| Objective: To explore the effect of hyperbaric oxygen therapy on apoptotic proteins in renal tissue during ischemia-reperfusion,so as to further clarify the therapeutic effect and mechanism of hyperbaric oxygen(HBO)therapy on renal ischemia-reperfusion injury(IRI)in mice model were established,and the changes of renal function,histopathology,apoptosis rate and apoptotic protein expression were observed in sham operation group,ischemia reperfusion group and hyperbaric oxygen treatment group.Methods: C57BL/6 mice were randomly divided into three groups,namely sham operation group,IRI group and HBO treatment group,each group was divided into four time points: 1h,3h,6h and 12 h,with 6 mice in each group.Sham operation group: the abdominal cavity was incised only,the renal pedicle was not blocked,and both kidneys and blood samples were cut at the corresponding time points;IRI group: the abdominal cavity was incised,the blood flow was opened after 45 min of renal pedicle occlusion,and both kidneys and blood samples were cut at 1 h,3 h,6 h,and 12 h after reperfusion;HBO treatment group: the abdominal cavity was incised,the blood flow was opened after 45 min of renal pedicle occlusion,and HBO treatment was performed for 1 h before occlusion and at 0 h,2 h,5 h,and 11 h after reperfusion,and both kidneys and blood samples were harvested after HBO treatment.Serum creatinine(SCr)was determined by fully automatic biochemical analyzer,HE method to observe the pathological changes of mouse renal tissue,TUNEL method to detect renal tissue cell apoptosis,the expression of Fas and FasL m RNA and protein were detected by RT-q PCR,the expression of protein Fas and FasL were detected by immunohistochemistry and Western blot.Results:(1)Changes in renal function: the SCr level of the renal IRI group was significantly higher than tant of the sham operation group at all time points(P<0.05),and it was significantly reduced after HBO treatment(P<0.05);there was no significant difference in the SCr level at each time point in the sham operation group;there was no statistically significant difference between 1h and 3h in the renal IRI group and HBO group,the SCr level at other time points was significantly different(P<0.05),and the SCr level gradually increased with the prolongation of reperfusion time.(2)Pathological changes of renal tissue: the morphology of renal tissue in the sham operation group was normal at each time point,and there was no obvious change.In the renal IRI group,turbidity and swelling of renal tubular epithelial cells were seen at 1h and3 h,cytoplasmic granular degeneration and the brush border disappeared were seen at 6h,a small amount of necrotic and shedding fragments and a small amount of hemorrhage in the tubular interstitium were seen in the lumen at 12 h,with the prolongation of reperfusion time,the lesions showed a trend of gradual aggravation.After HBO treatment,renal tubular epithelial cell swelling and interstitial hemorrhage were all relieved,which was most obvious in the early stage of reperfusion.(3)Apoptosis: TUNEL positive cells were occasionally seen in the kidney tissue of the sham operation group,while the number of TUNEL positive cells in the kidney tissue of the IRI group was significantly higher than that in the sham operation group(P<0.01),and the number of TUNEL positive cells was significantly reduced after HBO treatment(P<0.01);there was no significant difference in the number of positive cells at each time point in the sham operation group,while the difference between the IRI group and HBO group at each time point was statistically significant(P<0.05).(4)Immunohistochemical results: the expression changes of the Fas and FasL protein in renal tissues were consistent.Fas was mainly expressed in the cytoplasm of renal tubular epithelial cells,but no expression in the nucleus and glomeruli.FasL protein was mainly expressed in the cytoplasm of renal tubular epithelial cells,with a small amount of expression in the nucleus,but no expression in the glomeruli.Relative quantitative analysis showed that the expression levels of both in the renal IRI group at each time point were significantly higher than those in the sham operation group(P<0.05),and the expression levels of both were significantly reduced after HBO treatment(P<0.05);in sham operation there was no significant difference in the expression levels of the two proteins at each time point in the group,while the expression levels of the two proteins at each time point in the renal IRI group and the HBO treatment group were significantly different(P<0.05).With the extension of reperfusion time,the expression of the two proteins were gradually increasing.(5)The RT-qPCR results showed:the expression changes of Fas and FasL m RNA in renal tissues were also consistent,the expression levels of both in the renal IRI group at each time point were significantly higher than those in the sham operation group(P<0.05),and both after HBO treatment the expression level of were significantly reduced(P<0.05).There was no significant difference in the expression levels of the two genes at each time point in the sham operation group;the 12 h expression levels in the IRI group and the HBO treatment group were significantly higher than those at 1h,3h,and 6h(P<0.05),and with the extension of the reperfusion time,the expression of the audience gradually increased.(6)Western blot results showed that: the expression changes of Fas and FasL protein in renal tissues were also similar.The expression levels of both in the renal IRI group at each time point were significantly higher than those in the sham operation group(P<0.01).After HBO treatment,the expression level was significantly reduced(P<0.05).There was no significant difference in the expression levels of the two at each time point in the sham operation group,and the expression levels of the two proteins gradually increased with the prolongation of reperfusion time.There was no significant difference in the expression of the two proteins at 1h and 3h in the renal IRI group,and the difference was significant at other time points(P<0.05);the expression of Fas protein at each time point in the HBO treatment group was significantly different(P<0.05),while the amount of FasL protein table reached no significant difference between 1h and 3h,the other time points were significantly different(P<0.05).Conclusions:(1)Renal IRI may promote renal tissue cell apoptosis by activating the apoptosis Fas/FasL signaling pathway,and aggravate renal injury;(2)HBO treatment can reduce renal cell apoptosis by inhibiting the expression of apoptotic proteins Fas and FasL,thereby reducing renal injury. |
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