| Objective:To observe the use of low-dose glucocorticoid and specific caspase-3 inhibitors alone or in combination,renal protective effects on sepsis-associated acute kidney injury(SA-AKI)mice,and related mechanisms involved.Methods:SPF grade male C57BL/6 mice were established by cecal ligation and puncture(CLP)method.144 mice were randomly divided into three time groups(6 h,12 h,24 h),each time group was divided into six groups:sham operation group,CLP model group,DMSO organic solvent control group,Low-dose glucocorticoid(methylprednisolone)early intervention group(6.4mg/kg),Caspase-3 inhibitor early intervention group(4mg/kg),Low-dose glucocorticoid methylprednisolone and caspase-3 inhibitor combined early co-intervention group.All mice received routine fluid resuscitation(30mL/kg)after operation and were given 30 minutes after operation.Of the 8 mice in each group,4 were used for protein and gene detection in fresh tissue,and the other 4 were stained with tissue sections.The inflammatory reaction and apoptosis in SA-AKI mice were detected.The degree of kidney injury in SA-AKI mice was evaluated by Hematoxylin Eosin staining,to further clarify the acute kidney injury in septic mice.The inflammatory response was then detected:Detection of inflammatory factor IL-1? in serum of SA-AKI mice by ELISA.The expressions of GR-a protein was determined by Western blot,the gene change of GR-a mRNA and NF-?B mRNA was detected by Real-time relative fluorescence quantitative qPCR,and GR-a in renal tissue of SA-AKI mice was detected by immunofluorescence.Finally,the apoptosis was detected.The expression of apoptosis-related protein(cleaved caspase-3)and cytochrome c in kidney of SA-AKI mice was detected by Western blot.TUNEL fluorescence staining was used to label the cell apoptosis in renal tissue of SA-AKI mice.The fluorescence intensity of cleaved caspase-3 protein expression in kidney tissue of SA-AKI mice was detected by immunofluorescence.Results:1.The results of pathological score in SA-AKI mice were as follows:The increase of pathological score of renal tissue injury in SA-AKI mice was observed in all three time periods.Compared with CLP model group and DMSO organic solvent group,there was a significant difference between low-dose GC intervention group,caspase-3 inhibitor intervention group,combined intervention group(p<0.05),which indicated that clp method could induce obvious renal tissue damage in sepsis mice.At 12h and 24h groups,there was a significant difference between the low dose GC group and the combined intervention group(p<0.05).There was a significant difference between the caspase-3 inhibitor intervention group and the combined intervention group in 24 h group(p<0.05).2.Correlation detection of inflammatory reaction in SA-AKI mice:The results of measuring inflammatory factor IL-1? showed that the content of IL-1? in model group and organic solvent group increased gradually compared with each sham operation group.The contents of IL-1? in low-dose GC intervention group,caspase-3 inhibitor intervention group and co-intervention group were significantly lower than those in their respective CLP model groups(p<0.05).The results of glucocorticoid receptor protein and gene detection showed that compared with the sham-operated groups,the expression of GR-a protein in the 6h and 12h model group and 24h caspase-3 inhibitor group was significantly decreased The expression of GR-a mRNA in the three time periods model group and organic solvent DMSO group was significantly lower than that in the sham operation group.In low-dose GC group and 12 h,24h caspase-3 inhibitor group and 24h co-intervention group,the expression of GR-a mRNA gene also decreased significantly.Compared with the model groups at different time periods,the expression of GR-a in all low-dose GC group,6h and 12h intervention group was significantly higher.The expression of GR-a in 6h and 24h caspase-3 inhibitor group was lower than that in low-dose GC group,and it was significantly higher than that in co-intervention group(p<0.05).Compared with the model group and caspase-3 inhibitor group,the expression of GR-a mRNA gene was significantly increased in the 6h low dose GC group and in the 6h and 12h co-intervention group(p<0.05).The results showed that the expression of NF-?B mRNA gene in model group,organic solvent group at 6 h and 12 h group and caspase-3 inhibitor group at 12h was significantly higher than that in sham operation group.Compared with the respective model groups,the expression of NF-?B mRNA gene in the low-dose GC group and the co-intervention group and the caspase-3 inhibitor group at 6h and 12h was significantly lower(p<0.05).The results showed that both of the two early intervention drugs could inhibit the expression of NF-?B mRNA,and the common intervention group had the most significant effect.3.Detection of apoptosis in SA-AKI mice:The expression of cytochrome c protein in renal tissue of SA-AKI mice was detected.Compared with sham operation group,the expression of cytc in 6h model group and 24 h organic solvent group was significantly higher.Compared with the model group,the expression of cytc in the low-dose GC group at 6h.and 12h,caspase-3 inhibitor group and co-intervention group was significantly lower(p<0.05).The results of cleaved caspase-3 protein expression in kidney tissue of SA-AKI mice showed that the expression of cleaved caspase-3 in three time groups,6 h and 12 h of organic solvent DMSO group was significantly higher than that of their respective sham operation groups,and there was a significant apoptosis of the cells in these groups.Compared with the respective model groups,the expression of cleaved caspase-3 in the low-dose GC group at 6h and 12h,the caspase-3 inhibitor group at 12h and the co-intervention group at different time periods were significantly decreased(p<0.05).These results suggest that early intervention can inhibit the activation of caspase-3 protein to a certain extent,and thus reduce the occurrence of apoptosis.Conclusions:1.The early intervention of low-dose glucocorticoid alleviated the related inflammatory response by increasing the expression of GR-a protein and gene in the kidney tissue of SA-AKI mice,so as to play a protective role in the kidney.It can also inhibit the apoptosis of cells by inhibiting the gene expression of NF-?B.2.Caspase-3 inhibitor can directly inhibit the activation of caspase-3 and reducing the expression of cleaved caspase-3 protein and decrease the release of cytochrome c in mitochondrial-related apoptosis pathway,thereby reducing the apoptosis of SA-AKI mice to a certain extent.3.Compared with single use,when treated with low-dose glucocorticoid combined with caspase-3 inhibitor,the inhibition of inflammatory reaction and apoptosis was more obvious,and the renal protective effect on SA-AKI mice was improved. |
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