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Research Paper

Journal of Pharmacy And Pharmacology

Baicalin attenuates transforming growth factor-β1-induced human pulmonary artery smooth muscle cell proliferation and phenotypic switch by inhibiting hypoxia inducible factor-1α and aryl hydrocarbon receptor expression Shian Huanga, Puwen Chena, Xiaorong Shuib, Yuan Hea, Heyong Wangc, Jing Zhenga,d, Liangqing Zhanga, Jianwen Lib, Yiqiang Xuea, Can Chena and Wei Leia a

Laboratory of Cardiovascular Remodeling and Pharmaceutical Biotechnology, Department of Cardiovascular, The Affiliated Hospital, Guangdong Medical College, Zhanjiang, bVascular Surgery Laboratory, The Affiliated Hospital, Guangdong Medical College, Zhanjiang, cShanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China and dDepartment of Obstetrics and Gynecology, University of Wisconsin–Madison, Madison, WI, USA

Keywords aryl hydrocarbon receptor; baicalin; hypoxia inducible factor-1α; pulmonary arterial smooth muscle cell; transforming growth factor Correspondence Wei Lei, Department of Cardiovascular, The Affiliated Hospital of Guangdong Medical College, 57 Renmin Road, Xiashan, Zhanjiang, Guangdong 524000, China. E-mail: [email protected] Received January 23, 2014 Accepted April 13, 2014 doi: 10.1111/jphp.12273

Abstract Objectives Baicalin, a natural flavone, has antithrombotic, antihyperlipidemic and antiinflammortory activity. It can also inhibit cancer cell proliferation and reduce brain cell apoptosis. This study aimed to elucidate the effect of baicalin on the excessive proliferation of human pulmonary arterial smooth muscle cells (HPASMCs) induced by transforming growth factor-β1 (TGF-β1) and to investigate the roles of hypoxia inducible factor-1α (HIF-1α) and aryl hydrocarbon receptor (AhR) in mediating this TGF-β1-induced excessive proliferation of HPASMCs. Methods TGF-β1-induced proliferation of HPASMCs was assayed using the CCK8 method. The cellular phenotype was identified by immunocytochemical staining. Expression of HIF-1α and AhR mRNA was determined by real-time quantitative PCR. Key findings TGF-β1 promoted significantly HPASMC proliferation (P < 0.05) and induced a phenotypic switch from the contractile to synthetic type. Baicalin inhibited this TGF-β1-induced phenotypic switch and consequently the excessive growth of HPASMCs in a time-dependent and dose-dependent manner (P < 0.05). Furthermore, baicalin attenuated the abnormal proliferation of HPASMCs through suppression of the HIF-1α and AhR pathways. Conclusions Our study shows that baicalin has the potential to be used as a novel drug in the treatment of pulmonary arterial hypertension pathology by antagonizing HIF-1α and AhR expression and subsequently decreasing HPASMC proliferation and the phenotypic switch.

Introduction Pulmonary arterial hypertension (PAH), a severe heart– lung circulation syndrome, is characterized by progressively increasing pulmonary vascular resistance and right ventricular failure.[1] The aetiology of PAH is complex, involving chronic, mild inflammation and the abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs).[2] Quiescent PASMCs normally stay in the contractile phenotype. However, when microenvironmental hypoxia causes endothelium lesions and the stimulation of inflammatory cytokines, PASMCs will switch to an active

synthetic phenotype, leading to uncontrolled proliferation.[3] This phenotypic shift of smooth muscle cells is associated with changes in the expression of differentiation markers, which has been shown to result in decreased abundance of contractile proteins such as α-smooth muscle cell actin (α-SMA) and increased expression of synthetic proteins such as osteopontin (OPN).[4] PAH inflammation is an alternative immune response linked to a shift of arginine metabolism from the nitric oxide synthase (NOS) to the arginase pathway.[5]

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1469–1477

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Baicalin inhibits HPASMCs proliferation

Shian Huang et al.

Transforming growth factor (TGF-β1) is one of the cytokines responsible for this shift[6] and has been shown to play important roles in regulating cell growth, tissue fibrosis and inflammation.[7] For example, TGF-β1 expression is increased in the airway tissues of asthma patients, where it promotes airway remoulding by participating in the inflammation response, inducing tissue fibrogenesis, promoting extracellular matrix protein synthesis and stimulating smooth muscle cell proliferation and hypertrophy. Other studies have revealed the regulatory functions of TGF-β1 on both normal and abnormal cell development,[8] but little is known about the relationship between TGF-β1 and PAH, in particular regarding the effect of TGF-β1 on PASMC growth and pulmonary vascular remodelling. The formation and development of a hypoxic environment in the pulmonary arterial lumen is one of the most critical conditions of PAH pathogenesis. Hypoxia inducible factor-1α (HIF-1α) is the core transcription factor that responds to decreases in available oxygen within the cellular environment, which is closely associated with PAH, especially hypoxic PAH. The target genes of HIF-1α include erythropoietin, vascular endothelial growth factor, inducible NOS and endothelin-1.[9] HIF-1α is also an inflammatory mediator that causes the uncoupling of the endothelial NOS (eNOS) reaction leading to superoxide production.[10] HIF-1α is activated under hypoxic conditions and participates in the regulation of cellular growth by recognizing the receptor HIF-1β.[11] The aryl hydrocarbon receptor (AhR) is a potential competitive transcriptional factor of HIF-1α because its activation and signal transduction depends on the HIF-1β subunit.[12] In the field of toxic stress and immune responses mediated by inflammatory modulators, AhR is linked to alterations in cell proliferation, apoptosis, adipose differentiation, tumour promotion, immune and reproductive function, vascular remodelling and atherosclerosis.[13] Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or inflammatory factors alters the gene expression mediated by toxicity and diseases, and the mechanism of transcriptional activation by TCDD-activated AhR has been extensively characterized.[14] However, the negative regulation of the AhR signalling pathway is poorly understood, although it is also considered a feasible strategy for preventing abnormal cell proliferation and disease occurrence.[15] Taken together, the development of AhR and HIF-1α antagonists appears to be useful for the prevention and treatment of diseases related to hypoxic cell impairment such as PAH. Baicalin (C21H18O11, Figure 1) is one of the main active ingredients extracted from Huangqin (Skullcap), the dried root of the medicinal herb Scutellaria baicalensis. As a natural metabolite with high value and extensive efficacy, baicalin has numerous pharmacological effects and has 1470

O

OH OH

HO O

OH O

O

HO OH O Figure 1

Chemical structure of baicalin.

been used in clinical applications of traditional Chinese medicine for thousands of years.[16] Recent pharmacological studies show that baicalin is a good antithrombotic and antihyperlipidemic and that it reduces inflammation. It has even been used as anti-arrhythmic and antihypertensive drug in clinical trials.[17,18] Baicalin previously inhibited cancer cell proliferation by decreasing the expression of the apoptosis genes bcl-2 and bcl-6 and the proapoptosis genes p53 and bax,[19] as well as blocking the expression of superoxide dismutase and HIF-1α in lung cancer cells.[20] It also reduces brain cell apoptosis in hypoxic-ischaemic damaged brains by inhibiting HIF-1α mRNA expression levels; this effect was proportional to the concentration and time of the drug treatment.[21] Moreover, baicalin is an effective inhibitor of AhR. After the detailed screening of 20 herbal medicines, Scutellariae radix was shown to be responsible for the antagonistic effect of AhR activation against TCDD. Among the major constituents of this herb extract, baicalin was found to effectively block activation of AhR triggered by cigarette smoke, a strong AhR activator.[22] Therefore, baicalin could inhibit PASMC proliferation by down-regulating HIF-1α and AhR expression as a potential antagonist. The purpose of this study was to investigate the following topics: (1) the expression and localization of HIF-1α and AhR in human PASMCs; (2) the regulation mechanism of TGF-β1 on PASMC proliferation; and (3) the effect of baicalin on PASMC proliferation and HIF-1α and AhR expression.

Materials and Methods Reagents Baicalin (purity >99%) was purchased from Sigma Aldrich (St. Louis, MO, USA) and recombinant rat TGF-β1 was from Peprotech (Rocky Hill, NJ, USA). A 40-mM stock solution was prepared by dissolving baicalin in dimethyl sulfoxide. Antibodies against α-SMA, OPN, HIF-1α, AhR, fluorescein isothiocyanate (FITC) and phycoerythrin (PE) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1469–1477

Shian Huang et al.

Cells culture Primary human PASMCs (HPASMCs) were purchased from the European Collection of Cell Cultures (no. 06090734, ECACC, Porton Down, UK) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin according to the supplier’s instructions. Cells at passages 3–8 were used for experiments.

Baicalin inhibits HPASMCs proliferation

Table 1 Primer experiment

sequences

in

the

real-time

quantitative

PCR

Gene

Primer sequence

AhR

5′-ATTGTGCCGAGTCCCATATC-3′ 5′-AAGCAGGCGTGCATTAGACT-3′ 5′-GCAAGACTTTCCTCAGTCGACACA-3′ 5′-GCATCCTGTACTGTCCTGTGGTGA-3′ 5′-GGCACAGTCAAGGCTGAGAATG-3′ 5′-ATGGTGGTGAAGACGCCAGTA-3′

HIF-1α β-actin

AhR, aryl hydrocarbon receptor; HIF-1α, hypoxia inducible factor-1α.

Cells proliferation assays To evaluate the effects of baicalin on HPASMC proliferation, an appropriate proliferation model of HPASMCs based on TGF-β1 induction was first established, including the appropriate time and treatment dosage. Hence, different concentrations of TGF-β1 (0, 1, 10 and 100 μg/l) were incubated with cultured HPASMCs for 24 h, 48 h or 72 h. When this model was established, we seeded HPASMCs in 96-well plates (2 × 104 cells/well) before synchronizing with FBS starvation. The medium was replaced with DMEM supplemented with 0.1% FBS for 24 h to arrest mitosis, and the cells were subsequently incubated with fresh DMEM containing 10% FBS (CK group) or a combination of CK and TGF-β1 with different concentrations of baicalin (0, 5, 10, 20 and 40 μg/l) for 6, 12, 24 and 48 h. Each group was replicated in six separate wells. A CCK8 cell proliferation assay kit (Beyotime, Shanghai, China) was used to measure HPASMC proliferation according to the manufacturer’s instructions, and cell viability and proliferation were determined by reading the absorbance at 450 nm.

Immunocytochemical assay HPASMCs were grown on slides for 24 h and then fixed for immunocytochemical analysis. Slides were incubated with primary antibodies against human α-SMA (1 : 100) and OPN (1 : 100), then with FITC or PE secondary antibodies, respectively, for 2 h to specifically identify contractile and synthetic HPASMC phenotypes. Cellular antibodies against HIF-1α and AhR, conjugated with a PE-secondary antibody, were used for the corresponding target proteins in the same way. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain nuclei as previously described,[23] and cells were then observed under a laser scanning confocal microscope (Leica, TCS SP5 II, Wetzlar, Germany) with a 60× oil objective lens to detect the subcellular distribution of target proteins. The fluorescent signals were collected through the corresponding filters with excitation wavelengths of 488 nm (FITC), 488 nm (DAPI) and 543 nm (PE).

Real-time quantitative PCR detection Total RNA from cultured HPASMCs was extracted using Trizol reagent (Invitrogen, Grand Island, NY, USA), and reverse-transcribed into complementary DNA. The constitutively expressed β-actin gene served as an internal control. mRNA levels of the target genes HIF-1α and AhR were quantified by real-time quantitative PCR (RT-qPCR) using SYBR premix Ex Taq (Takara, Dalian, Liaoning, China). Primer sequences are listed in Table 1, and primers were synthesized by Sangon Biotechnology (Shanghai, China). The amplification reactions were performed using the LightCycler 480 II real-time PCR System (Roche Diagnostics, Penzberg, Germany), and data were collected and analysed using LightCycler 480 software SW 1.5 (Roche Diagnostics).

Data analyses PRISM software ver. 5.0 (GraphPad, La Jolla, CA, USA) and Microsoft Excel 2003 (Microsoft, Redmond, WA, USA) was used for statistical analyses with differences taken to be significant at P < 0.05. Data are shown as means ± standard errors and were analysed using the Kruskal–Wallis test followed by Dunn’s multiple comparison tests, which returned P-values of