| Objective:To study the protective mechanism of growth hormone-releasing peptide-6(GHRP-6) on myocardial cells of rats with congestive heart failure by observation the effects of GHRP-6 on heart functions and PI3K/Akt/Caspase-9 signaling pathway. Methods:There are 140 two/three-months age healthy SD rats, weight(188.17±21.86)g. The rats were divided into 4 groups randomly: Control group(A), Sham group(B), heart failure group(C) and GHRP-6 treated group(D). The left anterior descending coronary artery of rats of C and D groups were ligated to produce heart failure model. For sham group, the operations were applied on the rats but didn’t do ligation. 4 weeks later, D group rats were injected 100μg?kg-1?d-1 GHRP-6 by caudal vein and the other three groups were injected equal volum vehicle for 4 weeks. The heart functions of rats were detected by ECG and type-B ultrasonic. The expression of PI3 K p110α, Akt p-Ser308, Akt p-Thr473, Caspase-9 and Caspase-3 were detected by immunohistochemical method. Flow cytometry was applied to detect the apoptosis of left ventricle myocardial cell. Results:1. Heart functions:(1) Single channel standard postoperative limb lead II electrocardiogram showed: the ECG of A and B group were normal. ST segments of C and D group were decreased significantly with the inferior oblique shape and T wave inversion.. Some of rats in C and D groups shows pathological Q wave. ST segment of D group was decreased closer to normal level, the inversed T wave was less than 0.1 mV. The inversed T wave of C group was larger than 0.1 mV.(2) Type-B ultrasonic showed: The activity of anterior left ventricular wall in C and D groups were decreased. The movements of anterior ventricular wall of C group almost disappeared and left ventricle expanded clearly. Compared to A and B group, the percentage of LVEDD, LVESD, LVEDV, LVESV of C and D groups were increased(P<0.05),LVEF were improved(P<0.05). LVEDD, LVESD, LVEDV and LVESV of D were improved than that of C group, LVEF were increased(P<0.05).2. The results of histology: Under the optical microscope, the nucleus of cardiac myocyte were located in the middle of cell, most of them are spindle-shaped and the longitudinal axis were the same as myofibrils in group A and B groups. Myocardial cell of A and B groups were well arranged, transverse striation and intercalated disk can be seen clearly. In C group, the cardiac myocytes of infarct anterior left ventricular wall were disappeared, inflammatory cells Infiltrated, and the original structure of cardiac myocytes were replaced by proliferated of collagen fibers. The cells in posterior left ventricle and interventricular septum were disorganized and shows hypertrophy, the branches of cell increased, transverse striation and intercalated disk disappeared, there are many fibroplasia in interstitial tissue. After treated by GHRP-6 for 4 weeks, these symptoms were improved in D group.3. The results of immunohistochemistry: The expression of PI3 K p110α in left ventricular posterior wall and interventricular septum of C group is decreased than that of A and B groups(P<0.05). The expression of PI3 K p110α in D group is higher than that of A, B and C groups(P<0.05). Akt p-Thr308 and Akt p-Ser473 in C group were lower than that of in A and B group(P<0.05), and the expression of Akt p-Thr308 and Akt p-Ser473 in D group increased than that of C group(P<0.05), but there are no significant different between D group and A/B groups(P>0.05). The results of caspase-9 immuno-staining shows that C group higher than that of A/B groups(P<0.05) and the expression level in D group lower than that of C group(P<0.05). The same results was observed in the expression of caspase-3(P<0.05).4. The results of Flow cytometry: The apoptosis rate of left ventricular posterior wall and interventricular septum were(5.30±1.13)% in A group,(5.67±0.99)% in B group,(14.81±1.86)% in C group and(10.49±1.38)% in D group. Compare with A/B, the apoptosis rate of C group was increased(P<0.05). There are significant different between C and D group, the apoptosis rate of D group was decreased significantly(P<0.05). Conclusion:GHRP-6 can inhibit the apoptosis of cardiac myocyte via activating PI3K/Akt/Caspase-9/ Caspase-3 signaling pathway. |
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