| Methods (1) Human renal proximal tubular cell line (HK-2) was used as target cell. (2)The serum of neonates in one day after asphyxia(Apgar Scoring less than 4),whose concentration were 20% (volume fraction),were applied as attacking factor. (3) The experiment were divided as control group,asphyxia group, group of pretreatment with EPO (erythropoietin),group of pretreatment with EPO and 5-HD (5-Hydroxydecanoic acid sodium salt). (4) The following indicators were detected.The changes of morphology were observed under inverted microscope; The expression of Omi/HtrA2, caspase-3 and XIAP in cytoplast were detected by the use of immunohistochemical method; The apoptosis rate of HK-2 cells was detected by flow cytometer; The mitochondria membrane potential(Δψm) was examinted by confocal microscopy; The transposition of Omi/HtrA2 from chondriosome to endochylema by indirect immunofluorescence double staining was observed under confocal microscopy.(5)The statistical significance between treatments was analyzed using one-way ANOVA, where p<0.05 was considered significant.Results (1)Under inverted microscopy,HK-2 cells in control group were significantly increased,sticked to each other tightly and grew very quickly.Their adhesion were better,multy-ang applan,and refraction raised.The form and quantity of HK-2 cell was normal.Compared with control group,the changes in morphology of HK-2 were most serious and obvious in asphyxia group,the cells grew slowly,the mount decreased,form of the cells changed from typical multy-ang applan to off-normal round or ellipse.Refraction rate was decreased,and contour enhanced.The vacuolus,lipid droplet and granulation appeared in the kytoplasm.There was much cell debris in accrescent intercellular space.But compared with asphyxia group,the changes in morphology of HK-2 were obviously improved in the groups of pretreatment with EPO.However, those protective effects can be eliminated by 5-HD. (2) Compared with controls group(22.75±4.03,32.13±5.46,34.88±4.39),the expression of Omi/HtrA2(51.25±3.69) and caspase-3(62.88±4.70) were significantly increased and the expression of XIAP(21.63±2.77) were significantly decreased in asphyxia group; But compared with asphyxia group, the expression of Omi/HtrA2(34.75±3.54) and caspase-3 (47.38±5.18)were significantly decreased and the expression of XIAP(30.50±3.89) were significantly increased in EPO group;but those effects can be also inhibited by 5-HD(P<0.05).(3) Compared with control group(26.40±2.23%),the apoptosis rate of cells were obviously increased in the asphyxia group(68.82±3.63%);Compared with asphyxia group, the apoptosis rate of cells were obviously decreased in the group treated with EPO(37.18±0.57%);but those effects can be also inhibited by 5-HD (40.75±0.70%)(P<0.05)(4) Compared with control group(2.84±0.11),the JC-1 fluorescence ratios (590/530nm) (0.97±0.08)were obviously decreased in the asphyxia group. But compared with asphyxia group,the JC-1 fluorescence ratios in group of pretreatment with EPO(2.67±0.11)were significantly increased. but those effects can be also inhibited by 5-HD (2.18±0.13()P<0.05).(5) Compared with control group(11.50±2.88),the intracelluar translocation rate(%) of Omi/HtrA2 were obviously increased in the asphyxia group(31.38±5.50). But compared with asphyxia group, the intracelluar translocation rate of Omi/HtrA2 in group of pretreatment with EPO were significantly decreased. But those effects can be also inhibited by 5-HD (40.75±0.70%)(P<0.05).Conclusion EPO could relieve the apoptosis of human renal tubular cells induced by postasphyxial-serum in neonates,and it's intracellular signal transduction mechanisn were presumably involved in various stages of mitochondrial.It is concerned with NF-κB,mitochondrial membrane potential and mitochondrial membrane permeability. It is related to the decrease of Omi/HtrA2 translocation and the increase of XIAP.
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