Protective effect of baicalin against pulmonary arterial hypertension vascular remodeling through regulation of TNF-α signaling pathway

Abstract

Pulmonary arterial hypertension (PAH) is a progressive cardiovascular disease with high mortality. However, there were no efficient medical drugs for PAH to enormously improve the survival and quality of life measures. The present study aimed to explore the protective effect of baicalin against experimental PAH in vivo and vitro. All the experimental rats received intraperitoneal injection of monocrotaline (MCT) to induce PAH model. Baicalin was given by intragastric administration from 2 days after MCT injection. Forty animals were randomly divided into four groups: Control, MCT, saline-, and baicalin-treated groups (n = 10 in each). Post-operation, hemodynamic data, and index of right ventricular hypertrophy (RVHI) were recorded to evaluate the inhibition of baicalin on MCT-induced PAH. Furthermore, pulmonary artery smooth muscle cells (PASMCs) model induced by tumor necrosis factor-α (TNF-α) was used to observe the inhibition of vascular cells proliferation in vitro. The results demonstrated that baicalin significantly attenuated MCT-induced right ventricular systolic pressure (RVSP), the index of right ventricular hypertrophy, and vessel wall thickness; inhibit inflammatory and cell proliferation induced by MCT or TNF-α, respectively. In addition, we found that baicalin might protect against experimental PAH via regulating the TNF-α/BMPR2 signaling pathway.

Keywords: BMPR2; Baicalin; PAH; TNF-α; Vascular remodeling.

FIGURE 1

Effect of baicalin on MCT‐induced pulmonary hypertension. A comparison of the HR, SBP, RVSP, and RV/LV+S in each group. (B) A comparison of the medial thickness of the pulmonary arterial walls in each group. (C) Hematoxylin and eosin staining. (D) Massons staining. (E) A comparison of the OD value in each group. The data are present as mean ± SD; *p < .05 and **p < .01 compared with Control group; #p < .05 compared with MCT or Saline. Red bar =100 µm. HR, heart rate; MCT, monocrotaline; OD, optical density calibration; RV/LV+S, the ratio of right ventricular weight to left ventricle plus septum; RVSP, right ventricular systolic pressure; SBP, systemic blood pressure; WA%, the percent of vascular wall area (WA)/total vascular area (TA); WT%, the percent of the vascular wall thickness (WT)/vascular external diameter (ED)

FIGURE 2

Effects of baicalin on MCT‐induced pulmonary artery smooth muscle. (A) Immunohistochemical analysis of the protein expression of α‐smooth muscle actin (a‐SMA). (B) Immunofluorescence analysis of the protein expression of a‐SMA. (C) A comparison of optical density (OD) value by immunohistochemical and immunofluorescence n = 10 rats per group; *p < 0.05 compared with Control group; ^# p < 0.05 compared MCT or Saline group; the data are present as mean ± SD. Red bar =100 µm

FIGURE 3

Effect of baicalin on inflammatory in the lung. Quantitative real‐time polymerase chain reaction analysis of the mRNA level of tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β and IL‐6. (B) Western blots analysis of the protein expression of TNF‐α, phosphorylated Nuclear factor‐κB‐p65 (p‐NF‐κB‐p65), total NF‐κB‐p65, intercellular cell adhesion molecule‐1 (ICAM1), and vascular cell adhesion molecule (VCAM). (C) A comparison of the fold change of the TNF‐α, the ratio of p‐NF‐κB‐p65 to total NF‐κB‐p65, VCAM‐1 and ICAM in each group. n = 10 rats per group; *p < .05 compared with Control group; ^# p < .05 compared MCT or Saline group; The data are present as mean ± SD

FIGURE 4

Effect of baicalin on BMPR2 signaling pathway in the lung. (A) Western blots analysis of the protein expression of bone morphogenetic protein type II receptor (BMPR2), p‐Smad1/5/8, and ID1 in lung tissue. (B) A comparison of the fold change of BMPR2, Smad1/5/8, p‐Smad1/5/8, and ID. n = 10 rats per group;*p < .05 compared with Control group; ^# p < .05 compared MCT or Saline group; The data are present as mean ± SD

FIGURE 5

Effect of baicalin on TNF‐α–induced PASMC. MTT assay. (B) Transwell assay. (C) Comparison of number of cells. (D) The protein expression of Cyclin D1 and p27 analysis by western blot. *p < .05 compared with Control group; ^# p < .05 compared with TNF‐α group; The data are present as mean ± SD. Red bar =100 µm

FIGURE 6

Effect of baicalin on BMPR2 signaling pathway in vitro. The protein expression of BMPR2, Smad1/5/8, p‐Smad1/5/8, and ID1 analysis by western blot. (B) Normalized band intensity quantification showing the fold change of BMPR2, Smad1/5/8, p‐Smad1/5/8, and ID. *p < .05 compared with Control group; ^# p < 0.05 compared with TNF‐α group; The data are present as mean ± SD