Luteolin limits infarct size and improves cardiac function after myocardium ischemia/reperfusion injury in diabetic rats

Abstract

Background: The present study was to investigate the effects and mechanism of Luteolin on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in diabetic rats with myocardial ischemia/reperfusion (I/R) injury.

Methodology/principal findings: Diabetic rats underwent 30 minutes of ischemia followed by 3 h of reperfusion. Animals were pretreated with or without Luteolin before coronary artery ligation. The severity of myocardial I/R induced LDH release, arrhythmia, infarct size, cardiac function impairment, cardiomyocyte apoptosis were compared. Western blot analysis was performed to elucidate the target proteins of Luteolin. The inflammatory cytokine production were also examined in ischemic myocardium underwent I/R injury. Our results revealed that Luteolin administration significantly reduced LDH release, decreased the incidence of arrhythmia, attenuated myocardial infarct size, enhanced left ventricular ejection fraction and decreased myocardial apoptotic death compared with I/R group. Western blot analysis showed that Luteolin treatment up-regulated anti-apoptotic proteins FGFR2 and LIF expression, increased BAD phosphorylation while decreased the ratio of Bax to Bcl-2. Luteolin treatment also inhibited MPO expression and inflammatory cytokine production including IL-6, IL-1a and TNF-a. Moreover, co-administration of wortmannin and Luteolin abolished the beneficial effects of Luteolin.

Conclusions/significance: This study indicates that Luteolin preserves cardiac function, reduces infarct size and cardiomyocyte apoptotic rate after I/R injury in diabetic rats. Luteolin exerts its action by up-regulating of anti-apoptotic proteins FGFR2 and LIF expression, activating PI3K/Akt pathway while increasing BAD phosphorylation and decreasing ratio of Bax to Bcl-2.

Figure 1. Luteolin reduces LDH release and I/R induced arrhythmia in diabetic rats.

Diabetes increased LDH release compared with the non-diabetic group. Luteolin reduced the LDH release after I/R injury in diabetic mice. The PI3K inhibitor wortmannin pretreatment statistically increased the release of LDH compared with the Luteolin group (a). Luteolin decreased the incidence of PVB, VT and VF attacks as compared with the I/R group. Wortmannin abolished the anti-arrhythmia effects of Luteolin (b). PVB: premature ventricular beats; VT: ventricular tachycardia; VF, ventricular fibrillation. The columns and errors bars represent means and SD. * p<0.05 vs Non-DM, ^# p<0.05 vs Sham, ^§ p<0.05 vs Luteolin.

Figure 2. Luteolin decreases infarct size in diabetic rats subjected to I/R injury.

Representative illustrations of infarct size as stained by Evans Blue and TTC (a). Luteolin decreased infarct size 3 h after I/R injury compared with the I/R group and the wortmannin group (b). The risk of area had no statistical difference between the groups (c). The columns and errors bars represent means and SD. * p<0.05 vs Non-DM, ^# p<0.05 vs Sham, ^§ p<0.05 vs Luteolin.

Figure 3. Luteolin enhances left ventricular function evaluated by hemodynamic measurements and echocardiograpy.

Hemodynamic measurements indicated that diabetes decreased the ± LV dp/dt max compared with the non-diabetic group. I/R induced significant decrease of ± LV dp/dt max in diabetic rats. Luteolin enhanced the ± LV dp/dt max compared with the I/R group. The PI3K inhibitor wortmannin abolished the effects of Luteolin on the ± LV dp/dt max (a, b). Luteolin administration significantly enhanced LVEF as compared with the I/R group and the wortmannin group (c). Luteolin significantly inhibited the increase of LVESV and LVEDV compared with the I/R group and the wortmannin group (d, e). LVEF: Left ventricular ejection fraction; LVESV: Left ventricular end-systolic volume; LVEDV: Left ventricular end-diastolic volume. The columns and errors bars represent means and SD. * p<0.05 vs Non-DM, ^# p<0.05 vs Sham, ^§ p<0.05 vs Luteolin.

Figure 4. Luteolin decreases cardiomyocytes apoptosis in diabetic rats with I/R Injury.

Representative photomicrograph showed that TUNEL-positive cardiomyocytes were more frequently observed in the I/R group and the wortmannin group compared with the Luteolin group (a). The apoptotic rate was higher in the diabetic group compared with the diabetic group. The number of TUNEL-positive cardiomyocytes (in green, DAPI in blue) was significantly reduced in the Luteolin group than in the I/R group and the wortmannin group (b). Luteolin treatment also significantly decreased caspase-3 activity compared with the I/R group and the wortmannin group (c). Luteolin decreased caspase-3 and cleaved caspase-3 expression as compared with the I/R group and the wortmannin group (d, e).Scale bar: 25 ìm. The columns and errors bars represent means and SD. * p<0.05 vs Non-DM, ^# p<0.05 vs Sham, ^§ p<0.05 vs Luteolin.

Figure 5. Western blot analysis.

Western blot analysis revealed that Luteolin treatment increased the expression of antiapoptotic proteins FGFR2 and LIF as compared with the I/R group and wortmannin group (a, b). Luteolin also enhanced phosphorylation of Akt and BAD, increased Bax expression while decreased Bcl-2 expression resulted in decreased Bax/Bcl-2 ratio in cardiac tissue that were exposed to I/R injury (c, d, e). The PI3K inhibitor wortmannin abolished the effects of Luteolin on antiapoptotic proteins expression. The columns and errors bars represent means and SD. * p<0.05 vs Non-DM, ^# p<0.05 vs Sham, ^§ p<0.05 vs Luteolin.

Figure 6. Luteolin alleviates leukocyte infiltration and reduces cytokine levels after I/R injury in diabetic rats.

Luteolin reduced the MPO activity compared with the I/R group and the wortmannin group (a). Luteolin reduced the levels of IL-6, IL-1α and TNF-α production compared with the I/R group and the wortmannin group (b, c, d). The columns and errors bars represent means and SD. * p<0.05 vs Non-DM, ^# p<0.05 vs Sham, ^§ p<0.05 vs Luteolin.