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. 2021 Nov 2;16(1):1182-1192.
doi: 10.1515/biol-2021-0116. eCollection 2021.

The molecular mechanisms underlying arecoline-induced cardiac fibrosis in rats

Chang-Wen Ku  1   2 , Cecilia Hsuan Day  3 , Hsiu-Chung Ou  4 , Tsung-Jung Ho  1   2   5 , Ray-Jade Chen  6 , Velmurugan Bharath Kumar  7 , Wen-Yuan Lin  8 , Chih-Yang Huang  9   10   11   12   13
Affiliations

The molecular mechanisms underlying arecoline-induced cardiac fibrosis in rats

Chang-Wen Ku et al. Open Life Sci. .

Abstract

The areca nut is one of the most commonly consumed psychoactive substances worldwide, with an estimated consumption by approximately 10% of the world's population, especially in some regions of South Asia, East Africa, and the tropical Pacific. Arecoline, the major areca nut alkaloid, has been classified as carcinogenic to humans as it adversely affects various organs, including the brain, heart, lungs, gastrointestinal tract, and reproductive organs. Earlier studies have established a link between areca nut chewing and cardiac arrhythmias, and yet research pertaining to the mechanisms underlying cardiotoxicity caused by arecoline is still preliminary. The main purpose of this study is to test the hypothesis that arecoline causes cardiac fibrosis through transforming growth factor-β (TGF-β)/Smad-mediated signaling pathways. Male Wistar rats were injected intraperitoneally with low (5 mg/kg/day) or high (50 mg/kg/day) doses of arecoline for 3 weeks. Results from Masson's trichrome staining indicated that arecoline could induce cardiac fibrosis through collagen accumulation. Western blot analysis showed that TGF-β and p-Smad2/3 protein expression levels were markedly higher in the arecoline-injected rat hearts than in those of the control rats. Moreover, arecoline upregulated other fibrotic-related proteins, including SP1-mediated connective tissue growth factor expression. Tissue-type plasminogen activator and its inhibitor, plasminogen activator inhibitor, and matrix metalloproteinase (MMP) 9 were upregulated, and the inhibitor of MMP9 was downregulated. This study provides novel insight into the molecular mechanisms underlying arecoline-induced cardiac fibrosis. Taken together, the areca nut is a harmful substance, and the detrimental effects of arecoline on the heart are similar to that caused by oral submucous fibrosis.

Keywords: MMP9; Smad; TGF-β; arecoline; cardiac fibrosis.

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Conflict of interest statement

Conflict of interest: The authors state no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of arecoline, the principal alkaloid in the areca nut.
Figure 2
Figure 2
Administration of arecoline to animals.
Figure 3
Figure 3
(a) Pathological changes in the left ventricles of experimental rats. MT staining of cardiac tissues from control, low-dosage arecoline, and high-dosage arecoline rats. Collagen accumulation is shown in blue. The myocardial architecture images were magnified at ×400. The scale bar is 200 µm. (b) Semiquantitative grade morphormetric fibrosis scoring for trichrome slides of left ventricular cardiac section. * Significant difference (P < 0.05).
Figure 4
Figure 4
TGF-β1 and phosphorylated Smad2/3 protein expression levels in arecoline-treated rats. (a) TGF-β1 and p-Smad2/3 protein expression levels were examined by Western blot analysis of left ventricular samples from control, low-dosage, and high-dosage arecoline-treated rats. (b) Data were quantified densitometrically and expressed as mean ± SEM. Protein expression was normalized to β-actin expression. *Significant difference (P < 0.05).
Figure 5
Figure 5
SP1 and CTGF protein expression levels in arecoline-treated rats. (a) SP1 and CTGF protein expression levels were examined by Western blot analysis of left ventricular samples from control, low-dosage, and high-dosage arecoline-treated rats. (b) Data were quantified densitometrically and expressed as mean ± SEM. Protein expression was normalized to β-actin expression. *Significant difference (P < 0.05).
Figure 6
Figure 6
tPA and its inhibitor, PAI protein expression levels in arecoline-treated rats. (a) PAI and tPA protein expression levels were examined by Western blot analysis of left ventricular samples from control, low-dosage, and high-dosage arecoline-treated rats. (b) Data were quantified densitometrically and expressed as mean ± SEM. Protein expression was normalized to β-actin expression. *Significant difference (P < 0.05).
Figure 7
Figure 7
MMP9 and its inhibitor, TIMP2, protein expression levels in arecoline-treated rats. (a) MMP9 and TIMP2 protein expression levels were examined by Western blot analysis of left ventricular samples from control, low-dosage, and high-dosage arecoline-treated rats. (b) Data were quantified densitometrically and expressed as mean ± SEM. Protein expression was normalized to β-actin expression. *Significant difference (P < 0.05).
Figure 8
Figure 8
Schematic showing how arecoline induces cardiac fibrosis through upregulation of the TGF-β1/Smad2/3/CTGF as well as tPA/MMP9 signaling pathways.
See this image and copyright information in PMC

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