Deciphering The Mechanism Of Compound Diclofenac Sodium Chlorphenamine Maleate Tablets Induced Acute Kidney Injury Rats Based On Metabolomics

Compound diclofenac sodium chlorphenamine maleate tablets(CDCT),a common cold medicine,was used to relieve headache,fever,cough,and pharyngodynia caused by cold.Hematuria was the most common toxic and side effect of CDCT used in clinical,which limited its application in clinical.Objective:In this study,metabolomics was adpoted to reveal the mechanism of CDCT-induced nephrotoxicity and to provide theoretical basis for clinical use.Methods:(1)In this study,refer to the dosage of CDCT prescription,adopting ACB,DS and CPM replicates the rat hematuria symptom model.The time of hematuria and survival time of rats were observed and recorded.The pathological changes of renal tissue were observed by HE staining.The level of renal function index(BUN and CRE)and inflammatory factors(IL-1β,IL-6 and TNF-α)were detected by biochemical analysis kit.(2)Screening the potential biomarkers in blood and urine of CDCT-induced acute kidney injury rats based on metabolomics.Progensis Qi was used to preprocess the raw data,SIMCA-P software was used for multivariate statistical analysis(PCA,OPLS-DA),HMDB,Metlin and other databases were used for qualitative analysis of variables.Metaboanalyst software was used to draw the pathway of enrichment map.(3)The content of LTB4 was quantitative by ELISA assay based on the potential biomarkers were screened by metabolomics,the level of renal tissue apoptosis by Tunel,the expression level of Bax,Bcl-2,Caspase-3,Caspase-9,p65,IκBα,p-p65,pIκBα protein were detected by Western blot.These proteins were involved in apoptosis and the NF-κB signaling pathway,which were relatived with the characteristic of CL and LTB4.Results:(1)Rats was administered with CDCT,hematuria symptom appear on the third day and died,indicating renal toxicity.The levels of creatinine and urea nitrogen were abnormally elevated indicated kidney function deficit.Renal histopathology showed renal tubules degeneration and necrosis of epithelial cells,dilation of the lumen and glomerular atrophy,occasional hyperemia,accompanied by necrosis and infiltration of inflammatory cells.IL-6 and IL-1 β levels in the medicine group increased significantly,indicating inflammation has happened.(2)The results of PCA and OPLS-DA in serum and urine metabolic profile showed a good separation between the medicie group and the control group;28 metabolites were screened out.Ultimately,cardiolipin and LTB4 as key metabolites of CDCT-induced acute kidney injury were screened and conformed based on VIP,FC,AUC,FDR and P value combined with literature.(3)The pro-apoptotic protein Bax was significantly upregulated in the medicine group,while the anti-apoptotic protein Bcl-2 was significantly down-regulated.With the increase of Cyt-c expression,the expression of caspase-3 and caspase-9 content also increased sharply.In the end,CDCT induces cardiolipin release to interact with Cyt-c,which accelerates its release from mitochondria to the cytoplasm,leading to an increase in pro-apoptotic protein levels.The expression of p-p65 and nuclear p65 was significantly increased,while the expression of IκBα and p-IκBα was down-regulated.In turn,that promotes a cascade of inflamatory response.Conclusion:(1)CDCT can induce acute kidney injury in rats with nephrotoxicity.(2)Cardiolipin and LTB4 are key metabolites of CDCT-induced acute kidney injury.(3)CDCT-induced nephrotoxicity is mediated through apoptosis and the NF-κB signaling pathway.