Crocin Alleviated Renal Injury Induced By Sever Hemorrhagic Shock In Rats Acutely Exposed To High Altitude:Effects And Possible Mechanisms

Objective:The SPF grade SD rats were established the model of severe hemorrhagic shock under acutely exposured to high altitude,to further study the protective effect of crocin on renal injury and possible mechanism in rats by severe hemorrhagic shock under acutely exposured to high altitude.So the effects of crocin on survival time,survival rate,changes of renal function,renal pathological injury,oxidative stress,cytokines and NF-κB-NLRP3 signal pathway protein content in rats with severe hemorrhagic shock under acutely exposured to high altitude were determined.Methods:Thirty healthy male SD rats of SPF grade were randomly divided into5 groups:control group（C group）,hemorrhagic shock+normal saline group（NaCl group）,hemorrhagic shock+40mg/kg crocin group（LC group）,hemorrhagic shock+60mg/kg crocin group（MC group）and hemorrhagic shock+80mg/kg crocin group（HC group）with 6 rats in each group.All rats were transported from Lanzhou,Gansu Province（altitude 1503m）to Yushu,Qinghai Province（altitude 4010m）within 24hours.After the model of severe hemorrhagic shock in rats was successfully established,40 mg/kg,60 mg/kg,and 80 mg/kg of crocin were respectively injected intraperitoneally in the LC group,MC group,and HC group,and the NaCl group was given the same volume of normal saline,C group only catheterization without bloodletting.All groups of rats were resuscitated with crocin for 3h and then centrifuged 2m L of heart blood and the serum was stored in-20℃refrigerator.The left kidney was fixed with 10%formaldehyde,and the right kidney was stored in a sterile freezer at-80℃.The survival curve was draw after we observed the survival time of the rats in each group for 3 hours,observed the MAP and Sp O₂of rats in each group at different time points and serum from rats in each group was used to measure Scr and BUN by ELISA.The left kidney of the rats in each group were fixed with10%formaldehyde,stained with HE,dehydrated,embedded in paraffin,sliced,and then evaluated under the microscope according to the corresponding scoring standards for the pathological changes of the rat kidneys.Part of the right kidney tissue of the rats in each group was taken to measure the activities of oxidative stress indicators GSH and SOD as well as the levels of MDA and MPO by ELISA method,part of the right kidney tissue of the rats in each group was taken to measure the inflammatory factors TNF-α,IL-6,IL-18,IL-1β,NF-κB,NLRP3,caspase-1,parts of the right kidney tissues of rats in each group were used to detect the protein expressions of NF-κB,NLRP3,caspase-1 by Western Blot.Results:1.Compared with the NaCl group,the 1h survival rates of the LC,MC,HC and C groups were higher than NaCl group（P<0.05）,and the survival times and 2h and3h survival rates of the MC,HC and C groups were higher than NaCl group（P<0.05）;compared with the LC group,the MC and HC groups showed a dose-dependent prolongation of the survival time of rats after crocin intervention（P<0.05）.2.Compared with C group,MAP in NaCl group,LC group,MC group and HC group decreased after shock（at resuscitation）,0.5 h and 1 h after resuscitation（P<0.05）.3.Compared with the NaCl group,the Sp O₂in the MC group,HC group,and C group at 0.5h of resuscitation was increased（P<0.05）,and the Sp O₂in the LC group,MC group,HC group,and C group at 1h of resuscitation was increased（P<0.05）.Compared with the C group,the Sp O₂in the NaCl group,LC group,MC group,and HC group after shock（at resuscitation）,at 0.5h of resuscitation,and at 1h of resuscitation decreased（P<0.05）,Sp O₂in LC group,MC group and HC group at 0.5h and 1h of resuscitation increased after crocin intervention（P<0.05）4.The serum levels of Scr and BUN were significantly lower in LC group,MC group,HC group,and C group compared with NaCl group（P<0.05）.Scr and BUN levels were significantly higher in the NaCl group,LC group,and MC group compared with the C group（P<0.05）,and the serum levels of Scr and BUN were dose-dependently decreased in the LC group,MC group and HC group after crocin intervention（P<0.05）.5.After intervention with crocin,the pathological changes of renal tissue in LC group,MC group and HC group were significantly alleviated in a dose-dependent manner（P<0.05）.6.Compared with NaCl group,the activities of GSH and SOD in renal tissue of LC group,MC group,HC group and C group were significantly increased,while the levels of MPO and MDA were significantly decreased（P<0.05）.Compared with C group,the activities of GSH and SOD in NaCl group,LC group and MC group were significantly lower and the levels of MPO and MDA in renal tissue were significantly higher（P<0.05）.After treatment with crocin,the activities of GSH and SOD in renal tissue in LC group,MC group and HC group increased in a dose-dependent manner,while the levels of MPO and MDA decreased in a dose-dependent manner（P<0.05）.7.The levels of TNF-α,IL-6,IL-18,IL-1β,NF-κB,NLRP3,caspase-1 in the renal tissues of LC group,MC group,HC group,and C group were significantly lower compared with the NaCl group（P<0.05）.Compared with C group,the levels of TNF-α,IL-6,IL-18,IL-1β,NF-κB,NLRP3,caspase-1 in the renal tissues of NaCl group,LC group and MC group were significantly increased（P<0.05）,the levels of TNF-α,IL-6,IL-18,IL-1β,NF-κB,NLRP3,caspase-1 in the renal tissues of LC group,MC group and HC group after crocin intervention were significantly decreased（P<0.05）.8.Compared with the NaCl group,NF-κBp65,NLRP3,caspase-1 protein expression was decreased in the renal tissues of LC group,MC group,HC group and C group（P<0.05）.NF-κBp65,NLRP3,caspase-1 protein expression in the renal tissues of rats in LC group,MC group and HC group were decreased in a dose-dependent manner after crocin treatment（P<0.05）.Conclusion:Crocin was able to reduce the mortality of rats with hemorrhagic shock in the acute exposure to high altitude,and reduce the degree of renal injury in rats with severe hemorrhagic shock in the acute exposure to high altitude,and the protective effect was more obvious with the increase of crocin dose.The protective mechanism may be through reducing the inflammatory response and oxidative stress level,inhibiting the expression of NF-κB,NLRP3 and caspase-1 proteins in NF-κB-NLRP3 signaling pathway,reducing the inflammatory response,reducing the oxidative factors MPO and MDA level and increasing the activity of GSH and SOD to reduce oxidative stress,thus exerting the renal protective effect.