Study On The Therapeutic Effect Of Glucocorticoid On Acute Kidney Injury In Rats Exposed To Diquat

BackgroundWith the ban of paraquat,the sales volume of diquat pesticides in the herbicide market has increased sharply,making it more widely used in agricultural production in China,and the incidence of acute diquat poisoning incidents has also increased year by year.However,the poisoning mechanism of diquat has not been fully clarified,and the treatment of diquat poisoning has become another challenge and problem faced by the emergency field and even the public health field.ObjectiveThis study designed and carried out an acute toxicity experiment on diquat-exposed rats,in order to preliminarily explore whether glucocorticoids have a protective effect on diquatinduced acute kidney injury.Based on the experimental results and previous research literatures,we will summarize and discuss,in order to provide a strong experimental basis for the research on diquat poisoning,and also hope to provide new ideas and theoretical basis for the basic research of diquat poisoning.MethodsIn this study,150 Wistar rats were randomly divided into 6 groups:DQ group,GC group,Ctrl group,DQ+L-GC group,DQ+M-GC group,DQ+H-GC group,with 25 rats in each group.Each group was further divided into five subgroups,24h,3d,7d,14d,and 21d after exposure,according to the feeding time and the course of treatment,with 5 animals in each subgroup.Rats in DQ group,DQ+L-GC group,DQ+M-GC group and DQ+H-GC group were respectively given 115.5mg/kg diquat plus water for injection diluted to 1ml by one-time gavage.The rats in the Ctrl group and the GC group were given 1 ml of water for injection by one-time gavage.30 minutes after gavage,the rats in the DQ+L-GC group were given 2.1 mg/kg of dexamethasone diluted to 1 ml intraperitoneal injection,and the rats in the DQ+M-GC group were given 4.2 mg/kg dexamethasone diluted to 1 ml by intraperitoneal injection,8.4mg/kg dexamethasone was diluted to 1ml intraperitoneal injection for rats in DQ+H-GC group and GC group.Rats in the Ctrl group and DQ group were intraperitoneally injected with 1 ml of water for injection,and then the rats in each group were intraperitoneally injected with dexamethasone or water for injection as before.After 7 days,the intraperitoneal injection of dexamethasone was changed to 6.3 mg/kg prednisone by intragastric administration,and then 7 days later,it was changed to 3.15 mg/kg prednisone by intragastric administration until the end of the experiment on 21 days.After the start of the experiment,the changes in the conditions of the rats in each group were observed at a fixed time every day,and the changes in the body weight of the rats were monitored at the same time,and the death of the rats was recorded.24h,3d,7d,14d,and 21d after exposure,the rats were sacrificed by intraperitoneal injection of 100 mg/kg sodium pentobarbital overdose.Blood was collected by puncture of the inferior vena cava.After centrifugation,Cr and BUN were measured,and TNF-α,TLR4,MyD88,NF-κB were measured by Elisa.The upper segment of the left kidney was taken for histopathological examination of the kidney.Elisa was used to measure NGAL and KIM-1 in the lower segment of left kidney.The right kidney was used to detect the expression of TLR4,Myd88,IκBα,NF-κB p65 by Western Blot.Results(1)General condition:Most of the rats in the DQ group,DQ+L-GC group,DQ+M-GC group,and DQ+H-GC group developed symptoms such as lethargy,irritability,piloerection,and irritability,and a small number of them showed fatigue.Unsteady walking and reduced eating.After 1-3 days,shortness of breath,cyanosis,oliguria or even anuria gradually appeared.Around 7d,there were more bloody secretions in the nasal cavity and eyes of the rats.Most of the rats also had diarrhea,and the coat color gradually turned yellow after 14 days,and the degree gradually increased with time.After the experiment,the Ctrl group and the GC group were in good mental state,with sensitive activities,quick movements,normal food intake and water intake,and no symptoms of poisoning and related signs.(2)Weight monitoring results:The weight of rats in Ctrl group and GC group increased slowly during the test period(P>0.05).Compared with the Ctrl group,the body weight of the rats in the DQ group,DQ+L-GC group,DQ+M-GC group and DQ+H-GC group continued to decrease after self-injection(P<0.05).Body weight dropped to the lowest point around 7d,and gradually increased from 7d to 21d.Compared with DQ group,there was no significant difference in body weight between DQ+L-GC group,DQ+M-GC group and DQ+H-GC group in the first 7 days(P>0.05).From 7d to 21d,the weight of rats in DQ+L-GC group,DQ+M-GC group and DQ+H-GC group increased(P<0.05).(3)Renal histopathological changes:There was no obvious abnormality in the Ctrl group,and a small amount of capillary congestion in the medulla was observed after 7 days in the GC group,which gradually increased.The DQ group showed tubular atrophy,edema of the epithelium,and over time,the tubules were seen to dilate and become irregular in shape;there was also a large amount of capillary congestion in the renal cortex and medulla.The renal injury in the DQ+L-GC group was less than that in the DQ group.DQ+H-GC group had no obvious injury before 7d,but more renal tubules were seen in DQ+H-GC group from 7d to 14d,with irregular shape,lysis and necrosis of epithelial cells.(4)Kidney tissue damage indicators:BUN content:The serum BUN content of 24hDQ+M-GC group,DQ+H-GC group and 3dDQ+L-GC group,DQ+M-GC group was not significantly different from Ctrl group.+L-GC group,DQ+M-GC group,DQ+H-GC group increased serum BUN(P<0.05).Compared with the DQ group,except for the 14dDQ+M-GC group and the DQ+H-GC group,the serum BUN content of the DQ+L-GC group,DQ+M-GC group and DQ+H-GC group was different at other time nodes.degree of reduction(P<0.05).Serum Cr content:Compared with the Ctrl group,serum Cr in the DQ group,DQ+L-GC group,DQ+M-GC group and DQ+H-GC group increased at each time node(P<0.05).Compared with DQ group,24hDQ+L-GC group;3dDQ+L-GC group,DQ+M-GC group,DQ+H-GC group;7dDQ+M-GC group;14dDQ+M-GC group,DQ+Serum Cr in H-GC group had no significant change compared with DQ group(P>0.05).The serum Cr content in the glucocorticoid treatment group decreased to varying degrees at other time points(P<0.05).NGAL content:Compared with Ctrl group,DQ group,DQ+L-GC group,DQ+M-GC group,DQ+H-GC group increased NGAL content in kidney tissue at each time node(P<0.05).Compared with DQ group,the content of NGAL in kidney tissue of DQ+L-GC group,DQ+M-GC group and DQ+H-GC group was lower than that of DQ group at each time node(P<0.05).KIM-1 content:Compared with the Ctrl group,the KIM-1 content in the kidney tissue of the DQ group,DQ+L-GC group,DQ+M-GC group,and DQ+H-GC group increased at each time node(P<0.05).Compared with the DQ group,the KIM-1 content in the kidney tissue of the DQ+L-GC group,DQ+M-GC group and DQ+H-GC group at each time node was lower than that of the DQ group(P<0.05).(5)Inflammatory factors activate NF-κB pathway related protein expression:TNF-αcontent:Compared with Ctrl group,the content of TNF-α in kidney tissue of DQ group,DQ+LGC group,DQ+M-GC group and DQ+H-GC group increased at each time node(P<0.05).Compared with DQ group,the content of TNF-α in kidney tissue of DQ+L-GC group,DQ+MGC group and DQ+H-GC group was lower than that of DQ group at each time node(P<0.05).TLR4 content:Compared with Ctrl group,the expression of TLR4 in DQ group,DQ+L-GC group,DQ+M-GC group and DQ+H-GC group increased at each time node(P<0.05).Compared with the DQ group,the expression of TLR4 in the kidney tissue of the glucocorticoid treatment group was decreased(P<0.05).MyD88 content:Compared with the Ctrl group,the expression levels of MyD88 in the DQ group,DQ+L-GC group,DQ+M-GC group and DQ+HGC group increased at each time node(P<0.05).Compared with the DQ group,the expression of MyD88 in the kidney tissue of the glucocorticoid treatment group was decreased(P<0.05).NF-κB content:Compared with Ctrl group,the expression of NF-κB in DQ group,DQ+L-GC group,DQ+M-GC group and DQ+H-GC group increased at each time node(P<0.05).Compared with the DQ group,the expression of NF-κB in renal tissue in the glucocorticoid treatment group was decreased(P<0.05).(6)Expression of NF-κB-related proteins in kidney tissue at each time point:Compared with the Ctrl group,the NF-κB pathway-related proteins TLR4,Myd88,IκBα,NF-There was no significant change in the expression of κB p65.The expressions of NF-κB pathway-related proteins TLR4,Myd88,and NF-κB p65 in the kidney tissue of the rats in the DQ group,DQ+LGC group,and DQ+M-GC group were significantly increased,while the expression of IκBαwas significantly decreased(P<0.05)..At 3d and 7d,the expression levels of NF-κB pathway-related proteins TLR4,Myd88,IκBα and NF-κB p65 in the kidney tissue of rats in the GC group did not change significantly.The expressions of NF-κB pathway-related proteins TLR4,Myd88,NF-κB p65 and IκBα were significantly increased in the kidney tissue of the DQ group,DQ+L-GC group,DQ+M-GC group,and DQ+H-GC group.decreased(P<0.05).After diquat exposure,compared with the DQ group,at 24h,3d,7d,14d,and 21d,the renal tissue NF of the rats in the DQ+L-GC group,DQ+M-GC group,and DQ+H-GC group The expressions of-κB pathway related proteins TLR4,Myd88 and NF-κB p65 were decreased,and the difference was not statistically significant(P>0.05).Except for 7dDQ+H-GC group,14d,21dDQ+M-GC group and DQ+H-GC group,the expression of IκBα protein increased(P<0.05).Conclusions(1)Diquat can cause kidney damage in rats,mainly manifested as renal tubular atrophy,epithelial cell edema,capillary congestion and dilation,and the renal function damage indicators have increased in varying degrees.(2)Glucocorticoids have therapeutic effects on acute kidney injury in rats exposed to diquat.During the treatment of acute kidney injury with diquat,the efficacy of glucocorticoids after reaching 4.2 mg/kg did not increase with increasing doses.(3)TLR4 receptor-mediated TLR4/Myd88/NF-κB signaling pathway is involved in the inflammatory response of acute kidney injury in diquat poisoning rats.Glucocorticoids can inhibit the inflammatory response,thereby affecting the expression of TLR4/Myd88/NF-κB signaling pathway-related proteins.