Melissa officinalis L. Supplementation Provides Cardioprotection in a Rat Model of Experimental Autoimmune Myocarditis

Abstract

Due to existing evidence regarding antioxidant and anti-inflammatory effects of Melissa officinalis extracts (MOEs), this study was aimed at investigating the potential of ethanolic MOE to prevent the development of myocarditis and its ability to ameliorate the severity of experimental autoimmune myocarditis (EAM) by investigating MOE effects on in vivo cardiac function, structure, morphology, and oxidative stress parameters. A total of 50 7-week-old male Dark Agouti rats were enrolled in the study and randomly allocated into the following groups: CTRL, nontreated healthy rats; EAM, nontreated rats with EAM; MOE50, MOE100, and MOE200, rats with EAM treated with either 50, 100, or 200 mg/kg of MOE for 3 weeks per os. Myocarditis was induced by immunization of the rats with porcine myocardial myosin (0.5 mg) emulsion on day 0. Cardiac function and dimensions of the left ventricle (LV) were assessed via echocardiography. Additionally, the blood pressure and heart rate were measured. On day 21, rats were sacrificed and the hearts were isolated for further histopathological analyses (H/E and Picrosirius red staining). The blood samples were collected to determine oxidative stress parameters. The EAM group characteristically showed greater LV wall thickness and lower ejection fraction (50.33 ± 7.94% vs. 84.81 ± 7.74%) and fractional shortening compared to CTRL (p < 0.05). MOE significantly improved echocardiographic parameters (EF in MOE200 81.44 ± 5.51%) and also reduced inflammatory infiltrate (by 88.46%; p < 0.001) and collagen content (by 76.39%; p < 0.001) in the heart tissues, especially in the MOE200 group compared to the EAM group. In addition, MOEs induced a significant decrease of prooxidants production (O₂ ^-, H₂O₂, and TBARS) and improved antioxidant defense system via increase in GSH, SOD, and CAT compared to EAM, with medium and high dose being more effective than low dose (p < 0.05). The present study suggests that ethanolic MOEs, especially in a 200 mg/kg dose, improve cardiac function and myocardial architecture, possibly via oxidative stress mitigation, thus preventing heart remodeling, development of dilated cardiomyopathy, and subsequent heart failure connected with EAM. MOEs might be considered as a potentially helpful adjuvant therapy in patients with autoimmune myocarditis.

Figure 1

Representative M-mode echocardiograms. (a) CTRL, (b) EAM, (c) MOE50, (d) MOE100, and (e) MOE200. CTRL: control group; EAM: rats with experimental autoimmune myocarditis; MOE50, MOE100, and MOE200: groups of rats treated with either 50, 100, or 200 mg/kg of M. officinalis extract.

Figure 2

Effects of MOE on prooxidant parameters. (a) Superoxide anion radical (O₂⁻), (b) hydrogen peroxide (H₂O₂), (c) nitrites (NO₂⁻), and (d) index of lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS). CTRL: control group; EAM: rats with experimental autoimmune myocarditis; MOE50, MOE100, and MOE200: groups of rats treated with either 50, 100, or 200 mg/kg of Melissa officinalis extract. Data are presented as means ± standard deviation. Statistical significance at the level p < 0.05: A, compared to CTRL; B, compared to EAM; C, compared to MOE50; D, compared to MOE100; and E, compared to MOE200.

Figure 3

Effects of MOE on antioxidant parameters. (a) Superoxide dismutase (SOD), (b) catalase (CAT), and (c) reduced glutathione (GSH). CTRL: control group; EAM: rats with experimental autoimmune myocarditis; MOE50, MOE100, and MOE200: groups of rats treated with either 50, 100, or 200 mg/kg of Melissa officinalis extract. Data are presented as means ± standard deviation. Statistical significance at the level p < 0.05: A, compared to CTRL; B, compared to EAM; C, compared to MOE50; D, compared to MOE100; and E, compared to MOE200.

Figure 4

Representative heart tissue sections of H/E staining. Magnification 20x scale bar = 50 μm. (a) CTRL: control group; (b) EAM: experimental autoimmune myocarditis group; (c) MOE50: rats with EAM treated with M. officinalis extract in 50 mg/kg; (d) MOE100: rats with EAM treated with M. officinalis extract in 100 mg/kg; and (e) MOE200: rats with EAM treated with M. officinalis extract in 200 mg/kg. (f) Effects of MOE on heart inflammatory infiltrate density. Data are presented as means ± standard deviation. Statistical significance at the level p < 0.05: A, compared to CTRL; B, compared to EAM; C, compared to MOE50; D, compared to MOE100; and E, compared to MOE200.

Figure 5

Representative heart tissue sections of Picrosirius red staining. Magnification 40x scale bar = 25 μm. (a) CTRL: control group; (b) EAM: experimental autoimmune myocarditis group; (c) MOE50: rats with EAM treated with M. officinalis extract in 50 mg/kg; (d) MOE100: rats with EAM treated with M. officinalis extract in 100 mg/kg; and (e) MOE200: rats with EAM treated with M. officinalis extract in 200 mg/kg. (f) Effects of MOE on collagen content in heart tissue. Data are presented as means ± standard deviation. Statistical significance at the level p < 0.05: A, compared to CTRL; B, compared to EAM; C, compared to MOE50; D, compared to MOE100; and E, compared to MOE200.

Figure 6

Effects of MOE treatment on morphometric parameters. CTRL: control group; EAM: experimental autoimmune myocarditis group; MOE50, MOE100, and MOE200: rats with EAM treated with M. officinalis extract in either 50 mg/kg, 100 mg/kg, or 200 mg/kg. Data are presented as means ± standard deviation. Statistical significance at the level p < 0.05: A, compared to CTRL; B, compared to EAM; C, compared to MOE50; D, compared to MOE100; and E, compared to MOE200.