Abstract
Purpose: To investigate the protective effect of the acetone extract of Rumex japonicas Houtt. (AER) on rat myocardial cells. Methods: R. japonicas was extracted with 75 % aqueous ethanol by reflux to afford total extract (TER). TER was suspended in water and then extracted with acetone to afford acetone fraction of R. japonicas (AER). High performance liquid chromatography (HPLC) combined with standard substances was carried out to analyze the major constituents of AER. Apoptosis in myocardial H9c2 cell line was induced by H 2 O 2 (100 μmol/L). The cells were treated with AER (50, 100 and 200 μg/mL, and cell viability was evaluated by the 3-（4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, while oxidative stress level in H9c2 cells was evaluated by determining levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), creatinine kinase (CK), superoxide dismutase (SOD), and catalase (CAT). Furthermore, apoptotic proteins (caspase-3, Bax and Bcl-2) in H9c2 cells were analyzed by using western blot assay. Results: Results revealed that the main components of AER are aloe-emodin, rhein, emodin, chrysophanol and physcion. AER (50, 100 and 200 μg/mL) inhibited the cell viability reduction of the H9c2 cells induced by H 2 O 2 (p < 0.05, p < 0.01, p < 0.01, respectively). AER (50, 100 and 200 μg/mL) decreased LDH and CK contents of H9c2 cells (p < 0.01). The levels of SOD (p<0.01) and CAT (p < 0.01) were increased by AER treatments (100 and 200 μg/mL); in addition, AER (50, 100 and 200 μg/mL) decreased MDA levels (p < 0.01). Besides, the present results also revealed that AER could down-regulate caspase-3 and Bax, but up-regulated Bcl-2. Conclusion: AER alleviates apoptosis induced by H 2 O 2 in myocardial H9c2 cells via inhibition of oxidative stress and mitochondria-mediated apoptosis. This finding suggests that AER can potentially be developed for the treatment of myocardial apoptosis. Keywords: Rumex japonicas Houtt., Myocardial cells, Apoptosis, H9c2 cell, Oxidative stress
Highlights
Plants in the Rumex genus belong to the family of Polygonaceae, and are annual or perennial herbs distributed throughout the world [1,2]
This study is designed to investigate the protective effects of the acetone extracts of R. japonicas (AER) on myocardial cells using a H2O2-induced myocardial apoptosis H9c2 cell model
AER treatments (50, 100 and 200 μg/mL) significantly inhibited the H2O2 induced cell viability reduction in H9c2 cells (p < 0.05, p < 0.01, p < 0.01, respectively), compared to control group. These results indicated that AER could enhance the cell viability of the H2O2 stimulated myocardial cell
Summary
Plants in the Rumex genus belong to the family of Polygonaceae, and are annual or perennial herbs distributed throughout the world [1,2]. In Chinese folk medicine, the whole herb of R. japonicas is commonly used as an ingredient of prescriptions for treating heart diseases based on the function of promoting blood circulation for removing blood stasis [2,12]. IP cell lysis buffer, BCA protein assay kit, and Caspase-3, Bax and Bcl-2 primary antibodies were purchased from the Beyotime Co. The dried whole herb of R. japonicas (purchased from the Tongrentang traditional Chinese medicine drug store, Tianjing, China) was powdered and extracted with 75 % aqueous ethanol by reflux (3 times, each for 2 h). After 24 h treatment, MTT assay was carried out according to the previous report [15] and absorbance (A) was detected at 570 nm using a 96-well plate reader (BioTek Instruments, Inc, Burlington, VT, USA). Total proteins were extracted with IP cell lysis buffer, and protein concentration was determined using the BCA protein assay kit. Thereafter, protein bands were quantitated using a Bio-Rad Chemi Doc XRS gel imaging system (Hercules, CA, USA)
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