| Objective: This study aims to investigate changes in renal function,histopathology,and rate of apoptosis,along with the expression of stress pathway-related apoptotic proteins in the endoplasmic reticulum by establishing a mouse model of renal ischemia-reperfusion injury(IRI).Also,it explores the effects of hyperbaric oxygenation(HBO)on renal tubule expression of PERK and CHOP proteins,and HBO therapy’s regulatory effect on the endoplasmic reticulum stress pathway in renal IRI.Methods: C57 BL / 6 mice were divided into three groups randomly: sham operation group,IRI group,and IRI + HBO group(HBO group).Each group was divided into four subgroups according to reperfusion time: 1h,3h,6h,and 12 h,with 6 mice in each subgroup.In the sham operation group,only the abdominal cavity was cut open without the bilateral renal pedicle,and both kidneys were removed and blood was collected at 1h,3h,6h,and 12 h after operation.In the IRI group,the blood reflows after abdominal incision and clamp off bilateral renal pedicle for 45 minutes.Both kidneys were removed and blood was collected at 1h,3h,6h,and 12 h after reperfusion.In the HBO group,HBO treatment was given at 0 h,2 h,5 h,and 11 h after reperfusion for 1 h,and the kidneys were removed and blood was collected after treatment.The serum creatinine was detected by automatic biochemical analyzer,the pathological changes of renal tissue were observed by HE staining,the apoptosis of renal tissue was detected by TUNEL,the m RNA expression of PERK and CHOP was detected by RT-q PCR,and the protein expression of PERK and CHOP was detected by immunohistochemistry and Western blot.Results:1.Renal function test results: the SCr values of the IRI group and HBO group at each time point were significantly higher than that of the sham operation group(P < 0.05),while the SCr values of the HBO group were significantly lower than that of the IRI group at the same time point(P < 0.05).2.Pathological changes of renal tissue: the structure of renal tissue in the sham operation group was normal.In the IRI group,the glomerular volume increased significantly,and the epithelial cells of renal tubules swelled at 1 h and 3 h,granular degeneration in cytoplasm and unclear boundary between renal tubules could be seen at 6 h,and necrosis debris could be seen in the lumen of renal tubules at 12 h,with more hemorrhage in the interstitium of renal tubules and infiltration of inflammatory cells.After HBO treatment,the glomerular volume decreased,the renal tubulointerstitial hemorrhage,tubular epithelial cell swelling,and necrosis were alleviated.3.TUNEL assay results: TUNEL positive cells were occasionally seen in the sham operation group,and the number of positive cells in the IRI group increased significantly compared with the sham operation group(P < 0.01);after HBO treatment,the number of apoptosis cells decreased compared with IRI group,and the apoptosis rate decreased significantly(P < 0.05).4.Immunohistochemical results: PERK and CHOP proteins were mainly expressed in the cytoplasm of renal tubular epithelial cells,showing brown granules.Results of the relative quantitative analysis:(1)the expression of PERK and CHOP protein in the IRI group at each time point was significantly higher than that in the sham operation group(P < 0.05),and the expression was decreased after HBO treatment significantly(P < 0.05);(2)the expression of PERK in IRI group gradually increased with the prolongation of reperfusion time,and was significant difference at each time point(P < 0.05);in HBO group,there was no significant difference between 3 and 6 hours,but there was a significant difference at other time points(P < 0.05).While the expression of CHOP protein in the IRI group was no significant difference at 3 and 6 hours,the expression at other time points were significantly different(P<0.05).The expression in the HBO treatment group was significantly different at each time point(P <0.05).5.The results of RT-q PCR:(1)the expression of PERK and CHOP m RNA in the IRI group at each time point was significantly higher than that in the sham operation group,but it was lower significantly after HBO treatment(P < 0.05);(2)the expression of PERK in IRI group and HBO group at 6 hours was significantly higher than that in other time points(P < 0.05);the expression of CHOP m RNA in the IRI and HBO group at each time point was statistically significant(P < 0.05).6.The results of Western blot: the expression levels of PERK and CHOP protein in the IRI group at each time point were significantly higher than those in the sham operation group(P < 0.01),and the levels were significantly decreased after HBO treatment(P < 0.05).The expression of PERK in the IRI group and HBO group were significantly different at each time point(P < 0.05).The difference of CHOP expression in the IRI group at each time point was statistically significant(P < 0.05).While there was no significant difference in the HBO group at 1 and 3 hours,the difference was statistically significant at other time intervals(P < 0.05).Conclusions:1.Renal ischemia-reperfusion injury can aggravate the renal injury by up-regulating signal pathway of PERK-CHOP of the apoptosis cell.2.Hyperbaric oxygen therapy can inhibit apoptosis by down-regulating the expression of renal tubule apoptotic proteins PERK and CHOP,which in turn decrease renal ischemia-reperfusion injury. |
