| Chapter 1.Literature studyThis chapter mainly explains the definition,staging and harm of chronic kidney disease(CKD),summarizes the relationship between enterogenic uremic toxins and the progression of CKD;summarizes the sources of various enterogenic uremic toxins,their synthesis in the body and the damage to the body;analyzes the relationship between enterotoxin and intestinal flora;sorts out a variety of treatment methods to interfere with the production,transport and excretion of enterogenic uremic toxins;summarizes the research status of Huang-Kui-Si-Wu Formula(HKSWF)in the treatment of CKD,provides a theoretical basis for later exploration of the effect of HKSWF on the production and transport of nterogenic uremic toxins.Chapter 2.Regulation and mechanism of HKSWF on gut microbiota-mediated uremic toxin metabolism pathwayHKSWF is composed of four traditional Chinese medicines of abelmoschus manihot,astragalus mongholicus,polygonum cuspidatum and curcumae longae.Previous studies have found that HKSWF could significantly reduce kidney inflammatory cell infiltration,glomerular basement membrane deposition,and the degree of renal fibrosis in CKD model rats;Meanwhile,it also could significantly inhibit reduce the accumulation of uremic toxin p-cresyl sulfate(PCS)in plasma,liver and kidneys of CKD model rats,Additionally,it also significantly inhibited the synthesis of p-cresol in the intestine,suggesting that the effect of HKSWF on protecting CKD may be related to its inhibition of p-cresol synthesis in the intestinal flora and the reduction of p-cresol sulfate in the body.This chapter is mainly based on the overall animal level and anaerobic culture of intestinal bacteria in vitro to explore the regulation of HKSWF on the structure of intestinal flora of model animals,and further observe the effect of HKSWF on the synthesis of p-cresol by intestinal bacterial.The results showed that HKSWF had a small regulation effect on the intestinal flora of CKD rats,but did not show significant differences,suggesting that HKSWF inhibited PC production not by reducing the abundance of harmful bacteria,may be by inhibiting the synthesis of p-cresol.the intestinal flora can metabolize food-sourced tyrosine to produce p-cresol in the gut.In order to clarify the effect of HKSWF on the synthesis of p-cresol by intestinal flora,an in vitro anaerobic culture system and the method to detect p-cresol were established.Since HPLC-FLD could hardly detect p-cresol produced by normal intestinal bacteria,tyrosine was added to promote the synthesis of p-cresol by intestinal bacteria.On this basis,after adding HKSWF,results showed that HKSWF could significantly inhibit the production of p-cresol in gut bacteria in vitro,and its inhibitory capacity was dose-dependent.In order to explore the mechanism by which HKSWF inhibited the production of p-cresol by intestinal flora,we supplemented and improved the tyrosine metabolism pathway in the intestinal flora through literature research.Specifically,the catabolism of Tyr could be classified into two pathways,the oxidative pathway and the reductive pathway.In the oxidative pathway,HPPA,a primary metabolite of Tyr,was transformed to p-hydroxyphenylacetate(PHA)by p-hydroxyphenylpyruvate oxidase,and PHA is further metabolized to p-cresol by p-hydroxyphenylacetate decarboxylase.In the reductive pathway,HPPA is further metabolized to generate p-hydroxyphenyllactic acid(PPA)and then p-hydroxyphenylpropionic acid(PHPA).Based on this metabolic pathway,we co-cultured tyrosine or its primary metabolite HPPA with intestinal bacteria.The result showed that HKSWF could inhibit PC production by switching tyrosine metabolism from oxidative pathway to reductive pathway.Next,HKSWF could directly inhibit the oxidative pathway of tyrosine and reduce the decomposition of PHA,thereby inhibiting PC production,when PHA,a p-cresol precursor in the oxidation pathway,was co-cultured with intestinal bacteria.In order to clarify which components in HKSWF inhibited the synthesis of p-cresol in iltestinal bacterial,the total extract of four herbs were co-cultured with intestinal bacteria.The results showed that the total extract of four herbs could inhibit the p-cresol production.The total extract was composed of the alcohol-soluble components and the water-soluble polysaccharide components of each herb.Therefore,we further investigated the effects of the alcohol-soluble components and the water-soluble polysaccharide components of one herb on p-cresol production in intestinal bacterial.In order to study effect of the alcohol-soluble components of each herb on the p-cresol production in intestinal bacterial,the alcohol-soluble parts of each drug were co-cultured with intestinal bacteria.The results showed that the alcohol-soluble parts of Abelmoschus manihot,Polygonum cuspidatum and Curcumae longae could significantly inhibit the p-cresol production.In order to confirm which components in the alcohol-soluble parts of Abelmoschus manihot,Polygonum cuspidatum and Curcumae longae alcohol solution played a role in inhibiting the production of p-cresol,we screened out the eight main alcohol-soluble compounds in HKSWF and co-cultured them with intestinal bacteria.The results found that Isoquercitrin,Hyperoside,Quercetin,Curcumin,Emodin,polydatin and Resveratrol could significantly inhibit the metabolism of PHA by intestinal bacteria,thereby inhibiting the production of p-cresol.Among them,p-hydroxyphenylacetate decarboxylase is a key enzyme for intestinal bacteria to convert PHA to p-cresol.Therefore,through molecular docking technology,we further discovered that this seven compounds also have strong binding ability with p-hydroxyphenylacetate decarboxylase,which can be used as an inhibitor of the enzyme.The main component of the water-soluble parts of Chinese medicines is polysaccharides,and the Chinese medicine polysaccharides have the effect of regulating and protecting intestinal and kidney diseases.Therefore,the effects of each herb the water-soluble polysaccharide components on p-cresol production in intestinal bacterial were studied.The results showed that the water-soluble polysaccharide components of Astragalus mongholicus and Polygonum cuspidatun could significantly inhibit the production of p-cresol,and the inhibition rate of Astragalus mongholicus was close to 100%.Considering that the polysaccharide of single herb had a significant inhibitory effect on p-cresol production,and Astragalus mongholicus is a dual-use traditional Chinese medicine,we proposed whether food-derived polysaccharides also could inhibit the production of p-cresol.Finally,food-derived fructo-oligosaccharide(FOS)was selected for subsequent experimental research.The results found that FOS could significantly inhibit the production of p-cresol in intestinal bacteria.In addition,we found that FOS could decrease PC production through modulating gut microbiota mediated aromatic amino acid metabolism pathway.Specifically:1.FOS greatly promoted the conversion of Tyr metabolism from the oxidative pathway to the reduction pathway;2.FOS promoted the bioavailability of Tyr by promoting the proliferation of intestinal bacteria;3.FOS could also inhibit PC production by directly blocking the transformation of PHA to PC in the oxidative pathway.Chapter 3.HKSWF regulates host cell-mediated uremic toxin metabolic pathway and its mechanismThe previous research found that HKSWF could interfere with the process of p-cresol synthesis by intestinal bacteria in the PCS synthesis process.Based on this,this chapter used CKD animals to investigate the effects of HKSWF on the conversion of p-cresol to PCS and the transport of PCS to the kidney.The results of UPLC-TQ/MS detection showed that HKSWF could significantly inhibit the accumulation of PCS in the liver,and also showed a certain decrease in PCS levels in plasma and kidney,indicating that HKSWF could interfere with the accumulation of uremic toxin molecule PCS in host cells.In order to clarify the effect of HKSWF on the conversion of p-cresol to PCS in the synthesis of PCS,corresponding research experiments were made for its synthetic pathway.Mice were treated with HKSWF for 28 days,and were administrated with p-cresol on the last day to promote the PCS production in vivo.UPLC-TQ/MS was used to detect the PCS levels in the plasma,colon and liver.The results showed that HKSWF could significantly inhibit the accumulation of PCS in the plasma of mice,and had a tendency to inhibit the PCS content in the liver,but it did not affect the PCS level in the intestinal tract.When examining the enzyme activity and protein expression of SULT1A1 in the colon and liver,it was found that HKSWF did not affect the SULTlAlexpression in the intestine;it also did not affect the SULT1A1 enzyme activity in the liver;but HKSWF could significantly inhibit SULTIAI mRNA/protein expression levels in the liver.The above results indicated that HKSWF could significantly inhibit the expression of SULTIA1I thereby inhibiting the conversion of p-cresol to PCS,and ultimately inhibiting the PCS production in the body.In order to clarify the effect of HKSWF on the transport of PCS to renal cells via the kidney transporter OAT1/3,UPLC-TQ/MS was used to detect the PCS levels in plasma and kidney of mice injected with PCS.It was found that HKSWF did not affect PCS levels in plasma and kidneys of mice.We re-verified in normal rats.The results indicaded that HKSWF did not affect the PCS levels in the plasma and kidneys of rats,and also did not affect the expression of OAT 1/3 in the kidney.It was also found in CKD rats that HKSWF did not affect the expression of OAT 1/3 in the kidneys of model rats.The above results indicated that HKSWF inhibited the PCS accumulation in the kidney is not achieved by affecting the transport of PCS into the kidney.The above research showed that HKSWF could reduce PCS accumulation in vivo by regulating the intestinal flora-mediated p-cresol metabolic pathway and interfering with the expression of specific metabolic enzymes in the liver. |
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