Effective Constituents And Mechanism Of The Surface Layer Of Poria Cocos Against Renal Fibrosis

The surface layer of Poria cocos?Schw.?Wolf?SLPC?has been used for treatment of edema and dysuria because of its promoting water excretion and reducing edema in clinic.Numerous studies have revealed that triterpenes,diterpenoids,steroids,flavonoids,anthraquinones and lignans are the main components of SLPC.Modern pharmacological studies showed SLPC had diuretic,renoprotective,anti-inflammatory,anti-oxidative and anti-tumor effects.Tubulo-interstitial fibrosis?TIF?is the final manifestation when various chronic kidney disease?CKD?inevitably reach end-stage renal disease.Therefore,inhibition of the TIF progression is the key for prevention and treatment of CKD.In this study,we systematically isolated and identified the components of the SLPC,and indicated that 3,4-seco-lanostan type triterpenes and lanostan type triterpenes effectively attenuated TIF by inhibiting renin-angiotensin system?RAS?,Wnt/?-catenin and TGF-?/Smad pathways.Objective:We systematically isolated and identified the components of the SLPC to reveal the effective bioactive components and action mechanism of SLPC against renal fibrosis.This study will provide a theoretical and experimental foundation for reasonable utilization of SLPC and pharmaceutical research and development used for retarding renal fibrosis.Method:1.Dried SLPC was pulverized and extracted with ethanol,and the solvent was removed under reduced pressure.The extracts was subsequently dissolved in water and successively extracted with petroleum ether,ethyl acetate and n-butyl alcohol for three times,respectively.2.The ethyl acetate fraction was fractionated and purified by using MCI GEL CHP 20P,silica gel,Sephadex LH-20,ODS RP-18 and semi-preparative high performance liquid chromatography?HPLC?.The chemical structures of compounds were identified by IR,HRESIMS,1D and 2D NMR techniques.3.HK-2 cells and podocytes were treated with transforming growth factor-?1?TGF-?1?and angiotensin II?Ang??respectively.Based on the different skeletons and functional groups of poricoic acids,we evaluate their intervention effects of one tricyclic terpane poricoic acid ZF?PZF?,three 3,4-seco-lanostan type triterpenes poricoic acid ZC?PZC?,poricoic acid ZD?PZD?and poricoic acid ZG?PZG?,two lanostan type triterpenes poricoic acid ZE?PZE?and poricoic acid ZH?PZH?on the injury of HK-2 cells or podocytes induced by TGF-ed byic eff.4.The analyses of qRT-PCR,Western blot and immunofluorescence were used to evaluate the inhibitory effects of PZC,PZD,PZE,PZF,PZG and PZH on the TGF-?1-and AngII-induced activation of RAS and Wnt/?-catenin pathways in HK-2 cells and podocytes.5.We investigated the inhibitory effects of PZC,PZD,PZE,PZF,PZG and PZH on the TGF-?/Smad signaling pathway in HK-2 cells and podocytes induced by TGF-?1 and AngII.We further evaluated the effects of PZC,PZD,PZE,PZF,PZG and PZH on the expression of Smad2,Smad3,p-Smad2,p-Smad3,Smad4 and Smad7.6.Unilateral ureteral occlusion?UUO?method was used to induce TIF in mice.H&E staining was used to observe kidney damage,and Masson's staining was used to observe fibrotic effect.The qRT-PCR and Western blot methods were used to determine protein expression to illuminate the influence of PZC,PZD and PZE to RAS,Wnt/?-catenin and TGF-?/Smad signaling pathways in the obstructed kidney of UUO mice.Result:1.Systematic identification of the compositions of SLPC.100 compounds had been isolated and identified from ethyl acetate fraction of SLPC,including 36 3,4-seco-lanostan type triterpenes:poricoic acid A?FLP-1?,poricoic acid B?FLP-2?,poricoic acid E?FLP-5?,poricoic acid ZA?FLP-6?,poricoic acid D?FLP-7?,poricoic acid F?FLP-8?,poricoic acid ZB?FLP-9?,PZC?FLP-10?,poricoic acid G?FLP-12?,26-hydroxy-poricoic acid DM?FLP-13?,PZD?FLP-14?,poricoic acid AM?FLP-17?,poricoic acid C?FLP-23?,16-deoxyporicoic acid B?FLP-24?,PZG?FLP-27?,3,4-seco-lanosta-7,9?11?,24-trien-21-oicacid3-oate?FLP-30?,15?-hydroxylanosta-8?9?,24?25?-diene-3,21-dioic acid?FLP-32?,poricoic acid ZJ?FLP-34?,poricoic acid ZK?FLP-35?,poricoic acid AE?FLP-39?,6,7-dehydroporicoic acid H?FLP-41?,poricoic acid CE?FLP-44?,poricoic acid CM?FLP-51?,poricoic acid ZM?FLP-58?,poricoic acid ZO?FLP-63?,25-methoxy-poricoic acid A?FLP-66?,poricoic acid ZQ?FLP-71?,poricoic acid ZR?FLP-72?,poricoic acid ZS?FLP-73?,poricoic acid ZT?FLP-76?,poricoic acid ZU?FLP-80?,poricoic acid BM?FLP-82?,poricoic acid ZV?FLP-83?,poricoic acid GE?FLP-87?,16?-hydroxy-3,4-seco-lanosta-4?28?,7?9?,11,24-tetraene-3,21-dioic acid-3-ethyl ester?FLP-89?,poricoic acid GM?FLP-97?;38 lanostance tetracyclic triterpenoids:dehydrotrametenolic acid?FLP-3?,dehydroeburicoic acid?FLP-4?,3-epi-dehydrotumolosic acid?FLP-11?,3?,16?-dihydroxylanosta-7,9?11?,24-triene-21-oic acid?FLP-16?,3-O-acetyl-16?-hydroxyl dehydrotrametenolic acid?FLP-18?,dehydropachymic acid?FLP-19?,pachymic acid?FLP-20?,eburicoic acid?FLP-21?,PZE?FLP-22?,trametenolic acid?FLP-26?,PZH?FLP-28?,16?-hydroxytrametenolicacid?FLP-29?,poricoicacidZI?FLP-31?,16?-hydroxy-3-oxo-24-methyl-lanosta-5,7,9?11?,24?31?-tetraen-21-oic acid?FLP-33?,poricoic acid ZL?FLP-36?,3?,15?-dihydroxy-lanosta-7,9?11?,24-trien-21-oic acid?FLP-37?,3-epi-dehydropachymic acid?FLP-38?,pohyporenic acid C?FLP-40?,3?-O-acetyl-hydroxyl pachymic acid?FLP-43?,25-hydroxypachymic acid?FLP-45?,15?-hydroxy-dehydrotumulosic acid?FLP-47?,5?,8?-peroxydehydro-tumulosic acid?FLP-48?,6?-dehydropolyporenic acid C?FLP-49?,3?-O-acetyl-16?-hydroxy-lanosta-8,24-diene-21-oic acid?FLP-52?,3?-O-acetyl-lanosta-7,9?11?,24?31?-trien-21-oic acid?FLP-55?,3-oxo-16?-hydroxy-lanosta-7,9?11?,24?31?-trien-21-oic acid?FLP-61?,poricoic acid ZN?FLP-62?,3?,16?-dihydroxy-24-oxolanost-7,9?11?-dien-21-oic acid?FLP-64?,3?,15?-dihydroxy-lanosta-8,24-diene-21-oic acid?FLP-65?,poricoic acid ZP?FLP-67?,16?,27-dihydroxy-dehydrotrametenoic acid?FLP-68?,3?,16?-dihydroxy-7-oxo-24-methyllanosta-8,24?31?-dien-21-oic acid?FLP-69?,dehydro-trametenonic acid?FLP-77?,3-oxol-anosta-8?9?,24-diene-21-oic acid?FLP-78?,dehydroeburiconic acid?FLP-79?,3?,15?,16?-trihydroxy-lanosta-7,9?11?,24?31?-trien-21-oic acid?FLP-81?,poricoic acid ZW?FLP-84?and 3-?2-hydroxy-acetoxy?-5?,8?-peroxydehydrotumulosic acid?FLP-98?;one tricyclic terpane:poricoic acid ZF?FLP-25?;five diterpenoids:dehydroabietinol?FLP-53?,dehydroabietlc acid?FLP-54?,7-oxol-15-hydroxydehydroabietic acid?FLP-75?,dehydroabietic acid?FLP-85?,1?,16-dihydroxy-dehydroabietic acid?FLP-91?;five flavonoids:3',4',5,6,7,8-hexamethoxy-flavone?FLP-74?,4-hydroxy-3',5,6,7,8-pentamethoxy flavone?FLP-90?,wogonin?FLP-92?,4',5,6,7,8-pentamethoxyflavone?FLP-95?,4',7-dihydroxy isoflavone?FLP-100?;three steroids:ergosterol?FLP-46?,?-sitosterol?FLP-56?,ergosta-4,6,8?14?,22-tetraen-3-one?FLP-59?;one lignan:matairesinol?FLP-60?;one anthraquinone:aloe-emodin?FLP-94?;and ten other small molecule compounds:1,2-benzenedicarboxylic acid-1,2-dibutyl ester?FLP-15?,dibutyl phthalate?FLP-42?,1,2-benzenedicarboxylic acid-1,4-bis?6-methylheptyl?ester?FLP-50?,palmitic acid?FLP-57?,vanillin?FLP-70?,4,5-dihydroxy-9-methanol-12-?14,15-dimethyl?-methanol-1,7-diphenyl?FLP-86?,3,4,5-trihydroxy-benzaldehyde?FLP-88?,maltose?FLP-93?,4-hydroxy-2-nonenoic acid?FLP-96?,4,5-dihydroxy-10-methyl-11-?14,15-dimethyl?-methanol-1,7-diphenyl?FLP-99?.Among them,twenty-five compounds were new compounds,including FLP-6,9,10,14,22,25,27,28,31,34,35,36,58,62,63,67,71,72,73,76,80,83,84,86 and 99.Furthermore,seventeen known compounds including FLP-32,37,43,60,61,74,75,79,88,90,91,92,93,94,95,96 and 100 were isolated from Poria Cocos for the first time.2.Poricoic acids attenuated TGF-?1-and AngII-induced injury in HK-2 cells and podocytes,and ameliorated TIF in UUO mice.3,4-seco-lanostan type triterpenes including PZC,PZD and PZG and lanostan type triterpenes including PZE and PZH significantly inhibited the expression of fibrogenic proteins including collagen I,fibronectin and?-SMA,as well as maintained E-cadherin expression in TGF-?1-and AngII-induced HK-2 cells.Besides,they significantly enhanced the protein expression of podocyte markers including podocin,nephrin,podocalyxin and synaptopodin.In the obstructed kidney of UUO mice,poricoic acids PZC,PZD and PZE reduced inflammatory cell infiltration and extracellular matrix accumulation,and downregulated the expression of profibrotic proteins,including collagen I,fibronectin and?-SMA.3.Poricoic acids inhibited activation of RAS.Western blot,qRT-PCR and immunofluorescence results showed that both TGF-?1 and AngII activated RAS and upregulated expression of AGT,renin,ACE and AT₁R at both mRNA and protein levels in HK-2 cells and podocytes.Poricoic acids significantly inhibited the activation of RAS induced by TGF-?1 and AngII in both HK-2cells and podocytes.Ureteral obstruction resulted in upregulation of protein expression RAS components in renal tissue of the UUO mice,while activation of RAS was significantly inhibited after the treatment of poricoic acids.Poricoic acids significantly inhibited the activation of RAS in vitro and in vivo experiments,and can be used as a novel RAS inhibitor in new drug development.4.Poricoic acids inhibited activation of Wnt/?-catenin signaling pathway.TGF-?1 and AngII activated transcription of?-catenin and mediated expression of its downstream gene,including Snail1,Twist,MMP-7,PAI-1,and FSP-1 in HK-2 cells and podocytes.Furthermore,poricoic acids significantly inhibited the TGF-?1-and AngII-induced activation of Wnt/?-catenin signaling pathway both in HK-2 cells and podocytes.The expressions of Wnt1 and downstream proteins were significantly up-regulated in the obstructed renal tissues of UUO mice,while poricoic acids significantly inhibited the activation of Wnt/?-catenin pathway and showing a significant anti-fibrotic effect in vivo.5.Poricoic acids selectively inhibited Smad3 phosphorylation.TGF-?1 was the most important profibrotic factor,which contribute to TIF and glomerulosclerosis through a myriad of signaling pathways.TGF-?/Smad signaling pathway would be considered as a therapeutic target for anti-fibrotic drug development.Both TGF-?1 and AngII activated TGF-?/Smad signaling pathway in HK-2 cells and podocytes and upregulated the expression of p-Smad2,p-Smad3 and Smad4,while down-regulated Smad7 expression.Poricoic acids including PZC,PZD,PZE,PZG and PZH selectively inhibited the phosphorylation of Smad3,while they hardly reversed the abnormality of Smad4,Smad7 and p-Smad2.Ureteral bstruction induced the activation of TGF-?/Smad signaling pathway in renal tissue of mice,while poricoic acids selectively inhibited the phosphorylation of Smad3.6.The carboxyl group at C-3 and the hydroxyl group at side branch of poricoic acids was the bioactive functional group against renal fibrosis.PZD showed a stronger inhibitory effect on the TGF-?1-and Ang II-induced excessive extracellular matrix accumulation than PZC,while PZC was stronger than PZE,and PZG showed a strong inhibitory effect than PZH,which indicating the transformation from A ring of lanostance tetracyclic triterpenoids to carboxyl group contributed to their inhibitory effect on renal fibrosis.Moreover,the increasing number of hydroxyl groups at side branch?C-24 and C-31?contributed to the inhibitory effect on renal fibrosis.Conclusion:In this study,we isolated and indentified 100 compounds from SLPC.Among them,25 new compounds and 17 known compounds were first isolated and indentified from Poria Cocos.3,4-seco-lanostan type triterpenes?PZC,PZD and PZG?and lanostan type triterpenes?PZE and PZH?were illuminated to attenuate renal fibrosis by inhibiting activation of RAS and Wnt/?-catenin signaling pathway,as well as selectively inhibiting Smad3 phosphorylation level in HK-2 cells,podocytes and UUO mice.This study showed the effective components of SLPC and action mechanisms of SLPC against renal fibrosis in vivo and in vitro.The results in this study provided the lead compounds against renal fibrosis by selectively inhibiting Smad3phosphorylation,and also provided theoretical and experimental foundation for the application of SLPC.