Modulation of NRF-2 Pathway Contributes to the Therapeutic Effects of Boswellia serrata Gum Resin Extract in a Model of Experimental Autoimmune Myocarditis

Abstract

Myocarditis is a clinically dangerous disease that can result in death. Oxidative stress as well as inflammatory and immune responses play important roles in the development of myocarditis. Presently, more research has been carried out on anti-inflammatory treatment using natural compounds. The aim was to evaluate the anti-inflammatory and antioxidant effect of Boswellia gum resin extract in an experimental autoimmune myocarditis (EAM) and the involvement of molecular pathways. Rats were immunized with porcine cardiac myosin to ascertain EAM. The EAM rats were treated orally with Boswellia extract or vehicle for 21 days. EAM caused macroscopic and microscopic alterations with necrosis, inflammatory cell infiltration, fibrosis of the heart tissues, as well as clinical biochemical changes, cytokines release, altered immune response, and oxidative stress. Oral treatment with Boswellia markedly reduced myocardial damage, decreased inflammatory infiltrate, fibrosis, biochemical markers, such as lactate dehydrogenase and the creatine kinase, and heart weight/body weight ratio. In addition, low nitric oxide and malondialdehyde levels together with the upregulation of antioxidant nuclear factor erythroid 2-related factor 2 NRF-2 pathway were observed in EAM rats treated with Boswellia. Thus, Boswellia could be considered as a new natural extract to combat heart pathologies, such as autoimmune myocarditis.

Keywords: Boswellia gum extract; NRF-2; cytokines; immunity; inflammation.

Figure 1

Preliminary data of anti-inflammatory effects of Boswellia extract at different doses 10, 50, and 100 mg/kg. Values are means ± SEM of 6 animals for each group; ## p < 0.01 vs. CAR. # p < 0.05 vs. CAR.

Figure 2

Effect of Boswellia extract on mean blood pressure, heart rate, Hw/Bw and biochemical parameters. Mean blood pressure and heart rate (A,B), serum levels of LDH (C), CK-MB (D), Body weight, Hw and Hw/Bw (E–G) measures at last day of experiment. Boswellia extract administration was able to ameliorate all these parameters. Values = means ± SEM of 6 animals for each set; *** p < 0.001 vs. sham; ^### p < 0.001 vs. EAM ^## p < 0.01 vs. EAM ^# p < 0.05 vs. EAM.

Figure 3

Effect of Boswellia extract on macroscopic and microscopic scores. Macroscopic panel (A), Histological and Masson stainings were evaluated in heart tissues of sham (B,E), EAM + vehicle (C,F) EAM + Boswellia gum extract (D,G). Macroscopic and microscopic scores were shown (H,I). Percentage of Inflammatory cells area/total area (J). Boswellia extract administration was able to reduce macroscopic and microscopic scores and inflammatory infiltrate. Values = means ± SEM of 6 animals for each set. *** p < 0.001 vs. sham; ^### p < 0.001 vs. EAM. ^# p < 0.05 vs. EAM.

Figure 4

Effect of Boswellia extract on α-sma and TGF-β expressions. (A,D) shams, (B,E), EAM + vehicle, (C,F), EAM + Boswellia gum extract. Boswellia extract administration was able to reduce α-sma and TGF-β expressions. The results are expressed as % of positive pixels (G,H). Values = means ± SEM of 6 animals for each set; *** p < 0.001 vs. sham, ^### p < 0.001 vs. EAM. Scale bar: 75 μm. Magnification (40×).

Figure 5

Effect of Boswellia extract on cytokines levels. Serum levels of TNF-α (A), IL-6 (B), IL-17 (C), IL-2 (D), IL-10 (E), IL-4 (F). Boswellia extract administration was able to reduce proinflammatory cytokines and increase anti-inflammatory cytokines. Values = means ± SEM of 6 animals for each set. *** p < 0.001 vs. sham; ^## p < 0.01 vs. EAM. ^### p < 0.001 vs.EAM. ^# p < 0.05 vs. EAM.

Figure 6

Effect of Boswellia extract on CD4 and CD8 expressions. Immunofluorescence for CD4 and CD8 (red) (see yellow arrows) in heart sections of sham animals (A,D), EAM + vehicle (B,E) and EAM + Boswellia gum extract (C,F). Boswellia extract administration was able to reduce CD4 and CD8 expressions.. Scale bar: 75 μm. Magnification (40×).

Figure 7

Effect of Boswellia extract on CD45 and CD11β expressions. Immunofluorescence for CD45 “red” and CD11β “green” (see yellow arrows) in heart sections of sham (A,D), EAM + vehicle (B,E) and EAM + Boswellia gum extract (C,F). Boswellia extract administration was able to reduce CD45 and CD11β expressions. Scale bar: 75 μm. Magnification (40×).

Figure 8

Effect of Boswellia extract on NRF-2 pathway and evaluation of NO release and MDA levels. NRF-2 immunohistochemistry for (A) sham group, (B), EAM + vehicle group, (C) EAM + Boswellia gum extract. The result is expressed as % of positive pixels (D). Scale bar: 75 μm. Magnification (40×). Western blot for NRF-2 (E,E1), HO-1 and SOD (F,F1). Exposed is a characteristic blot of lysates from 6 animals/set, together with a densitometric evaluation for all (E1,F1). Nitrite levels (G), MDA levels (H). Boswellia extract administration was able to activate NRF-2 pathway as well as reduce nitrite and MDA levels. Values = means ± SEM of 6 animals for each set. *** p < 0.001 vs. sham. ** p < 0.01 vs. sham. * p < 0.05 vs. sham ^## p < 0.01 vs. EAM. ^### p < 0.001 vs. EAM.

Figure 9

Summary of the effects of Boswellia gum extract during EAM.