| Purpose:Toxic herbal medicines can be reduced and increased in efficacy by pairing.Based on the chemical composition,effectiveness and toxicity of Radix et Rhizoma Glycyrrhizae before and after the combination,the project team showed that Radix et Rhizoma Glycyrrhizae with Glycyrrhizae intervened in rats with nephrotic syndrome and showed sound toxicity reduction and efficacy enhancement,identified biomarkers related to bile acid metabolism through urine metabolomics,suggesting that the mechanism of action of Radix et Rhizoma Glycyrrhizae combination in rats with nephrotic syndrome is related to bile acid metabolism.In this study,we established a rat model of nephrotic syndrome and analyzed the changes of short-chain fatty acids and bile acids in faeces and serum by cross of Radix et Rhizoma-Glycyrrhiza glabra,and explored the mechanism of toxicity reduction and potency of the combination.Material and method:1.After 60 SD rats were acclimatized in SPF level environment for one week,ten rats were taken as a blank group,and the rest rats were injected with adriamycin at a dose of 6.5 mg/kg in a single tail vein to induce nephrotic syndrome model,and urine protein was collected in a metabolic cage for 24 h on the 14 th day after modelling to determine the establishment of the nephrotic model.The rats were randomly divided into the model group,thundergongteng administration group(TPW group),thundergongteng with licorice administration group(TPW-GLY group),Chinese medicine positive control group and western medicine positive control group,and 10 rats in each group were administered with 240 mg/kg of thundergongteng extract by gavage in the thundergongteng administration group and 240mg/kg of thundergongteng extract with licorice administration group by gavage in the thundergongteng administration group.The rats in the blank and model groups were given saline in a volume of 10 m L/kg by gavage once daily for 14 d.During the experiment,body weight,diet and water intake were recorded daily.During the experiment,the weight,diet,water consumption,urine and faeces were recorded every day,and after the last administration,the faeces and serum were collected and stored at-80℃.2.Using metabolomic methods,we investigated the effects of Radix et Rhizoma Glycyrrhizae on endogenous substances in serum and feces of rats with nephrotic syndrome,and applied UPLC-Q/TOF-MS to identify the metabolite profiles of serum samples and fecal samples,combined with PCA and OPLS-DA for pattern recognition of the metabolite fingerprints of the samples,to screen and identify Radix et Rhizoma with Glycyrrhizae The biomarkers of intervention in nephrotic syndrome and the related characteristics of metabolomics were identified.3.To establish a quantitative method for the analysis of short-chain fatty acids such as acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and hexanoic acid in faeces by GC technique,including: selectivity,standard curve,accuracy and precision,extraction recovery and stability.To quantify the short-chain fatty acids such as acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and hexanoic acid in rat feces,and to investigate the effect of the administration of Radix et Rhizoma-Glycyrrhizae in combination on intestinal short-chain fatty acids.4.The quantitative analysis of bile acids such as CA,DCA,CDCA,UDCA,GCA,GCDCA,TCA,LCA,TCDCA in serum was established by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS)technique,including: selectivity,standard curve,accuracy and precision,extraction recovery,matrix effect,stability.The bile acid components of CA,DCA,CDCA,UDCA,GCA,GCDCA,TCA,LCA and TCDCA in serum and faeces of rats were quantitatively analyzed to investigate the effects of Ragweed-Glycyrrhiza glabra combination administration on bile acids in serum and feces of rats with nephrotic syndrome.Results:1.The metabolic profiles of rat sera were significantly different.24 biomarkers with significant differences were identified between the blank and model groups,21 biomarkers were identified and back-regulated in the TPW-GLY group,and 15 biomarkers were back-regulated in the TPW group.Seven endogenous biomarkers were finally screened and identified,namely lysophosphatidylcholine(22:6 [4Z,7Z,10 Z,13Z,16 Z,19Z]),phosphatidylethanolamine(24:1 [15Z]/15:0),oleamide,arachidonic acid,linoleic acid,palmitic acid,and oleic acid.2.The metabolic profiles of rat feces were significantly different.48 biomarkers with significant differences were identified between the blank and model groups,25 biomarkers were identified and back-regulated in the TPW-GLY group,and 24 biomarkers wereback-regulated in the TPW group.Six endogenous biomarkers were finally screened and identified,namely protoporphyrin IX,docosahexaenoic acid,prostaglandin A1,5-hydroxytryptamine,7-dehydrocholesterol,and allocholinic acid.3.The methodological validation results of the GC method for the determination of short-chain fatty acids in feces showed that the separation of the analytes to be measured and the internal standard met the requirements,and the method selectivity was good;the linearity was good in the range of 1-50 mg/m L for acetic acid,0.6-30 mg/m L for propionic acid,isobutyric acid and butyric acid,and 0.4-20 mg/m L for isovaleric acid,valeric acid and capric acid;the intra-and inter-batch accuracy was within ± 15 % of the indicated value,and the intra-and inter-batch precision was not more than 15 %.The intra-and inter-batch accuracy was within ± 15 % of the indicated value,and the intra-and inter-batch precision was not more than 15 %.The levels of acetic acid in the feces of rats with nephrotic syndrome increased,and the administration of TPW-GLY and TPW decreased the levels of acetic acid;the levels of propionic acid increased,and the administration of TPW-GLY decreased the levels of butyric acid;the levels of butyric acid and valeric acid increased,and the administration of TPW-GLY and TPW decreased the levels of butyric acid,and the administration of TPW-GLY decreased the levels of valeric acid.The level of butyric acid was reduced by TPW-GLY and TPW administration,and the level of valeric acid was reduced by TPW-GLY administration.4.The methodological validation results of the LC-MS method for the determination of bile acids in serum showed that: the separation of the analytes and the internal standard met the requirements,the endogenous substances in serum did not interfere with the determination of the analytes and the internal standard,and the method selectivity was good;the linearity of GCA,TCA,TCDCA was within 10-10000 ng/m L,GDCA was within 40-40000 ng/m L,UDCA,CA,LCA,CDCA The linearity of UDCA,CA,LCA,CDCA and TCDCA was within the linear range of 20-2000 ng/m L with good linearity;the intra-and inter-batch accuracy was within ±15% of the indicated values,and the intra-and inter-batch precision was not more than 15 %;the extraction recovery,matrix effect and stability all met the requirements.In the nephrotic syndrome model rats,the level of CA in serum was elevated,and TPW and TPW-GLY could significantly decrease the level of CA in serum;the level of LCA in serum of model rats tended to increase,and the administration of TPW and TPW-GLY could decrease the level of LCA in serum;the level of DCA in serum of model rats was elevated,and the administration of TPW and TPW-GLY could decrease the level of DCA levels were increased in the serum of model rats,and TPW and TPW-GLY administration decreased thelevels of DCA.In the model of nephrotic syndrome,the level of GCDCA in the feces of rats was significantly increased,and TPW and TPW-GLY both reduced the level of GCDCA significantly;the level of UDCA in the feces of model rats was significantly increased,and the administration of TPW-GLY reduced the level of UDCA significantly;the level of CA in the feces of model rats was significantly increased,and the administration of TPW-GLY reduced the level of CA significantly;the level of CA in the feces of model rats was significantly increased,and the administration of TPW-GLY reduced the level of CA significantly.The level of CDCA in the feces of model rats was significantly increased,and TPW-GLY administration significantly decreased the level of CDCA;the level of LCA in the feces of model rats was significantly increased,and TPW and TPW-GLY administration significantly decreased the level of LCA;the level of DCA in the feces of model rats was significantly increased,and TPW and TPW-GLY administration significantly decreased the level of DCA;the level of DCA in the feces of model rats was significantly increased,and TPW and TPW-GLY administration significantly decreased the level of DCA.The level of DCA in the feces of model rats was significantly increased,and the level of DCA was significantly decreased by TPW and TPW-GLY administration.Conclusion:1.In the intervention of Radix et Rhizoma Glycyrrhizae in nephrotic syndrome,the TPW-GLY group may improve phospholipid metabolism,fatty acid biosynthesis and metabolism,arachidonic acid metabolism,linoleic acid metabolism,tryptophan metabolism,PPAR signaling pathway,unsaturated fatty acid biosynthesis and metabolism,steroid biology,glycerophospholipids in rats with nephrotic syndrome by comparing with TPW group.Metabolism and other processes,more significant and play a role in reducing toxicity and increasing efficiency.2.In the intervention of nephrotic syndrome with Radix et Rhizoma Glycyrrhizae,by comparing the TPW-GLY group with the TPW group,the TPW-GLY group was more significant and exerted the effect of toxicity reduction and potentiation in regulating the levels of intestinal acetic acid,propionic acid,butyric acid and valeric acid,and the levels of CA,DCA and LCA in serum and GCDCA,UDCA,CA,CDCA,LCA and DCA in feces. |
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