| Objective: This study aims to explore the role and possible mechanism of Hypoxic preconditioning(HPC)combined with Remote ischemic preconditioning(RIPC)in the process of RIRI through the establishment of a renal ischemia reperfusion injury(RIRI)model on rats.Methods:(1)In this study,fourty male rats were choosed and stochastically seperated into five groups(eight rats in one group):the sham operation group,only removed the right kidney(Sham group),the ischemia reperfusion injury group(I/R group),hypoxic preconditioning intervention before the I/R injury model(HPC intervention group),remote ischemic preconditioning intervention before the I/R injury(RIPC intervention group),and hypoxic preconditioning combined with remote ischemic preconditioning intervention before the I/R injury model(HPC+RIPC intervention group).sham group: Right kidney was cuted and left renal artery was dissociated.i/r group: Renal artery on the left was closed with a noninvasive artery clamp for fourty five minutes,the right kidney of rats was completely resected before that,and then the artery clamp was loosened to make the I/R injury model.HPC intervention group: Before making I/R injury model,the experimental rats were first placed in a hypoxic chamber with constant temperature and humidity,under normal light,and Inject a mixture of nitrogen and oxygen into the chamber in order to maintain the oxygen at 12%,15 hours a day(17:00 to 08:00)for 4 weeks,the remaining steps completely according to the I/R group.RIPC intervention group: 24 hours before I/R injury model,the left leg of rats was subjected to circular compression for 10 minutes ischemia with medical elastic bandage,and then release for 10 minutes,totally cycle 3 times,the remaining steps completely according to the I/R group.HPC+RIPC intervention group: 24 hours before I/R injury model,firstly start following the HPC intervention group,then follow the RIPC intervention group,lastly completely follow the I/R group.In the above groups,operate on the rats first and routinely feed them for 24 hours.After anesthesia,firstly the blood was collected from the postcava,then the chest of rats was opened and the kidney was repeatedly lavaged through the left ventricle with ice normal saline until the color was pale.Lastly the left kidney was cuted off,and completelly dipped in 4% paraformaldehyde,last for 48 hours.(2)Automatic biochemical analyzer were used to evaluate the levels of urea nitrogen(BUN)and creatinine(SCR).(3)Pathological examination: The pathological damage of kidney in each group was evaluated.by Hematoxylin-eosin(HE)staining and Paller score(4)Tunel staining was applyed to evaluate the apoptosis which come from renal tubular and the apoptotic index(AI)was calculated.(5)Immunohistochemical method was applyed to evaluate the expression levels of Caspase-3 and Bcl-2.Results:(1)Blood biochemical test: Contrast with Sham group,the other groups increased more about the levels of BUN and SCR.contrast with i/r group,the HPC intervention group,RIPC intervention.group and HPC+RIPC intervention.group decreased more significantly.about the levels of scr and.bun.But Contrast with HPC intervention.group and RIPC intervention.group,.the levels of scr.and bun in HPC+RIPC intervention.group decreased more obviously..(2)The pathological.examination.results of renal tissue by.HE staining: The score was estimated according to the renal Paller scoring standard..In the Sham group,.the glomerular structure.was standard,the epithelial cells were closely arranged,the brush border was intact,and there were no necrotic exfoliated cells or tubular formation observed in the lumen;In the.I/R group,.the structure was obviously.disorganized,the lumen.of the renal tubules were.clearly dilated,the epithelial.cells showed different.degrees of swelling and vacuolar.degeneration,part of the brush.border disappeared,some necrotic.exfoliated cells and tubular.formation were observed.in the lumen,the renal interstitial hyperemia and infiltrating.inflammatory cell can.be observed,and the pathological injury of kidney was serious;contrast with the I/R group,in the HPC intervention group or RIPC intervention group,the dilated degree of renal tubules was lower,there were less necrotic exfoliated cells or tubular formation,the renal interstitial hyperemia and infiltrating inflammatory cell was observed occasionally,the pathological injury of kidney was alleviated;While contrast with the HPC intervention group and RIPC intervention group,the pathological injury was relieved more obviously in HPC+RIPC intervention group,the renal tubules were slightly dilated,and necrotic exfoliated cells were rarely observed.(3)Apoptosis and Apoptosis Index(AI)of the epithelial cells from renal tubules by TUNEL staining: There were few apoptotic in the the Sham group;But in the I/R group,the epithelial cells of renal tubules showed serious apoptotic degree,numerous apoptotic cells were seen;Both of the HPC intervention group and RIPC intervention group can reduce the levels of apoptosis caused by ischemia reperfusion injury;The apoptosis decreased more in the HPC and RIPC intervention group.(4)Immunohistochemical: The expression of caspase-3 increased more and the bcl-2 decreased more.in the.I/R group contrast.with the Sham group;.The bcl-2 increased more and.caspase3 decreased more.in the HPC and RIPC intervention.group contrast with the.I/R group,and the difference.was significant;what,s more,in the HPC and RIPC intervention group,the expression.of bcl-2.increased more and.caspase-3.decreased more contrast with the HPC and RIPC intervention group.Conclusion: Both the hypoxic preconditioning and the remote ischemia preconditioning can alleviate renal injury caused by ischemia reperfusion injury in rats.And there is synergy between the two methods.The mechanism may be related to the inhibition of apoptosis,which work by decrease the expression of Caspase3 and increase the expression of Bcl-2. |
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