| Cyclophosphamide,a broad-spectrum chemotherapeutic agent,is widely used in the treatment of many malignant tumors,but severe side effects such as hepatotoxicity limit its clinical application.According to diagnostic theory of Traditional Chinese Medicine,the adverse symptoms of patients treated by cyclophosphamide could be classified as asthenia syndrome.Therefore,cyclophosphamide is often used together with ginseng by doctors of integrated Chinese and Western Medicine in clinical treatment,beneficial to alleviate cyclophosphamide-induced hepatotoxicity.However,the specific mechanisms remain unexplored.This study aimed to discuss the potential mechanisms involved in intestinal bacteria of ginseng to alleviate the hepatotoxicity of cyclophosphamide at the animal level,mainly by evaluation of hepatotoxicity with classical pharmacodynamics and pathology,combined with the microbiology method based on 16S rRNA gene sequencing technology to characterize intestinal bacteria,UPLC-QTOF-MS/MS-based untargeted metabolomic method and GC-TQ-MS/MS-based targeted quantitative technology to analyze intestinal and hepatic metabolites and the proteomic method based on Tandem Mass Tag technology to measure hepatic proteins.This will benefit clarify the scientific connotation of the combined application of ginseng and cyclophosphamide,provide a theoretical basis for its clinical application and new ideas for innovative research on the combined use of Chinese and Western medicines.The research could be divided into the following parts:1.Preparation and quality characterization of ginseng decoctionAccording to the clinical scheme,ginseng decoction was prepared.And,its quality control was carried out by UPLC-QTOF-MS/MS with 16 kinds of main ginsenosides as representative components,to ensure the repeatability and controllability of experimental results.The results showed that ginsenosides with large polarity,such as ginsenoside Re,Rg1,Rb1,Ro and Rd,were dominant in ginseng decoction.But,the contents of ginsenosides with small polarity,such as ginsenoside 20(R)-Rh1,F1 and CK,were rare.2.The alleviation of cyclophosphamide-induced hepatotoxicity by ginsengMale Wistar rats were divided into control group,cyclophosphamide group,cyclophosphamide combined with ginseng low and high-dose groups.Serum biochemical indexes and hepatic pathological slices of each group were compared to evaluate the effect of ginseng on the hepatotoxicity of cyclophosphamide.The results showed that ginseng can significantly alleviate cyclophosphamide-induced hepatotoxicity,mainly by decreasing the levels of serum alanine transferase(ALT)and aspartate transferase(AST)as well as improving the injury of hepatocytes.3.The improvement of cyclophosphamide-induced intestinal dysbacteriosis by ginsengThe composition of intestinal bacteria was characterized by 16S rRNA gene sequencing technology,and its diversity and potential physiological function in each group were further analyzed by bioinformatics.The results showed that ginseng can significantly improve the intestinal dysbacteriosis induced by cyclophosphamide,mainly by increasing the diversity level of bacterial communities and the relative abundance of Bacteroides spp.,Bifidobacterium spp.and Faecalibaculum spp.Besides,energy metabolism,lipid metabolism,and amino acid metabolism participated in intestinal bacteria were also regulated.4.The amelioration of cyclophosphamide-induced intestinal metabolic disorders by ginsengEndogenous metabolites and short-chain fatty acids in the intestine of rats were analyzed by UPLC-QTOF-MS/MS-based untargeted metabolomic method and GC-TQ-MS/MS-based targeted quantitative technology,respectively.The results showed that ginseng can significantly ameliorate intestinal metabolic disorders caused by cyclophosphamide.It was mainly manifested in the callback of the relative abundance of 25 potential biomarkers such as a-dimorphic acid,13S-hydroxyoctadecadienoic acid and tetradecyl carnitine,which mainly related to linoleic acid metabolism and a-linolenic acid metabolism.Besides,the total contents of short-chain fatty acids in the intestine were increased,specifical for propionic acid and butyric acid.5.The amelioration of cyclophosphamide-induced hepatic metabolic disorders by ginsengEndogenous metabolites and short-chain fatty acids in the liver of rats were analyzed by UPLC-QTOF-MS/MS-based untargeted metabolomic method and GC-TQ-MS/MS-based targeted quantitative technology,respectively.The results showed that ginseng can significantly ameliorate the hepatic metabolic disorders caused by cyclophosphamide.It was mainly manifested in the callback of the relative abundance of 17 potential biomarkers such as Taurodeoxycholic acid,tauroursodeoxycholic acid,phospholipids and lysophospholipids,which mainly related to glycerophospholipid metabolism,linoleic acid metabolism and a-linolenic acid metabolism.Besides,the contents of hepatic propionic acid and butyric acid were conspicuous increased.6.The adjustment of cyclophosphamide-induced unbalanced expression of hepatic proteins by ginsengThe expression of hepatic proteins in each group was evaluated by proteomic method based on Tandem Mass Tag technology,and differential proteins and biological process involved were further selected and analyzed by bioinformatics.The results showed that ginseng can significantly adjust the unbalanced expression of hepatic proteins caused by cyclophosphamide.It was mainly manifested in the callback of glutathione S transferase,cytochrome C oxidase and bradykinin.These differential proteins were mainly related to linoleic acid metabolism,NF-κB signaling pathway and cholesterol biosynthesis.7.The enhancement of ginseng intestinal metabolism in the case of cyclophosphamide treatmentHealthy rats were divided into control and cyclophosphamide groups.The intestinal metabolic profile of the two groups administrated with ginseng decoction was analyzed by UPLC-QTOF-MS/MS-based metabolomic method,and the differential metabolic markers of ginseng were also screened and identified.The results show that the intestinal metabolism of ginseng was enhanced in the case of cyclophosphamide treatment,mainly by increasing the relative contents of ginsenosides metabolites,including ginsenoside F2,Rg3,Rd,Rh1,Rg1,CK,Rg2 and S-PPT.In conclusion,ginseng can significantly alleviate the hepatotoxicity caused by cyclophosphamide,and mainly by improving the relative abundance of producing short-chain fatty acid bacteria such as Bifidobacterium spp.and Faecalibaculum spp.,increasing the contents of intestinal a-dimorphic acid,13 S-hydroxyoctadecadienoic acid and hepatic propionic acid,butyric acid as well as enhancing the expression of hepatic anti-inflammatory proteins such as glutathione S transferase and cytochrome C oxidase.Besides,the intestinal metabolism of ginseng was enhanced in the case of cyclophosphamide treatment,and increasing the relative contents of secondary ginsenosides such as Rg3,Rh1 and CK maybe more conducive to hepatoprotective effect. |
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