Azathioprine pretreatment ameliorates myocardial ischaemia reperfusion injury in diabetic rats by reducing oxidative stress, apoptosis, and inflammation

Figures

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  FIGURE 1. General characteristics. (A) Measurement of body weight and (B) blood glucose before and after the establishment of the DM model, 5 days after administration of AZA and after the establishment of the I/R model. (C) Determination of serum ALT and (D) AST levels in the sham, control +I/R, DM +I/R and DM +I/R + AZA groups. *p < 0.05; ****p < 0.0001 and ns: not significant. ALT, alanine aminotransferase; AZA, azathioprine; DM, diabetes mellitus; I/R, ischaemia reperfusion

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  FIGURE 2. The effects of AZA pretreatment on ECG changes for diabetic rats following I/R. (A) Sham group; (B) control +I/R group; (C) DM +I/R group; (D) DM +I/R + AZA group. AZA, azathioprine; DM, diabetes mellitus; ECG, electrocardiogram; I/R, ischaemia reperfusion

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  FIGURE 3. The effects of AZA pretreatment on myocardial infarction size and myocardial injury in diabetic rats following I/R. (A) Representative images of heart sections by TTC staining. Bar = 2 mm. (B) Quantitative results of infarct size. (C) H&E staining of heart sections. Bar = 20 μm. ****p < 0.0001. AZA, azathioprine; I/R, ischaemia reperfusion; TTC, 2,3,5‐Triphenyltetrazolium chloride

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  FIGURE 4. The effects of AZA pretreatment on serum CK‐MB, MDA, MPO and SOD levels in diabetic rats following I/R. Assessment of (A) serum CK‐MB, (B) MDA, (C) MPO and (D) SOD levels by ELISA. *p < 0.05; **p < 0.01 and ns: not significant. AZA, azathioprine; CK‐MB, creatine kinase isoenzyme; I/R, ischaemia reperfusion; MDA, malonaldehyde; MPO, myeloperoxidase; SOD, superoxide dismutase

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  FIGURE 5. Western blot for the effects of AZA pretreatment on apoptosis and inflammation in diabetic heart under I/R. (A) Representative images. Determination of (B) Caspase8, (C) Caspase3, (D) BAX, (E) Bcl‐2, (F) Bcl‐2/Bax ratio, (G) TLR4 and (H) TNF‐α expression in heart tissues of the sham, control +I/R, DM +I/R and DM +I/R + AZA groups. ***p < 0.001 and ****p < 0.0001. AZA, azathioprine; DM, diabetes mellitus; I/R, ischaemia reperfusion

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  FIGURE 6. Immunohistochemistry for the effects of AZA pretreatment on apoptosis and inflammation in diabetic rats following I/R. (A) Representative images of immunohistochemistry. Bar = 20 μm. Assessment of (B) Caspase3, (C) Caspase8, (D) BAX, (E) Bcl‐2, (F) TLR4 and (G) TNF‐α expression in heart tissues of the sham, control +I/R, DM +I/R and DM +I/R + AZA groups. **p < 0.01; ***p < 0.001 and ****p < 0.0001. AZA, azathioprine; DM, diabetes mellitus; I/R, ischaemia reperfusion

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  FIGURE 7. The effects of AZA pretreatment on cardiac AKT1/GSK3β in diabetic rats following I/R. (A) Representative images of western blot. Evaluation of (B) AKT1, (C) p‐AKT1, (D) GSK3β and (E) p‐GSK3β expression in heart tissues of the sham, control +I/R, DM +I/R and DM +I/R + AZA groups. (F) Immunohistochemistry results of heart sections. Bar = 20 μm. Determination of (G) AKT1, (H) p‐AKT1, (I) GSK3β and (J) p‐GSK3β expression in heart tissues of each group. **p < 0.01; ***p < 0.001 and ****p < 0.0001. AZA, azathioprine; DM, diabetes mellitus; I/R, ischaemia reperfusion

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  FIGURE 8. Immunofluorescence for the effects of AZA pretreatment on cardiac Caspase8, GSK3β and p‐GSK3β expression in diabetic rats following I/R. (A) Immunofluorescence of heart sections. Detection of cardiac (B) Caspase8, (C) GSK3β and (D) p‐GSK3β expression in the sham, control +I/R, DM +I/R and DM +I/R + AZA groups. **p < 0.01; ***p < 0.001 and ****p < 0.0001. AZA, azathioprine; DM, diabetes mellitus; I/R, ischaemia reperfusion