Cardioprotective Effects And Mechanisms Of Allitridum In Anoxia/Reoxygination And Ischemia/Reperfusion Injury In Rats

Part IPreconditioning with Allitridum Protect Myocardium through against apoptosis in Neonatal Rat Anoxia/ Reoxygenation ModelObjectivePrevious studies had shown that allitridum can protect myocardium from ischemia/reperfusion injury. The present study was to investigate whether allitridum has the effect of preconditioning and to evaluate whether allitridum has inhibitory effect on the myocardium apoptosis. MethodsNeonatal SD rats myocytes were cultured, then were randomly divided into four groups: Control (CON) group; Anoxia/Reoxygenation (A/R) group; Anoxia Preconditioning (AP) group and Allitridum pretreatment (A) group. CON group were cultured in normal condition; AP group were pretreated with anoxia for 45min 24h before A/R operation, A group were pretreated with allitridum 24h before A/R operation. Then A/R, AP, A groups were cultured in anoxia condition for 3 hours, then deoxygenated for 1 hours. Malondialdehyde (MDA), Lactate Dehydrogenase (LDH), Superoxide dismutase (SOD) were measured in all groups. The apoptosis index (AI) by TUNEL staining and DNA Fragmentation agarose gel electrophoresis were used todetect myocardium apoptosis in all groups.ResultsA and AP groups resulted in reduction in LDH, MDA release and SOD increase compared with A/R group in response to A/R injury (P<0.01). AI of A/R group was higher than AP, A and CON groups (18.61±2.02% vs. 3.49±0.37%, 5.38±1.32%, 1.22±0.27%, respectively, P<0.01) . The presention of myocardium apoptosis in each group by TUNEL is consistent with DNA Fragmentation agarose gel electrophoresis. The AI of A group and AP group were no significant different. ConclusionsThis study indicates that allitridum has the effect of protecting myocytes against anoxia/reoxygenation injury in cultured neonatal rat myocytes. The effect was through decreasing the myocardial apoptosis probably.Part II Preconditioning with Allitridum protect myocardium in Rats ofischemia/reperfusion model and the mechanismObjectivePrevious studies have shown that allitridum could protect cardiomyocytes from anoxia/reoxygenation injury model of neonatal rat in the first part of our research. In this part the aims were to investigate whether allitridum has the effect of protecting myocardium from ischemia/reperfusion injury in vivo, moreover, to indicate whether the protective effect was through the p38MAPK (mitogen-activated protein kinase p38) pathway. MethodsPentobarbital sodium-anesthetized Sprague-Dawley rats underwent 30 min of left anterior descending (LAD) coronary occlusion and 120 min reperfusion to make ischemia/reperfusion (I/R) injury model in vivo. Sixty-nine rats were divided into five groups randomly: Control (CON), Ischemia/reperfusion injury (I/R) group, Allitridum pretreatment (A) group, Allitridum+SB203580 (A+SB) group and SB203580 (SB) group. CON, I/R and SB groups with saline, A and A+SB groups with allitridum were administrated 24h before operation. CON group underwent only the sham operation; other four groups underwent I/R operation. All but CON groups measured infarcted size (IS/ AAR %). Malondialdehyde (MDA), Creatine kinase isoenzyme-MB (CK-MB ), Superoxide dismutase (SOD) , the apoptosis index (AI) by TUNEL staining, DNA Fragmentation agarose gel electrophoresis, tissue total p38MAPK, phospho-p38 MAPK by western blotting were measured in each group. Results: A group resulted in reduction when compared with I/R, A+SB, SB groups in IS/ AAR% [(21.85 ± 1.49)% vs. (44.65 ± 4.65)%, (41.17 ± 3.16)%, (40.98±7.62)%, respectively P < 0.01], CK-MB [(986.40 ± 94.01) U/L vs. (2044.25 ± 107.28 )U/L, (2004. 75 ± 206. 43)U/L, (2044. 33 ± 144. 13) U/L , respectively P < 0.01)] and MDA [(3.26 ± 0.35) nmol/mg pro vs. (4. 96 ± 0. 46) nmol/mg pro, (4. 79 ± 0. 41) nmol/mg pro, (4. 75 ± 0. 35) nmol/mg pro, respectively P<0. 01)] , then SOD was higher than I/R , A+SB, SB group[(140. 20+12. 89) U/mg pro vs. (73. 16 ± 11. 22)U/mg pro, (73. 93+8. 46) U/mg pro, (74.35+4.66) U/mg pro, respectively P< 0. 01)]. AI of A group was decreased than I/R, A+SB, SB groups [ (6.97 ± 1.23) % vs. (13.99 ± 3.05) %, (13.55 ± 2.54)%, (14. 16 ± 3. 70)%, respectively P< 0. 01], was consistent with the result of DNA Fragmentation agarose gel electrophoresis. The amount of p-p38MAPK of A group was higher than that of I/R, A+SB, SB groups, meanwhile p38MAPK was no significant different in all groups. ConclusionsThese data indicate that allitridum has the effect of protecting myocardium against I/R injury through against apoptosis. The effect against apoptosis was through p38MAPK cell signal pathway. Part IIIAllitridum Attenuates Ischemia/Reperfusion induced Apoptotie Cell Death by Modulating Expression of Bcl-2, Bax and NF-κBObjectiveOur previous studies have shown that allitridum could protect myocardium from anoxia/reoxygenation and ischemia/reperfusion (I/R) injury, moreover, allitridum has the effect of anti-apoptosis in our previous report. The aim of present study was to investigate the role of Bcl-2, Bax, and NF-κB in the anti-apoptosis effect of Allitridum. MethodsPentobarbital sodium-anesthetized Sprague-Dawley (SD) rats underwent 30 min of left anterior descending (LAD) coronary occlusion followed by 120 min of reperfusion. Following the part II of this study, Control (CON, n=6), I/R (I/R, n=6) group, Allitridum pretreatment (A, n=6) groups were chosen to measure the expression of Bcl-2 and Bax protein by immunohistochemistry and the DNA binding activity of NF-κB by Electrophoretic Mobility Assay (EMSA) in each group. ResultsThere was few immunoreactivity of Bcl-2 in CON group. Bcl-2 immunoreactivity was weakly expressed in I/R group and strongly expressed in the peri-necrosis zone of the A group. In contrast, the expression of Bax was significantly increased in I/R group, but was weaker in A group. When expressed with positive expression index (PEI %), compared with I/R group the PEI of Bcl-2 in A group increased significantly (1.30±0.19 vs. 5.47±0.49, P<0.01), and PEI of Bax in A group decreased significantly (6.10±0.90 vs. 1.80±0.22, P<0.01). DNA binding activity of NF-κB was present at low levels in CON group but significantly increased in I/R group. Allitridum markedly inhibited NF-κB DNA binding activity in the ischemia/reperfusion. ConclusionThe protective effect of allitridum was probably achieved through decreasing myocardium apoptosis in I/R injury and modulating expression of Bcl-2, Bax, binding activity of NF-κB. Bcl-2, Bax and the binding activity of NF-κB plays vital role in decreasing myocardium apoptosis of allitridum preconditioning.