Icariin Attenuates Monocrotaline-Induced Pulmonary Arterial Hypertension via the Inhibition of TGF-β1/Smads Pathway in Rats

Background Pulmonary artery remodeling is important in the development of pulmonary artery hypertension. The TGF-β1/Smads signaling pathway is activated in pulmonary arterial hypertension (PAH) in rats. Icariin (ICA) suppresses the TGF-β1/Smad2 pathway in myocardial fibrosis in rats. Therefore, we investigated the role of icariin in PAH by inhibiting the TGF-β1/Smads pathway. Methods Rats were randomly divided into control, monocrotaline (MCT), MCT + ICA-low, and MCT + ICA-high groups. MCT (60 mg/kg) was subcutaneously injected to induce PAH, and icariin (50 or 100 mg/kg.d) was orally administered for 2 weeks. At the end of the fourth week, right ventricular systolic pressure (RVSP) was obtained and the right ventricular hypertrophy index (RI) was determined as the ratio of the right ventricular weight to the left ventricular plus septal weight (RV/LV + S). Western blots were used to determine the expression of TGF-β1, Smad2/3, P-Smad2/3, and matrix metalloproteinase-2 (MMP2) in lung tissues. Results Compared to the control group, RVSP and RI were increased in the MCT group (ρ < 0.05). Additionally, TGF-β1, Smad2/3, P-Smad2/3, and MMP2 expressions were obviously increased (ρ < 0.01). Compared to the MCT group, RVSP and RI were decreased in the MCT + ICA group (ρ < 0.05). TGF-β1, Smad2/3, P-Smad2/3, and MMP2 expressions were also inhibited in the icariin treatment groups (ρ < 0.05). Conclusions. Icariin may suppress MCT-induced PAH via the inhibition of the TGFβ1-Smad2/3 pathway.


Introduction
Pulmonary arterial hypertension (PAH) is a serious vascular disease characterized by increased pulmonary artery pressure, progressive right heart hypertrophy, and heart failure [1]. Recently, PAH has become increasingly recognized as a chronic proliferative disease, particularly because of the extensive vascular remodeling of pulmonary artery vasculature, leading to intimal fibrosis, medial hypertrophy, luminal stenosis, and obliteration in small pulmonary arteries [2,3]. Additionally, it is associated with poor prognosis, with a median survival time of 2 to 5 years from the point of diagnosis in most patients [4][5][6].
Icariin (ICA), isolated from Epimedium pubescens, is the main active flavonoid of Herba Epimedii [19,20]. Additionally, it inhibits TGFβ1/Smad2 signaling and alleviates myocardial fibrosis in rats [21]. us, this study was designed to elucidate the effects of icariin in PSMCs remodeling in PAH via the inhibition of the TGF-β1/Smads signal pathway.

Animals and Ethics Statement.
In this study, male Sprague Dawley (SD) rats (aged 6-8 weeks, weighing 250-300 g) were obtained from the Laboratory Animal Center of Zhejiang province (certificate no. SCXL (Zhe) 2019-002). e animals were provided with free access to a standard diet and tap water at a temperature of 21 ± 1°C and a humidity of 55 ± 5%. e experimental procedures were approved by the Ethics Review of Animal Use Application of Fifth Affiliated Hospital of Wenzhou Medical University and were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals.

Monocrotaline (MCT)
Treatment. MCT (Sigma, USA) was dissolved in 1 mol/L HCl, and the pH was adjusted to 7.20-7.40 with 1 mol/L NaOH. Rats in the MCT and MCT + ICA groups were subcutaneously injected with the MCT solution (diluted with 0.9% saline, 60 mg/kg) once. e control rats were injected with 0.9% saline (0.8 ml).

Treatment Protocol In Vivo.
All the animals were randomly assigned to four groups. e control group rats (control, n � 10) orally administered 0.9% saline. Model group rats (MCT, n � 20) received MCT with 0.9% saline. Icariin group rats (n � 20/group) received icariin (Plant Bio-Engineering Co., Ltd., Xi'an, China) at a dose of 50 or 100 mg/kg per day. After 2 weeks of MCT injection, the rats in ICA groups were orally maintained daily for 2 weeks with different doses of ICA. Body weight was measured weekly to adjust the dose accordingly.

Hemodynamic and Cardiac
Monitoring. Rats were anesthetized with isoflurane via the respiratory tract (induction with concentration of 5% for two minutes and a concentration of 2% for maintaining), followed by insertion of a catheter (PE50 tubule) into the right ventricular cavity through the right jugular vein. After measuring the right ventricular systolic pressure (RVSP, mmHg), the rats were sacrificed with 10% potassium chloride solution that was administered to the inferior vena cava under the anesthetized condition of isoflurane. e heart was dissected, and the right ventricular hypertrophy index (RI) was assessed by the ratio of the right ventricular weight to the left ventricular plus septal weight (RV/LV + S). Finally, the right lung was fixed in 10% formaldehyde for histopathology studies, and the remainder was stored at −80°C.

Histopathology.
After incubation for 72 h, the upper lobe of the right lung tissues was dehydrated via a graded alcohol series, embedded in paraffin, and cut into 3-5 μm thin sections. e tissue sections were stained with hematoxylin and eosin (HE) and Masson. en, the small arteries (diameter 25-100 µm) were visualized with a microscope. For each artery, the degree of wall thickness was calculated as follows: the ratio of the vascular wall thickness (WT%) � 100% × wall thickness/outer diameter. ree fields of six sections were randomly chosen for analysis in each group.
2.6. Immunohistochemistry. Lung tissue sections (4 μm thick) were dewaxed and rehydrated and then washed with PBS (pH 7.2-7.4). After antigen retrieval and blocking with 5% bovine serum albumin (BSA), the sections were incubated with anti-α-smooth muscle actin (α-SMA) antibody overnight at 4°C, followed by the secondary antibody. en, the sections were visualized with 3′3′-diaminobenzidine (DAB) and counterstained with hematoxylin. Afterwards, the stained sections were observed by the light microscope (Nikon, Japan).

Statistical Analysis.
e data are presented as means ± SEM. Significant differences were determined by one-way ANOVA using GraphPad Prism (version 7.0) software followed by Tukey's multiple comparisons test. Values were considered to be significant when ρ < 0.05.

Icariin Suppressed Pulmonary Small Artery Remodeling.
Under the microscope, the wall of small pulmonary artery was remarkably hypertrophic. Additionally, the WT (%) was obviously increased unlike those in the control group (ρ < 0.01). ese pathological changes were improved with icariin treatment in the MCT + ICA-low group (ρ < 0.05) and MCT + ICA-high group (ρ < 0.01) (Figure 2). Additionally, treatment of icariin partly inhibited the fibrosis in PSMC with Masson stain (Figure 3).

Discussion
PAH is a malignant vascular disease with poor prognosis. In our study, compared with the control group, RVSP and RI in the MCT group were significantly increased, indicating that the model of MCT-related PAH was successfully established. In this study, treatment with a different dose of icariin markedly suppressed pulmonary small artery remodeling, which ameliorated pulmonary hypertension and right heart hypertrophy. In addition, previous studies demonstrated that progressive thickening of the pulmonary artery wall contributed to the development of PAH [22][23][24][25]. erefore, the thickness of the vascular wall was observed by the HE stain and WT%. Additionally, it was proved that WT% was strongly increased in the MCT group, and treatment with icariin improved WT% in a dose-dependent manner. us, these results show that the inhibiting effect of icariin on PAH may be related to dose. us, treatment with icariin with a dose of 100 mg/kg·d was used to analyse the target proteins and to observe the changes in the small pulmonary artery by Masson stains, as well as α-SMA by immunohistochemical assay. Studies have shown that smooth muscle cell (SMC) and endothelial cell (EC) proliferation are involved in the pathology of pulmonary vascular remodeling in pulmonary hypertension [26][27][28]. In this study, we observed the media change in the small pulmonary artery with α-SMA, in accordance with the HE stain. Additionally, treatment with icariin reduced TGF-β1, Smad2/3, P-Smad2/3, and MMP2 expression.
e members of TGF-β family cause numerous cellular responses through different receptors and intracellular signal pathways and are significant mediators in pulmonary fibrosis and vascular remodeling [8,[29][30][31][32]. TGF-β1, which is one of three isoforms, is the most abundantly expressed one [33]. TGF-β1 binds to type I and II receptors on the cell surface, and the intracellular signaling induced by TGF-β ligands is then mediated by the Smad family proteins. Smad2 and Smad3 are the first identified substrates of the TbRI kinase, which are activated through carboxy-terminal phosphorylation. e receptor-activated Smads (R-Smads) are released from the receptor complex to form a heterotrimer of two R-Smads and one Smad4, which then translocates to the nucleus to regulate target gene expression [29]. As in previous studies, in the model of the MCT-induced PAH, the relative levels of TGF-β1, P-Smad/Smad2, and P-Smad3/Smad3 were increased significantly in lung tissues [34,35]. ese results indicated that the TGF-β1-Smad2/3 signal pathway was involved in the process of PAH. Additionally, treatment with icariin partly reduced the levels of these target proteins in lung tissues. us, we may speculate that icariin may improve the PAH via the inhibition of TGF-β1-Smad2/3 signal pathway at the molecular level, but it will be confirmed in vitro and in vivo research studies in the future.  Figure 4: Icariin reduces TGF-β1, Smad2/3, and P-Smad2/3 protein levels in rat lung tissues. Western blot was performed to determine the expression levels of target proteins. * * ρ < 0.01, * ρ < 0.05, ns � no significant, N � 6/group (P-Smad3, Smad3, n � 4/group).  Evidence-Based Complementary and Alternative Medicine ECM represents a key component of pulmonary vascular remodeling and regulates the metabolism by the matrix metalloproteinases (MMPs). Imbalance in MMPs and ECM metabolism induces pulmonary hypertension. TGF-β1 increases ECM deposition during vascular remodeling, mainly collagen type I and fibronectin deposition [36,37]. Moreover, TGF-β1, Smad2, and Smad3 can increase the synthesis of fibronectin, collagen, and proteoglycans and can reduce the decomposition of collagen protein, further resulting in an imbalance of the extracellular matrix and the deposition of collagen. Further, extracellular TGF-β1 may be activated by proteases, such as MMP2 [38]. Our results show that the lung tissues of MCT-induced rats display a higher expression of MMP2. is result demonstrates the complex interactions between the TGF-β1/Smad2/3 pathways and ECM metabolism in the pathology of vascular remodeling in MCT-induced PAH. In addition, treatment with icariin suppressed the expression of MMP2 in lung tissues. Additionally, it indicated that icariin may inhibit the complex reaction between TGF-β1/Smad2/3 pathway and MMP2.

Conclusion
In conclusion, our study showed that icariin ameliorated MCT-induced pulmonary hypertension. e possible mechanism is likely mediated via the suppression of TGF-β1/Smad2/3 signaling.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.