Effective Constituents And Mechanism Of Alismatis Rhizoma Against Renal Fibrosis

Alismatis Rhizoma（AR）is the dried tuber of Alisma orientatle（Sam.）Juzep.,which has been used to treat edema,oliguria and hyperlipidemia,etc.in numerous clinical scenarios.Numerous studies revealed that triterpenoids,sesquiterpenes and alkaloids are the dominant chemical constituents of AR.Among these components,triterpenoids are considered to be the most effective components.Alisol B 23-acetate（ABA）has been used as the only marker of AR since Pharmacopoeia of the People’s Republic of China（volume1,2010 edition）.Triterpenoids are deemed as bioactive components of AR.Recent pharmacological studies showed that ABA exhibits multiple bioactivities including diuresis,renoprotection,hepatoprotection,antihyperlipidemia and antitumor.AR is commonly used to treat chronic kidney disease（CKD）according to the principle of traditional Chinese medicine.However,the medical studies of AR are inadequate,especially in the field of the effective constituents and mechanism of AR against renal fibrosis,which not only restrains the development of AR as a novel drug,but also prevents the modernization and internationalization of traditional Chinese medicine.Therefore,we investigated the bioactive ingredients and action mechanisms of AR against renal fibrosis in this study.Objective:1.We investigate the chemical constituents of AR,screen the diuretic fractions and systematically isolate and identify the chemical constituents from active fractions and further elucidate the renoprotection components of AR.2.We focus on evaluating the effective components of RA against renal fibrosis,then revealing its action mechanisms,providing numerous references and experimental basis for discovering new drug and therapies of CKD via AR.Methods:1.AR was pulverized and then extracted with water and ethanol to obtain aqueous extract（AE）and ethanol extract（EE）.EE was then successively extracted with petroleum ether（PE）,ethyl acetate（EA）and n-butanol（NB）to get fraction PE（Fr.PE）,fraction EA（Fr.PE）,fraction NB（Fr.NB）and fraction residue（Fr.RE）.Firstly,the diuretic activities of AE and EE were assessed via a Saline-induced rat model of water retention.Subsequently,the diuretic activities of Fr.PE,Fr.EA,Fr.NB as well as Fr.RE were investigated,respectively.Normal rats were orally administrated saline（5 mL/100 g）and all the rats were randomly divided into several groups（n=10/group）30 minutes later.The rats in the corresponding groups were give oral administration of indicated drugs.The urine of each rat was collected within 6 h.The volume,pH value as well as concentrations of Na⁺,K⁺and Cl^-of the urine were determined.2.Fr.EA of AR was firstly separated by middle chromatogram isolated gel.The pure compounds were further isolated from the obtained fractions by using the chromatographic technologies including silica gel,RP-18,sephadex gel as well as semipreparative.The structures of the compounds were identified through nuclear magnetic resonance spectroscopy and mass spectrometry.3.The effect of ABA against renal fibrosis.Firstly,in vivo experiment was used to evaluate the anti-fibrotic activity of ABA.5/6 Nephrectomy（NX）and unilateral ureteral obstruction（UUO）rat models were established.ABA was then administrated to NX and UUO rats.At the end of the experiment,urine within 24 h,blood and kidney were collected.The physiological and biochemical parameters including body weight,systolic blood pressure,serum creatinine and urea as well as urine protein were determined.The collagen deposition and histomorphology of the kidney were analyzed by histopathology.To assess the anti-fibrotic effect of ABA,expressions ofα-SMA,collagen I as well as fibronectin and the proteins related to transforming growth factorβ/Smad（TGF-β/Smad）signaling pathway were analyzed by Western blot and quantitative real time polymerase chain reaction,etc.The mechanism of ABA for the inhibition of Smad3 phosphorylation was explored by immunofluorescence staining,siRNA and immunoprecipitation.4.The bioactivity of the new tetracyclic triterpenoid 25-O-methylalisol F（MAF）against renal fibrosis.Firstly,the effects of MAF on the protein expressions of collagen I、fibronectin,α-SMA,vimentin as well as E-cadherin in TGF-β1-and angiotensin II（Ang II）-induced NRK-52E and NRK-49F cells were determined by Western blot,quantitative real time polymerase chain reaction and immunofluorescence staining,etc.Secondly,the expressions of the components in renin-angiotensin system（RAS）and the proteins related to TGF-β/Smad and Wnt/β-catenin signaling pathways were also analyzed.The mechanism of MAF for the inhibition of Smad3 phosphorylation was explored by immunofluorescence staining,siRNA and immunoprecipitation.Results:1.AR has both diuretic and antidiuretic effects.EE exhibited a diuretic effect at the low doses while exerted an antidiuretic effect at the high doses.Fr.EA had the similar trends in diuretic and antidiuretic activity,indicating Fr.EA was the diuretic and antidiuretic extract of AR.2.Triterpenoids are the primary chemical constituents of AR from Fr.EA of AR.We isolated 63 compounds from Fr.EA,including 38 triterpenoids,2 steroids,1 diterpenoid,7sesquiterpenoids,3 aromatic derivatives,2 alkaloids,1 flavone,1 furan derivative and 8fatty acids,respectively.Triterpenoids include alisol A,alisol A 23-acetate,alisol A24-acetate,11-deoxyalisol A,25-O-methylalisol A,25-O-ethylalisol A,alisol B,alisol B23-acetate,11-deoxyalisol B,11-deoxyalisol B 23-acetate,16β-hydroxyalisol B 23-acetate,16β-methoxyalisol B 23-acetate,16β-ethoxyalisol B 23-acetate,16-oxo-alisol A,16-oxo-alisol A 24-acetate,alisol C,alisol C 23-acetate,11-deoxy-alisol C,11-deoxy-alisol C 23-acetate,16β-hydroperoxyalisol B 23-acetate,alisol G,25-anhydroalisol A-11-acetate,25-anhydroalisol A-24-acetate,16-oxo-11-anhydro-alisol A,alisol L,alisol L 23-acetate,alisol X,16-oxo-alisol A 24-acetate,alismanol A,alismanol B,alismanol C,alismanol E,25-O-methylalisol F,alisol F,alisol Z,25-anhydro-alisol F as well as alisol I.Steroids includeβ-sitosterol and stigmasta-5,22-dien-3β-ol-7-one.Diterpenoid is16（R）-（-）-kaurane-2,12-dione.Sesquiterpenoidsincludealismoxide,2β,4β,10α-trihydroxy-1αH,5βH-guaia-6-ene,orientalol A,orientalol B,orientalol C,orientalol E as well as alismol.Aromatic derivatives include cinnamic acid,dibutyl phthalate and 2-diethylhexyl phthalate.Alkaloids include neochinulin A and11,14-dihydroxylneochinulin E.Flavonoid is tangeretin.Furan derivative is5-hydroxymethyl furfural.Fatty acids include glycerol monostearate,linoleic acid,hexadecanoic acid-2-hydroxy-1,3-propanediyl ester,hexadecanoic acid,methyl hexadecanoate,methyl（7E,9E）-6,11-dioxononadeca-7,9-dienoate,myristic acid as well as stearic acid.Among them,16β-ethoxyalisol B 23-acetate,alisol Y,25-O-methylalisol F and alisl Z are novel compounds.3.ABA exerts the effect against renal fibrosis through inhibiting the up-regulation of p-Smad3 and down-regulation of Smad7 mediated by NX and UUO.Our study demonstrated ABA could significantly lower the levels of serum creatinine and urea and urine proteins in NX rats and significantly down-regulate the protein expressions ofα-SMA,collagen I,fibronectin as well as FSP1 etc.in the kidney tissues of NX and UUO rats.The further study showed ABA could also obviously down-regulate the expression of p-Smad3 and simultaneous up-regulate the expression of Smad7 in the kidney tissues of NX and UUO rats.However,ABA had no significant effect on the expression of other Smad proteins.These results were further validated in TGF-β1-induced NRK-52E cells.The inhibitory activity of ABA on the relevant protein expressions was markedly reduced in the cells infected by siRNA Smad3.This study also revealed ABA could inhibit the binding between TGFβRI and Smad3 as well as SARA and Smad3 but promot the interactions of TGFβRI and Smurf2,TGFβRI and Smad7 as wells as Smurf2 and Smad7 to exert the effect against renal fibrosis.4.MAF has the effect against renal fibrosis via regulating RAS,TGFβ/Smad and Wnt/β-catenin signaling pathways.MAF significantly down-regulated the protein expressions of collagen I,fibronectin as well asα-SMA etc.in TGF-β1-and Ang II-induced NRK-52E and NRK-49F cells.The results showed that MAF could decrease the levels of RAS components including angiotensinogen,renin,ACE and AT₁R.Meanwhile,MAF could also obviously down-regulate the expression of p-Smad3 and up-regulate the expression of Smad7 but have no effect on the expressions of other Smad proteins.In addition,MAF was able to significantly down-regulate the protein expression of Wnt1 and activeβ-catenin and its downstream proteins including Snail1,Twist,MMP-7,PAI-1 and FSP1.Conclusion:The present study demonstrated that Fr.EA is the diuretic and antidiuretic extract of AR and triterpenoids are the main and active components in AR.Our results illustrated the triterpenoids from AR exert the antifibrotic effect by blocking RAS,down-regulating the expression of p-Smad3 but up-regulating the expression of Smad7 and inhibiting Wnt/β-catenin signaling pathway.We found triterpenoids from AR have the effect against renal fibrosis and explored its molecular mechanisms.This study further demonstrated the importance of ABA used as the index constituent for the quality control of AR and will provide the experimental evidence and theoretical basis on development and applications of AR.