| Objective To investigate the protective role and it's mechanism of recombinant human erythropoietin (rhEPO) in relieving the injury of renal tubular cells (HK-2) induced by postasphyxial-serum of neonate.Methods (1) Human renal proximal tubular cell line (HK-2 cells) were 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) Firstly,the experiment was designed as control group and asphyxia group to research whether the morphological changes of mitochondria were related with the injury of renal tubular cells (HK-2) induced by postasphyxial-serum in neonate. Secondly,the experiment was designed as control group,asphyxia group and the group treated with rhEPO, which were investigated the protective role and it's mechanism of rhEPO in relieving the injury of renal tubular cells (HK-2) induced by postasphyxial-serum of neonate. (4) The following indicators were detected: the changes of morphology were observed under inverted microscope,the cell viability was measured by CCK-8 methods,The expression of DRP1 and OPA1 in cytoplast was detected by the use of immunohistochemical method. The morphological changes of mitochondria were observed under transmission electron microscope (TEM). (5) All parameters were expressed as the mean value±standard difference(±S),statistical analysis were carried out by the use of t test and one-way ANOVA, LSD test. Difference was considered significance when the p.value was less than 0.05.The statistics work were finished by SPSS 13.0 software.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. Compared with asphyxia group,the changes in morphology of HK-2 were obviously improved in the group treated with rhEPO.(2) The cell viability was measured by CCK-8 methods:Compared with control group(0.74±0.08),the cell viability (optical density,OD) were obviously decreased in the asphyxia group(0.41±0.06).Compared with asphyxia group, the cell viability (OD) were obviously increased in the group treated with rhEPO (0.60±0.08,P<0.05),but not to the same as that in the control group. (3) Compared with control group (0.24±0.03),the expression of DRP1(OD) were significantly increased in asphyxia group(0.51±0.03,P<0.05). Compared with asphyxia group, the expression of DRP1 were obviously decreased in the group treated with rhEPO (0.38±0.03,P<0.05),but not to the same as that in the control group. (4) Compared with control group (0.47±0.02),the expression of DRP1(OD) were significantly increased in asphyxia group(0.21±0.02,P<0.05). Compared with asphyxia group, the expression of DRP1 were obviously decreased in the group treated with rhEPO (0.36±0.02,P<0.05),but not to the same as that in the control group. (6) Under TEM, mitochondria in asphyxia group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae; but in control and rhEPO group, mitochondrial structure was integrated, with uniform matrix and visible mitochondria cristae.Conclusion RhEPO could play the role in relieving the injury of renal tubular cells induced by postasphyxial-serum in neonate. The pretreatment with rhEPO could activate OPA1 and inhibit DRPl,then inhibit the morphological changes of mitochondria induced by postasphyxial-serum. |
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