth muscle cell proliferation via inhibiting inflammatory response. Chin J Physiol [serial online] 2019 [cited 2022 Sep 30];62:157-65. Available from: https://www.cjphysiology.org/text.asp?2019/62/4/157/265790
Zhi Chang, Peng Zhang, and Min Zhang contributed equally to this work.
| Introduction|| |
Pulmonary arterial hypertension (PAH) is a refractory disease characterized by elevated pulmonary vascular resistance and arterial pressures, driven by a progressive pulmonary vasculopathy that leads to right ventricular hypertrophy and ultimately right ventricular failure and even death. Pulmonary arterial vascular smooth muscle cells (PASMCs) proliferation is a pivotal part in the pathological vascular remodeling of PAH. The primary cellular mechanism underlying vascular remodeling, which has been well documented, involves structural alterations in the small arteries caused by excessive proliferation, apoptosis resistance, and the migration of PASMCs.,
It has been reported that the prevalence of PAH varied from 5 to 52 cases/million population. Despite the advent of new biological-based therapies and increased insight into the pathobiology of primary pulmonary hypertension, the actuarial survival of patients has demonstrated only modest improvement, as 50% of all people with primary PAH die within 7 years of diagnosis. No effective preventative or curative treatments are available. When PAH is superimposed on diseases such as bronchopulmonary dysplasia, a chronic lung disease of infancy, congenital heart disease, cystic fibrosis, or rheumatologically disease, the prognosis is dramatically worsened. Thus, the need to generate new knowledge that might be translated into a therapeutic tool has never been more palpable.
The mechanism of PAH is complex and is generally assumed as a process of the interaction of multiple factors. In the past decade, an increasing number of studies have addressed the molecular pathway involved in the development of PAH. Increasing evidence has pointed toward an inflammatory component being important in the development of PAH. The role of inflammation is increasingly recognized in the pathogenesis of PAH. In fact, recent studies described inflammation as a characteristic feature of many forms of PAH in both humans and animals. Levels of proinflammatory cytokines and chemokines are increased in lung tissue and blood of patients with PAH. In addition, the effect of inflammation in PAH development is further confirmed by the facts that clinical improvements were observed in patients after steroid treatment or immune suppressor administration. Nuclear factor-κB (NF-κB) and tumor necrosis factor-alpha (TNF-α) signaling pathways became hot spots for research and have been documented to play a predominant role in the pathogenesis of PAH.,, The primary importance of inflammation is illustrated by successful therapies using an interleukin-1 (IL-1) receptor antagonist and antibodies to monocyte chemotactic protein-1 in monocrotaline (MCT)-induced PAH model. Epithelial dysfunction induced by inflammation is believed to be the early event in the development of PAH., Stimuli such as cytokines and growth factors contribute to the abnormal PASMCs proliferation involved in vascular remodeling, correspondingly, cell-based studies of normal pulmonary arterial smooth muscle cells show that NF-κB is activated by inflammatory or proliferative stimuli. NF-κB is activated in pulmonary macrophages, lymphocytes, endothelial, and PASMCs in patients with end-stage idiopathic PAH. At the same time, there are studies show that NF-κB was activated in the PASMCs on various stimuli and then disassociated from IκB, leading to the translocation of NF-κB active forms p50 and p65 to the nucleus, where they regulate gene transcription and ultimately promote PASMCs proliferation. Thus, the proliferation of PASMCs was accompanied by the increased activation of NF-κB., Therefore, combine the above facts, we designed a series of experiments to investigate whether Aloperine can inhibit proliferation of PASMCs through negatively regulating NF-κB signaling pathway.
Sophora alopecuroides L. (Leguminosae) is a commonly used traditional Chinese herb, which is widely distributed in the northwestern region of China and is generally used for anti-inflammatory, antipyretic, and analgesic purposes.,, Aloperine, one of the most important alkaloids isolated from S. alopecuroides L, possesses a variety of pharmacological activities such as anti-inflammatory, anticancer, anti-microbial, antiviral, and anti-allergic effects., Considerably, the biological properties and potential therapeutic mechanisms of Aloperine have been widely tested in laboratory-based studies. Mounting evidence indicates that Aloperine possesses outstanding anti-inflammatory effects in neuropathic pain, cerebral ischemia, hydrogen peroxide-induced injury, and IR-induced acute renal injury., Recently, in vitro and in vivo experiments have shown that Aloperine confers beneficial effects on cell proliferation and apoptosis in HCT116 human colon cancer cells.,, In addition, the most important thing is that our group found Aloperine inhibits MCT-induced pulmonary hypertension in rats by the regulation of Rho A/ROCK pathway in previous researches. This study supports the opinion that this alkaloid is helpful for the treatment of pulmonary hypertension. Combined with the above observations, the current studies were undertaken to explore the relationship between the protective effects of Aloperine and its anti-inflammatory potential on PAH.
| Materials and Methods|| |
Materials and experiment design
Aloperine (98% purity as determined by high-performance liquid chromatography analysis) was ordered from Shanghai Yuanye Medical Technology Development Co., Ltd. (Shanghai, China). In vitro, the human pulmonary artery smooth muscle cells (HPASMCs) were grown to 70%–80% confluency and then subjected to serum starvation for 24 h before being used for the experiments. Cells were treated with different concentrations of Aloperine or 100 nM sildenafil (Pfizer, Sandwich, Kent, UK), which is known to inhibit PASMC proliferation for 24 h after the stimulation with platelet-derived growth factor-BB (PDGF-BB) (20 ng/ml).,,, The cells used in this study were taken between passages three and five. Recombinant human PDGF-BB was ordered from ProsPec-Tany TechnoGene Ltd. (Rehovot, Israel). Cell counting kit-8 (CCK-8) was obtained from TRANSGEN BIOTECH Inc. (Beijing, China). A cell proliferation ELISA, BrdU (colorimetric) kit was purchased from Roche (Roche Diagnostics, Mannheim, Germany). The antibodies IκBα (Cat NO: Ab-32536), IκBα (phospho Y42) (Cat NO: Ab-24783), IKKα (Cat NO: Ab-32041-100), IKKα (phospho S176) (Cat NO: Ab-138426), NF-κB (Cat NO: Ab-32536), TNF-α (Cat NO: Ab-9635), Cyclin E1 (Cat NO: Ab-33911), and p27kip1 (Cat NO: Ab-32034) were purchased from Abcam Biotechnology (MA, USA, Cat NO: Ab-129068). HPASMCs (Cat NO: 3110), purchased from Cell Research of Zhong Qiao Xin Zhou Institute, Shanghai, People's Republic of China, were cultured in smooth muscle cell medium smooth muscle cell growth medium (Cat NO: 1101) containing 10% fetal bovine serum (FBS) (Cat NO: 0010), and antibiotics (1% penicillin/streptomycin).
Measurement of cell proliferation
Whether Aloperine suppresses the growth of HPASMCs is currently unknown. To determine the effect of Aloperine on HPASMCs proliferation, the effect of different doses of Aloperine (0.125–1 mM) on proliferation in 24 h was investigated using the CCK-8 cell proliferation assay, according to the manufacturer's protocol. The cells were treated with PDGF-BB (20 ng/ml) for 24 h and then in the presence of Aloperine or sildenafil for another 24 h, and incubated with CCK-8 for the final 4 h. Cell proliferation was determined by measuring the absorbance at 450 nm.
Measurement of DNA synthesis
BrdU incorporation assay was performed to test cell proliferation according to the instructions of the BrdU ELISA Kit. Briefly, PASMCs were spread in a 96-well plate at a density of 3 × 103 cells/well and serum-deprived (1% FBS in Dulbecco's Modified Eagle's Medium) overnight. During the final 24 h of treatment, BrdU was added to label the cells at 37°C. The labeling medium was then removed, and cells were treated with FixDenat solution for 30 min and subsequently treated with anti-BrdU monoclonal antibody conjugated to peroxidase for 90 min at room temperature. After that, substrate solution was added to each well, and the absorbance was measured at 370 nm using a microplate reader that was normalized to the initial reading to verify equal cell numbers at the start of assay absorbance.
Cell cycle analysis via flow cytometry
Cell cycle progression was determined using a cell cycle analysis kit (Beyotime Institute of Biotechnology, Haimen, China), in accordance with the manufacturer's instructions, and fluorescence activated cell sorting. On reaching 70%–80% confluency in the six well plates, the HPASMCs were subjected to serum starvation for 24 h. The cells were then preincubated with PDGF-BB (20 ng/ml) for 24 h and subsequently treated with Aloperine (0.5 mM) for 24 h before analysis. The proportion of cells in the G0/G1, S and G2/M phases was investigated by flow cytometry. Cells were fixed with 70% ethanol, centrifuged at 800 rpm for 5 min and resuspended in staining buffer before detection. The cells were washed with PBS again and stained with 5 μg/ml RNase and 20 μg/ml of propidium iodide (PI) in the dark at 37°C for 30 min after overnight incubation at 4°C, and analyzed by flow cytometry (FACScalibur; BD Biosciences, Germany). All experiments were performed in triplicate. At least 10,000 events were counted for each sample.
Apoptosis assays via flow cytometry
Programmed cell death rates were assessed with a commercially available apoptosis assay kit (Becton Dickinson, Franklin Lakes, NJ, USA). On reaching 70%–80% confluency in the six well plates, the HPASMCs were subjected to serum starvation for 24 h. The cells were then preincubated with PDGF-BB (20 ng/ml) for 24 h and subsequently treated with Aloperine (0.5 mM) for 24 h prior to analysis. Then, the cells were harvested with 0.25% trypsin without ethylenediaminetetraacetic acid and Ca2+, washed twice with ice-cold PBS, and resuspended in 500 μl binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Subsequently, 1 × 105 cells were harvested and stained with 10 μl FITC Annexin V and 10 μl PI in the dark for 10 min at room temperature and detected using flow cytometry. The percentage of apoptotic cells was determined in three independent experiments.,
Protein was extracted from HPASMCs in lysis buffer using protein extraction reagent (BCA Protein Quantitative Kit, Kaiji, Nanjing, China). Lysates were centrifuged (2 × 104 rpm) at 4°C for 10 min. Protein expression (40 μg/L) was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8, 15% gel), and proteins were electro-blotted onto nitrocellulose membranes. The blots were probed with a monoclonal antibody against human. Equal amount of cell lysates were loaded to SDS-PAGE, and the separated proteins were next transferred onto a polyvinylidene difluoride (PVDF) membrane, followed by blocking with phosphate buffer saline (PBS) containing 5% nonfat dry milk powder under room temperature for 2 h. The PVDF membrane was then incubated with rabbit polyclonal antibody at 4°C overnight, followed by incubation with peroxidase-conjugated affinipure goat anti-rabbit IgG (H + L) secondary antibodies β-actin (Cat No. 20536-I-AP), β-Tubulin (Cat No. 10094-I-AP) and GAPDH (Cat No. 10494-I-AP) purchased from (Proteintech, Rosemont, IL, USA), respectively, at room temperature for 2 h and washing for 10 min/wash for three washes. The primary antibodies used were as follows: IκBα (1:1000), IκBα (phospho Y42) (1:1000), IKKα (1:10000), NF-κB (1:50000), TNF-α (1:500), Cyclin E1 (1:1000), and p27kip1 (1:1000), secondary antibodies were diluted into 1:1000. An enhanced chemiluminescence detection system (AZURE, USA) was used for protein detection.
SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All data were expressed as mean ± standard deviation. Group comparisons were performed using one-way analysis of variance followed by Dunnett post hoc test (between the control group and treatment groups) or S-N-K (Student–Newman–Keuls) post hoc test (among different groups). P ≤ 0.05 was considered to be statistically significant.
| Results|| |
Aloperine inhibits the proliferation of platelet-derived growth factor-BB-induced human pulmonary artery smooth muscle cells
To assess the role of Aloperine in modulating HPASMCs proliferation, the cells were administered different concentrations of Aloperine (0.125–1 mM). We observed that Aloperine inhibited HPSMCs proliferation in a dose-dependent manner, with a maximal effect elicited by a 0.5 mM dose. As compared with the control cells, PDGF-BB significantly increased the optical density of the cells after 24 h [Figure 1]a. Similarly, the results of BrdU-incorporation assays confirmed that Aloperine treatment (0.125–1 mM) significantly suppressed the increase in DNA synthesis in a dose-dependent manner in HPASMCs [Figure 1]b. According to the results above, the greatest level of suppression of proliferation was induced by Aloperine at a concentration of 0.5 mM.
|Figure 1: Effects of Aloperine on PDGF-BB induced HPASMCs proliferation and DNA synthesis. HPASMCs were pre-cultured in serum free medium for 24 h, cells were treated with different concentrations of Aloperine (0.125–1 mM) for 24 h after the stimulation with PDGF-BB (20 ng/ml). Cell proliferation was examined using (a) the Cell Counting kit 8 test, and (b) bromodeoxyuridine incorporation was determined using an enzyme linked immunosorbent based assay. Data are expressed as the mean OD ± standard deviation. ##P < 0.01 versus the control group; *P < 0.05, **P < 0.01 versus cells exposed to PDGF-BB alone; n = 6. PDGF: Platelet-derived growth factor, HPASMCs: Human pulmonary artery smooth muscle cells, OD: Optical density|
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Aloperine blocks platelet-derived growth factor-BB-induced cell cycle progression through G0/G1 to S phase cell cycle arrest
Increased proliferation and decreased apoptosis of PASMCs played an important role in pulmonary vascular remodeling. PDGF-BB has been previously shown to enhance HPASMCs proliferation. To explore whether Aloperine modulates the progression of PAH, Aloperine concentration of 0.5 mM was adopted as previously reported., The effect of Aloperine on cell cycle progression was analyzed using flow cytometric analysis. PDGF-BB treatment alone significantly increased the percentage of cells in S phase while decreasing the G0/G1 populations [Figure 2]. By contrast, Aloperine treated cells showed significant suppression of cell cycle progression. Aloperine at a dose of 0.5 mM reduced the percentage of cells in S phase and increased the G0/G1 populations among the PDGF-BB Aloperine cells. This suggests that Aloperine affects the G0/G1 to S phase transition rather than being involved in the S or G2/M phases.
|Figure 2: Effects of Aloperine on PDGF-BB-induced cell cycle progression. HPASMCs were treated with 20 ng/ml PDGF-BB for 24 h in the absence or presence of Aloperine (0.5 mM). Quantification of HPASMCs in the G0/G1, S and G2/M phases was determined using flow cytometric evaluation. Values are expressed as the mean ± standard deviation. ##P < 0.01 versus the control group; **P < 0.01 versus PDGF-BB treated cells (n = 6). (a): Control group; (b): PDGF-BB group; (c): PDGF-BB + Aloperine. PDGF: Platelet-derived growth factor, HPASMCs: Human pulmonary artery smooth muscle cells|
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Aloperine promotes platelet-derived growth factor-BB-induced human pulmonary artery smooth muscle cells apoptosis
To determine whether the decreased proliferation of HPASMCs occurs as a result of apoptosis, we investigated the apoptotic effects of Aloperine in HPASMCs. In the present study, as displayed in [Figure 3], treatment with Aloperine increased the proportion of early [Figure 3]d, late [Figure 3]e and total [Figure 3]f apoptotic cells. Collectively, Aloperine-inhibited proliferation of HPASMCs was due to cell apoptosis.
|Figure 3: Effects of Aloperine on PDGF-BB-induced apoptosis progression in HPASMCs. (a-c) Flow cytometry dot plots at 24 h; (d-f) Results of a quantitative analysis of apoptotic HPASMCs treated with Aloperine. Values are expressed as the mean ± standard deviation. P >0.05 versus the control group; **P < 0.01 versus PDGF-BB treated cells (n = 6). (a): Control group; (b): PDGF-BB group; (c): PDGF-BB + Aloperine; (d): Ratio of early apoptosis; (e): Ratio of late apoptosis; (f): Total ratio of apoptosis. PDGF: Platelet-derived growth factor, HPASMCs: Human pulmonary artery smooth muscle cells|
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Nuclear factor-κB signaling pathway involves in platelet-derived growth factor-BB-induced proliferation in human pulmonary artery smooth muscle cells
To elucidate the mechanism by which Aloperine inhibited proliferation and induced apoptosis, the expression levels of proteins involved in the proliferation and apoptosis pathway were examined using Western blot analysis. Because the NF-κB signaling pathway was associated with cell proliferation and apoptosis, we assumed that the effects of Aloperine on the proliferation and apoptosis of HPASMCs were associated with the NF-κB signaling pathway. Therefore, we examined the effects of Aloperine on PDGF-BB-induced NF-κB regulation. As shown in [Figure 4]a and [Figure 4]b, PDGF-BB stimulated IKKα activation as demonstrated by increased level of phosphorylated IKKα, but had no effect on total IKKα level. As shown in [Figure 4]c and [Figure 4]d, PDGF-BB stimulated IκBα activation as demonstrated by increased level of phosphorylated IκBα, but had no effect on total IκBα level. As shown in [Figure 4]e, [Figure 4]f, [Figure 4]g, [Figure 4]h, the stimulation of PDGF-BB increased levels of NF-κB p65 and downstream protein TNF-α, Cyclin E1, and decreased the expression of p27kip1. Furthermore, treatment with Aloperine attenuated the levels ofp- IKKα, p- IκBα, NF-κB p65, TNF-α, Cyclin E1 and improved the level of p27kip1 in HPASMCs stimulated with PDGF-BB, but did not exhibit any inhibitory effects on IKKα and IκBα. Collectively, these results demonstrated that NF-κB signaling pathway was involved in the inhibition activity of the proliferation and induction of apoptosis in HPASMCs induced by PDGF-BB.
|Figure 4: Effects of Aloperine on the inactivation of signaling pathways in PDGF-BB stimulated HPASMCs. HPASMCs were treated with Aloperine (0.5 mM) for 24 h after treatment with 20 ng/ml PDGF-BB. The protein expression levels of (a) IKKα, (b) p-IKKα, (c) IκBα, (d) p-IκBα, (e) NF-κB p65, (f) TNF-α, (g) Cyclin E1 and (h) p27kip1 were determined by Western blot analysis. One representative image out of three independently performed experiments is shown. Values are expressed as the mean ± standard deviation. ##P < 0.01, #P < 0.05 versus the control group; **P < 0.01, *P < 0.05 versus the PDGF-BB treated cells. Vehicle; the PGDF-BB group: Platelet-derived growth factor-BB; HPASMCs: Human pulmonary artery smooth muscle cells|
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| Discussion|| |
Current treatment paradigms for PAH mainly include four therapeutic categories by drug effects: endothelia-receptor antagonist pathway, phosphodiesterase type-5 inhibitor pathway, prostacyclin (PGI2) pathway, and circulating guanylate cyclase stimulator pathway.,, While currently approved drugs for PAH have some benefits on the quality of life in patients, limitations of short dosing intervals, peripheral vasodilation, and unwanted side effects make patients who are diagnosed with this disease still suffer from the lack of satisfactory treatment strategies to prolong survival. Moreover, the morbidity and mortality of PAH remain extremely high with no curative therapy. The limited success of PAH treatment is mainly due to the poor understanding of its pathophysiology and to the lack of effective empiric therapeutic regimens. Meanwhile, these vasodilators have very limited efficacy for eliminating pulmonary vascular remodeling and inflammatory response in the lung. Therefore, novel drugs are urgently required to exert better therapeutic effects on PAH.
Aloperine possesses beneficial properties against inflammation, tumor growth, and infection in the traditional Chinese medicine. By combining the results to our previous study, we found that Aloperine can reduce the expression of the nicotinamide adenine dinucleotide phosphate oxidase (NOX-2, NOX-4) and oxidative stress and obviously prevent the progression of PAH in rats and reverse the remodeling of the right ventricle., According to our preexperimental results, we found that using Aloperine alone (0.5 mM) did not affect cell viability. In the present study, we explored that in vitro, Aloperine induced apoptosis and inhibited the proliferation of HPASMCs through induction of G0/G1 phase arrest, one potential mechanism through inhibiting the NF-κB pathway and significantly upregulating the expression of p27kip1, as well as downregulating Cyclin E1, which coincided with its anti-inflammatory, anti-proliferation, and anti-apopototic activity.
NF-κB is a central regulator of inflammation. The activation of the classical NF-κB pathway induces expression of various genes encoding proinflammatory cytokines and chemokines, such as TNF-α and IL-1β; therefore, NF-κB can promote vascular inflammation by chemotaxis of leukocytes., Some studies showed that the role of NF-κB in PAH is of great importance: pulmonary artery smooth muscle cells and macrophages derived from patients with idiopathic PAH show increased NF-κB activation, and NF-κB inhibition ameliorates MCT-induced PAH.,
TNF-α is a proinflammatory cytokine with potent modulatory effects on the pulmonary circulation., Elevated serum TNF-α level was observed in patients with pulmonary hypertension secondary to chronic thromboembolic disease and connective tissue disease. In animal studies, TNF-α was shown to increase pulmonary vascular reactivity, to reduce PGI2 production in pulmonary artery smooth muscle cells, and to potentiate platelet-activating factor-induced pulmonary vasoconstriction. The over expression of TNF-α has been shown to result in severe pulmonary hypertension and emphysema in mice. Moreover, the suppression of TNF production by high doses of pentoxifylline reduces both systemic and pulmonary vascular resistance. Taken together, these data provide strong experimental evidence that TNF-α plays an important role in pulmonary vascular physiology.
One consequence of PDGF treatment is the activation of NF-κB. NF-κB activity can be induced by numerous and various stimuli. In unstimulated cells, NF-κB is retained in the cytoplasm through an interaction with inhibitory proteins known as IkB. The canonical mechanism of NF-κB activation involves signal-inducible degradation of IkB, which causes the translocation of NF-κB to the nucleus and transcription activation of targeted genes., Numerous studies have demonstrated that the pathological proliferation of PASMCs is associated IKK/IκB/NF-κB signaling pathway.,
To address these issues, the activation of NF-κB pathway was assessed in HPASMCs stimulated with PDGF-BB, and further examined the levels of Cyclin E1 and p27kip1 protein, two molecules serving as biomarkers of cell proliferation, and the results showed that after the stimulation of PDGF-BB in HPASMCs, cell proliferation was promoted through the activation of NF-κB p65 and subsequent upstream factorsp-IKKα and p-IκBα, downstream factors, such as TNF-α, p27kip1, and Cyclin E1. The results of the present study suggested that the treatment of HPASMCs with Aloperine upregulated the relative level of NF-κB pathway-associated protein p27kip1, while downregulating the relative expression levels of p-IKKα, p-IκBα, NF-κB p65, TNF-α, and Cyclin E1. These results are in line with anti-inflammation ability of Aloperine. Therefore, it was indicated that HPASMCs proliferation was inhibited by Aloperine through regulation of the NF-κB pathway. In addition, Aloperine remarkably declined the expression of Cyclin E1 and increased the expression of p27kip1 induced by PDGF-BB, suggesting the anti-proliferative capacity of Aloperine in HPASMCs under PDGF-BB stimuli. The downregulation of Cyclin E1 is a necessary step required for DNA repair. Cyclin E1 is considered as a key regulator of the G1 phase of the cell cycle and its inhibition results in the cell cycle arrest. p27kip1, a cyclin kinase inhibitor, is a major suppressor of apoptosis, which has been shown to prevent DNA-damaged cells from entering the cell cycle. p27kip1 is rapidly accumulated at sites of DNA damage, similarly to DNA repair factors. p27kip1 is required to displace DNA replication enzymes, thereby blocking processive DNA synthesis.
Increased expression of PDGF and PDGF receptor (PDGFR) were found in lung tissue of patients and animals with PAH. PDGFR is a receptor tyrosine kinase, which exerts its actions through binding PDGF and then activating a downstream signaling pathway to promote PASMCs proliferation and migration. PDGF-BB induced the proliferation of vascular smooth muscle cells and has been proposed to function in the development of atherosclerosis, lung fibrosis, PAH, and chronic thromboembolic pulmonary hypertension. Increased expression of PDGF and PDGFR were found in lung tissue of patients and animals with PAH. Furthermore, the levels of PDGF in the blood and lung tissues of patients with PAH are increased, further suggesting that PDGF plays a critical role in the development of pulmonary vascular remodeling and the increase in pulmonary arterial pressure. Consistently, our results showed that exogenous PDGF-BB promotes PASMCs proliferation, which further demonstrates that PDGF and PDGFR play an important role in the pathogenesis of PAH associated with abnormal PASMCs proliferation.
PAH occurrence and development are associated not only with abnormal proliferation of PASMCs, but are also with the suppression of apoptosis. The present study indicated a significant inhibitory effect of Aloperine on the proliferation and cell cycle of HPASMCs. To explore whether Aloperine modulates the progression of HPASMCs proliferation, Aloperine concentration of 0.5 mM was adopted. The results as illustrated showed PDGF-BB led to an augmented HPASMCs proliferation, which was markedly restrained by Aloperine treatment. Aloperine blocks HPASMCs proliferation and DNA synthesis induced by PDGF-BB, while not exerting any negative effects on human normal PASMCs. PAH is characterized by unbalanced proliferation/apoptosis axis, to investigate the impact of Aloperine on HPASMCs proliferation, it was observed that HPASMCs underwent cell cycle arrest in G0/G1 phase after treatment with Aloperine. Cell cycle arrest has also been linked to the inhibition of the proliferation of HPASMCs. Decreased apoptotic activity has been found to exist in HPASMCs from PAH patients. To assess the impact of Aloperine on HPASMCs apoptotic response, the results showed that the Aloperine notably accelerated PDGF-BB-mediated apoptosis, increased the proportion of early, late, and total apoptotic cells, indicating that Aloperine may promote HPASMCs apoptosis. These results collectively suggest that Aloperine suppresses cell proliferation and promotes apoptosis of HPASMCs under PDGF-BB condition.
| Conclusions|| |
Taken together, our novel findings suggested that aloperine induced apoptosis and inhibited the proliferation of HPASMCs through the induction of G0/G1 phase arrest, one potential mechanism through inhibiting the NF-κB pathway and significantly upregulating the expression of p27kip1, as well as downregulating Cyclin E1, which coincided with its anti-inflammatory, anti-proliferation, and anti-apopototic activity. Furthermore, our study provided more evidence of endothelial protection and attenuated collagen fibers and anti-inflammatory by Aloperine before it can be applied to the clinical treatment of PAH.
We are indebted to the staff in the Animal Center and the Science and Technology Center who provided assistance in the study.
Financial support and sponsorship
This project was supported by the 2017 Ningxia Medical University youth backbone talent cultivation selected project, National Natural Science Foundation of China (Grant No. 81402990), 2017 Ningxia Hui autonomous region science and technology innovation leader training project (Grant No. KJT2017005), 2017 Ningxia Medical University youth backbone talent cultivation selected project, the National Natural Science Foundation of China project based on the IKK-IkB-NF-κB signaling pathway to study the role and mechanism of oxidized saponin against cerebral ischemic injury (Grant No. 81660674) and the Personnel training projects of the Chinese Academy of Sciences “Western Light” (Grant No. XAB2015A12).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gao H, Cheng Y, Zong L, Huang L, Qiao C, Li W, et al.
Aspirin attenuates monocrotaline-induced pulmonary arterial hypertension in rats by suppressing the ERK/MAPK pathway. Clin Exp Hypertens 2017;39:34-41.
Hu XJ, Chen XL, Chen C, Ai J, Li J, Han XJ, et al.
Alterations in pulmonary arterial reactivity during pulmonary arterial hypertension at the early-stage of pulmonary fibrosis in rats. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2011;27:110-4.
Boucherat O, Vitry G, Trinh I, Paulin R, Provencher S, Bonnet S, et al.
The cancer theory of pulmonary arterial hypertension. Pulm Circ 2017;7:285-99.
Dai YP, Bongalon S, Hatton WJ, Hume JR, Yamboliev IA. ClC-3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery. Br J Pharmacol 2005;145:5-14.
Dang Z, Zhu L, Lai W, Bogerd H, Lee KH, Huang L, et al.
Aloperine and its derivatives as a new class of HIV-1 entry inhibitors. ACS Med Chem Lett 2016;7:240-4.
Wu F, Hao Y, Yang J, Yao W, Xu Y, Yan L, et al.
Protective effects of aloperine on monocrotaline-induced pulmonary hypertension in rats. Biomed Pharmacother 2017;89:632-41.
Fuji S, Matsushita S, Hyodo K, Osaka M, Sakamoto H, Tanioka K, et al.
Association between endothelial function and micro-vascular remodeling measured by synchrotron radiation pulmonary micro-angiography in pulmonary arterial hypertension. Gen Thorac Cardiovasc Surg 2016;64:597-603.
Wang H, Yang S, Zhou H, Sun M, Du L, Wei M, et al.
Aloperine executes antitumor effects against multiple myeloma through dual apoptotic mechanisms. J Hematol Oncol 2015;8:26.
Goncharov DA, Kudryashova TV, Ziai H, Ihida-Stansbury K, DeLisser H, Krymskaya VP, et al.
Mammalian target of rapamycin complex 2 (mTORC2) coordinates pulmonary artery smooth muscle cell metabolism, proliferation, and survival in pulmonary arterial hypertension. Circulation 2014;129:864-74.
Gruber HE, Hoelscher GL, Ingram JA, Bethea S, Cox M, Hanley EN Jr., et al.
Proinflammatory cytokines modulate the chemokine CCL2 (MCP-1) in human annulus cells in vitro
: CCL2 expression and production. Exp Mol Pathol 2015;98:102-5.
Hu S, Zhang Y, Zhang M, Guo Y, Yang P, Zhang S, et al.
Aloperine protects mice against ischemia-reperfusion (IR)-induced renal injury by regulating PI3K/AKT/mTOR signaling and AP-1 activity. Mol Med 2016;21:912-23.
Barst RJ, Beghetti M, Pulido T, Layton G, Konourina I, Zhang M, et al.
STARTS-2: Long-term survival with oral sildenafil monotherapy in treatment-naive pediatric pulmonary arterial hypertension. Circulation 2014;129:1914-23.
Huang Z, Liu Z, Luo Q, Zhao Z, Zhao Q, Zheng Y, et al.
Glycoprotein 130 inhibitor ameliorates monocrotaline-induced pulmonary hypertension in rats. Can J Cardiol 2016;32:1356.
Gossmann J, Burkhardt R, Harder S, Lenz T, Sedlmeyer A, Klinkhardt U, et al.
Effect of angiotensin II infusion with and without angiotensin II Type 1 receptor blockade on nitric oxide metabolism and endothelin in human beings: A placebo-controlled study in healthy volunteers. Clin Pharmacol Ther 2000;68:501-9.
Kadowaki M, Mizuno S, Demura Y, Ameshima S, Miyamori I, Ishizaki T, et al.
Effect of hypoxia and beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: The role of p27kip1. Respir Res 2007;8:77.
Lee JH, Park BK, Oh KS, Yi KY, Lim CJ, Seo HW, et al.
Aurotensin II receptor antagonist, KR36676, decreases vascular remodeling and inflammation in experimental pulmonary hypertension. Int Immunopharmacol 2016;40:196-202.
Lin WC, Lin JY. Five bitter compounds display different anti-inflammatory effects through modulating cytokine secretion using mouse primary splenocytes in vitro
. J Agric Food Chem 2011;59:184-92.
Liu C, Fang C, Cao G, Liu K, Wang B, Wan Z, et al.
Ethyl pyruvate ameliorates monocrotaline-induced pulmonary arterial hypertension in rats. J Cardiovasc Pharmacol 2014;64:7-15.
Marsh LM, Jandl K, Grünig G, Foris V, Bashir M, Ghanim B, et al.
The inflammatory cell landscape in the lungs of patients with idiopathic pulmonary arterial hypertension. Eur Respir J 2018;51:1701214.
Medoff BD, Okamoto Y, Leyton P, Weng M, Sandall BP, Raher MJ, et al.
Adiponectin deficiency increases allergic airway inflammation and pulmonary vascular remodeling. Am J Respir Cell Mol Biol 2009;41:397-406.
Liu HL, Chen XY, Li JR, Su SW, Ding T, Shi CX, et al.
Efficacy and safety of pulmonary arterial hypertension-specific therapy in pulmonary arterial hypertension: A meta-analysis of randomized controlled trials. Chest 2016;150:353-66.
Wang X, Jiang B, Li L, Yao H, Deng H. Effects of total alkaloid of Sophora alopecuroides
on serum IL-1beta and IL-4 expression in mice with acute ulcerative colitis. Zhongguo Zhong Yao Za Zhi 2010;35:1177-80.
Wort SJ, Woods M, Warner TD, Evans TW, Mitchell JA. Endogenously released endothelin-1 from human pulmonary artery smooth muscle promotes cellular proliferation: Relevance to pathogenesis of pulmonary hypertension and vascular remodeling. Am J Respir Cell Mol Biol 2001;25:104-10.
Wu F, Yao W, Yang J, Zhang M, Xu Y, Hao Y, et al
. Protective effects of aloperin on monocroline-induced pulmonary hypertension via regulation of Rho A/Rho kinsase pathway in rats. Biomed Pharmacother 2017;95:1161.
Xu YQ, Jin SJ, Liu N, Li YX, Zheng J, Ma L, et al.
Aloperine attenuated neuropathic pain induced by chronic constriction injury via anti-oxidation activity and suppression of the nuclear factor kappa B pathway. Biochem Biophys Res Commun 2014;451:568-73.
Wang Y, Wang B, Guerram M, Sun L, Shi W, Tian C, et al.
Deoxypodophyllotoxin suppresses tumor vasculature in HUVECs by promoting cytoskeleton remodeling through LKB1-AMPK dependent rho A activatio. Oncotarget 2015;6:29497-512.
Song Y, Wu Y, Su X, Zhu Y, Liu L, Pan Y, et al.
Activation of AMPK inhibits PDGF-induced pulmonary arterial smooth muscle cells proliferation and its potential mechanisms. Pharmacol Res 2016;107:117-24.
Yeager ME, Belchenko DD, Nguyen CM, Colvin KL, Ivy DD, Stenmark KR, et al.
Endothelin-1, the unfolded protein response, and persistent inflammation: Role of pulmonary artery smooth muscle cells. Am J Respir Cell Mol Biol 2012;46:14-22.
Xiao Y, Peng H, Hong C, Chen Z, Deng X, Wang A, et al.
PDGF promotes the warburg effect in pulmonary arterial smooth muscle cells via activation of the PI3K/AKT/mTOR/HIF-1α signaling pathway. Cell Physiol Biochem 2017;42:1603-13.
Yi B, Cui J, Ning J, Gu J, Wang G, Bai L, et al.
CGMP-dependent protein kinase iα transfection inhibits hypoxia-induced migration, phenotype modulation and annexins A1 expression in human pulmonary artery smooth muscle cells. Biochem Biophys Res Commun 2012;418:598-602.
Yi B, Cui J, Ning JN, Wang GS, Qian GS, Lu KZ, et al.
Over-expression of PKGIα inhibits hypoxia-induced proliferation, akt activation, and phenotype modulation of human PASMCs: The role of phenotype modulation of PASMCs in pulmonary vascular remodeling. Gene 2012;492:354-60.
Zeng J, Yi B, Wang Z, Ning J, Wang X, Lu K, et al.
Effect of annexin A2 on hepatopulmonary syndrome rat serum-induced proliferation of pulmonary arterial smooth muscle cells. Respir Physiol Neurobiol 2013;185:332-8.
Zhang WF, Zhu TT, Xiao-Yue GE, Xiong AZ, Chang-Ping HU. Calpain mediated pulmonary vascular remodeling in hypoxia induced pulmonary hypertension. Chin J Pharmacol Toxicol 2016;41:929-36.
Zhang L, Zheng Y, Deng H, Liang L, Peng J. Aloperine induces G2/M phase cell cycle arrest and apoptosis in HCT116 human colon cancer cells. Int J Mol Med 2014;33:1613-20.
Wang RC, Jiang FM, Zheng QL, Li CT, Peng XY, He CY, et al.
Efficacy and safety of sildenafil treatment in pulmonary arterial hypertension: A systematic review. Respir Med 2014;108:531-7.
Li XW, Li XH, Du J, Li D, Li YJ, Hu CP, et al.
Calcitonin gene-related peptide down-regulates bleomycin-induced pulmonary fibrosis. Can J Physiol Pharmacol 2016;94:1315-24.
Zheng YX, Zhang L, Deng HZ, Liang L. Effects of sophoridine, matrine and aloperine on the contents of cytokine IL-6 and TNF-α in LPS-induced IEC-6 cells inflammatory model. Chin J Exp Tradit Med Formulae 2014;20:276-83.
Zurlo G, Piquereau J, Moulin M, Da Silva JP, Gressette M, Ranchoux B, et al.
Sirtuin 1 regulates pulmonary artery smooth muscle cell proliferation: Role in pulmonary arterial hypertension. J Hypertens 2018;36:1164-77.
Zhang H, Xu Y, Zhang Z, Xu S, Ni W, Chen S, et al.
Effect of nuclear factor-kappa B on vascular endothelial growth factor mRNA expression of human pulmonary artery smooth muscle cells in hypoxia. J Huazhong Univ Sci Technolog Med Sci 2004;24:9-12, 18.
Nogueira-Ferreira R, Vitorino R, Ferreira R, Henriques-Coelho T. Exploring the monocrotaline animal model for the study of pulmonary arterial hypertension: A network approach. Pulm Pharmacol Ther 2015;35:8-16.
Polonio IB, Acencio MM, Pazetti R, Almeida FM, Silva BS, Pereira KA, et al.
Lodenafil treatment in the monocrotaline model of pulmonary hypertension in rats. J Bras Pneumol 2014;40:421-4.
Ren D, Ma W, Guo B, Wang S. Aloperine attenuates hydrogen peroxide-induced injury via anti-apoptotic activity and suppression of the nuclear factor-κB signaling pathway. Exp Ther Med 2017;13:315-20.
Shao J, Wang P, Liu A, Du X, Bai J, Chen M, et al.
Punicalagin prevents hypoxic pulmonary hypertension via anti-oxidant effects in rats. Am J Chin Med 2016;44:785-801.
Sun LY, Cai ZY, Pu J, Li J, Shen JY, Yang CD, et al
. 5-aminosalicylic acid attenuates monocrotaline-induced pulmonary arterial hypertension in rats by increasing the expression of nur77. Inflammation 2017;40:806-17.
Soon E, Crosby A, Southwood M, Yang P, Tajsic T, Toshner M, et al.
Bone morphogenetic protein receptor Type II deficiency and increased inflammatory cytokine production. A gateway to pulmonary arterial hypertension. Am J Respir Crit Care Med 2015;192:859-72.
Vengethasamy L, Hautefort A, Tielemans B, Belge C, Perros F, Verleden S, et al.
BMPRII influences the response of pulmonary microvascular endothelial cells to inflammatory mediators. Pflugers Arch 2016;468:1969-83.
Hwang YS, Lee J, Zhang X, Lindholm PF. Lysophosphatidic acid activates the rhoA and NF-κB through akt/IκBα signaling and promotes prostate cancer invasion and progression by enhancing functional invadopodia formation. Tumour Biol 2016;37:6775-85.
Liu HM, Jia Y, Zhang YX, Yan J, Liao N, Li XH, et al.
Dysregulation of miR-135a-5p promotes the development of rat pulmonary arterial hypertensionin vivo
and in vitro
. Acta Pharmacol Sin 2019;40:477-85.
Rieg AD, Suleiman S, Anker C, Verjans E, Rossaint R, Uhlig S, et al.
PDGF-BB regulates the pulmonary vascular tone: Impact of prostaglandins, calcium, MAPK and PI3K/AKT/mTOR signalling and actin polymerisation in pulmonary veins of Guinea pigs. Respir Res 2018;19:120.
Qian Z, Li Y, Chen J, Li X, Gou D. miR-4632 mediates PDGF-BB-induced proliferation and antiapoptosis of human pulmonary artery smooth muscle cells via targeting cJUN. Am J Physiol Cell Physiol 2017;313:C380-91.
Bai Y, Li ZX, Zhao YT, Liu M, Wang Y, Lian GC, et al.
PCPA protects against monocrotaline-induced pulmonary arterial remodeling in rats: Potential roles of connective tissue growth factor. Oncotarget 2017;8:111642-55.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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