| Background and objectivesAsphyxia of newborn is one of the important factors which caused death and disability in infant. Asphyxia anoxia lead to a series of changes to internal environment of the body, which include different degrees of hypoxia-ischemia damage of various organs such as heart, brain, kidneys and other important organs,and its incidence rate can be as high as82%[1-2].Moreover, structure and function of newborn kidneys are immature, which adjustment range is naarow to steady state of internal environment [3],so the kidney are highly vulnerable to primary and reperfusion injury caused by hypoxic-ischemic.More and more scholars and clinicians pay attention to the research to renal damage of neonatal asphyxia,but it is still short of in-depth exploration of the molecular level.Hypoxia inducible factor-1(HIF-1)is a key transcription factor mediating cellular hypoxia adaptility, which a subunit (HIF-la) has much important biological functions,that can regulate the expression of a series of downstream target genes and participation in a variety of mechanism which are energy metabolism and oxygen utilizationin of hypoxic cells when the body appeared anoxic signal. Vascular endothelial growth factor (VEGF) is a basic factor of regulation angiogenesis, and is one of important target genes of HIF-1a. Capillary network of the kidney is very rich,which hypoxic-ischemic disease often associated with microvascular damage, so we can conjecture that VEGF may play an important role in oxygen deficiency disease of the kidney. These should be further researched later.Creatine phosphate is the human body’s own active substances,and it is one of the important substances involved in cellular energy metabolism,which was discovered in1927by Eggleton P etc. Main function of Creatine phosphate is not just a "buffer" keeping constant ATP concentration in high-energy-consuming cell, but also a carrier of energy in the cell.At present, Creatine phosphate Sodium(PCr) is a high efficiency and low toxicity synthetic medicine commonly used the myocardial protection drug, has been widely used in the treatment of various diseases in the heart and has a good curative effect. The kidney is also one of the most sensitive organs to hypoxia-ischemia,whether it has a protective effect to renal hypoxic-ischemic disease is rarely reported in the literature.We will explore the influence of asphyxia hypoxia on kidney and protective effect of PCr to hypoxic kidney injury by observing renal tissue morphology changes and effects of PCr on expressions HIF-1a and VEGF in this experiment,so that provide the laboratory evidence for the clinical treatmentof renal anoxic diseases.Methods1Experimental animals and groupsA total of24young rats were randomly divided into three groups, asphyxia anoxia with creatine phosphate sodium intervention group(PCr group), asphyxia anoxia with normal saline model group(model group) and normal control group,8rats in each group, which were healthy SD young rats of3-4weeks ages, weight70-85g, regardless of gender.In animals of each group,the rats of PCr group and model group were made of asphyxia anoxia model, normal control group without above treatment.2Drug applicationThe young rats of PCr group were injected intraperitoneally with creatine phosphate sodium on the0.5h before asphyxia anoxia, and after asphyxia anoxia immediate, by the dose of1.4mg/g.At the same time phase, equal normal saline were injected respectively in young rats of model group. Nothing was given the normal control group.3Specimens collection and processingAfter asphyxia anoxia6h, took8young rats of each group respectively, which were cut out one side kidney by narcotizing of intraperitoneal injection after weighing and were divided the kidney into two parts along axis vertical.the half of the renal tissue fixed in4%paraformaldehyde to make the pathological section and immunohistochemical staining (SP), respectively using HE staining to observe shape and structure of the kidney were observed and immunohistochemistry staining technique to detect the expressions of HIF-1a and VEGF protein; the other half wern frozen in liquid nitrogen for1hour, then were placed in-80℃refrigerator to reserve for later, using reverse transcription polymerase chain reaction (RT-PCR) method to detect the expressions of HIF-1a and VEGF mRNA.4Statistical analysisTo analyze all the data by SPSS17.0statistical software, the relative content of HIF-la and VEGF mRNA and protein expressions in renal tissue among the3groups were used One-way ANOVA. LSD-t was used for multiple comparison. Each goup data used Non-parametric statistics for heterogeneity of variance. Correlation analysis of HIF-la and VEGF mRNA and protein in model group used Pearson correlation analysis. The difference was statistically significant when P<0.05.Results1Compared with normal control group, pathological changes of renal lesions was observed in renal tissue of both PCr and Model group in HE staining, serious injury of Model group were obviously observed. Turgidity of renal tubular epithelial cells was alleviative and shape and structure of glomerulus was close to normal in PCr group than model group, but there were still varying degrees pathological changes compared with normal control group.2The expressions of HIF-1a and VEGF mRNA in renal tissue among3groups had significant differences (F=11196.805,32484.734P<0.001),which were higher in both PCr (0.522±0.012,0.601±0.007) and model groups (0.731±0.007, 0.821±0.006) than those in normal control group(0.090±0.007,0.039±0.005),and lower in PCr group than those in model group(P<0.001).3The expressions of HIF-la and VEGF protein in renal tissue among3groups had significant differences(F=90.410,79.329P<0.001),which were higher in both PCr(115.78±4.66,106.79±2.95) and Model groups (124.45±5.58,116.72±3.61) than those in normal control group (94.53±3.17,95.96±3.30),and lower in PCr group than those in model group(P<0.001).4The results of correlation analysis of Model group that relative expression content of both HIF-la and VEGF mRNA and protein were positively correlation (r were respectively0.934and0.928, P<0.01).Conclusion1Renal tissue of young rats took place pathological morphology changes after asphyxia anoxia. Meanwhile,the expressions of relative amounts of HIF-1a and VEGF significantly increased, and the experimental results has shown positive correlation between the relative expression levels of both.This suggested that HIF-la and VEGF involve in the course which renal tissue of rats produced pathological changes of hypoxia ischemia.2Exogenous PCr can compound ATP and quickly add the energy required for hypoxic tissue through releasing high-energy phosphate bond,so that it increased the utilization of oxygen in anoxic tissues, and relieved oxidative damage, then can alleviate the pathological changes in asphyxia rats, drop the expressions of HIF-la and VEGF to play a protective role in kidney damage of asphyxia young rats. |
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