| Objectives In this study,Srague Dawley(SD)rats were used as the research objects.The protective effect of Preconditioning Buyang Huanwu Decoction on kidney and its mechanism were discussed.It aimed to provide a theoretical basis for kidney protection of brain death.Materials and Methods 1.Materials 30 SPF male SD rats,each weighing 200-250 g.2.Grouped Thirty male SD rats were randomly assigned numbers of 1-30,of which 1-10 were called as mock surgical group(D1): After successful anesthetize,only femoral artery cannulation and tracheal intubation are performed,but not induced to brain death.The numbers of 11-20 were recognized as brain death group(D2): After successful anesthetize,inducing the rats to brain death by Progressive pressurization after femoral artery cannulation and tracheal intubation performed.The numbers of 21-30 were called as Buyang Huanwu Decoction and brain death induction group(D3): 1 week before surgery,each rat was given the Buyang Huanwu by Gastrointestinal administration at this dose(0.6ml/100g)once a day for 7 consecutive days.Rats brain death were induced 30 minutes after gastrointestinal administration on day 7,and the rest process was same as the group D2.3.Methods 3.1 Establishing brain death models The brain death model was established by progressive pressure method,and the international brain death criterion was used to judge whether the models was successful.3.2 Apoptosis detected by tunel in renal tissue samples from different groups The apoptosis of the kidneys from the three groups was detected by TUNEL,and the apoptosis rate of each group was calculated.Qualitative analysis of apoptosis using immunofluorescence.3.3 Evaluations protein expression of Bcl-2,Bax,Caspase-3 and Caspase-9 in apoptotic pathway Western blot was used to detect the protein expression of Bcl-2,Bax,Caspase-3 and Caspase-9 in the renal tissues from the three groups.4.4 Pathological examination Morphological and structural changes of kidneys in each group were observed under light microscope after HE staining.4.Statistical analysis All statistical analyses were carried out by SPSS 13.0.The results were presented as mean±standard deviation(SD).P value<0.05 was considered to be significant.Differences were determined by t test and analysis of variance(ANOVA).Results 1.Establisheing 12 rat models of brain death successfully: The groups of D2 and D3 induced brain death of 6 rats successfully,brain death can be maintained by 6h,and the mean arterial pressure(MAP)of rats is above 80 mmHg.2.The results of TUNEL showed that the apoptotic rate of D2 group was significantly higher than that of D1(0.02±0.01)group(P<0.01).The apoptosis rate of D3(0.15±0.01)group was significantly lower than that of D2 group,higher than D1 group(P<0.01).3.WB results showed that the protein expression of Bax,Caspase-3 and Caspase-9 were significantly increase and Bcl-2 was significantly decrease in D2 group compared with D1 group(P<0.01).Compared with D2 group,the protein expression of Bax,Caspase-3 and Caspase-9 in D3 group were significantly decrease(P<0.01),the protein expression of Bcl-2 significantly increased(P<0.05).4.HE staining showed that there was no abnormality in the morphological structure in D1 group;There is renal tubular epithelial cell edema,granule degeneration,infarction,stenosis and cast,and glomerular atrophy in D2 group;in D3 group,The tubules are slightly edematous,but the cells are generally neatly arranged,The Cell degeneration is lighter than D2 group,and without Morphological and structural changes in Morphological and structural changes.Conclusions 1.The method of Progressive intracranial compressionis a reliable method for establishing BD model with high success rate 2.After BD in rats,apoptosis is involved in the process of kidney injury;3.Pretreating with Buyang Huanwu Decoction can inhibit cell apoptosis and reduce kidney damage in BD model of rats;4.Buyang Huanwu Decoction may inhibit apoptosis via mitochondrial apoptosis pathway. |
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