Protective Effect And Mechanism Of Huang-Kui-Si-Wu Formula On Chronic Kidney Disease

1.literatureThis chapter systematically summarizes the definition of chronic kidney disease,the stage of chronic kidney disease,the cause of the disease,and clinical intervention strategies.This chapter also summarizes Indoxyl sulfate(IS)and P-cresyl sulfate(PCS)in enteric bacteria and intestinal uremic toxins,and their sources,physicochemical properties,processes of in vivo production and metabolism,and their role in the progression of chronic kidney disease.The third section of this chapter summarizes the research status of the chemical constituents and pharmacological effects of Huang Kui and its formula,and provides the basis for later research on the efficacy and mechanism of the drug.2.Study on the Protective Effect and Mechanism of Huang-Kui-Si-Wu Formula on CKD ratsThe partial nephrectomy model is currently the ideal and most widely used model of chronic progressive nephropathy.Mainly because the pathophysiological changes in the body are highly consistent with the progression of chronic kidney disease.Rats were modeled after 5/6 nephrectomy and given Huang Kui capsule or Huang-Kui-Si-Wu Formula(HKSW)of different doses(1.25 g/kg,2.5 g/kg,5 g/kg)for 8 weeks.During the different time periods of modeling and administration,rat plasma and urine samples were collected,and multi-functional microplate readers,determining the 24-hour urinary protein excretion,serum creatinine,blood urea nitrogen,renal pathological changes and other indicators to evaluate the efficacy of HKSW by multifunctional enzyme marker,HPLC-FLU and other instruments.The results showed that:(1)HKSW can significantly reduce the 24-hour urinary protein excretion of model rats,and has a significant reduction effect at the beginning of administration;(2)HKSW of different doses can significantly reduce the serum creatinine and blood urea nitrogen in rats.Rat renal tissue was stained with HE to evaluate the extent of inflammatory cell infiltration in kidney tissue.The results showed that:(3)a large number of inflammatory cells infiltrated in the renal interstitium of model rats,and the score of pathological injury was close to 3 points;However,HKSW can significantly reduce the infiltration of inflammatory cells in the kidneys of model rats.When the HKSW was given 1.25 g/kg,the pathological damage score was close to 1 point,which was a slight injury.(4)B7-1 and PAI-1 are indicators of renal podocyte injury and renal fibrosis,and have high expression in the injured kidney;WT1 and Podocin were highly expressed in the podocytes of normal mature renal tubules,and their expression was decreased after injury.Treatment with HKSW can significantly improve the expression of B7-1,PAI-1,WT1 and Podocin in renal protein of 5/6 nephrectomized rats.Based on the above four indicators,it is proved that the efficacy of HKSW is exact.Literature studies have found that patients with chronic kidney disease commonly exist abnormal metabolism of amino acid and protein,and their amino acid levels of tissue and intestinal are significantly abnormal compared with healthy people.Upon this reminder,we used UPLC-TQ/MS to quantitatively determine the amino acid content in the intestine of rats.The results showed that amino acid metabolism of the intestine was significantly abnormal and showed periodic changes in 5/6 nephrectomized renal failure rats for the experimental period of modeling of 2 weeks and administration of 8 weeks.At the 4th week after model establishment,the contents of tryptophan and tyrosine were significantly different in the intestine of the blank group and the model group.At the 6th week after model establishment,the contents of 6 amino acids such as tryptophan,tyrosine,phenylalanine were significantly different in the intestine of the blank group and the model group.At 8 weeks after modeling,the amino acid content of 8 amino such as tryptophan,tyrosine,phenylalanine,and glutamic acid were significant differences between the two groups of rats.At 10 weeks of modeling,the amino acid content of 9 amino such as tryptophan,tyrosine,phenylalanine,and serine were significant differences between the two groups of rats.Compared with the blank group,the amino acid metabolism in the intestine of the model group was more and more disordered as the model time increased.Intestinal amino acids are synthesized by intestinal microbes via various pathways.Therefore,we performed 16S rDNA sequencing on the gut microbiota in feces of two groups of rats in different modeling cycles.As a result,it was found that the abundance of the gut microflora among the gut microorganisms gradually changed with the increase of the modeling time.The changes of abundance of intestinal microflora were most obvious in the second week and the tenth week,such as Chlamydiales,Rhodospirillales,Rhodobacterales,Rhizobiales,Xan-thomonadales,Lactobacillales,Legionellales,Rhodocyclales,etc.Most of these intestinal bacteria are related to the decomposition of proteins or the production of amino acids.The amino acid is a raw material for the production of certain urea toxin precursors.The above results suggest that during the progression of chronic kidney disease,the intestinal flora abundance and its metabolic process,especially the amino acid metabolism are significantly changed in the model rats,and the intestinal microbes will gradually change.In order to find out the regulatory role of HKSW on endogenous metabolites in model rats,we used a UPLC-TOF/MS technique and a MassLynx workstation to qualitatively analyze differential metabolites in plasma of 5/6 nephrotic nephropathy rats based on the metabolomics research strategy.Among the identified various endogenous metabolites between the blank group and the model group,urinary toxins such as indoxyl sulfate(IS)and sulfuric acid p-cresol(PCS)were significantly different metabolites between the blank group and the model group.At the same time,semi-quantitative analysis found that HKSW has a significant reduction in the content of these two uremic toxin molecules.Therefore,it was determined that the HKSW regulates the intestinal uremic toxins IS,PCS and its precursors as the research direction,to evaluate the specific action mechanism of HKSW in the treatment of chronic kidney disease.We used UPLC-TQ/MS and HPLC-FLU to quantitatively analyze the content of IS,PCS and its precursor molecules(threonine,p-cresol)in model animals(plasma,liver,kidney).The results showed that after 8 weeks of administration,HKSW significantly reduced the content of IS and PCS in plasma at a dose of 1.25 g/kg,significantly reduced the synthesis of IS and PCS in the liver,and significantly reduced IS,PCS transfer to the kidney;It is suggested that HKSW has a significant inhibitory effect on the synthesis of the liver and the transport of kidneys for IS and PCS.At the same time,at the dose of 1.25 g/kg,HKSW can significantly reduce the content of precursor molecules indole and p-cresol of IS and PCS in feces.After 4 weeks of administration,the contents of indole and p-cresol in the feces showed a significant decrease in the administration group rats.At the 8th week after administration,the HKSW administration group had significantly reduced the effects of indole and p-cresol in the feces of rats.3.HKSW on adenine-induced nephropathy rats’ kidney injury protection and mechanismPrevious study found that HKSW had the best efficacy at the then-set minimum dose(1.25 g/kg).At this dose,HKSW can significantly reduce the renal damage in 5/6 nephrectomized rats with renal failure,and can significantly reduce 24-hour urinary protein excretion in model rats.The HKSW did not show better efficacy at higher doses.It is suggested that HKSW does not show a positive dose-dependent relationship at 1.25 g/kg-5 g/kg.In order to find the best dose ofHKSW,we took 1.25 g/kg as high dose and observed the efficacy of HKSW at lower doses(0.31 g/kg,0.63 g/kg).Rats were treated with 5/6 nephrectomy and given HKSW(low dose:0.31 g/kg;middle dose:0.63 g/kg;high dose:1.25 g/kg)for 8 weeks.Urine,plasma,and other samples were collected during this period.Rat urine protein,serum creatinine,urea nitrogen,and IS,PCS in vivo were quantitatively analyzed.Renal pathological sections were stained with HE,PAS,and MASSON,and pathological indexes were scored.The results showed that rats in the model group had severe renal pathological lesions and 24-hour urinary protein excretion significantly increased.The urinary protein excretion at 24 h was significantly lower than that of the model group and the other dose groups at the dose of 1.25 g/kg.HE staining showed that there were obvious organic lesions in the kidneys of model rats,and there was a large number of inflammatory cell infiltration and renal interstitial fibrosis in the renal interstitium.The mean lesion score was 2.57.In the HKSW group at the dose of 1.25 g/kg,there was a small amount of inflammatory cell infiltration in renal interstitium,and the area of renal interstitial fibrosis was significantly reduced.The average lesion score was 1.33.At the doses of 0.31 g/kg and 0.63 g/kg,the mean lesion scores were 2.22 and 2.00,respectively.PAS staining showed that the kidneys of the model group had obvious organic lesions and the glomerular basement membrane thickened.At the dose of 1.25 g/kg,there was only mild thickening of rat kidney glomerular basement membrane.MASSON staining showed that the percentage of renal fibrosis area in the model group was close to 50%,while at the dose of 1.25 g/kg,the percentage of renal fibrosis area was only 18%.At the same time,the blood urea nitrogen and serum creatinine levels of the rats were significantly reduced at a dose of 1.25 g/kg.The expression of B7-1,PAI-1,WT1 and Podocin in renal proteins was optimally regulated at a dose of 1.25 g/kg.The levels of IS and PCS in plasma,liver,and kidney were also the lowest in each dose group.Based on the above data,it is proved that for the 5/6 nephrectomized renal failure model rat,1.25 g/kg is the best dose to be administered HKSW.Adenine renal failure model can well simulate the course and characteristics of the occurrence and development of human chronic kidney disease.Adenine renal failure model can well simulate human chronic kidney disease.Its advantages lie in its easy replication,good stability,low mortality,and multiple complications of CKD.The clinical similarity is high,and it can better meet the needs of CKD progression mechanism and the prevention and treatment of complications.In order to further verify the efficacy of HKSW,adenine model was selected to observe the optimum dose of HKSW.After adenine model was established in rats,it was treated with HKSW(low dose group:0.31 g/kg;middle dose group:0.63 g/kg;high dose group;1.25 g/kg)for 8 weeks.Feces,urine,plasma and other samples of rats were collected during the period.24h Urinary protein,serum creatinine,urea nitrogen and other indicators were quantitatively analyzed using microplate reader,HPLC-FLU,UPLC-TQ/MS and other instruments.At the same time,renal pathological sections were stained by HE,PAS,MASSON and scored.The results showed that 1.25 g/kg was still the best dose to be administered HKSW.At this dose,the 24-hour urinary protein excretion of rats was significantly lower than that of other groups.The pathological lesion scores of rat kidneys stained with HE,PAS and MASSON also showed that the kidneys had the lowest degree damage at 1.25 g/kg,with the smallest degree of inflammatory cell infiltration,the smallest area of renal interstitial fibrosis,and the minimum thickness glomerular basement membrane;At the same time,the urea nitrogen and serum creatinine levels in rats were significantly reduced,and the expression of B7-1,PAI-1,WT1 and Podocin in the kidney protein had the best positive regulation.The contents of IS and PCS in plasma and kidney were also the lowest in the administration groups,and the treatment effects of HKSW on 5/6 nephrectomized renal failure rats were basically the same.It is suggested that HKSW has the best dose to treat chronic kidney disease rats at a dose of 1.25 g/kg.Based on the above data,the following conclusions can be drawn:(1)HKSW has exact efficacy and can significantly reduce urinary protein excretion;HKSW significantly improved renal damage,reduce renal inflammatory response,reduced renal interstitial fibrosis,and reduced glomerular basement membrane deposition;HKSW significantly reduced serum creatinine and blood urea nitrogen in rats,significantly beneficial regulated podocyte injury and expression of renal fibrosis markers in renal proteins.(2)The effective mechanism of HKSW can be summarized as follows:HKSW has many links to the generation and translocation of intestinal uremic toxins IS and PCS.For example,the precursors(indole,p-cresol)of IS,PCS in the intestinal tract are all significantly inhibited.(3)The best dose of HKSW is 1.25 g/kg.