| Objective: Cyclosporin A Cyclosporine A,CsA)is a powerful and effective immunosuppressive agent commonly used clinically.It is commonly used in immune diseases and human organ transplantation.However,with the extensive use of CsA,its toxic and side effects have become increasingly apparent.The toxic effects of major organs including kidneys and liver have been reported successively.However,after clinical organ transplantation,the operator needs to take a high dose of cyclosporine A for a long time to resist immune rejection,which provides an induction condition for the occurrence of drug-induced toxicity.Although the clinical use of CsA in patients is accompanied by monitoring of blood drug concentration,the side effects of damage can be controlled only by reducing the dose of CsA and affecting the patient's postoperative recovery and quality of life.This study intends to explore biomarkers in vivo of CsA induced hepatic and renal toxicity by metabolomics techniques,discovering the metabolic pathways associated with the toxic effects of CsA,and combining current preclinical conditions for the use of schisandrin preparations in clinical practice and the study of Schisandra in our previous researches to clarify the mechanism of ethanol extract of schisandra chinensis(SCE)on the protective mechanism of liver and kidney toxicity of cyclosporine A,and provided a reference for rational application of CsA.Methods: 1.Establishment of Fingerprint of SCE By comparing the chromatograms at different mobile phase systems,column temperatures,flow rates,and detection wavelengths,the chromatographic conditions were optimized and determined;and the chromatograms of the 10 batches of SCE were evaluated using a Chinese herbal fingerprint chromatographic fingerprint similarity evaluation system to establish the SCE HPLC fingerprint.2.Metabonomics study of hepatic and renal toxicity induced by CsA in rats SD rats were treated 50 mg/kg·d CsA for 1 day,3 days,and 7 days,respectively,to observe the effects of CsA on the rats in each group.Serum glutamic-pyruvic transaminase(ALT)and aspartate aminotransferase(AST)levels were measured in rats in each group to assess the level of liver function;serum creatinine(SCr)and urea nitrogen(BUN)were measured in rats in each group to evaluate renal function in rats.Level.Rats liver and kidney tissues were stained with HE to observe histopathological damage.In 24 hours before sacrifice,the urine of each group of rats was collected for metabolomic analysis.LC-MS/MS was used to collect metabolic ion information in urine.The mass spectrometry data were analyzed by XCMS software.Metabolite identification and metabolic pathway analysis were based on the database KEGG(http://www.kegg.jp/).3.Mechanism of protective effect of SCE on hepatic and renal toxicity induced by CsA in rats On the basis of the second chapter,this study added CsA treatment for 14 days to verify the effect of SCE on CsA hepatotoxicity and nephrotoxicity.50 mg/kg·d CsA was given to SD rats,and 216 mg/kg·d of Schisandra communis was administered for 3 days,7 days,and 14 days,respectively.Each group of rats affected.Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured in rats in each group to evaluate the rat liver function;serum creatinine(SCr)and urea nitrogen(BUN)were measured in each group for evaluation.Rat kidney function level;Rat liver and kidney tissue were stained with HE to observe histopathological damage;Malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),and glutathione(GSH)were detected in liver and kidney tissues to evaluate the level of oxidative stress in rats;Western Blot was used to detect the expression levels of 4HNE,Nrf2,HO-1,NQO1,GCLM,Casepase-3,Bax and Bcl-2 in rat liver and kidney.Results: 1.A HPLC fingerprint of SCE was established.The liquid chromatography chromatograms of 10 batches of SCE indicated 15 common peaks,and four common peaks were identified,which are Schisandrol A,Schisantherin A,Schizandrin A,and Schizandrin B.The similarity of 10 batches of SCE was above 0.942.2.CsA 50 mg/kg·d treated for 1 day,3 days,and 7 days,respectively,and the traditional hepatic and nephrotoxicity index indicated that the liver function(ALT,AST)and the renal function(SCR,BUN)have no signifiacat change observed in the rats of the 1 day group,compared to Vehicle groups.Significant differences occurred in liver and kidney function were observed in the 3 and 7 days group.At the same time,pathological results showed that there was no change in the liver and kidney tissues in the 1 day group,but inflammatory cell infiltration was observed in the 3 days group.Obvious pathological changes were observed to indicate the formation of liver and kidney toxicity in 7 days group.Urine from each rat was collected for 24 hours,and the collected urine was analyzed for metabolites using LCMS/MS.After LC-MS/MS detection,a total of 11,555 identified ions were obtained by analyzing mass spectrometry data.After standard deviation analysis(RSD ? 30%),7657 differential ions were screened out,of which 3469 were in the 1st day group,including 2532 up-regulated ions and 937 down-regulated ions;3403 in the 3rd day group,containing 2360 up-regulated ions and 1043 One down-regulated ions;5393 ions in the 7-day group,containing 2283 up-regulated ions and 3110 down-regulated ions.Using the analysis of multiples of differences and t-tests to analyze data from the threeexperimental groups compared with the blank group,697 common metabolites were obtained.In 697 identified differential metabolites,15 metabolites were selected as possible metabolites by KEGG enrichment analysis and HMDB enrichment analysis,including Uric acid ? Serine phosphoethanolamine?D-4'-Phosphopantothenate?d CDP?L-Diiodotyrosine?Citicoline?d ATP ? d ITP ? UDP-N-acetylmuramic acid ? Amino Aspartate ? d UDP ? CMP-2-aminoethylphosphonate?CDP-ethanolamine?Adenylosuccinic acid?NADP.Metabolic pathways involved in 15 metabolic markers include pyrimidine metabolism,purine metabolism,amino acid metabolism,glycerophospholipid metabolism,and glutathione metabolism.3.To verify the effects of SCE on liver and kidney toxicity induced by CsA,rats were given CsA 50 mg/kg·day for 3 days,7 days,and 14 days,respectively,and 216 mg/kg SCE was given at the same time as CsA administration,of which the hepatic and renal toxicity of rats given CsA alone increased with time,but the liver and kidney toxicity of rats given CsA and SCE was significantly reduced,manifested in liver and kidney function and pathology.In the 14-day group experiment,under the condition that CsA can precisely cause the pathological changes of liver and kidney,SCE can significantly delay the pathological changes of liver and kidney tissues.At the same time SCE can improve the level of MDA,SOD,CAT,GSH in liver and kidney.Western Blot results showed that SCE can upregulate the expression levels of Nrf2,NQO1,HO-1,GCLM,and Bcl-2,and down-regulate expression levels of Casepase-3,Bax in tissues.Conclusion: In this study,the fingerprint of SCE was successfully established,which can guarantee the extraction method and quality control of fructus schisandra chinensis alcohol extract.Using the method of LC-MS /MS metabolomics,the biomarkers of CsA-induced hepatorenal toxicity in rats were successfully screened out,providing a reliable basis for the diagnosis of early hepatic and renal toxicity of CsA.The protective mechanism of SCE on hepatic and renal toxicity induced by rat CsA may be realized by activating Nrf2 signaling pathway to regulate oxidative stress and inhibit the occurrence of apoptosis of tissue cells. |
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