| Chapter 1.Literature studyIn this chapter,literatures are reviecied the definition,staging and the harm of the chronic kidney disease(CKD);the relationship between the intestinal microecological disorder and the progress of the CKD;the origin,synthesis and metabolism of enterogenic uremic toxins and their precursors,their important role in the progression of CKD and their research progress;the progress of targeted intestinal microbe or enterotoxin in the treatment of CKD;the pharmacological action and research status ofAbelmoschus Manihot(HK)and its main flavonoids.Chapter 2.Effect of HK on uremic toxins production in model animals.The disorder of intestinal microecology is closely related to the progression of CKD.Previous studies found that the bioavailability of flavonoid glycosides,the main active component of HK,was low after administration,and the residence time in the intestinal tract was long,so that the okra can act on the intestinal microecology to play a drug effect.It is suggested that the HK may play a role in the intestinal micro-ecology.Literature studies have found that enterobacteriaceae bacteria are rich in tryptophanase and are capable of metabolizing amino acids to form indoxyl sulfate(IS),bifidobacteria bacteria can metabolize amino acids to finally synthesize p-cresol sulfate(pCS),both of the families have significant abundance amplification in the intestine of model rats.IS and pCS are both very important uremic toxins.In order to observe the regulation of the HK extract on the metabolite of the intestinal bacteria in CKD,UPLC-TQ/MS was used to detect the content of IS and pCS in plasma,liver and kidney of rats.The results showed that the plasma IS and pCS levels in the model rats were significantly higher than those in the normal rats.After 4 weeks of HK administration,the IS content in the plasma,liver and kidney of the model rats was significantly decreased,but there was no significant effect on the plasma pCS content.The synthesis of IS consists of two steps:in the first step,intestinal bacteria metabolizes tryptophan to IS precursor indole by tryptophanase;in the second step,indole enters the liver through the portal vein and cytochrome in the host liver cells.After hydroxylation of the P450 enzyme,IS is formed by the action of a sulfotransferase.In order to confirm the role of HK in the synthesis of IS in vivo,corresponding research experiments were carried out on its synthetic pathway.Firstly,indole and HK were directly administered to rats.UPLC-TQ/MS was used to detect the content of IS in rat plasma and kidney,and the effect of HK on the transformation process of indole to IS was observed.The results showed that HK did not reduce the increase of IS in rat plasma and kidney caused by indole,suggesting that HK does not affect the metabolic process of IS in the host.Subsequently,in order to examine the effect of HK on the metabolic process of IS in the intestinal bacteria,HPLC-FLD was used to detect the content of IS precursor indole and it was found that HK can significantly reduce the cause of chronic kidney disease.High indole levels in the intestine.The above results indicate that HK mainly reduces the intestinal indole level by interfering with the synthesis of IS precursor indole in intestinal bacteria,and finally reduces the content of urinary toxin in the systemic circulation.In order to evaluate the regulation of HK extract on the intestinal bacteria of CKD,a model of CKD in 5/6 nephrectomized rats was established.After the successful modeling,the HK extract was given for 8 weeks,and the fresh feces of each group were collected for 16S rDNA sequencing to compare the differences of different groups of rats.The results showed that the structure of intestinal bacteria in the model rats was disordered,which was mainly reflected in the significant expansion of the bacterial abundance of the proteobacteria,and the detection of five bacterial families with significant abundance differences with normal rats.The abundance of enterobacteriaceae,bifidobacteria,enterococcus and corynebacterium in the intestinal tract of rats was up-regulated,and the abundance of bacillus licheniformis was down-regulated.AThe HK extract only inhibited the expansion of the corynebacterium in the intestine of the model rats,but it did not significantly affect other family's bacteria,especially the enterobacteriaceae which showed inflammation in the intestine of rats with renal failure.This result suggests that the HK extract does not interfere with intestinal flora disturbance caused by CKD.In order to research the effect of HK on the indole production in intestinal bacteria,E.coli was selected as model bacteria.Based on anaerobic workstation,HPLC-FLD and multi-function microplate reader,the in vitro determination of HK and its flavonoids inhibited the intestine.An in vitro assay system for the determination of HK and its flavonoids in inhibiting intestinal bacteria indole production was established.In vitro experiments showed that HK can inhibit the E.coli indole production in a dose-dependent manner,and is time-dependent.400 ?g/ml HK does not affect the growth of E.coli.The above results were verified in the stool mixed bacteria experiment.Chapter 3.Study on the mechanism of the effect of HK on indole productionThe previous study found that HK can effectively reduce the content of IS in model rats by regulating the intestinal bacteria indole production.Based on this experiment,this chapter further clarifies the mechanism of action of flavonoids in HK on bacteria indole production.The main flavonoids of isoquercitrin,myricetin,quercetin and hyperoside in HK were selected for activity screening.It was found that all four flavonoids can inhibit the indole production of E.coli.The activities from high to low:isoquercitrin,myricetin,quercetin,and hyperoside,the effect on bacterial growth from low to high is:isoquercitrin,hyperoside,quercetin,myricetin.Each compound inhibited the bacterial indole production is in a time-dependent manner.It is worth noting that the flavonoid glycoside isoquercetin(ISO)is non-fatal and effective in inhibiting bacterial indole production.In order to verify the activity of ISO inhibiting bacterial indole production,different concentrations of ISO were co-incubated with E.coli to detect the effects on bacterial growth and indole secretion.It was found that ISO can reduce the concentration of E.coli cells and culture supernatant indole concentration in a dose-dependent and time-dependent manner.To confirm whether ISO is non-specifically inhibiting E.coli indole synthesis,we collected cecal contents and feces from normal C57BL/6 mice and SD rats,and incubated the four intestinal mixed bacteria with ISO.The results showed that ISO had significant inhibitory effects on the indole production of intestinal mixed bacteria from various sources,and had no effect on the growth of bacteria.In addition,in order to study the effect of ISO on indole synthesis in the intestine,E.coli was implanted into normal or model mice by antibiotic cocktail method,and the fecal indole content of the ISO group was significantly lower than that of the control group.The above experiments show that ISO can effectively inhibit intestinal bacteria indole production in vitro and in vivo.The synthesis of inodle in bacteria requires two key steps,namely the transfer of exogenous tryptophan into the bacteria,and tryptophanase in bacteria converts tryptophan to indole.In order to study the effect of flavonoid glycosides on the key aspects of indole synthesis,the effects of flavonoid glycosides on bacterial tryptophanase activity and tryptophan transport were investigated.Firstly,it was investigated whether the flavonoid glycosides affect the activity of tryptophan metabolism enzymes in bacteria.The enzymatic reaction was used to determine the effect of flavonoid glycosides on the activity of tryptophanase.It was found that flavonoid glycosides did not significantly affect the activity of ryptophanase compared with the control group.The results indicate that flavonoid glycosides do not directly interfere with the conversion process of tryptophan to indole.To investigate whether flavonoid glycosides interfered with the transport of tryptophan into bacteria,we used UPLC to detect the residual content of tryptophan in the supernatant of bacterial culture.The results showed that in the presence of flavonoid glycosides,the concentration of tryptophan in the supernatant of bacterial culture was significantly higher than that of the control group,that is,the amount of tryptophan transported by the flavonoid glycoside group to the inside of the bacteria was significantly lower than that of the control group.The above findings were verified by UPLC-TQ/MS test for the determination of isotope-labeled tryptophan in bacteria and culture supernatants.The above results indicate that flavonoid glycosides do not directly inhibit tryptophanase activity,but reduce the indole production of bacteria by directly inhibiting the transport of exogenous tryptophan into the interior of bacteria.The transport of tryptophan into the interior of the bacteria requires a co-transport of the extramembranous proton kinetic(proton gradient),which in turn is produced by the on-membrane electron transport chain.In order to study the effect of flavonoid glycoside on the extracellular proton gradient of bacteria,the cell membrane potential was analyzed by fluorescence microscopy and membrane potential probe carbocyanine dye DiOC2(3).It was found that the bacteria showed weak red after co-incubation between bacteria and flavonoids.Fluorescence intensity indicates a decrease in membrane potential,that is,a decrease in the proton potential energy outside the membrane.This result indicates that flavonoid glycosides can disrupt the bacterial membrane potential and reduce the proton potential of the bacterial membrane gap.The effect of flavonoid glycoside on the electron transport chain was evaluated by the electron transfer chain complex I activity assay.The results showed that the activity of the bacterial electron transport chain complex I was significantly lower than that of the control group after co-incubation of Escherichia coli with flavonoid glycosides.The activity of complex I can be expressed by the ratio of NADH/NAD.The results show that the ratio of NADH/NAD in bacteria is significantly increased in the presence of flavonoid glycosides,indicating that the conversion process of NADH to NAD is inhibited,resulting in a decrease in proton release during the process.The extramembrane proton gradient is weakened.The proton gradient drives ATP production by ATP synthase,which provides energy for bacterial life activities.The results show that the presence of flavonoid glycosides significantly reduces ATP concentration in bacteria.The above results suggest that flavonoid glycosides reduce the transport of tryptophan into the bacteria by interfering with the establishment of proton potential energy outside the membrane.In order to further verify the above viewpoints,an excessive electron transfer chain terminal electron acceptor is added to the bacterial culture system to accelerate the electron transport chain process and accelerate the establishment of proton potential energy.The results showed that the presence of terminal electron acceptors such as oxygen,DMSO and TMAO significantly attenuated the inhibitory effect of flavonoid glycosides on tryptophan transport and reduced the inhibition rate of bacterial sputum synthesis.Chapter 4.Study on the structure-activity relationship of small molecule compounds on indole productionThis chapter focuses on the structure-activity relationship analysis of the ability of flavonoid glycosides and flavonoid glycosides to inhibit the synthesis of strontium.In addition,an extended experiment on the structure-activity relationship analysis of other small molecule compounds was carried out.In order to research the effect of flavonoid glycosides after hydrolysis,UPLC was used to detect the concentration of flavonoid glycosides and hydrolyzed glycosides in bacterial culture medium.The results showed that during incubation with E.coli,the concentration of flavonoid glucoside decreased gradually with the prolongation of incubation time,and the concentration of glycoside increased with the prolongation of culture time.Glucoside flavonoid glycosides,which have a stronger inhibitory effect on indole production,are hydrolyzed gradually after incubation with bacteria,while other glycosides,which have been present in the form of prototypes,have very low ability to inhibit indole production.It was found that the inhibitory effect of glucoside hydrolysate combined with glycoside quercetin on indole secretion was significantly lower than that of flavonoid glycoside alone.These results suggest that flavonoid glycosides inhibit the synthesis of indole by bacteria in three ways:including a glycoside hydrolysis process that consumes extracellular protons of the bacterial membrane,release of flavonoid aglycans such as quercetin,which inhibits the activity of the electron transport chain,and inhibition of the release of glucose by tryptophanase by the metabolite repression effect.In order to study the structure-activity relationship of flavonoid aglycones,the concentration of ruthenium in the culture solution based on quercetin-based flavonoid aglycone and bacteria was determined by HPLC-FLD.The results of comprehensive experiments can be used to speculate the structure-activity relationship of flavonoid aglycones:when the number of flavonoid aglycone phenolic hydroxyl groups is consistent,the inhibition ability of bacteria indole secretion is flavonols>flavones>flavanonols>flavan-3-ols,planar molecular activity is stronger than non-planar molecules;when the main structure of flavonoid aglycon is consistent,the more A and B cyclic phenolic hydroxyl groups,the stronger the ability to inhibit bacterial synthesis of hydrazine.And the following basic structure is obtained:flavonoid aglycon is a key group for inhibiting bacterial synthesis of hydrazine:the carbonyl group on the C-ring binds to the 2,3-double bond,the o-diphenolic hydroxyl group in the B-ring,and the 3-position hydroxyl group in the C-ring.The key functional groups are all important components of flavonoid aglycone to inhibit the activity of bacteria indole synthesis.At last,in order to investigate the structure-activity relationship of other types of traditional Chinese medicine small molecule compounds in the inhibition of indole production in bacteria,HPLC-FLD was used to detect the concentration of indole in the culture medium after the five compounds co-incubated with bacteria.The results showed that the stilbene compounds had the strongest ability to inhibit the synthesis of indole,followed by the diketone curcumin.But it's different with flavonoid glycosides,the cells growth was significant inhibited after coincubated with this compounds.The remaining classes of small molecule compounds such as anthraquino nes,phenylpropanoids and triterpenoids did not exhibit significant inhibition on bacteria indole synthesis. |
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