| Objective:Hyperuricemia is a metabolic disorders,caused by the increased uric acid synthesis or the decreased uric acid excretion.Gouty nephropathy is mainly caused by excessive uric acid generation or decreased excretion of uric acid in the kidney,which results in damage of renal tissue.The main pathological features are urate deposition in the renal interstitium and renal tubula,renal tubular epithelial cell are swelling and necrosis.Hyperuricaemia is the physical and chemical basis of Gouty nephropathy.The main pathogenes is that uric acid transfer protein are aberrant expression.Therefore,we consider to take uric acid transport protein gene,protein,renal interstitial fibrosis of renal damage protein regulation for drug target to research Clerodendranthus spicatus for hyperuricemia and gouty nephropathy renal protective effects and mechanism.Clerodendranthus spicatus is an herb with the effect of heat-cleaning and dampness-eliminating,which the taste was characterized with sweet,light,taste,bitter,cold bitter dryness of dampesss.In southeast Asian countries,including Indonesia and Xishuangbanna Autonomous Prefecture,Yunnan Province.It has significant effect on the hyperuricemia,gout and kidney disease.We observed the protection and molecular mechanism of Clerodendranthus spicatus to the hyperuricemia and gouty nephropathy on the animal models.To investigate the role of Clerodendranthus spicatus in reducing the injury of human renal proximal tubular cells(HK-2 cells)by inducing renal interstitial fibrosis model with uric acid.Method:1.Collcet different habitats Clerodendranthus Status and establish Clerodendranthus spicatus fingerprint by HPLC analysis for 15 batches of different habitats Clerodendranthus spicatus,to establish rosemary acid and caffeic acid in Clerodendranthus spicatus by HPLC.Select the best quality of habitats Clerodendranthus spicatus in a Box-Behnken response surface method to optimize the extraction and purification process.2.KM mice were chosen as the hyperuricaemia animal model for experimental research.We evaluate the effect of Clerodendranthus spicatus in hyperuricaemia mice on serum uric acid,urea nitrogen,creatinine.Observing the role of the kidney pathological morphology in mice with HE staining.The uric acid of the members of the family of organic anion transporters(OAT1),NGAL,TIMP-1 expression were tested.Renal cortical URAT1 and OAT1 gene expression in mice were detected by real-time fluorescent quantitative PCR.Investigating Clerodendranthus Statu on mice kidney cortex TIMP-1,the influence of URAT1,OAT1 protein expression in mice by Western Blot.3.The gouty nephropathy rat models were caused by yeast and adenine.24 hour urine was collected using metabolic cage for the determination of urine protein.The serum uric acid,urea nitrogen,creatinine and the content of NGA in renal cortical were detected to evaluate the effect of Clerodendranthus spicatus in gouty nephropathy rat.Observing the effect of Clerodendranthus spicatus on the kidney pathological morphology in rats with HE staining.Detect renal cortical URAT1 and OAT 1 gene expression in rats by real-time fluorescent quantitative PCR.4.Using double antibody protein chip clip art technology for gouty nephropathy rats of kidney damage factor screening,and by immunohistochemical and protein imprinting method(Western Blot)for verification.Using western blot to investigate renal tea renal cortex of rat uric acid transporters URAT1,OAT1 protein expression.5.The uric acid stimulate proximal renal tubular epithelial cells(HK-2).HK-2 cell damage and renal interstitial fibrosis cell model is set up.The cell were treated with Clerodendranthus spicatus extract and its main monomers.To explore the cell vitality of Clerodendranthus spicatus on the cell vitality of HK-2 cell by CCK8.Apoptosis and the changes of cell cycle were detected by flow cytometry.6.The xanthine oxidase system was established in vitro.The system of enzymatic reaction were determined by HPLC method in the substrate xanthine in content changes before and after the reaction,using HPLC to detect the content of high uric acid hematic disease in mice liver xanthine to examine Clerodendranthus Status inhibition of xanthine oxidase.The aging model of rats made by D-galactose was employed in this experiment to study the antioxidant effect of Clerodendranthus Status.Results:1.Determine the Clerodendranthus spicatus xishuangbanna yunnan is the optimal quality,optimizing the best extraction and purification technology of Clerodendranthus spicatus2.Clerodendranthus spicatus alcohol extract can effectively reduce the high uric acid hematic disease in serum uric acid,serum urea nitrogen,creatinine of mice.HE staining pathology observation results showed that Clerodendranthus spicatus can protect mice kidney,immunohistochemical results showed that tea can adjust mice kidney uric acid of the members of the family of protein OAT1 expression,protein NGAL lowered renal injury,the expression of TIMP-1,real-time fluorescent quantitative PCR results showed that Clerodendranthus spicatus can cut high uric acid hematic disease mice kidney cortex URAT1 mRNA expression,raised high uric acid hematic disease mice OAT1 mRNA expression.The Clerodendranthus spicatus alcohol extract can reduce high blood uric acid kidney tissues of mice TIMP-1,the expression of URAT1 protein expression.The Clerodendranthus spicatus total flavones in middle and high dose and the Clerodendranthus spicatus aextract can raise the protein OAT1 expression in hyperuricemiamice model mice kidney.3.Clerodendranthus spicatus alcohol extract can effectively reduce the gouty nephropathy rats serum uric acid,urea nitrogen,creatinine,renal cortex NGA content,improve the renal function of rats,pathological observation HE staining results showed that tea can protect the rat kidney,real-time fluorescent quantitative PCR results showed that Clerodendranthus spicatus high dose group can increase the OAT1 mRNA expression in hyperuricemiamice model mice.4.The results of Protein chip double mono-cloned antibody were that there are differences among the three kinds of kidney injury protein expression.We found that alcohol extract of Clerodendranthus spicatus high doses can significantly reduce the gouty nephropathy rat kidney NGAL,TIMP-1,KIM-1 protein expression.Immunohistochemistry and Western Blot showed a consistency of the results of the validation.5.The extract of Clerodendranthus spicatus and rosemary acid to HK-2 cell damage induced by uric acid has certain protective effect.6.The extract of Clerodendranthus spicatus has a certain inhibitory effect of XOD in vitro and in vivo.It can improve SOD vigor in the blood and reduce MDA content,have better oxidation of resisting in vivo.Summary:The extract of Clerodendranthus Spicatus can reduce renal damage in hyperuricaemia mice and gouty nephropathy rats. |
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