| Objective To investigate the protective effect and mechanism of Erythropoietin(EPO) 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) was used as target cell. (2)The serum of neonates 1 day after asphyxia, whose concentration was 20% (volume fraction),was applied as attacking factor;(3) The first experiment was designed as:control group,asphyxia group,the group of pretreatment with EPO.The group of pretreatment with EPO had five subgroups,which were researched whether EPO had the protective effect,and the best concentration.(4)In the second experiment, the cells were pretreated with EPO of the best concentration. The experiment was designed as:blank control group,the group of pretreatment with EPO ;at 15 minutes,1hour,2hours, the experiment was designed as:the serum attacking group and each time point group after serum attacking when the cells had been preconditioned with EPO firstly.(5) The following indicators were detected :In the first experiment , the changes of morphology were observed under inverted microscope,the cell viability was measured by MTT methods,and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.In the second experiment,the nuclear translocation rate of NF-κB were observed using indirect immunofluorescence under confocal microscope,Western blot was used to detect the mount of I-κBα.(6)All parameters were expressed as the mean value±standard difference( x±S),statistical analysis was carried out by the use of one-way ANOVA. Between two groups ,statistical analysis was carried out by the use of the least significant difference.Difference was considered significance when the p.value was less than 0.05.The statistics work was finished by spss12.0 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.But compared with asphyxia group, the changes in morphology of HK-2 were obviously improved in the groups of pretreatment with EPO in a dose-dependent manner, especially in the group of 50IU/ml,100IU/ml.(2)Compared with control group(63.49±1.30%),the leakage rate of LDH was significantly increased in asphyxia group(76.37±1.32%),statistical significance existed between EPO group and asphyxia group(P<0.05)except for 1IU/ml of EPO group(76.04±1.50%).The protective effect of EPO increased in a dose-dependent manner.There was no statistical significance among 50IU/ml of EPO,100IU/ml of EPO and control group.(3)Compared with control group(0.642±0.018),the cell viability (optical density,OD) was obviously decreased in the asphyxia group(0.240±0.036).Compared with asphyxia group, statistical significance existed between EPO group and asphyxia group except for 1IU/ml of EPO group(0.240±0.044).The protective effect of EPO was increased in a dose-dependent manner.There was no statistical significance among 50IU/ml of EPO,100IU/ml of EPO and control group.(4)Before the attack , when compared with the blank control group(9.7±2.1%),the nuclear translocation rate of NF-κB was increased in group of pretreatment with EPO(20.0±2.6%).After the attack, when compared with the blank control group ,the nuclear translocation rate of NF-κB was increased in every group,especially at 1 hour; compared with the serum attacking group, the nuclear translocation rate of NF-κB was decreased in each moment after attack in group of preconditioning with EPO;(5)Before the attack , when compared with the blank control group(0.52±0.05),the rate of I-κBα/β-actinIOD was decreased in group of pretreatment with EPO (0.26±0.03); After the attack, when compared with the blank control group ,the rate of I-κBα/β-actinIOD decreased in every group,especially at 1 hour; compared with the serum attacking group ,the rate of I-κBα/β-actinIOD was increased in each moment after attack in group of preconditioning with EPO .Conclusion EPO could play the role in relieving the injury of renal tubular cells induced by postasphyxial-serum in neonate. The pretreatment with EPO could activate NF-κB,then inhibit the activation of NF-κB induced by postasphyxial-serum. Objective To investigate the protective effect and mechanism of astragalus 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) was used as target cell.(2)The serum of neonates 1 day after asphyxia, whose concentration was 20% (volume fraction), was applied as attacking factor.(3) The first experiment was designed as:control group, asphyxia group,the group of pretreatment with astragalus.The group of pretreatment with astragalus had five subgroups,to study whether astragalus had the protective effect,and the best concentration.(4) In the second experiment,the cells were pretreated with astragalus of the best concentration. The experiment was designed as:blank control group, the group of pretreatment with astragalus. At 15 minutes,1hour,2hours, the experiment was designed as:the serum attacking group and each time point group after serum attacking when the cells were preconditioned with astragalus firstly.(5) The following indicators were detected :In the first experiment , the changes of morphology were observed under inverted microscope,the cell viability was measured by MTT methods,and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.In the second experiment,the nuclear translocation rate of NF-κB was observed using indirect immunofluorescence under confocal microscope,Western blot was used to detect the mount of I-κBα.(6)All parameters were expressed as the mean value±standard difference( x±S),statistical analysis was carried out by the use of one-way ANOVA. Between two groups ,statistical analysis were carried out by the use of the least significant difference.Difference was considered significance when the p.value was less than 0.05.The statistics work was finished by spss12.0 software.Results (1)Under inverted microscopy, HK-2 cells in control group were significantly increased, sticked to each otcher 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, the 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 group of pretreatment with astragalus in a dose-dependent manner.The protective effect was obviously to seen when the concentration was over 20ug/ml,and the protective effect of 40ug/ml group was obvious.(2)Compared with control group(63.50±1.30%),the leakage rate of LDH was significantly increased in asphyxia group(76.37±1.32%), statistical significance existed between astragalus groups and asphyxia group(P<0.05).The protective effect of astragalus was increased in a dose-dependent manner between 5ug/ml and 40ug/ml.The protective effect of 40ug/ml group was obvious , and the leakage rate of LDH was minimum(65.29±1.34%),but still had statistical significance compared with control group. When the concentration of astragalus was over 40ug/ml, the protective effect was decreased.(3)Compared with control group(0.642±0.018),the optical density was significantly decreased in asphyxia group ( 0.240±0.036 ) .Statistical significance existed between astragalus groups and asphyxia group(P<0.05).The protective effect of astragalus was increased in a dose-dependent manner between 5ug/ml and 40ug/ml.The protective effect of 40ug/ml group was obvious (0.593±0.044), and the optical density was maximum ,but still had statistical significance compared with control group. When the concentration was over 40ug/ml, the protective effect was decreased. (4)Before serum attacking , the nuclear translocation of NF-κB didn't change in group of pretreatment with astragalus(10.0±2.00%) ,when compared with the blank control group(9.7±1.2%); after serum attacking, the nuclear translocation of NF-κB was increased in every group,when compared with the blank control group ,especially at 1 hour(75.7±4.7%); compared with the serum attacking group ,the nuclear translocation of NF-κB were decreased in each moment group of preconditioning with astragalus(5)Before serum attacking , the rate of I-κBα/β-actinIOD didn't change in group of pretreatment with astragalus(0.54±0.10), when compared with the blank control group ( 0.54±0.09 ) ; after serum attacking, the rate of I-κBα/β-actinIOD was decreased in every group,when compared with the blank control group ,especially in the 1 hou(r0.02±0.01); compared with the sersum attacking group ,the rate of I-κBα/β-actinIOD was increased in each moment after serum attacking group of preconditioning with astragalus.Conclusion Astragalus could play the role in relieving the injury of renal tubular cells induced by postasphyxial-serum in neonate. The astragalus could inhibit the activation of NF-κB induced by postasphyxial-serum. |
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