The protective of baicalin on pulmonary hypertension vascular remodeling through regulation of TNF-α signaling pathway

Xia Xue Shandong University Cheeloo College of Medicine Wen Jiang Shandong University Cheeloo College of Medicine Chao Sun Shandong University Cheeloo College of Medicine Jue Wang Shandong University Cheeloo College of Medicine Qian Xin Shandong University Cheeloo College of Medicine Kaili Li Shandong University Cheeloo College of Medicine Tonggang Qi Shandong University Cheeloo College of Medicine Yun Luan (  luanyun@sdu.edu.cn ) Shandong University


Introduction
Pulmonary hypertension (PH) is a life-threatening disease, is characterized by excessive proliferation of pulmonary vascular cells, eventually leads to pulmonary vascular resistance, vascular remodeling, right ventricular hypertrophy and failure [1,2]. Various types of PH prevalence exceed 30-50/1 million every year with onset around the middle-aged (75%, 20-40 years old). The pathogenesis of PH is complicated, so far, there is no effective treatment for it and the mortality rate is still high.
A large number of reports [3][4][5] showed that excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important pathogenesis in PH vascular remodeling process. Bone morphogenetic protein type II receptor (BMPR2) is a key factor in the process of PH pulmonary remodeling occurs [6,7]. BMPR2 levels in pulmonary vascular was signi cant reduced in non-genetic forms of PH. More than 70% of heritable and 20% of idiopathic PH cases have the BMPR2 gene mutations. Recent studies have found that in ammation is thought to be strong trigger factors and promotes the development of PH in Bmpr2 +/mice, in ammatory play an important role in PAH [7][8][9], in ammatory factors was signi cantly increased in clinical PH patient. In ammatory mediator tumor necrosis factor-α (TNF-α) could further promote the development of PH by reducing BMPR2 expression in PASMCs.
In the present study, we tested the hypothesis that baicalin have the therapeutic effect on PH through regulation of TNF-α/BMPR2 signaling pathway and further inhibiting pulmonary vascular remodeling.
These will provide theoretical foundation for the clinical treatment of PH.

Materials And Methods
Animals experimental design Baicalin (purity > 95%) was purchased from Sigma (St. Louis, MO, USA) and was dissolved in dimethyl sulfoxide (DMSO). The PH model was induced by intraperitoneal injection of 60 mg/kg MCT for 6 weeks (Sigma-Aldrich, USA) as our previously described with modi cations [15]. 100 mg/kg baicalin or the same amount of saline solution was given by intragastric administration from 2 days after MCT injection. Forty animals were randomly assigned to 4 groups: Control, MCT, saline and baicalin treated groups ( n = 10 in each). Hemodynamic and the right ventricular hypertrophy assessment All animals were anaesthetized by iso urane inhalation (1.5-2%) and then euthanized by cervical dislocation. Hemodynamic data were recorded after operation as previously described with some modi cations. Via femoral vein access, a 5F Swan-Ganz catheter (Edwards Lifesciences Corp, Irvine, CA) was advanced into the pulmonary artery for determination of heart rate (HR), systemic blood pressure (SBP) and right ventricular systolic pressure (RVSP). For assessment of right ventricular hypertrophy, the left ventricle (LV) plus the septum (LV + S) were harvested, and the weight ratio of the RV to LV + S weight calculated to quantify the right ventricular hypertrophy. The right ventricular hypertrophy index (RVHI) by the formula: RV/(LV + S) × 100.

Immunological and immunohistochemical analyses
Post-operation, the lung and heart were quickly harvested and xed in 4% paraformaldehyde and embedded in para n, the serially sectioned at a thickness of 4-5 µm were stained with hematoxylineosin (H&E). To evaluate pulmonary artery structural remodeling, the vascular wall thickness (WT), vascular external diameter (ED), vascular wall area (WA) and total vascular area (TA) to calculate WT% (WT/ED) and WA% (WA/TA) were measured as previously study. Fibrosis area analysis by Masson's trichrome staining, and then the sections were captured as digital images. The vascular was counted in blind on 30 sections by using a light microscope at a × 400 magni cation. The average of the 10 highpower elds (hpf) was randomly selected, and positively stained areas were padded with a single color and converted into pixels through optical density (OD) calibration.
Pulmonary artery proliferation were then carried out to analysis the expression of smooth muscle actin (a-SMA) by immunohistochemistry and immuno uorescence. Brie y, after blocking unspecifc protein binding with 5% bovine serum albumin for 30 min at room temperature, the lung sections were incubated overnight at 4 °C with antibodies. Images were taken with an Eclipse 90i microscope (Nikon, Tokyo, Japan). Staining was quanti ed using Image Pro Plus 6.0 image analysis software (Media Cybernetics, Rockville, MD). All experiments were performed by two examiners blinded to treatment assignment.
The migratory function of PAMSC was evaluated using a modi ed Boyden chamber (Transwell; Corning Life Sciences, Inc., Tewksbury, MA, USA) assay with a polycarbonate lter with 8-µm pores placed between the upper and lower chambers. In brief, at 0 and 24 h following TNF-α pre-treatment, cells were treated with baicalin containing 1% FBS and added to the upper chamber. The lower chamber was lled with complete medium in the presence of 10% FBS. After a 48-h incubation at 37˚C under 5% CO 2 , cells that had not migrated were removed, whereas migrated cells were xed in 4% paraformaldehyde for 10 min at room temperature and stained with the Crystal Violet Staining Solution kit (Solarbio, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The number of migrated cells was counted using a Nikon Eclipse 90i microscope. RNA preparation and quantitative reverse transcription-PCR Total RNA was extracted using the RNeasy Mini Kit with DNAse digestion (Qiagen, West Sussex, UK) from lung tissues and cultured PAMSCs. Quantitative real-time polymerase chain reaction(qRT-PCR) analysis was performed using a M × 3000P System.1.5% agarose gel electrophoresis in the presence of ethidium bromide (Sigma-Aldrich) was used to ampli cation fragments, and β-actin as and internal control.

Ethics
All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Also, all experiments were approved by the Institutional Animal Care and Use Committee of Shandong University. All animals were anaesthetized by iso urane inhalation (1.5-2%) and then euthanized by cervical dislocation. patients. The work was done in accordance with the Helsinki Declaration's guidelines.

Statistical analysis
All data are expressed as mean ± SD. Comparisons of parameters between 2 groups were made with unpaired Student t test. Comparisons of parameters among 3 groups were made with one-way analysis of variance (ANOVA), followed by the Scheffe post hoc test. Statistical analysis was carried out by using the SPSS 19.0 software. P < 0.05 was regarded as signi cant statistical difference.

Effect of baicalin on PH vascular remodeling
Six weeks later, we evaluated MCT-induced lung and heart injury by detecting RVSP and right ventricular hypertrophy index RV/(LV + S), as shown in Fig. 1A, a signi cant increased of RVSP and RV/(LV + S) in MCT administration group as compared with control (P < 0.05), these indicated that we successfully established PH model in rats. However, RVSP and RV/(LV + S) were signi cantly inhibited in baicalin treated rats than that in MCT rats (P < 0.05). There was no signi cantly different in HR and SBP between groups (P > 0.05).
Lung sections were stained with H&E and Masson's trichrome was used to analysis the medal thickness of pulmonary arterial walls and the degree of brosis. As shown in Fig. 1B and 1C, WT% and WA% of muscular arteries with an external diameter of 15 to 50 µm were signi cantly increased in MCT group than that in control, but notably decreased in baicalin group (P < 0.05). Masson's stained results showed that the lung brosis was signi cantly reduced in baicalin treated group than that in MCT group ( Fig. 2A and 2B, P < 0.05).
Effect of baicalin on smooth muscle cells In this study, smooth muscularization cells marker α-SMA were analyzed by immunohistochemistry and immuno uorescence. The results showed that the expression of α-SMA were signi cantly increased in MCT group than that in control, however, which were signi cantly lower in baicalin treated group (P < 0.05, Fig. 3A and 3B).

Effect of baicalin on in ammatory response
To explore the underlying mechanisms of baicalin against pulmonary vascular remodeling, the mRNA levels of interleukin (IL)-1β and IL-6 were analyzed by qRT-PCR, the proteins expression of TNF-α, VCAM-1 and ICAM were detected by western blot. The results showed that the in ammatory factors TNF-α, VCAM-1, ICAM and the ratio of phosphorylated to total NF-κB-p65 levels were obviously increased in lung tissue when the animals were subjected to MCT, but which were signi cantly reduced in baicalin treated group rats as compared with MCT rats (P < 0.05, Fig. 4A-4C).
Effect of baicalin on BMPR2 signaling pathway in vivo BMPR2 signaling playing an important role in the remodeling in PH, in the present study, we detected the protein expression of BMPR2, Smad1/5/8, p-Smsd1/5/8 and ID1 by western blot. Our results showed that an obviously up-regulation of protein expression of BMPR2, Smad 1/5/8, p-Smsd1/5/8 and ID1 in baicalin treated group than those in MCT groups (P < 0.05, Fig. 5).

Effect of baicalin on PAMSC proliferation and migration
The effect of baicalin on proliferation and migration abilities of PAMSC were analyzed in vitro. Brie y, after cells were treated with TNF-α for 48 h and following treated with 100 µg/ml baicalin for 24 h, 48 h and 72 h. MTT assay results exhibited that the cells viability rate was signi cantly increased in TNF-α group as compared with the normal groups. However, when the cells were pretreatment with baicalin, which was signi cantly inhibited (P < 0.05, Fig. 6A).
Transwell assay was performed to observe the effect of baicalin on cells migratory ability. As demonstrated in Fig. 5B, the migratory ability was signi cantly enhanced when the cells were induced by TNF-α for 48 h, but which was obviously inhibited by baicalin (P < 0.05, Fig. 6B and 6C).
Furthermore, western blot results showed that the protein expression of Cyclin D1 was signi cantly increased, but the protein expression of P27 kip1 was signi canlty decreased in MCT group than that in control. However, baicalin administration could signi cantly restore these results than that in MCT rats (P < 0.05, Fig. 6D).

Effect of baicalin on TNF-α induces BMPR2 signaling
To further explore the underlying mechanisms, the proteins expression of BMPR2 signaling were analyzed by immuno uorescence and western blot in vitro. The results showed that the protein expression of BMPR2, ID1 and p-Smad1/5/8 were signi cantly suppressed in TNF-α induced group, however,which were restored when the cells were treated with baicalin (P < 0.05, Fig. 7).

Discussion
Pulmonary hypertension (PH) is a kind of refractory rare lung diseases, distal pulmonary arterial remodeling is the characteristic of it [16][17][18]. The pathogenesis of PH is not clear yet, and no effective therapy is available for it. Previous studies [14,15] suggested the baicalin has the potential to inhibition of vascular remodeling in PH. In the present study, our data con rmed that administration of baicalin could signi cantly reduce RVSP and RV/(LV + S) as compared with the MCT rats, which is considered as a novel potential therapeutic approach for PH.
In ammatory was associated with the pathogenesis of PH [9,19], which play an main role in initiating and maintaining vascular remodeling in PH animal models, and therapeutics targeting of in ammation maybe attenuate the development of PH [20]. Reports showed that TNF-α levels was signi cantly higher in heritable and idiopathic PH cases as compared with healthy people [9,21,22]. The present study showed signi cantly decreased the mRNA and protein levels of TNF-α, IL-1β, IL-6 and NF-κB in baicalin group than that in MCT-induced PH group. These results indicate that baicalin could suppress the in ammatory response in MCT-PH rats. We therefore analysis the protein expressions of the adhesion molecules VCAM-1, ICAM and ratio of phosphorylated to total NF-κB-p65 levels which were also involved in in ammatory processes [23,24]. Collectively, these results showed that baicalin attenuated MCT-induced in ammation as evidenced by decreased expressions of adhesion molecules.
Bone morphogenetic proteins (BMPs) and their receptors were required for PH-induced right ventricular hypertrophy, which playing an important role in the remodeling of pulmonary resistance vessels in the process of PH occurs [8,25,26]. BMPR2 was signi cantly decreased in MCT and chronic hypoxic induced rat PH models [27,28]. BMP signaling through receptor-mediated phosphorylation and p-Smad1/5/8 transcription factors and alterations in gene transcription to regulation the occurrence of pulmonary brosis process. In addition to BMPR2, BMP2, BMP4, BMP6 and BMP9 also have regulatory role for pulmonary vascular cell proliferation [29][30][31][32]. In the present study, we found that the protein expression of BMPR2, Smad1/5/8, p-Smad1/5/8 and ID1 were signi cantly up-regulation by baicalin. Takentogether, these results con rmed that baicalin could signi cantly repair of MCT-induced PH pulmonary vascular remodeling through regulation of the BMP signaling pathways.
Recent study showed that the expression of BMPR2 was inhibited by TNF-α in pulmonary vascular cells [33] and pulmonary artery endothelial cells [34]. Many cytokines involved in the pathogenesis of PH, such as TNF-α, IL-1β, IL-6 and IL-8 are implicated in the pathogenesis of PH, only TNF-α selectively reduced BMPR2 expression in distal PASMCs and PAECs. On the other hand, TNF-α could promote pulmonary vascular remodeling in the setting of BMPR2 de ciency, increase BMP6 expression caused upregulation in transient p-Smad1/5/8 responses in PASMCs [35].
To further explore the underlying mechanism of baicalin for PH. We used TNF-α induced PAMSC injury in vitro in the presence or absence of baicalin. The effect of baicalin on proliferation and migration abilities of PAMSC were also analyzed, the MTT and Transwell results showed that the viability and proliferation rate were signi cantly increased after cells were treated with TNF-α as compared with the normal cells. However, baicalin administration could signi cantly inhibit the cells proliferation and migration. Furthermore, TNF-α induce PAMSC proliferation was also observed by detecting the protein expression of Cyclin D1 and P27, the results showed that the expression of CyclinD1 was signi cantly higher, but P27 was signi cantly lower in baicalin group than that in TNF-α group. More-importantly, in the present study, our results showed that the proteins expression of BMPR2 signaling were signi cantly up-regulation in baicalin administration group than that in TNF-α group. Therefore, our data provide a strong evidence for the mechanism of baicalin inhibition PH vascular remodeling was to through down-regulation TNF-a drives PH by suppressing the BMPR2 signaling.
In summary, the present study demonstrated for the rst time that baicalin attenuated PH pulmonary vascular remodeling through inhibition of in ammatory response, but increased the vascular remodeling associated BMPR2 signaling pathway.   (C)Normalized band intensity quanti cation showing the fold change of the TNF-α, the ratio of P to Total NF-κB-p65 (P/TNF-κB-p65), VCAM-1 and ICAM in each group. the data are present as mean ± SD; *P<0.05 compared with control group;#P<0.05 compared with MCT or saline group. Effect of baicalin on BMPR2 signaling pathway in the lung (A) Western blots analysis the protein expression of bone morphogenetic protein type II receptor (BMPR2), p-Smad1/5/8 and ID1 in lung tissue.

Declarations
(B) Normalized band intensity quanti cation showing the fold change of BMPR2, Smad1/5/8, p-Smad1/5/8 and ID. the data are present as mean ± SD; *P<0.05 compared with control group;#P<0.05 compared with MCT or saline group.