| ObjectivesNeonatal asphyxia may induce multi-organ dysfunction (MODS) if resuscitation is delayed. But after resuscitation, reperfusion injury may exacerbate organ damages. Brain and heart are two organs which are more sensitive to hypoxic ischemic damage than the other organs. Many pathological factors are involved in the process of hypoxic ischemic/ reperfusion myocardial injury. Mitochondria are the powerhouse providing over 90% of adenosine triphosphate (ATP) through oxidative phosphorylation for the survival of the cell. The mitochondrial injury may take part in the process of myocardial necrosis and apoptosis. Recent researches had found that opening of the mitochondrial permeability transition pore (MPTP) might be the key event of cell death and MPTP could be influenced by the opening of mitochondrial ATP-sensitive K+ channels (mitoKATP). But the exact mechanisms of the interaction of MPTP and mitoKATP are still unclear.This study was designed to explore the mechanisms of protective effects of Diazoxide, a mitoKATP opener (KCO), on mitochondrial functions through detection of the opening of MPTP, mitochondrial membrane potentials (ψm) and mitochondrial reactive oxygen species (ROS) in hypoxic ischemic myocardial damage model induced by asphyxia in neonatal SD rats. Through protection of mitochondrial functions, Diazoxide may alleviate myocardial injury after asphyxia, which may decrease the morbidity and mortality and improve the prognosis of neonatal asphyxia. This research provides a new strategy of treatment to the myocardial injury after neonatal asphyxia.MethodsCesarean sections were undertaken in female SD rats at the 21st day after pregnancy and the neonatal rats were used in this research. The pregnant rats were divided randomly into two groups, normal operation and clamping group. The uterine arteries were isolated but not clamped in normal operation group and the pups of this group were recorded as control group. The uterine arteries were isolated and clamped for 30 minutes followed by releasing for 1 hour in the rats at the clamping group and the survivors were divided randomly into four groups, asphyxia, Diazoxide (Dia), Glibenclamide (Gli) and solvent (NaOH) group. No drugs were used in control and asphyxia groups. Diazoxide was injected intraperitoneally (i.p.) in dose of 3mg/kg to the neonatal rats in Diazoxide group. Diazoxide (3mg/kg) and Glibenclamide (300 g/kg) were injected i.p. to the rats in Glibenclamide group. 0.5ml NaOH (0.1mM) was injected i.p. to the rats in solvent group. There were 30 neonatal rats in each group and all the neonatal rats were sacrificed 24h after birth. In each group, 10 rats were used for collection of heart blood samples and isolation of myocardial mitochondria, 10 rats were used for TTC staining of myocardial tissues and the other 10 rats were used for preparation of paraffin section and ultrathin section of myocardial tissues. Serum cardiac troponin I (cTnI) levels were detected by ELISA. Cardiac apoptosis inducing factors (AIF) were detected by immunohistochemical staining. The ultrastructures of myocardial cells were observed by transmission electron microscope. The opening of MPTP, mitochondrialψm and ROS was detected by fluorospectrophotometry. The results were recorded as mean±SD ( x±s) and analyzed by SPSS for Windows 13.0 software.ResultsSerum cTnI levels were 0.08±0.04, 0.40±0.29, 0.10±0.04, 0.33±0.17 and 0.34±0.20( g/L)in control, asphyxia, Diazoxide, Glibenclamide and solvent group respectively (P<0.01). It was increased significantly in asphyxia group compared with control group. After treatment of Diazoxide, cTnI levels were decreased. The myocardial ischemic areas in control, asphyxia, Diazoxide, Glibenclamide and solvent group were 8.01±3.48, 42.50±15.90, 14.79±3.98, 31.51±20.86 and 28.37±14.36(%)respectively (P<0.01) detected by TTC staining. The myocardial ischemic areas in asphyxia group were higher than those in control group. After treatment of Diazoxide, the myocardial ischemic areas were decreased, but still higher than those in control group. In HE staining slices, the myocardial cells in asphyxia group were disarranged and edematous. Compared with asphyxia group, the array of the myocardial cells was improved and the cellular edema was abated. Observed by the electron microscope, there were karyopyknosis, caryorrhexis and mitochondrial vacuolar degenerations and ruptures in myocardial tissues from asphyxia group. In Diazoxide group, the morphology of myocardial mitochondria and nuclei was improved, but those improvements were not seen in Glibenclamide and solvent group. The AIF-staining positive cells in myocardial tissue slices were 9.70±3.06, 77.50±11.81, 44.60±17.49, 70.70±14.36 and 73.60±15.19(%)in control, asphyxia, Diazoxide, Glibenclamide and solvent group respectively (P<0.01). It was increased significantly in asphyxia group compared with control group and it was decreased after Diazoxide treatment. In control, asphyxia, Diazoxide, Glibenclamide and solvent group, the opening degrees of MPTP were 118.10±19.10, 79.40±10.57, 106.40±14.61, 72.50±11.21 and 76.20±3.79 (RFU) (P<0.01), the mitochondrialψm were 1.61±0.08, 2.01±0.09, 1.86±0.15, 1.96±0.27 and 2.10±0.29 (RFU) (P<0.01) and ROS were 237.10±53.33, 875.30±206.62, 308.50±103.12, 787.40±261.35 and 892.00±151.45 (RFU) (P<0.01) respectively. The opening degrees of MPTP were increased, the mitochondrial membrane potentials were decreased and mitochondrial ROS were increased in asphyxia group compared with control group. After treatment of Diazoxide, the opening degrees of MPTP were decreased and the mitochondrial ROS were decreased. The correlation coefficient of serum level of cTnI and opening degree of myocardial MPTP was -0.384 (P<0.01) and the correlation coefficient of expression value of AIF and the opening degree of myocardial MPTP was -0.725 (P<0.01) from all groups. As lower RFU indicated higher opening degree of MPTP, the serum level of cTnI and expression value of AIF was positive correlated with the opening degree of MPTP.ConclusionsThere are myocardial ischemia and necrosis, mitochondrial vacuolar degeneration and high levels of serum cTnI and myocardial AIF expressions in neonatal rats after asphyxia, which indicates that asphyxia, may cause myocardial ischemic injury. Opening of myocardial MPTP; loss of mitochondrial membrane potentials and accumulation of ROS, which may induce releasing of AIF, are the main causes of myocardial injury after asphyxia in neonatal rats. It highlights that higher degree of the opening of myocardial MPTP might cause more serious myocardial injury.After treatment of Diazoxide, the serum levels of cTnI are decreased, myocardial ischemic areas are lessened, shapes of myocardial mitochondria are improved and the expressions of myocardial AIF are diminished. It indicates that Diazoxide, a mitoKATP channel opener, could protect myocardial mitochondrial functions after myocardial hypoxic ischemic damage induced by asphyxia.The protective effects of Diazoxide on the myocardial mitochondria in neonatal rats after asphyxia, including inhibition of opening of MPTP and diminution of ROS releasing, can be counteracted by Glibenclamide, a mitoKATP channel inhibitor, which indicates that those protective effects are developed through opening of mitoKATP channel. The opening of mitoKATP channel might inhibit the opening of MPTP directly or through decreasing of ROS releasing. The exact mechanisms still need further researches. |
