| Objective:To evaluate the protective effect of low-dose cyclophosphamide?CTX?on renal ischemia-reperfusion injury?IRI?in rats,and to explore the correlation between its protective effect and high mobility group box 1 protein?HMGB1?,and to further elucidate the protective mechanism of CTX on renal IRI,providing a new strategy for clinical treatment of renal IRI.Methods:Eighty healthy male Sprague-Dawley?SD?rats of SPF grade,weighing 250300 g,were randomly divided into 8 groups of 10 rats each?n=10?.After passing the animal ethical review,the unilateral kidney?left kidney?I/R model of SD rats was established:the right kidney of rats was ligated to replace the right nephrectomy,and the left kidney was clamped with an atraumatic arterial clip for 45min and then the clip was released to restore blood perfusion for 24 h.Different doses(?5/10/20?mg·kg-1)of CTX and a uniform dose?100 ng/ml?of recombinant HMGB1?rHMGB1?were injected into rats via the tail vein 1 h before ischemia and at the moment of restoring blood perfusion,respectively.After 24 h of reperfusion,left renal and left renal venous blood samples were collected from rats in each group?n=6?under anesthesia.Parts of renal tissue were obtained from each kidney?left kidney?to make paraffin sections,and the rest were stored in-80°C freezer.Serum was extracted from venous blood after centrifugation,and the levels of blood urea nitrogen?BUN?and serum creatinine?Scr?in rat serum were measured using an automatic biochemical detector,and the expression levels of HMGB1,TNF-?,IL-1?,IL-6 and monocyte chemotactic protein 1?MCP-1?in serum were measured by ELISA kits.Paraffin sections of renal tissue were stained with HE,along with immunohistochemical?IHC?for HMGB1,CD68,and myeloperoxidase?MPO?.Renal mRNA levels of HMGB1,Toll-like receptor 4?TLR-4?,myeloiddifferentiation factor88?MyD88?,p65?a member of the NF-?appa B family?,TNF-?,IL-1?,IL-6,and MCP-1 were measured by qRT-PCR.After isolation of nucleoplasmic proteins from kidney cells,the expression of HMGB1 in the cytoplasm was detected by Western blotting?WB?technique.Results:All rats survived 24 h after reperfusion,except for two rats in the I/R group who were abandoned due to injury of perirenal tissues during modeling.The results of biochemical tests showed that the serum BUN and Scr levels of rats in the I/R group were significantly higher than those in the sham group,suggesting that the rat renal I/R model was successfully modeled.Pretreatment with different doses of CTX reduced renal function impairment in rats to varying degrees?as indicated by a decrease in BUN and Scr levels?.Among them,pretreatment with CTX?20 mg/kg?minimized renal function damage due to I/R.Injection of exogenous rHMGB1aggravated renal function injury in rats.In addition,CTX?20 mg/kg?and rHMGB1were successively injected through the tail vein of rats,and rHMGB1 reversed the protective effect of CTX on renal function in rats.We found that compared with the I/R group,the serum levels of BUN and Scr were significantly decreased in the CTX?20 mg/kg?pretreatment group;the serum levels of HMGB1,TNF-?,IL-1?,IL-6,and MCP-1 were significantly decreased;the mRNA expression of HMGB1,TLR-4,MyD88,p65,TNF-?,IL-1?,IL-6,and MCP-1 was significantly down-regulated in the renal tissue;the HMGB1 protein expression in the cytoplasm was significantly down-regulated;the infiltration of CD68 macrophages and MPO neutrophils was significantly decreased in the renal tissue sections;and the tubular structural destruction was significantly alleviated.IHC showed that HMGB1 in renal tissue sections of rats in the I/R group was mainly visualized in the cytoplasm with little or no staining of the nucleus,while HMGB1 in renal tissue sections of rats in the CTX pretreatment group was mainly visualized in the nucleus with little or no staining in the cytoplasm.Conclusion:HMGB1,as an early inflammatory factor,plays an important role in the occurrence and development of renal IRI;the addition of exogenous HMGB1 can aggravate renal IRI in rats;low-dose CTX can effectively protect rat kidney from IRI;exogenous HMGB1 can reverse the protective effect of CTX on renal IRI in rats;the protective effect of CTX on renal IRI in rats is achieved by preventing the nucleocytoplasmic translocation and extracellular release of HMGB1.The formation of adducts between CTX and DNA effectively blocked the nucleo-cytoplasmic translocation of HMGB1. |
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