The Imbalance Of Th17/Treg Cells And Intervention Of Ulinastatin On Rats With Acute Kidney Injury Induced By Crush Syndrome

Objective:We established a rat model of crush syndrome-induced acute kidney injury based on the previous works,and explored the effect of ulinastatin on Th17/Treg cells in an established rat model of crush syndrome-induced acute kidney injury(CSAKI),aiming to provide a new ideas for the pathogenesis for crush syndrome-induced acute kidney injury,and to find new targets for its intervention.Methods:Forty-six male Wistar rats were randomly assigned to five groups:the normal control(n=6),CSAKI model(n=10),CSAKI plus UTI1(50,000 U/kg)(n=10),CSAKI plus UTI2(100,000 U/kg)(n=10)and CSAKI plus UTI3(200,000 U/kg)(n=10).Animals in the CSAKI model and CSAKI plus UTI group were anesthetized with 2%sodium pentobarbital(3 mL/kg)intraperitoneally after being weighed,then placed in a prone position,and squeezed with a new type of digital extrusion platform(which featured an accurately set extrusion weight)to establish a rat model of CSAKI.The lower extremities were extrusion for 24 h and the pressure was 10 times the body weight of the rats.After undergoing pressure the indicated amount of time,rats were given50,000 U/kg,100,000 U/kg and 200,000 U/kg Ulinastatin in the CSAKI plus UTI1,UTI2,UTI3 groups,respectively.After 24 h of observation,peripheral blood samples were collected after anesthesia in the model group,UTI treatment group and normal control group.The peripheral blood samples were collected for detecting serum biochemical indicators and flow cytometric analysis to detect the ratio of Th17 cells and Treg cells.The levels of IL-6,IL-17,IL-10 and TGF-?1 in serum and renal tissues were detected by ELISA.One kidney of each rat and a piece of muscle tissue were fixed in 4%polyformalin for histological examination.The spleen was collected for real-time polymerase chain reaction(PCR)analysis.Results:1.Compared with the normal control group,the serum BUN,Mb,K~+,CK,Scr,IL-6,IL-17 and IL-10 levels in the CSAKI group were highly significant increased(P<0.05),while the levels of TGF-?1 were highly significant decreased(P<0.01).In the kidney tissues,the levels of IL-6,IL-17,IL-10 and TGF-?1 showed the same trend as in serum.HE staining showed that compared with the normal control group,some muscle fibers in the striated muscle tissue of the CSAKI group were disordered,sarcomere rupture,interstitial edema,inflammatory cell infiltration.Compared to normal control group,in the CSAKI group glomerular congestion and edema,renal tubular epithelial cells are swollen and degenerated,and inflammatory cells exudate forming a large number of myoglobin casts in some renal tubules.For Th17 cells analysis,the percentage of Th17 cells in peripheral blood and the expression of IL-17mRNA were significant increased(P<0.01).For Treg cells analysis,the expression of Foxp3 mRNA in CSAKI group were significantly decreased(P<0.01)when compared with the normal control group,and the proportion of Treg cells were significant decreased(P<0.05).2.Compared with the CSAKI group,the levels of BUN,Mb,CK,K~+and Scr in serum were significantly decreased in the CSAKI+UTI group(P<0.05).In serum and kidney tissue,the levels of IL-6 and IL-17 were significantly decreased(P<0.05).Ulinastatin treatment(100,000 and 200,000 U/kg)led to a significant increase in the serum concentration of TGF-?1,but no changes in serum level IL-10.For Th17 cells analysis,compared with CSAKI group,treatment with ulinastatin showed a lower percentage of Th17 cells and down-regulated IL-17 mRNA levels in spleen(P<0.01).However,after treatment with ulinastatin the percentage of Treg cells in CD4~+T cells was significantly increased.Meanwhile,up-regulated Foxp3 mRNA levels in spleen.Compared with the CSAKI group,after treatment with ulinastatin,the muscle fibers in the striated muscle tissue were aligned,some inflammatory cells were scattered in the muscle interstitial fibers,and the interstitial swelling was less than before.Besides,no large slices of rhabdomyolysis were observed.The histopathological changes of kidney were alleviated compared with the CSAKI group.The glomerular congestion and edema were lessened than before.The swelling of renal tubular epithelial cells were significantly improved,and the inflammatory cells exudation were less than before.Conclusion:The imbalance of Th17/Treg cells and its associated inflammatory factors may play an important role in mediating acute kidney injury associated with crush syndrome in rats.After ulinastatin treatment,the levels of inflammatory factors and the biochemistry indicators of kidney injury were decreased,indicating that ulinastatin attenuates crush syndrome-induced acute kidney injury,and the possibly mechanism alleviate inflammatory response in early stage may be due to regulating the balance between Th17/Treg cells.