| Background:Hyperuricemic nephropathy（HN）refers to renal injury caused by hyperuricemia（HUA）.The onset of HN is hidden and difficult to detect in the early stage of the disease.Allowing its development can cause irreversible damage to renal function,but the current therapeutic drugs can not meet the clinical needs.Clerodendranthus spicatus is the dry aboveground part of Clerodendranthus spicatus[Clerodendranthus spicatus（Thunb.）C.Y.Wu ex H.W.Li]in Labiatae[1].It is mainly distributed in Yunnan Province,Guangxi Province,Guangdong Province,Hainan Province,Fujian Province and Taiwan Province in China.Because Clerodendranthus spicatus has the effects of clearing away heat and dampness,expelling stones and promoting water,it is often used to treat rheumatism,arthralgia,stone drenching,heat drenching and other diseases.It is reported that Clerodendranthus spicatus can significantly improve hyperuricemia and gouty nephropathy,but its specific mechanism is still unclear[2].Objective:The purpose of this experiment is to explore the pharmacodynamic substances and mechanism of Clerodendranthus spicatus on hyperuricemic nephropathy（HN）,so as to lay a foundation for clinical rational drug use and drug research and development.Methods:（1）C57BL/6J mice were injected intraperitoneally with potassium oxonate（PO）to establish the hyperuricemia（HUA）model.The 70%ethanol extract of Clerodendranthus spicatus was administered by gavage.The serum biochemical indexes,the activity of liver xanthine oxidase（XOD）and the expression of renal uric acid transporter were detected to verify its effect on reducing uric acid in HUA mice.（2）The HN mouse model was established by intragastric administration of adenine combined with PO for 25 days,and phenylbromomalone,70%ethanol extract of Clerodendranthus spicatus was administered to mice at the same time.The serum biochemical indexes,the pathological changes of kidney,the activity level of XOD in liver,the expression of phosphoribosyl pyrophosphate synthase（PRPS）protein,and the protein expression of renal uric acid transporter,inflammatory factor,the protein expression of NLRP3、TLR4/My D88、NF-κB inflammatory pathway and the expression level of fibrosis related proteins were detected,so as to verify the uric acid lowering,anti-inflammatory and renal protective effects of Clerodendranthus spicatus on HN mice,and the regulatory effect of Clerodendranthus spicatus alcohol extract on intestinal bacteria was verified by 16S r DNA sequencing analysis of mouse intestinal bacteria.（3）Three broad-spectrum antibiotics,ampicillin,neomycin sulfate and vancomycin,were used to inhibit the distribution of intestinal bacteria in mice.The HN mouse model was constructed by adenine combined with PO.Through 16S r DNA sequencing analysis of Enterobacteriaceae,the effect of Enterobacteriaceae disorder on the activity of Clerodendranthus spicatus alcohol extract in the treatment of HN was verified.The serum biochemical indexes,renal pathological changes,liver PRPS,renal uric acid transporter,key proteins of inflammatory pathway,inflammatory factors and renal fibrosis related proteins were detected.（4）The monomer components of HN activity of Clerodendranthus spicatus set were screened through network pharmacological research,and 0.75 m M sodium urate was given to rat renal tubular epithelial cells（NRK-52E）to induce cell injury.The regulatory effects of Clerodendranthus spicatus active monomer on cell uric acid transporter,inflammatory factors,key proteins of inflammatory pathway and proteins related to fibrosis process were detected.Result:（1）Clerodendranthus spicatus alcohol extract could down regulate the level of serum uric acid（SUA）,inhibit the activity of liver XOD,up regulate the expression of renal uric acid transporter organic anion transporter 1（OAT1）protein and down regulate the expression of glucose transporter 9（GLUT9）protein in mice with hyperuricemia and hyperuricemic nephropathy;Moreover,for mice with hyperuricemia nephropathy,the ethanol extract of Clerodendranthus spicatus could also reduce the 24-hour clearance of uric acid（Cur）,down regulate the expression of liver PRPS and renal urate anion transporter 1（URAT1）protein,and up regulate the expression of renal ATP binding cassette G2（ABCG2）protein.（2）Adenine combined with PO could induce renal histological lesions,up-regulate the expression of renal and serum inflammatory factor proteins,and activate NLRP3,TLR4/MYD88 and NF-κB inflammatory pathway.Administration of Clerodendranthus spicatus alcohol extract could improve renal injury,inhibit the release of inflammatory factors and the activation of inflammatory pathway.（3）Adenine combined with PO could induce the increase of serum creatinine（Scr）and blood urea nitrogen（BUN）,the decrease of creatinine clearance rate（Ccr）and the decrease of the protein expression ofα-SMA was up-regulated and the protein expression of E-cadherin was down-regulated;Clerodendranthus spicatus alcohol extract could up-regulate Ccr,down-regulate Scr and BUN,and down-regulate the renal protein expression ofα-SMA,up-regulated the protein expression of E-cadherin.（4）The alcohol extract of Clerodendranthus spicatus could regulate the flora structure of intestinal microorganisms in HN mice,reduce the relative abundance of Bacteroides and Alistipes,and increase the relative abundance of Lachnospiraceae＿NK4A136＿group and Akkermansia.（5）16S r DNA sequencing of mouse enterobacteria showed that antibiotics could inhibit the relative abundance of most enterobacteria except proteobacteria.（6）After administration of antibiotics,SUA,the protein expression of PRPS in liver,the protein expression of GLUT9and URAT1 in kidney were up-regulated,the protein expression of OAT1 and ABCG2 in kidney were down-regulated,and Cur were also down-regulated in HN mice;at this time,Clerodendranthus spicatus alcohol extract had no significant regulatory effect on mouse SUA,which could reduce the protein expression of PRPS in liver,down-regulate the expression of GLUT9 and URAT1 in kidney,and up-regulate the expression of OAT1 and ABCG2 in kidney.（7）After administration of antibiotics,HN mice developed renal histological lesions,up-regulated the protein expression inflammatory factor in kidney,and NLRP3,TLR4/MYD88 and NF-κB inflammation related pathways were activated.Administration of Clerodendranthus spicatus alcohol extract can improve the damaged renal structure and renal inflammatory response.（8）After antibiotic administration,Scr and BUN of HN mice were up-regulated,Ccr was down-regulated and the protein expression ofα-SMA was up-regulated and the protein expression of E-cadherin was down regulated in kidney;Clerodendranthus spicatus alcohol extract could significantly reverse the above biochemical indexes and renal protein expression.（9）The results of network pharmacology showed that 5,3’-dihydroxy-7,4’-dimethoxyflavone,4’,5,6,7-tetramethoxyflavone and sinensetin might be the monomer components of Clerodendranthus spicatus in the treatment of HN.（10）In the NRK-52E cell modelinducedbysodiumurate,5,3’-dihydroxy-7,4’-dimethoxyflavone,4’,5,6,7-tetramethoxyflavone and sinensetin significantly down-regulated uric acid transporter GLUT9 and URAT1,up-regulated the protein expression levels of OAT1 and ABCG2,and inhibited the expression level of inflammatory factor,interleukin-1β（IL-1β）、interleukin-18（IL-18）、interleukin-6（IL-6）、monocyte chemoattractant protein-1（MCP-1）、tumor necrosis factor-α（TNF-α）,and inhibited the activation of NLRP3,TLR4/MYD88 and NF-κB pathway,significantly down-regulated the protein expression of Collagen 1、α-SMA.Conclusion:（1）70%ethanol extract of Clerodendranthus spicatus could significantly reduce the level of blood uric acid in PO induced hyperuricemia mice.（2）70%ethanol extract of Clerodendranthus spicatus could regulate the structure of intestinal flora in mice with hyperuricemic nephropathy,inhibit the activity of XOD in liver and the protein expression of PRPS,regulate the protein expression of uric acid transporter in mouse kidney,so as to reduce the level of blood uric acid,and inhibit NLRP3 pathway,TLR4/My D88pathway and the activation of NF-κB pathway in kidney,reduce the expression level of inflammatory factors,slow down the process of renal fibrosis and improve renal injury.（3）Antibiotics reduced the diversity of intestinal bacteria in mice and inhibited the distribution level of most intestinal bacteria except proteobacteria.In this case,70%ethanol extract of Clerodendranthus spicatus could down regulate the relative abundance of pathogenic bacteria Proteobacteria and Firmicutes in the intestine of HN mice,up regulate the relative abundance of probiotic verrucomicrobia,and regulate the intestinal bacterial homeostasis of mice.However,the up regulation of the relative abundance of Bacteroides hindered the improvement of mouse renal function.（4）The active monomers 5,3’-dihydroxy-7,4’-dimethoxyflavone,4’,5,6,7-tetramethoxyflavone and sinensetin in kidney tea could significantly improve the damage of NRK-52E cells induced by sodium urate and reduce the accumulation of intracellular uric acid. |
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