Basic Research Of TCM Incompatibility Of "Zao Ji Sui Yuan Ju Zhan Cao"

This work is one part of the National Basic Research Program of China(973 Program):Basic research of Chinese-medicine-incompatibility theory based on the "Eighteen Incompatible Medicament"(2011CB505300)-"Zao Ji Sui Yuan Ju Zhan Cao" basic research in compatibility relationships and toxicity-efficacy characterization(2011CB505303).The author focuses on the Gancao-Yuanhua incompatible herbal pair,and investigate their biological mechanisms from aspects of gut tissue damage,mucus secresion,gut microbiota and renal Aquaporins;meanwhile the author studied Gancao-Gansui/Jingdaji/Haizao incompatibility mechanisms in gut microbiota.The thesis is composed of four chapters;their contents or main results are as follows:Chapter 1.Literature studyIn this chapter,literatures are reviewed concerning 1)current situation of Traditional Chinese Medicine(TCM)-compatibility-safety research,2)progresses of TCM "Eighteen Incompatible Medicament" research,3)Gancao-Gansui/Jingdaji/Haizao and Gancao-Yuanhua incompatibility characterization and chemical basis.Chapter 2.Biological mechanisms of Gancao-Yuanhua incompatibility based on gut tissue/functional injury and gut microbiotaSection 1.In-vivo study of Gancao-Yuanhua incompatibility mechanisms in gut mechanical barrierPart 1.Impact of Gancao-Yuanhua combination on gut mucosa histopathology.In this part gut barrier function and mucosa tissue injury in normal mice administrated with Yuanhua,Gancao and Gancao-Yuanhua combination are reflected by biomarkers and gut histopathology staining.Results show that Gancao-Yuanhua combination significantly elevate serum diamine oxidase level,rather than Yuanhua or Gancao.Gancao-Yuanhua combination also increase serum lipopolysaccharide level compared to Yuanhua or Gancao.Other evidences suggest Yuanhua or Gancao induces slight duodenum mucosa injury,but does not impact other segments.Differently,Gancao-Yuanhua combination induces slight duodenum mucosa injury and obvious ileum injury,including ileum inflammation filtration,submucosa edema and epithelial cell deletion.Part 2.Influences of Gancao-Yuanhua combination on chemical phenotypes of mice gut mucus layer.Mice gut mucus layer was studied in the aspect of sialo-and sulfo-modification of mucus polysaccharides,which inflect mucus stability and its impact on microenvironment.Study shows that Yuanhua increases the neutral/acidic mucus ratio,and Gancao-Yuanhua combination further increases the ratio.Similarly,Yuanhua and Gancao both increase the sulfomucin/sialomucin ratio,and Gancao-Yuanhua combination more significantly increases this ratio.These changes of mucus chemical phenotypes may damage the stability of mucus and promote the proliferation of sulfate reducting bacteria.Part 3.Mucosal tight junction and mucus secretion related protein changed by Gancao-Yuanhua combination.Molecular mechanisms of mucosa mechanical injury were studied by MUC2 and ZO-1 protein expression.Yuanhua and Gancao have no effect on duodenum MUC2 or ZO-1 protein expression,but Gancao-Yuanhua combination significantly down-regulate duodenum MUC2 and ZO-1 expression,and also up-regulate interleukin-1? and interleukin-6 expression.In colon,Gancao-Yuanhua combination also down-regulate Occludin and ZO-1 expression,up-regulate interleukin-1? and interleukin-6 expression.Section 2.In-vitro study of Gancao-Yuanhua incompatibility mechanisms in gut mechanical barrierThis section studied the effect of Gancao-Yuanhua combination on gut epithelial cell proliferation and migration,as well as MUC2 and ZO-1 protein expression.Yuanhua,Gancao,Gancao-Yuanhua combination,as well as yuanhuacine and glycyrrhetinic acid all have limited effect on IEC-6 cell proliferation or cell migration.In LS174T cells,genkwanin,apigenin,glycyrrhizic acid and glycyronic acid-monoglucuronic acid all dose-dependently decrease MUC2 protein expression;yuanhuacine dose-dependently increase MUC2 protein expression,and its effect can be weakened by glycyrrhizic acid.Glycyrrhizic acid and genkwanin also down-regulate MUC2 gene transcription,and glycyrrhizic acid,glycyrrhetinic acid and yuanhuacine also up-regulate sufo-transferases gene transcription.In IEC-6 cells,genkwanin,apigenin,yuanhuacine,glycyrrhetinic acid and glycyronic acid-monoglucuronic acid all dose-dependently down-regulate ZO-1 protein expression.These data suggest that main components in Yuanhua and Gancao have similar effect on gut epithelial cells,down-regulating tight junction and mucin secretion related protein,which caused gut mechanical barrier damage.Section 3.Study of gut barrier function regulation by Gancao-Yuanhua combination based on cell metabolomics and proteomics.This section studied the metabolic profiles and proteomics of LS174T cells handled with genkwanin and glycyrrhizic acid or their combination,so as to investigate more biological processes through which the two chemicals interact with each other.Data suggests that genkwanin and glycyrrhizic acid both influence the glycerophospholipid metabolism,amino acid metabolism,and nucleoside metabolism.Proteomics study revealed that among the differential expressed proteins after genkwanin or glycyrrhizic acid treatment,about one half of them are shared between the two chemicals.Pathway analysis suggest genkwanin and glycyrrhizic acid both can regulate 8 same pathways among top 20 pathways in each treatment.The shared pathways include virus infection,pathogen infection,tight junction and cell damage etc.,these pathways are all related to gut barrier functons.Section 4.influences of Gancao-Yuanhua combination on gut microbiota and metagenomesThis section employed 16S rDNA sequencing to characterize Gancao-Yuanhua incompatibility in the aspect of gut microbiota and its harmful metabolites.Results showed that Gancao-Yuanhua combination significantly impaired the community structure of mice gut microbiota,compared to Yuanhua or Gancao.Yuanhua,Gancao and Gancao-Yuanhua combination respectively causes 3.2 and 13 differential bacteria genera;Gancao-Yuanhua combination rises the abundance of Desulfovibrio genus to 9 times than that of normal mice,and other Desulfovibrio related genera are also significantly changed.In mice metagenomes,Yuanhua,Gancao or Gancao-Yuanhua combination induces 2306 differential genes which correspond to 341 chemical reactions of pathways.In the top 20 differential reactions induced by Yuanhua,Gancao or Gancao-Yuanhua combination,hydrogen sulfide producing genes are elevated by all the three treatments.Finally,Gancao-Yuanhua combination,rather than Yuanhua or Gancao,significantly increases mice fecal hydrogen sulfide level and decreases mice serum hydrogen sulfide level,demonstrating that metabolic balance of mice gut hydrogen sulfide is broken.Chapter 3.Influences of Gancao-Gansui/Jingdaji/Haizao combination on gut microbiotaThis chapter continues to study the gut microbiota and applies it in characterizing other Gancao related incompatibilities,as revealed in TCM verse of "Zao Ji Sui Yuan Ju Zhan Cao".Further,the author also investigated if there's incompatibility between Chinese herb of Qianjinzi and Gancao in gut microbiota.Results show that although Gansui,Gancao or Gancao-Gansui combination does not cause gut mucosa injury,they still induce significant bacteria changes.Also,Gancao-Gansui combination makes abundance of Desulfovibrio genus rise 10 times compared to normal mice,and the extremely rare genus.Mycoplasma,becomes the most abundant genus of 15.5%.And in the metagenomes of Gancao-Gansui combination treated mice,carbon fixation pathway,energy metabolism pathway,ubiquinone and other terpenoid-quinone bosythesis are down-regulated;pentose phosphate pathway,phosphoteansferase system,glycerolipid metabolism are up-regulated.What's more,Gancao-Gansui combination treatment increases mice fecal hydrogen sulfide and cholesterol level,suggesting that gut microbiota,metagenomes and hydrogen sulfide/cholesterol metabolisms are synchronously disturbed.Gancao-Jingdaji and Gancao-Haizao combination both significantly change microbiota community structure,and interrupt their metabolisms.Gancao-Jingdaji combination increases ammonia/hydrogen sulfide-producing bacteria,and Mycoplasma genus.Addingly,Gancao-Jingdaji combination increases sporulation and lipid metabolism related genes,decreases tricarboxylic acid cycle related genes.Gancao-Haizao combination also increases Mycoplasma genus abundance,disturbs fructose metabolism,fatty acid metabolism and selenocompound metabolism functions.The combination of Gancao and Euphorbia factor LI in Qianjinzi exerts harmful effect on gut microbiota community,increases the genes related to phenylalanine and tryptophan metabolism,leucine metabolism,methane metabolism,sphingolipid metabolism,and chorismic acid;decreases protein repair and carbohydrate fermentation related genes.This changes may cause more harmful metabolites and less short chain fatty acid,against the stability of gut microbiota and microenvironment.Chapter 4.Biological mechanisms of Gancao-Yuanhua incompatibility based on water channel proteins(Aquaporins,AQPs)Section 1.Diuretic effect evaluation of Gancao-Yuanhua combinationIntraperitoneal water-loading mice model was used to test the diuretic effect of Yuanhua,Gancao and Gancao-Yuanhua combination,meanwhile study the key factors in urine production.Results show that within the pharmacopeia dose limits,Yuanhua has significant diuretic effect while Gancao has no effect;Gancao-Yuanhua combination shows weakened diuretic effect compared to Yuanhua.In this process,none of Yuanhua,Gancao,Gancao-Yuanhua combination influences glomerular filtration rate.Yuanhua significantly reduces urine concentrating rate,resulted in diluted urine,while Gancao or Gancao-Yuanhua doesn't.Yuanhua does not impact the reabsorption of urine electrolyte of Na+,K+ or Cl-,as well as arginine vasopressin and aldosterone.Gancao and Gancao-Yuanhua combination both induce K+ loss.Section 2.AQPs protein regulation effect of Gancao-Yuanhua combination.Based on water-loading mice model,renal AQPs expression level was studied by Western Blot experiments.Among the four main AQP subtypes of AQP 1,2,3 and 4,Yuanhua only significantly down-regulate AQP 2 protein level;Gancao oppositely up-regulate renal AQP 2 regulation,and Gancao-Yuanhua combination have no effect on AQP 2.Besides,none of Yuanhua,Gancao or Gancao-Yuanhua combination influences renal AQP 1,AQP 3 or AQP 4,as well as phosphorylated AQP 2(including p256-AQP 2,p261-AQP 2 and p269-AQP 2)and arginine vasopressin receptor AVPV2R.Further study reveals that yuanhuacine and genkwanin in Yuanhua both dose-dependently down-regulate AQP 2 protein expression in collecting duct cells,while glycyrrhetinic acid in Gancao dose-dependently up-regulate AQP 2 protein expression in collecting duct cells;and glycyrrhetinic acid weakens the effect of yuanhuacine and genkwainin.In-vivo study also suggests the diuretic effect of yuanhuacine and genkwanin and their regulation effects on renal AQP 2;and glycyrrhizic acid reduces the effect of yuanhuacine and genkwanin in diuresis and AQP 2 regulation.Section 3.Study of AQP 2 regulation mechanisms based on network pharmacology and molecular docking.Further study employed the method of network pharmacology and molecular docking to investigate the molecular mechanisms of AQP 2 regulation by yuanhuacine,genkwanin and glycyrrhetinic acid.Results show that yuanhuacine,genkwanin and glycyrrhetinic acid totally share 30 protein target and 49 pathways;and among the 11 target linked to glycyrrhetinic acid,9 targets are also linked to yuanhuacine or genkwanin.Among all the 49 pathways,MAPK pathway and corresponding target of MEK1 and FGFR1 are most significantly related to yuanhuacine,genkwanin and glycyrrhetinic acid,and MAPK pathway also participated in AQP 2 regulation.Molecular docking further validates the interactions between these chemicals and MEK1 or FGFR1 proteins.Following experiments on collecting duct cells finally confirmed that yuanhuacine,genkwanin or glycyrrhetinic acid regulate AQP 2 expression through changing p-ERK and p-CREB levels in MAPK pathway.