Fig 2: Cardiomyocyte injury in T. cruzi-infected rhesus monkeys.Cardiomyocyte damage was assessed by immunohistochemical detection of Cx43 in the myocardium of the left ventricle and CK-MB activity levels in the serum of noninfected and chronically T. cruzi-infected rhesus monkeys. (A) Photomicrograph of myocardium section of the left ventricle of the noninfected monkey #94 showing normal pattern of Cx43 expression in intercalated discs. (B) Photomicrograph of myocardium section of the left ventricle of the T. cruzi-infected monkey #64 (23 ypi) showing normal aspect. (C) Photomicrograph of left ventricle section of the T. cruzi-infected monkey #99 (20 ypi) showing normal Cx43 pattern. (D) Photomicrograph of section of left ventricle of the T. cruzi-infected monkey #90 (20 ypi) revealing Cx43 loss in myocardial area lacking inflammation. (E–F). Photomicrographs of left ventricle section of the cardiopatic T. cruzi-infected monkey #95 (20 ypi) showing Cx43 loss in area with (E) intense diffuse inflammation and (F) the substitution of cardiomyocytes by mesenchymal cells. (G) Frequency of stained Cx43 area in heart sections of noninfected and chronically T. cruzi-infected monkeys (20–23 ypi). (H) Detection of CK-MB activity in the serum of noninfected and chronically T. cruzi-infected monkeys (20–23 ypi). (I) Correlation between the number of iNOS/NOS2+ cells in heart tissue and CK-MB activity levels in serum of rhesus monkeys. Bar = 100 µm.

Fig 3: Effect of canthinone alkaloids 46 (a) and 57 (b) on the high expression of iNOS and COX-2 proteins. RAW 264.7 cells were treated by LPS (1 μg/ml) with or without canthinone alkaloids (12.5, 25, and 50 μM) for 24 h. The expressions of iNOS and COX-2 proteins were determined by Western blot analysis. Data were normalized on the basis of β-actin levels. Values were expressed as mean ± SD (n = 3). ##p < 0.01 vs. the untreated group. **p < 0.01 vs. the LPS treatment group

Fig 4: Effect of E2 on mRNA expression of Pd-l1, iNos, and Nrf2-mediated anti-oxidant enzyme genes. (A–H) mRNA expression of Pd-l1 (A, B), iNos (C, D), Ho-1 (E, F), and Nqo1 (G, H) in WT and Nrf2 KO male (A, C, E, G) and female (B, D, F, H) mice. Gapdh was used as an internal control. The P-values obtained from the Kruskal–Wallis test is shown in the figure; *P < 0.05 for comparison between two groups (Mann–Whitney test); # P < 0.05 for comparison between males and females (Mann–Whitney test). WT, wild-type; KO, knockout; AOM, azoxymethane; DSS, dextran sodium sulfate; Pd-l1, programmed death-ligand 1; iNos, inducible nitric oxide synthase; Ho-1, heme oxygenase 1; Nqo1, NAD(P)H dehydrogenase (quinone) 1; Gapdh, Glyceraldehyde 3-phosphate dehydrogenase.

Fig 5: iNOS/NOS2+ cells in the myocardium and NO in the serum of T. cruzi-infected rhesus monkeys.The presence of iNOS/NOS2+ cells in the myocardium of the left ventricles was immunohistochemically detected and NO concentration was evaluated by a Griess-based method. Photomicrograph of myocardium section of the left ventricle (A) and (B) of the noninfected monkey #81, which showed a few iNOS/NOS2+ cells. Photomicrograph of myocardioum section of the left ventricle (C) and (D) of the T. cruzi-infected monkey #95 (20 ypi). (E) Number of iNOS/NOS2+ cells in 100 microscopic fields in heart sections. (F) Concentration of NO in the serum of noninfected controls and chronically (20–23 ypi) T. cruzi-infected monkeys. (G) Concentration of NO in the serum of monkey #95 prior to infection (day 0), during the acute phase when parasitemia was positive (56 dpi) and negative (163 dpi) and during the chronic phase (16 ypi and 20 ypi). Bar = 100 µm in (A) and (C); Bar = 25 µm in (B) and (D).