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. 2021 Oct 26:12:755077.
doi: 10.3389/fphar.2021.755077. eCollection 2021.

Magnolol Attenuates Right Ventricular Hypertrophy and Fibrosis in Hypoxia-Induced Pulmonary Arterial Hypertensive Rats Through Inhibition of the JAK2/STAT3 Signaling Pathway

Minyi Fu  1   2   3 , Fangmei Luo  4 , Eli Wang  1   5 , Yueping Jiang  1   2   3 , Shao Liu  1   2   3 , Jun Peng  5   6 , Bin Liu  1   2   3
Affiliations

Magnolol Attenuates Right Ventricular Hypertrophy and Fibrosis in Hypoxia-Induced Pulmonary Arterial Hypertensive Rats Through Inhibition of the JAK2/STAT3 Signaling Pathway

Minyi Fu et al. Front Pharmacol. .

Abstract

Right ventricular (RV) remodeling is one of the essential pathological features in pulmonary arterial hypertension (PAH). RV hypertrophy or fibrosis are the leading causes of RV remodeling. Magnolol (6, 6', 7, 12-tetramethoxy-2,2'-dimethyl-1-β-berbaman, C18H18O2) is a compound isolated from Magnolia Officinalis. It possesses multiple pharmacological activities, such as anti-oxidation and anti-inflammation. This study aims to evaluate the effects and underlying mechanisms of magnolol on RV remodeling in hypoxia-induced PAH. In vivo, male Sprague Dawley rats were exposed to 10% O2 for 4 weeks to establish an RV remodeling model, which showed hypertrophic and fibrotic features (increases of Fulton index, cellular size, hypertrophic and fibrotic marker expression), accompanied by an elevation in phosphorylation levels of JAK2 and STAT3; these changes were attenuated by treating with magnolol. In vitro, the cultured H9c2 cells or cardiac fibroblasts were exposed to 3% O2 for 48 h to induce hypertrophy or fibrosis, which showed hypertrophic (increases in cellular size as well as the expression of ANP and BNP) or fibrotic features (increases in the expression of collagen Ⅰ, collagen Ⅲ, and α-SMA). Administration of magnolol and TG-101348 or JSI-124 (both JAK2 selective inhibitors) could prevent myocardial hypertrophy and fibrosis, accompanied by the decrease in the phosphorylation level of JAK2 and STAT3. Based on these observations, we conclude that magnolol can attenuate RV hypertrophy and fibrosis in hypoxia-induced PAH rats through a mechanism involving inhibition of the JAK2/STAT3 signaling pathway. Magnolol may possess the potential clinical value for PAH therapy.

Keywords: JAK2; magnolol; myocardial fibrosis; myocardial hypertrophy; right ventricle remodeling; stat3.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Magnolol prevented hypoxia-induced PAH and RV remodeling. (A) Representative images for right ventricular systolic pressure (RVSP) were measured by the right heart catheterization method. (B) The value of RVSP in each group. (C) The ratio of RV weight to left ventricular (LV) plus interventricular septum (IVS). (D) The ratio of RV weight to tibial length. (E) The ratio of RV weight to body weight. All values are presented as mean ± S.D. (n = 10 per group). **p < 0.01 vs. Control; + p < 0.05, ++ p < 0.01 vs. Control; ## p < 0.01 vs. Vehicle.
FIGURE 2
FIGURE 2
Magnolol improved RV dysfunction in hypoxia-induced PAH rats. (A) Representative images of right ventricular wall thickness in diastole and systole period. (B) Representative images of pulsed Doppler from pulmonary artery flow tract recorded in parasternal long axis, the pulmonary artery acceleration time (PAAT, green horizontal line), the pulmonary artery ejection time (PAET, red horizontal line). (C) The value of right ventricular wall thickness during the diastole period (RVDT). (D) The value of right ventricular wall thickness during the systole period (RVST). (E) The ratio of PAAT/PAET. (F) Heart rate. All values are presented as mean ± S.D. (n = 10 per group). **p < 0.01 vs. Control; + p < 0.05 vs. Control; # p < 0.05, ## p < 0.01 vs. Vehicle.
FIGURE 3
FIGURE 3
Magnolol attenuated RV hypertrophy in hypoxia-induced PAH rats. (A) Representative images of HE staining for heart tissues. (B) Representative images of WGA staining for RV tissues. (C) The right ventricular wall thickness (calculated from HE staining). (D) The cross-sectional area of cardiomyocytes (calculated from WGA staining). (E) The perimeter of cardiomyocytes (calculated from WGA staining). (F) The mRNA levels of ANP in RV tissues. (G) The mRNA levels of BNP in RV tissues. **p < 0.01 vs. Control; + p < 0.05, ++ p < 0.01 vs. Control; ## p < 0.01 vs. Vehicle.
FIGURE 4
FIGURE 4
Magnolol attenuated RV fibrosis in hypoxia-induced PAH rats. (A) Representative images of EVG staining for RV tissues. The collagen fibers are red, and the elastic fibers are purple-black. (B) Representative images of Masson staining for RV tissues. The collagen fibers are blue. (C) Representative images of Sirius red staining for RV tissues. The collagen fibers are red, and the background is yellow. (D) The mRNA levels of Collagen Ⅰ in RV tissues. (E) The mRNA levels of Collagen Ⅲ in RV tissues. (F) The mRNA levels of α-SMA in RV tissues. (G) Representative images of Western blot results for Collagen Ⅰ, α-SMA, and α-tubulin. (H, I) The ratio of optical density between Collagen Ⅰ or α-SMA and α-tubulin. All values are presented as mean ± S.D. (n = 6–10 per group). **p < 0.01 vs Control; + p < 0.05, ++ p < 0.01 vs. Control; ## p < 0.01 vs. Vehicle.
FIGURE 5
FIGURE 5
Magnolol blocks hypoxia-induced JAK2 and STAT3 phosphorylation in RV tissues. (A) Results from “SwissTargetPrediction” target prediction database. (B) Representative images of immunofluorescence staining for p-JAK2 in each group. (C) Representative images of Western blot results for p-JAK2, JAK2, p-STAT3, STAT3, and α-tubulin. (D) The ratio of optical density between p-JAK2/JAK2 or p-STAT3/STAT3. All values are presented as mean ± S.D. (n = 6 per group).**p < 0.01 vs. Control; + p < 0.05, ++ p < 0.01 vs. Control; # p < 0.05, ## p < 0.01 vs. Vehicle.
FIGURE 6
FIGURE 6
Magnolol attenuated hypoxia-induced cardiac hypertrophy of H9c2 through inhibition of JAK2/STAT3 signaling pathway. (A) Representative images for H9c2 cell morphology under the fluorescence microscope. The cells were incubated with primary antibodies against α-SMA followed by incubation with the secondary antibody of Alexa Fluor 488-labeled Goat Anti-Mouse IgG (green fluorescence). (B) The cross-sectional area of H9c2 cells. (C) The mRNA levels of ANP in H9c2 cells. (D) The mRNA levels of BNP in H9c2 cells. (E) Representative images of Western blot results for p-JAK2, JAK2, p-STAT3, STAT3, and α-tubulin in H9c2 cells. (F) The ratio of optical density between p-JAK2 and JAK2. (G) The ratio of optical density between p-STAT3 and STAT3. All values are presented as mean ± S.D. (n = 8 per group). **p < 0.01 vs. Control; + p < 0.05, ++ p < 0.01 vs. Control; ## p < 0.01 vs. Vehicle.
FIGURE 7
FIGURE 7
Magnolol attenuated hypoxia-induced myocardial fibrosis of cardiac fibroblasts through inhibition of JAK2/STAT3 signaling pathway. (A) The mRNA levels of Collagen Ⅰ in cardiac fibroblasts. (B) The mRNA levels of Collagen Ⅲ in cardiac fibroblasts. (C) The mRNA levels of α-SMA in cardiac fibroblasts. (D) Representative images of Western blot results for Collagen Ⅰ, α-SMA, p-JAK2, JAK2, p-STAT3, STAT3, and α-tubulin in cardiac fibroblasts. (E) The ratio of optical density between Collagen Ⅰ or α-SMA and α-tubulin. (F) The ratio of optical density between p-JAK2/JAK2 or p-STAT3/STAT3. All values are presented as mean ± S.D. (n = 8–10 per group). **p < 0.01 vs. Control; + p < 0.05, ++ p < 0.01 vs. Control; # p < 0.05, ## p < 0.01 vs. Vehicle.
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