Protective Effects of 2-Methoxyestradiol on Acute Isoproterenol-Induced Cardiac Injury in Rats

Abstract

Myocardial injury (MI) is an important pathological driver of mortality worldwide., and arises as a result of imbalances between myocardial oxygen demand and supply. In MI, oxidative stress often leads to inflammatory changes and apoptosis. Current therapies for MI are known to cause various adverse effects. Consequently, the development of new therapeutic agents with a reduced adverse event profile is necessary. In this regard, 2-methoxyestradiol (2ME), the metabolic end-product of oestradiol, possesses anti-inflammatory and antioxidant properties. The aim of this research is to assess the impact of 2ME on cardiac injury caused by isoproterenol (ISO) in rats. Animals were separated into six groups; controls, and those receiving 2ME (1 mg/kg), ISO (85 mg/kg), ISO + 2ME (0.25 mg/kg), ISO + 2ME (0.5 mg/kg), and ISO + 2ME (1 mg/kg). 2ME significantly attenuated ISO-induced changes in electrocardiographic changes and the cardiac histological pattern. This compound also decreased lactate dehydrogenase activity, creatine kinase myocardial band and troponin levels. The ability of 2ME to act as an antioxidant was shown by a decrease in malondialdehyde concentration, and the restoration of glutathione levels and superoxide dismutase activity. Additionally, 2ME antagonized inflammation and cardiac cell apoptosis, a process determined to be mediated, at least partially, by suppression of Gal-3/TLR4/MyD88/NF-κB signaling pathway. 2ME offers protection against acute ISO-induced MI in rats and offers a novel therapeutic management option.

Keywords: 2-Methoxyestradiol (2ME); Inflammation; Isoproterenol (ISO); Myocardial Injury (MI); Oxidative Stress.

Fig. 1

Effect of 2ME on ISO-induced electrocardiographic changes in rats.

Fig. 2

Effect of 2ME on ISO-induced histological changes in cardiac tissues stained with hematoxylin and eosin stain. Scale bars are 100 μm for the large micrograph and 25 μm for the insert micrograph. ISO; isoproterenol, 2ME; 2-methoxyesterdiol. ISO group showed severe cardiac fibrosis and mononuclear inflammatory cell infiltration (black arrow).

Fig. 3

Effect of 2ME on ISO-induced histopathological changes in cardiac tissues stained by Masson's trichrome (MT) and Sirius red (SR) stains. ISO group shows severe fibrosis (black arrow). Scale bar is 50 μm. ISO; isoproterenol, 2ME; 2-methoxyestradiol. Groups 1 (control) and 2 (2ME 1 mg/kg) show a normal cardiac pattern. Group 3 (ISO) shows abundant fibrosis. Groups 4 (ISO + 2ME 0.25 mg/kg) and 5 (ISO + 2ME 0.5 mg//kg) show moderate to low level of fibrosis. Group 6 (ISO + 2ME 1 mg//kg) evidences the least amount of fibrosis.

Fig. 4

Effect of 2ME on serum cardiac injury markers in ISO-treated rats. (A) LDH activity, (B) CK-MB activity, (C) troponin levels. Data are displayed as mean ± SD (n = 8). ISO; isoproterenol, 2ME; 2-methoxyestradiol. a: significantly different from the control group at p < 0.05. b: significantly different from the 2ME (1 mg/kg) group at p < 0.05. c: significantly different from the ISO group at p < 0.05. d: significantly different from ISO + 2ME (0.25 mg/kg) group at p < 0.05. e: significantly different from ISO + 2ME (0.5 mg/kg) group at p < 0.05.

Fig. 5

Effect of 2ME on oxidative status in ISO-induced cardiotoxicity in rats. (A) MDA, (B) GSH, (C) SOD activity. Data are displayed as mean ± SD (n = 8). ISO; isoproterenol, 2ME; 2-methoxyesterdiol. a: significantly different from the control group at p < 0.05. b: significantly different from the 2ME (1 mg/kg) group at p < 0.05. c: significantly different from the ISO group at p < 0.05. d: significantly different from ISO + 2ME (0.25 mg/kg) group at p < 0.05. e: significantly different from ISO + 2ME (0.5 mg/kg) group at p < 0.05.

Fig. 6

Effect of 2ME on expression of the pro-inflammatory mediators, COX-2, and iNOS, transcription factor, NF-κB, and cytokines, IL-6 and TNF-α, in ISO-induced cardiotoxicity in rats. ISO; isoproterenol, 2ME; 2-methoxyesterdiol. OD; relative density. a: significantly different from the control group at p < 0.05. b: significantly different from the 2ME (1 mg/kg) group at p < 0.05. c: significantly different from the ISO group at p < 0.05. d: significantly different from ISO + 2ME (0.25 mg/kg) group at p < 0.05. e: significantly different from ISO + 2ME (0.5 mg/kg) group at p < 0.05.

Fig. 7

Effect of 2ME on cardiac mRNA expression of (A) Bax, (B) Bcl-2, and (C) caspase-3 in ISO-treated rats. ISO; isoproterenol, 2ME; 2-methoxyestradiol. a: significantly different from the control group at p < 0.05. b: significantly different from the 2ME (1 mg/kg) group at p < 0.05. c: significantly different from the ISO group at p < 0.05. d: significantly different from ISO + 2ME (0.25 mg/kg) group at p < 0.05.

Fig. 8

Effect of 2ME on TLR4, MYD88, Galectin3, and NF-κB protein expression in ISO-induced cardiac injury in rats. (A) Western blot for different experimental groups, group 1 (control), group 2 (2ME 1 mg/kg), group 3 (ISO), group 4 (ISO + 2ME 0.25 mg/kg), group 5 (ISO + 2ME 0.5 mg//kg) and Group 6 (ISO + 2ME 1 mg/kg). The semi-quantification of (B) TLR4, (C) MYD88, (D) Galectin 3, and (E) NF-κB expression is shown as the relative density of protein bands normalized to β-actin and to controls. Data are expressed as mean ± SD (n = 3). a: significantly different from the control group at p < 0.05. b: significantly different from the 2ME (1 mg/kg) group at p < 0.05. c: significantly different from the ISO group at p < 0.05. d: significantly different from ISO + 2ME (0.25 mg/kg) group at p < 0.05.