| Objective: To establish the renal ischemia reperfusion injury(IRI)model in the syngenic(C57BL/6J)mice to observe the expression changes of vascular endothelial growth factor(VEGF),B-cell lymphoma-2(BCL-2),nuclear factor kappa B(NF-K B),hypoxia inducible factor-lalpha(HIF-1 alpha)and connective tissue growth factor(CTGF)in renal tissue of ischemia reperfusion and the effect of hyperbaric oxygen(hyperbaric oxygenation,HBO)on the expression of these factors,further study the pathophysiological changes of renal tissue after ischemia reperfusion as well as the therapeutic action and mechanism of HBO on renal ischemia reperfusion injury.Methods: C57 BL / 6J mice were randomly divided into normal control group,sham-operated group,renal ischemia group,HBO plus renal ischemia group,renal IR group and kidney IR plus HBO treatment group,and then IR group and IR group plus HBO treatment group was divided into 1h,3h,6h,12 h,24h group,6 mice are in each group.Once the mice in the normal control group were incised the abdominal cavity,then kill them immediately and took the double kidneys.The mice in the sham operation group were closed the abdominal cavity after opening the abdominal cavity.The renal ischemia group was opened the abdominal cavity and the bilateral renal pedicle was,after that mice were sacrificed to be blood sampling and taken out the kidneys.Ischemia plus HBO group: 1 hour of hyperbaric oxygen therapy was performed on the mice before the same treatment in the renal ischemia group.The mice in renal IR group were opened abdominal cavity,bilateral renal pedicle were interdicted 45 min after opening blood flow,then took out double kidneys and venous blood sampling respectively at 1,3,6,12,24 hours after reperfusion;IR plus HBO treatment group: open abdominal cavity,bilateral renal pedicle were interdicted 45 min after opening blood flow,respectively was treated at 0,2,5,11 and 23 hours with HBO treatment after perfusion.After 1 hours of treatment,the kidneys were taken and venous blood was taken.The bilateral renal pedicle was not blocked and the kidneys were taken and venous blood was taken at the corresponding time.The pathological changes of renal tissue were observed by HE staining.The expression ofVEGF,BCL-2,NF-?B and HIF-1? in renal tissue were detected by immunohistochemistry.Real-time PCR was used to detect the expression of VEGF and CTGF.Serum creatinine(SCr)was detected by fully automatic biochemical analyser.Results: 1.Changes in renal function: the results showed that the sham operation group and normal control group had no significant difference in serum creatinine,serum creatinine in renal ischemia group and HBO plus renal ischemia group was significantly higher than that of sham-operated group(P < 0.05),after HBO treatment,the renal function damage caused by renal ischemia has improved but not obvious.The damage of renal function aggravated after reperfusion,and gradually aggravated with the prolongation of reperfusion time.The most serious injury occurred during reperfusion of 24 h.HBO treatment significantly improved the renal function after reperfusion,especially after 1h,3h,6h and 12 h,and the protective effect of hyperbaric oxygen treatment on renal function decreased after 24 hours.2.The pathological changes of renal tissue: the normal control group and sham-operated group showed no obvious pathological changes in renal tubular cells,ischemia group and ischemia plus HBO group showed obvious swelling in renal tubular cell,swelling in renal tubular cell was slightly better after HBO treatment.In the IR group in 1h,in shows visible mesangial cell hyperplasia and kidney vascular mild hyperemia,glomerular volume increased slightly,mild swelling of renal tubular epithelial cells;in 3h,the renal tubular epithelial cell swelling further increased,showed granular degeneration and vascular congestion;in 6h,renal tubular cells has gradually lost the original cell morphology,swelling of renal tubular epithelial cells aggravated,with a small amount of neutrophil infiltration,in12 h and 24 h,it showed a large number of tubular cell damage,renal tubular cells of the integration,the cell structure is completely destroyed,and A small amount of nuclear fragmentation and karyolysis,and occurred a large number of neutrophilic infiltration.After hyperbaric oxygen treatment,the swelling of renal tubular cells was obviously reduced at any time point,and the cytoclasis was obviously reduced,and the phenomenon of karyolysis was also decreased.3.The expression of VEGF,BCL-2,NF-?B and HIF-1 in renal tissue detected by immunohistochemistry:(1)The expression changes of VEGF protein in renal tissue: VEGF protein is mainly expressed in cytoplasm of renal tubular epithelial cells,and express a little in nucleus.The expression of VEGF in nucleus and cytoplasm is increased after HBO therapy.The relative quantitative results showed that the expression of VEGF in renal ischemia group,HBO plus group,renal IR group and renal IR plus HBO group were significantly higher than the normal control group and sham-operated group(P < 0.05),and applying hyperbaric oxygen treatment before ischemia showed increased expression of VEGF(P < 0.05).After renal IR,expression of VEGF compared with the sham-operated group were continuously increased and reached the peak at 6h(P < 0.05),after 12 h it began to show a downward trend but still higher than the sham-operated group(P < 0.05);after HBO therapy,VEGF expression in renal IR plus HBO group compared to the renal IR group increased significantly and the most obvious in 6h(P < 0.05)at each corresponding time point.(2)Expression changes of HIF-1? protein in renal tissue: HIF-1? protein was mainly expressed in the cytoplasm of renal tubular cells,and was expressed a little in the nucleus.The relative quantitative results show that expression of HIF-1? protein in renal ischemia group,HBO plus renal ischemia group,renal IR group and renal IR plus HBO group was significantly higher than that in normal control group and sham operation group(P <0.05).The expression of HIF-1? was increased by hyperbaric oxygen therapy before renal ischemia,but there was no statistical significance(P> 0.05).The expression of HIF-1? increased continuously and peaked at 6 h(P <0.05).After the renal ischemia reperfusion injury,The expression of HIF-1? increased continuously and peaked at 6 h(P <0.05).After 12 h,the expression of HIF-1? was decreased and still higher than that of sham operation group(P <0.05).After treatment with hyperbaric oxygen,the expression of HIF-1? increased significantly compared with renal IR group(P <0.05)at each corresponding time point.(3)Expression changes of Bcl-2 protein in renal tissue: Bcl-2 protein was mainly expressed in the cytoplasm of renal tubular epithelial cells,and was expressed a little in the nucleus.The relative quantitative results show that the expression of Bcl-2 protein in renal ischemia group,HBO plus renal ischemia group,renal IR group and renal IR plus HBO group was significantly higher than that in normal control group and sham operation group(P <0.05)The expression of Bcl-2 was increased by hyperbaric oxygen therapy before renal ischemia,but there was no statistical significance(P> 0.05).After renal ischemia reperfusion injury,the expression of Bcl-2 increased continuously and peaked at 3-6 h(P <0.05).After 12 h,the expression of Bcl-2 was lower than that of sham-operated group(P <0.05).The expression of Bcl-2 protein at each corresponding time point after hyperbaric oxygen treatment was significantly higher than that of kidney IR group,and the most obvious in 3-6h(P <0.05).(4)The expression changes of NF-?B protein in renal tissue: NF-?B protein was mainly expressed in the cytoplasm of renal tubular epithelial cells and was expressed a little in the nucleus.The relative quantitative results show that the expression of NF-?B protein in renal ischemia group,HBO plus renal ischemia group,renal IR group and renal IR plus HBO group was significantly higher than that in normal control group and sham operation group(P <0.05).There was no significant difference in the expression of NF-?B by pretreatment of hyperbaric oxygen therapy after renal ischemia(P> 0.05).After renal ischemia reperfusion injury,the expression of NF-?B increased continuously and peaked at 6h,and decreased after 12 h,but still higher than that of sham operation group(P <0.05).After hyperbaric oxygen therapy,The expression of NF-?B was significantly lower than that in renal IR group at each corresponding time point,and decreased the most obvious in 6h(P <0.05).4.The expression changes of VEGF,m RNA,CTGF and m RNA in renal tissue was detected by real-time quantitative PCR(real-time PCR):(1)the expression changes of VEGF m RNA in renal tissue: the expression of VEGF m RNA in renal ischemia group,HBO plus renal ischemia group,renal IR and renal IR+HBO group was significantly higher than that in normal control group and sham-operated group(P <0.05),after hyperbaric oxygen therapy the expression of VEGF m RNA in HBO plus renal ischemia group was higher than renal ischemia(P < 0.05).The expression of VEGF m RNA increased gradually with the prolongation of time and reached the peak at 6h(P < 0.05)after renal IR,and the expression of that began to gradually decrease after 12 h.After hyperbaric oxygen therapy we can find the expression of VEGF m RNA in renal IR plus HBO group compared with renal IR group in 1h,3h,6h,12 h were significantly increased at each corresponding time point(P < 0.05),and showed no significant difference in 24h(P > 0.05).(2)The expression changes of CTGF m RNA in renal tissue: the expression of CTGF m RNA in renal ischemia group,HBO plus renal ischemia group,renal IR group and renal IR plus HBO group were significantly higher than those in normal control group and sham-operated group(P <0.05),But there was no significant difference(P> 0.05)of the CTGF m RNA expression by the pretreatment with hyperbaric oxygen after renal ischemia.After renal ischemia-reperfusion injury the expression of CTGF m RNA was significantly increased(P <0.05).After hyperbaric oxygen therapy,the expression of CTGF m RNA in renal IR group was significantly decreased at each corresponding time point(P <0.05).Conclusion: 1.HBO treatment can significantly improve the hypoxia of ischemia-reperfusion renal tissue through HIF-VEGF-Notch pathway,promoting the formation of neovascularization,thereby reducing renal ischemia-reperfusion injury.2.HBO treatment can reduce the expression of CTGF,and alleviate renal damage by reducing the expression of fibrosis factor in renal ischemia-reperfusion injury,protecting the kidney.3.HBO treatment can increase the expression of Bcl-2 and reduce the expression of NF-?B,and regulate the upstream factors of apoptosis,thus protecting the kidneys in ischemia-reperfusion injury.4.HBO treatment play a protective role in the early stage of renal ischemia and reperfusion,which had a significant effect on the formation of neovascularization,the inhibition of fibrosis factor and the inhibition of apoptosis factors,and decreased with the prolongation g of time.Thereby,hyperbaric oxygen therapy should be used as soon as possible in the prevention and treatment process of renal ischemia-reperfusion injury,for reducing ischemia and reperfusion damage to the kidneys. |
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