Effective Constituents And Mechanism Of Plantaginis Semen Against Aryl Hydrocarbon Receptor-Mediated Renal Fibrosis

Plantaginis Semen?PS?,the dried and matured seeds of Plantago asiatic L.or Plantago depressa Wild.,has been uesed for relieving pain,heat stranguria,cure edema,diarrhoea,hot eyes,heat cough,distending pain and sputum in clinical application.Numerous studies have revealed that phenylethanoid glycosides,iridoids,flavonoids,steroids,alkaloids,terpenes,amino acids and polysaccharides are the main components of PS.Modern pharmacological studies showed PS had diuretic,hypolipidemic,antiinflammatory,immunomodulatory,antioxidant,renoprotective and hepatoprotective effects.Tubulointerstitial fibrosis?TIF?is the final clinical manifestation of chronic kidney disease.TIF causes high morbidity and mortality worldwide due to the lack of effective treatment.Thus,it is one of difficulties in medical research to elucidate the mechanism of TIF,effectively prevent and treat TIF.Aryl hydrocarbon receptor?AhR?,a ligand-activated transcription factor,activated during renal diseases.Therefore,AhR shows a promising target for drug targeting intervention.It is revealed that endogenous aryl-containing metabolites activated AhR to promote TIF,systematically studied the chemical constituents of PS,and demonstrated that lignans and flavonoids in PS showed against TIF effect by inhibiting AhR signaling pathway in this study.Objective:1.The current aim focuse on the study of the biochemical and molecular mechanisms of TIF mediated by AhR,which will provide a novel target for the discovery of new drugs against TIF by antagonizing AhR.2.We further investigate the chemical constituents of PS,screen the antagonists of AhR from PS and further elucidate the mechanism of the antagonists of AhR against renal fibrosis by regulating AhR,providing a theoretical and experimental basis for clarifying the effect of PS against TIF.Methods1.PS?600 kg?was pulverized and extracted with 80%methanol to get the extract,which then was partitioned between water and organic solvents and then successively extracted with petroleum ether and ethyl acetate three times to yield petroleum ether and ethyl acetate fractions,respectively.2.The compounds was isolated from ethyl acetate fraction by using separated columns including MCI gel CHP 20P,silica gel,RP-18,Sephadex LH-20,TLC and HPLC.The structures of the compounds were identified by ¹H-NMR and ¹³C-NMR.3.Male C57BL/6 mice weighing 20-25g?n=32?were randomly divided into sham group and the UUO groups at weeks 1,2 and 3.UUO was used to make TIF in mice.Kidney tissue were collected for biochemical parmetars,histopathological analysis,and metabolomics at weeks 1,2 and 3 after UUO procedure.The antifibrotic effect of GFA was evaluated with UUO mice.UUO+GFA group was orally adiministerd GFA?20 mg/kg/d?dissolved in 0.5%CMC-Na solution by gastric irrigation for 2 weeks.Sham and UUO groups were only administered with 0.5%CMC-Na solution by oral gavage.Kidney tissues were obtained for biochemical parmetars,histopathological analysis,and metabolomics.4.Male SD rats weighing 190-210 g?n=80?were randomly divided into 10 groups,including sham group and the 5/6 nephrectomzied?NX?groups at weeks 0,1,2,3,4,5,6,9,12 and 20.Serum and kidney tissue were obtained for biochemical parmetars,histopathological analysis,and metabolomics at week 0 1,2,3,4,5,6,9,12 and 20 after NX procedure.The antifibrotic effects of PHF and BSA were evaluated with NX rats.NX+PHF and NX+BSA groups was orally adiministerd PHF?10 mg/kg/d?and BSA?10 mg/kg/d?dissolved in 0.5%CMC-Na solution by gastric irrigation from 9^thh week to 12^thh week.Sham and NX groups were only administered by oral gavage with 0.5%CMA-Na solution.Serum and kidney tissues were collected for biochemical parmetars,histopathological analysis,and metabolomics.5.The antifibrosis effect of GFA was evaluated with renal tubular adventitial cells?NRK-52E?induced by indoxyl sulfate.6.Metabolic profile of the kidney tissues from UUO mice and NX rats were determined by using ultra-performance liquid chromatography coupled with high-definition mass spectrometry?UPLC-HDMS?and multivariate pattern recognition.The important mass spectral data were selected through principal component analysis?PCA?and LASSO softwares,and the metabolites were identified by using mass spectromety fragment information,database searches and reference comparisons to the structure identificantion.7.AhR protein from mouse and rat were homologously modeled using SWISS.Autodock 4.0software was used to analyze the interaction between aryl-containing metabolites or compouds isolated from PS and AhR,and the interaction effect was analysised by using Discover Studio 4.5Client and Pymol softwares.8.We performed the expression analyses of AhR and its target genes?CYP1A1,CYP1A2,CYP1B1,COX-2?and probrotic genes including collagen I,?-SMA,fibronectin,vimentin,FSP1 and E-cadherin in the kidney tissue from UUO mice,NX rats and drug-treated mice or rats were based on pathological methods?HE,Masson trichrome,immunohistochemical staining?and molecular biology methods?PCR,Western blot,RNA overexpression and RNA interference?.Results1.Identification of the chemical constituents from PS33 compounds were isolated from the ethyl acetate fraction of 80%methanol extract of PS by column chromatography,including 6 steroids:?-sitosterol?CQZ-1?,7-oxo-?-sitosterol?CQZ-2?,5?-stigmast-3,6-dione?CQZ-3?,stigmast-4-en-6?-ol-3-one?CQZ-4?,6?-hydroxycampest-4-en-3-one?CQZ-5?,6?-hydroxycholest-4-en-3-one?CQZ-6?;6 phenols:vanillic acid?CQZ-7?,4-vinylguaiacol?CQZ-8?,,phloretic acid methy lester?CQZ-9?,methyldihydroferulate?CQZ-10?,vanillin?CQZ-11?,5,7-dihydroxychromone?CQZ-12?;2 lignans:erythro-guaiacylglycerol-?-ferulic acid ether?GFA,CQZ-13?,threo-guaiacylglycerol-?-ferulic acid ether?CQZ-14?;3 flavonoids:barlerisides A?BSA,CQZ-15?,5,7,3',4',5'-pentahydroxyflavanone?PHF,CQZ-16?,rhoifolin?CQZ-17?;3 phenylethanoid glycosides:isoacteoside?CQZ-18?,2-phenylethyl 3-O-?6-deoxy-?-L-mannopyranosyl?-?-D-glucopyranoside?CQZ-19?,calceolariosideB?CQZ-20?;5 alkaloids:indole-3-carbaldehyde?CQZ-21?,3-indoleethanol?CQZ-22?,indol-3-carboxylic acid?CQZ-23?,3-?Hydroxyacetyl?indole?CQZ-24?,acetyl tryptophan methyl ester?CQZ-25?;3 terpenes:3-oxo-?-ionol?CQZ-26?,3-oxo-7,8-dihydro-?-ionol?CQZ-27?,tricyclohumuladiol?CQZ-28?;5fatty acids:linoleic acid?CQZ-29?,stearic acid?CQZ-30?,eicosane acid?CQZ-31?,1-heneicosanol?CQZ-32?and dimethyl azelate?CQZ-33?.Among these compounds,18 compounds were firstly isolated from PS,including CQZ-2,-3,-4,-5,-6,-8,-9,-11,-14,-15,-16,-19,-22,-24,-25,-26,-27and-28.2.UUO causes renal metabolic disorders and activated AhR signaling pathwayMetabolomics showed significantly differences in metabolic profiles between UUO group and sham group at weeks 1,2,and 3.A total of 231 metabolites were identified in both positive and negative ion modes,and the levels of 50 metabolites were higher in UUO mice compared to sham group at weeks 1,2 and 3.In addition,The levels of 1-methoxypyrene,indoxyl sulfate?IS?,6-hydroxymelatonin and 5-methoxytryptamine were significantly higher than that of sham group in kidney tissue of UUO mice,and docking analysis revealed the 4 aryl-containing metabolites could interact with the ligand binding domain of AhR.3.AhR activation promoted EMT in kidney tissue of UUO mice.Firstly,the significantly increased AhR protein expression in nuclei and significantly decreased AhR protein expression in cytoplasm were observed in kidney tissue of UUO mice at weeks 1,2 and 3.In addition,the mRNA levels of AhR and its target genes CYP1A1,CYP1A2,CYP1B1 and COX-2 were correspondingly increased in a time-dependent manner in kidney tissue of UUO mice and were higher than that of sham group,which suggested that AhR was activated during TIF.Secondly,the level of1-methoxypyrene,IS,6-hydroxymelatonin and 5-methoxytryptamine were significantly increased in kidney tissue of AhR overpression mice with UUO,and significantly decreased in AhR deficiency mice with UUO compared to UUO mice.The mRNA expression levels of AhR and its target genes including CYP1A1?CYP1A2?CYP1B1 and COX-2 were significantly increased in kidney tissue of AhR overpression mice with UUO,and they were significantly decreased in AhR deficiency mice with UUO compared to UUO mice.Finally,the upregulated expressions of collagen I,?-SMA and fibronectin as well as downregulated expression of E-cadherin were observed in AhR overexpression mice with UUO compared to UUO group.Similarly,the downregulated collagen I,?-SMA and fibronectin expressions as well as upregulated E-cadherin expression were also observed in AhR deficiency mice with UUO compared to UUO mice.4.GFA showed an antifibrotic effect by suppressing AhR signaling pathwayFirstly,GFA treatment improved renal injury and reduced extracellular matrix deposition in UUO mice.GFA treatment significantly downregulated nuclear AhR expression and upregulated cytoplasmic AhR expression in UUO mice.GFA treatment significantly inhibited upregulated mRNA expressions of CYP1A1,CYP1A2,CYP1B1 and COX-2 in UUO mice compared to UUO mice.Moreover,GFA treatment inhibited upregulated expressions of collagen I,?-SMA and fibronectin,vimentin and FSP1 and enchanced downregulated E-cadherin expression.These results indicated that GFA inhibited AhR nuclear translocation in obstructed kidney of UUO mice,thereby inhibited TIF.Secondly,GFA inhibited the AhR nuclear translocation in IS-treated NRK-52E cells?10?M?.In addition,GFA reduced the mRNA expression of CYP1A1,CYP1A2,CYP1B1 and COX-2 and downregulated the expression of collagen I,?-SMA,fibronectin and upregulated E-cadherin expression.After specific AhR RNAi transfection,the inhibitory effect of GFA on AhR expression and EMT was partially weakened in NRK-52E cells.Finally,GFA significantly downregulated the expression of collagen I,?-SMA,fibronectin and upregulated E-cadherin expression in the obstructed kidney of AhR overpression mice with UUO.5.NX causes renal metabolic disorders and activated AhR pathwayFirstly,based on metabolomics of kidney tissue,PCA results showed that NX rats were significantly separated from sham-operated rats at weeks 1,2,3,4,5,6,9,12 and 20.Renal metabolic disorder presented a certain correlation with kidney damage throughout the entire study period.Secondly,206 differential metabolites were identified with LASSO procedure,and the levels of 6aryl-containing metabolites were significantly increased in the remant kidney of NX rats compared to sham rats.In addition,linear regression analysis showed that 1-hydroxypyrene,1-aminopyrene and 1-methoxypyrene revealed a positive correlation with serum creatinine level and a negative correlation with GFR.Significantly increased AhR protein expression in nuclei and significantly decreased AhR protein expression in cytoplasm were observed in the remant kidney of NX rats.Finally,docking analysis revealed that 6 aryl-containing metabolites bound to the active site of AhR and showed a strong interaction.6.Flavonoids incluidng PHF and BSA ameliorated renal fibrosis by suppressing AhR pathway PHF and BSA significantly reduced the level of serum creatinine,urea and proteinuria,and evlavated GFR in the remant kidney of NX rats.PHF and BSA treatment could significantly downregulate nuclear AhR expression and upregulate cytoplasmic AhR expression in the remant kidney of NX rats.In addition,PFH and BSA could bind to the ligand binding site of AhR,suggesting that PHF and BSA may improve renal function by competing for the AhR binding site.ConclusionThis study revealed that progressive renal fibrosis led to endogenous metabolite disorder in UUO and NX animal models and further demonstrated renal fibrosis was mediated through AhR signaling pathway activation induced by endogenous aryl-containing metabolites.Our study identified 33compounds from PS and 18 compounds were firstly identified from PS.This study revealed that GFA,PHF and BSA isolated from PS ameliorated renal fibrosis by antagonizing AhR.The study illuminated the renoprotective constituents of PS and revealed antifibrotic mechanism of biochemistry and molecular biology.This paper will provide lead compounds against renal fibrosis by antagonizing AhR,and provide theoretical basis and experimental reference for the development and application of PS.