Wa waco ni rwom me remo pa gin ma ki lwongo ni gut-derived tryptophan metabolite indole-3-propionic acid (IPA) tye piny i kom lutwo ma tye ki two ma mako adunu. I kwan man, wa kwedo transcriptome ki DNA methylome i kom dano ma kom gi pek ma lubu rwom me IPA i remo, kacel ki tic pa IPA i kelo tic pa hepatic stellate cells (HSCs) i vitro.
Kwano ni tye ki lutwo 116 ma kom gi pek ma pe ki two cukari (T2DM) (mwaka 46.8 ± 9.3 mwaka; BMI: 42.7 ± 5.0 kg/m2) ma gukato ki i ot yat ma ki lwongo ni bariatric surgery i Kuopio Bariatric Surgery Center (KOBS). Ki pimo rwom me IPA ma tye ka mol ki liquid chromatography-mass spectrometry (LC-MS), ki kwano lok ma mako adunu ki kwano RNA weny, ki kwano methylation me DNA kitimo kun kitiyo ki Infinium HumanMethylation450 BeadChip. Gin ma ki lwongo ni Human hepatic stellate cells (LX-2) ki otiiyo kwede me temo gin ma ki lwongo ni in vitro.
Kit ma IPA tye kwede i remo rwate ki nyutu pa gene ma tye i apoptotic, mitophagic, ki kwo mabor i adunu. Gin ma ki lwongo ni AKT serine/threonine kinase 1 (AKT1) gene onwongo tye ma pol ki ma loyo gene ma tiyo i kom adunu ki DNA methylation profiles. Yat me IPA okelo apoptosis, odwoko piny yweyo pa mitochondrial, ki oloko kit ma cell nen kwede ki kit ma mitochondrial tiyo kwede kun loko kit ma gene ma ki ngeyo ni gi doro fibrosis, apoptosis, ki kwo pa LX-2 cells.
Ka ki kwanyo ne kacel, ngec eni nyutu ni IPA tye ki adwogi maber ki twero kelo apoptosis ki loko kit ma HSC nen kwede me bedo ma pe tiyo, ma weko medo kero me gengo fibrosis me adunu kun balo tic pa HSC ki tic pa mitochondrial.
Kit ma two ma mako kom ki two ma mako kom dano tye ka nya kwede ki nongo ni tye ka mede ki two ma mako adunu ma mako kom dano (MASLD); two ni mako 25% onyo 30% pa dano weny [1]. Adwogi madit pa MASLD etiology tye liver fibrosis, gin ma tiime ma ki ngeyo ki dongo pa fibrous extracellular matrix (ECM) [2]. Cell ma pire tek ma tye i two ma mako adunu aye cell ma ki lwongo ni hepatic stellate cells (HSCs), ma nyutu kit angwen ma ki ngeyo: quiescent, activated, inactivated, ki senescent [3, 4]. HSCs twero tic ki loko gi ki i kit ma gi tye ka tic kwede me doko cell ma calo fibroblast ma mito teko madwong, ki medo rwom me α-smooth muscle actin (α-SMA) ki type I collagen (Col-I) [5, 6]. I kare me loko two ma mako adunu, HSCs ma ki tiyo kwede ki kwanyo gi woko ki i apoptosis onyo inactivation. Tic magi tye iye dwoko piny rwom me fibrogenic genes ki loko rwom me kwo pa genes (ma calo NF-κB ki PI3K/Akt signaling pathways) [7, 8], kacel ki alokaloka i kit ma mitochondrial tiyo kwede ki tic [9].
Kit ma remo tye kwede ikom gin ma ki lwongo ni tryptophan metabolite indole-3-propionic acid (IPA), ma ki yubu i ic, kinongo ni odok piny i two ma mako kom dano ma tye iye MASLD [10-13]. IPA rwate ki cam ma ki camo, ki ngeyo pi gengo two ki gengo two, ki dwoko piny two ma pe kelo kongo (NASH) ma bino pi cam i kom dano ki i kom dano [11-14]. Gin mogo ma ki nongo oa ki i kwan wa mukato, ma onyutu ni rwom me IPA i remo onongo tye piny i kom lutwo ma tye ki two ma mako adunu ma loyo lutwo ma kom gi pek ma pe ki two ma mako adunu i Kuopio Bariatric Surgery Study (KOBS). Medo ikom meno, wanyutu ni yat IPA twero dwoko piny nyutu pa gene ma obedo lanyut me cell adhesion, cell migration ki hematopoietic stem cell activation i human hepatic stellate cell (LX-2) model ki twero gwoko hepatoprotective metabolite [15]. Ento, pwod pe tye atir kit ma IPA kelo kwede two ma mako adunu kun weko HSC to ki mitochondrial bioenergetics.
Kany, wanyutu ni serum IPA rwate ki nyutu gene ma tye ki apoptosis, mitophagy, ki yoo me kwo pi kare malac i adunu pa dano ma kom gi pek ento pe ki two cukari me 2 (KOBS). Medo ikom meno, wanongo ni IPA twero kelo kwanyo ki balo jami ma ki lwongo ni activated hematopoietic stem cells (HSCs) kun wok ki i yoo me inactivation. Adwogi eni nyutu tic manyen pa IPA, ma miyo en bedo gin ma ki tiyo kwede me cango two ma mako adunu.
Kwan ma okato i dul pa KOBS onyutu ni lutwo ma tye ki two ma mako adunu onongo tye ki rwom me IPA ma piny ka kiporo ki lutwo ma pe ki two ma mako adunu [15]. Me kwanyo adwogi marac ma twero bedo tye i kom two cukari me dul me 2, wa coyo dano 116 ma kom gi pek ma pe ki two cukari me dul me 2 (mwaka ma i kine ± SD: 46.8 ± 9.3 mwaka; BMI: 42.7 ± 5.0 kg/m2) (Table 1) ki i kwan me KOBS ma tye ka wot anyim macalo wel dano ma ki kwano [16]. Dano weny ma gubedo i yub man gu miiyo cwak gi ma ki coyo ki yub me kwan ni ki yee ki komiti ma neno kit ma dano myero gukwo kwede i ot yat me North Savo County malube ki cik me Helsinki (54/2005, 104/2008 ki 27/2010).
Gin ma ki kwanyo ki i adunu ki nongo i kare me yat ma ki lwongo ni bariatric surgery ki ki kwano ki ludac ma ngeyo lok kom two ma lubu kit ma ki waco kwede con [17, 18]. Kit me temo ni ki coyo gi i Supplementary Table S1 ki dong ki waco con [19].
Kamu kene ma ki kwanyo ki i kom dano ma ki kwanyo ki i cam ki kwano ki untargeted liquid chromatography-mass spectrometry (LC-MS) pi metabolomics analysis (n = 116). Kamu kene ni ki kwano kun kitiyo ki UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) kit ma kiloko kwede con19. Ngeyo kongo ma ki lwongo ni isopropyl alcohol (IPA) onongo jenge i kom kare me gwoko ki poro MS/MS spectrum ki rwom ma opore. Kit ma IPA signal intensity (kabedo ma malo) ki keto i tic i kom jami ma ki medo [20].
Kit ma ki lwongo ni Whole liver RNA sequencing ni kitimo kun kitiyo ki Illumina HiSeq 2500 ki ngec ni ki yubu con kit ma kiloko kwede con [19, 21, 22]. Watimo kwan ma ki yubu me poko kin jami ma ki coyo ma gudo tic pa mitochondrial/biogenesis kun wa tiyo ki gene 1957 ma ki yero ki i MitoMiner 4.0 database [23]. Kwedo DNA me adunu ki timo kun kitiyo ki Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) kun kitiyo ki yoo acel ma kiloko iye con [24, 25].
Gin ma ki lwongo ni Human hepatic stellate cells (LX-2) ki miino ki bot Prof. Stefano Romeo ki ki pito gi ki gwoko gi i DMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 107-1060). Me yero rwom me tic pa IPA, LX-2 cells ki tiyo kwede ki rwom me IPA ma pat pat (10 μM, 100 μM ki 1 mM; Sigma, 220027) i DMEM/F12 pi cawa 24. Medo ikom meno, me kwedo kero pa IPA me juku tic pa HSCs, LX-2 cells ki tiyo kwede kacel ki 5 ng/ml TGF-β1 (R&D systems, 240-B-002/CF) ki 1 mM IPA i serum-free medium pi cawa 24. Pi jami ma ki tiyo kwede, 4 nM HCL ma tye ki 0.1% BSA ki tiyo kwede pi TGF-β1 ki 0.05% DMSO ki tiyo kwede pi IPA, ki gin aryo ni ki tiyo kwede kacel pi tic ma ki rubu.
Apoptosis ki temo kun kitiyo ki FITC Annexin V Apoptosis Detection Kit ki 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) kit ma lucat wil owaco kwede. Ki lok macek, LX-2 (1 × 105 cells/well) ki pito gi dyewor i 12-well plates ki dong ki tiyo kwede ki IPA onyo IPA ki TGF-β1 mapol. I nino ma lubo, ki kwanyo cell ma tye ka mol ki ma mako, ki lwoko ki PBS, ki dwoko i Annexin V binding buffer, ki ki keto i FITC-Annexin V ki 7-AAD pi dakika 15.
Mitochondria ma tye i cell makwo ki keto rangi i kom tic pa oxidative kun kitiyo ki MitotrackerTM Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Pi temo MTR, LX-2 cells ki gwoko gi i rwom ma rom ki IPA ki TGF-β1. Iyonge cawa 24, cell makwo ki trypsinized, ki lwoko ki PBS, ki dong ki keto i 100 μM MTR i serum-free medium i 37 °C pi 20 min kit ma kiloko kwede con [26]. Pi nyamo kit ma cell ma kwo tye kwede, dit pa cell ki kit ma cytoplasmic tek kwede ki kwano kun kitiyo ki forward scatter (FSC) ki side scatter (SSC) parameters, kit meno.
Ngec weny (30,000 ma otimme) ki nongo ki tic ki NovoCyte Quanteon (Agilent) ki ki kwano ki tic ki NovoExpress® 1.4.1 onyo FlowJo V.10 software.
Kit ma yamo tiyo kwede (OCR) ki kit ma cell tiyo kwede (ECAR) ki pimo i kare kikome kun kitiyo ki Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) ma ki keto iye Seahorse XF Cell Mito Stress kit ma lucat wil owaco kwede. Ki lok macek, 2 × 104 LX-2 cells/well ki keto i kom XF96 cell culture plates. Iyonge gwoko ne pi dyewor, ki tiyo ki isopropanol (IPA) ki TGF-β1 (Yoo me medo 1). Kwano ngec ni kitimo kun kitiyo ki jami ma ki lwongo ni Seahorse XF Wave software, ma tye ki Seahorse XF Cell Energy Phenotype Test Report Generator. Cake i kom lok man, ki kwano Bioenergetic Health Index (BHI) [27].
RNA weny ki coyo i cDNA. Pi yoo mogo ma ki ngeyo, nen gin ma ki coyo [15]. Ki tiyo ki rwom me mRNA pa dano ma ki lwongo ni Human 60S ribosomal acidic protein P0 (RPLP0) ki cyclophilin A1 (PPIA) ma calo jami ma ki tiyo kwede me doro gene. QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, The Netherlands) ki tiyo kwede ki TaqManTM Fast Advanced Master Mix Kit (Applied Biosystems) onyo Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), ki rwom me gene ma ki poro ki cyc jami ma ki keto (ΔΔCt) ki yoo me ∆∆Ct. Lok ma mako jami ma ki lwongo ni primers ni ki miyo i Supplementary Tables S2 ki S3.
Nuclear DNA (ncDNA) ki mitochondrial DNA (mtDNA) ki kwanyo kun kitiyo ki DNeasy blood and tissue kit (Qiagen) kit ma kiloko kwede con [28]. Wel ma rwate pa mtDNA ki kwano kun ki kwano ratio pa dul mtDNA acel acel ma ki yubu ki rwom me geometric mean pa dul DNA adek (mtDNA/ncDNA), kit ma ki waco kwede i Supplementary Methods 2. Lok ma mako primers pi mtDNA ki ncDNA ki miyo i Supplementary Table S4.
Cell makwo ki keto rangi i kom gi ki MitotrackerTM Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) me neno kin cell ki intracellular mitochondrial networks. LX-2 cells (1 × 104 cells/well) ki pito gi i kom gin ma ki lwongo ni glass slides ma tye ki glass-bottomed culture plates (Ibidi GmbH, Martinsried, Germany). Iyonge cawa 24, LX-2 cells makwo ki gwoko ki 100 μM MTR pi 20 min i 37 °C ki cell nuclei ki keto DAPI (1 μg/ml, Sigma-Aldrich) kit ma kiloko kwede con [29]. Mitochondrial networks ki neno kun kitiyo ki Zeiss Axio Observer inverted microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) ma ki keto iye Zeiss LSM 800 confocal module i 37 °C i yamo ma ngiic ki 5% CO2 kun kitiyo ki 63×NA 1.3 objective. Wanongo cal apar ma ki lwongo ni Z-series pi dul acel acel ma ki nyutu. Z-series acel acel tye ki dul 30, acel acel ki pek pa 9.86 μm. Pi labol acel acel, cal pa dul ma pat pat apar ki nongo ki tic ki ZEN 2009 software (Carl Zeiss Microimaging GmbH, Jena, Germany), ki kwan ma mako kit ma mitochondrial nen kwede ki tiyo ki ImageJ software (v1.54d) [30, 31] malube ki jami ma ki waco i Supplementary Methods 3.
Ki keto cell ni ki 2% glutaraldehyde i 0.1 M phosphate buffer, ki dong ki keto ki 1% osmium tetroxide solution (Sigma Aldrich, MO, USA), ki kwanyo pii ki acetone (Merck, Darmstadt, Germany), ki dong ki keto i epoxy resin. Ki yubu dul ma tidi tutwal ki ki keto rangi ki 1% uranyl acetate (Merck, Darmstadt, Germany) ki 1% lead citrate (Merck, Darmstadt, Germany). Cal ma nen maber ki nongo ki tic ki JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) ki teko ma tye ka mede ki 80 kV.
Kit ma LX-2 cells ma ki tiyo kwede ki IPA pi cawa 24 nen kwede ki kwano ki phase-contrast microscopy i 50x magnification kun kitiyo ki Zeiss inverted light microscope (Zeiss Axio Vert.A1 ki AxioCam MRm, Jena, Germany).
Ngec ma ki nongo ki i ot yat ki nyutu calo gin ma tye i kine ± apoka poka ma tye i kin piny (interquartile range: IQR). Kwedo yoo acel me apoka poka (jami ma mede ki mede) onyo χ2 test (jami ma ki poko) ki tiyo kwede me poro apoka poka i kin gurup adek ma ki kwano ni. Wel ma pi adaa (FDR) ki tiyo kwede me yubu pi temo mapol, ki gene ma tye ki FDR < 0.05 ki ngeyo ni tye ma pire tek. Ki tiyo ki Spearman correlation analysis me rwate ki CpG DNA methylation ki IPA signal intensity, ki wel p ma ki ngeyo (p < 0.05) ma ki coyo.
Kwedo yoo ni kitimo kun kitiyo ki gin ma ki lwongo ni web-based gene set analysis tool (WebGestalt) pi 268 transcripts (nominal p < 0.01), 119 mitochondria-associated transcripts (nominal p < 0.05), ki 4350 CpGs ki ikom 3093 liver transcripts ma onongo tye ki rwom me IPA ma tye ka mol i remo. Gin ma ki lwongo ni Venny DB (version 2.1.0) ma ki nongo labongo cul mo ki tiyo kwede me nongo gene ma rwate, ki StringDB (version 11.5) ki tiyo kwede me neno kit ma protein-protein gi rwate kwede.
Pi yub me LX-2, ki temo kamu kene me neno ni tye maber kun kitiyo ki D'Agostino-Pearson test. Ngec ni ki nongo ki ikom jami makwo ma romo adek ki ki keto i one-way ANOVA ki Bonferroni post hoc test. P-value ma tye piny ki 0.05 ki ngeyo ni pire tek. Ngec ni ki nyutu calo rwom me ± SD, ki wel jami ma ki temo ki nyutu i cal acel acel. Kwedo lok ki cal weng ki tiimo no tic ki GraphPad Prism 8 ma ki lwongo ni statistical software pi Windows (GraphPad Software Inc., version 8.4.3, San Diego, USA).
Mukwongo, wa kwedo rwate pa rwom me IPA i remo ki adunu, kom weny, ki jami ma ki coyo i kom dano. I kin jami ma ki coyo ni, gene ma tek loyo ma rwate ki rwom me IPA i remo obedo MAPKAPK3 (FDR = 0.0077; mitogen-activated protein kinase-activated protein kinase 3); i kom jami ma ki coyo ma mako mitochondria, gene ma tek loyo obedo AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Additional file 1 ki Additional file 2).
Wa dong wa nyamo jami ma ki coyo i wilobo (n = 268; p < 0.01) ki jami ma ki coyo ma mako mitochondria (n = 119; p < 0.05), ma lacen wa nongo ni apoptosis aye obedo yoo ma pire tek loyo (p = 0.0089). Pi coc ma mako mitochondrial ma rwate ki rwom me IPA i remo, wa keto tamwa i kom apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), ki TNF signaling pathways (FDR = 0.000006) (lanyut 1A, Table 2, ki Supplementary Figures 1A-B).
Kwedo ma rwate i kom jami ma ki coyo i wilobo, mitochondria-associated transcripts, ki DNA methylation i adunu pa dano ma rwate ki rwom me IPA i remo. A nyutu 268 global transcripts, 119 mitochondria-associated transcripts, ki DNA methylated transcripts ma ki keto i 3092 CpG sites ma rwate ki serum IPA levels (p values ​​< 0.01 pi global transcripts ki DNA methylated, ki p values ​​< 0.00 pi mitochondrial transcripts). Coc ma ki coyo ma rwate ki kin gi ki nyutu i dyere (AKT1 ki YKT6). B Map me rwate pa gene 13 ma tye ki rwom me rwate ma malo (0.900) ki gene mukene ki yubu ki gene 56 ma rwate (kabedo macol) ma onongo tye ki rwate ki rwom me IPA i remo kun kitiyo ki jami tic me intanet ma ki lwongo ni StringDB. Green: Gene ma ki keto i Gene Ontology (GO) dul kom: mitochondria (GO:0005739). AKT1 tye protein ma tye ki wel ma malo (0.900) pi rwate ki protein mukene malube ki ngec (ma lubu coc ma ki nongo, temo, database, ki co-expression). Kabedo ma ki lwongo ni network nodes nyutu proteins, ki edges nyutu rwate i kin proteins.
Kit ma gut microbiota metabolites twero doro epigenetic composition kun wok ki i DNA methylation [32], wa kwedo ka ce rwom me IPA i remo onongo tye ka rwate ki liver DNA methylation. Wanongo ni kabedo aryo ma pire tek ma rwate ki rwom me IPA i remo onongo tye cok ki proline-serine-rich region 3 (C19orf55) ki heat shock protein family B (small) member 6 (HSPB6) (Additional file 3). DNA methylation pa 4350 CpG (p < 0.01) onongo tye ki rwate ki rwom me IPA i remo ki onongo tye ki rwom me kwo ma lac (p = 0.006) (lanyut 1A, Table 2, ki Supplementary Figure 1C).
Me niang kit ma jami ma kwo tiyo kwede ma tye i kin rwom me IPA i remo, coc ma ki coyo i kom dano, coc ma ki coyo ma mako mitochondria, ki DNA methylation i adunu pa dano, watimo kwedo ma rwate i kom gene ma kinongo i yoo ma okato ni (lanyut 1A). Adwogi me kwedo rwom me dongo pa gene 56 ma gi rwate (i kin dul macol i cal 1A) onyutu ni yoo me apoptosis (p = 0.00029) onyutu gene aryo ma rwate ki kwedo adek ni: AKT1 ki YKT6 (YKT6 v-SNARE homolog), kit ma ki nyutu kwede i Venagram ki cal 2 1A). Ma omyera, wanongo ni AKT1 (cg19831386) ki YKT6 (cg24161647) onongo tye ki rwate maber ki rwom me IPA i remo (Additional file 3). Me ngeyo kit ma protein twero rwate kwede i kin jami ma ki yubu ki gene, wayero gene 13 ma tye ki rwom ma malo loyo (0.900) i kin gene 56 ma rwate calo gin ma ki keto iye ki wa yubu map me rwate. Malube ki rwom me gen (gen ma nok), AKT1 gene ma tye ki wel ma malo (0.900) onongo tye malo (lanyut 1B).
Malube ki kwedo yoo, wanongo ni apoptosis obedo yoo ma pire tek, ki dong wa kwedo ka ce yat IPA obi gudo apoptosis pa HSCs i vitro. Wa onyutu con ni rwom ma pat pat me IPA (10 μM, 100 μM, ki 1 mM) onongo pe kelo peko i kom LX-2 cells [15]. Kwano man onyutu ni tic ki IPA i 10 μM ki 100 μM omedo wel cell ma kwo ki ma otoo. Ento, ka kiporo ki dul ma ki temo, kwo pa cell odok piny i 1 mM IPA concentration, ma nongo cell necrosis rate odong ma pe oloko (lanyut 2A, B). Ma lubo, me nongo rwom ma opore me kelo apoptosis i LX-2 cells, wa temo 10 μM, 100 μM, ki 1 mM IPA pi 24 h (lanyut 2A-E ki cal me medo 3A-B). I yo me miti, IPA 10 μM ki 100 μM odwoko piny rwom me to pa dano (%), kadi bed kumeno, IPA 1 mM omedo rwom me to pa dano (%) ka kiporo ki ma ki temo ki dong ki yero pi temo mukene (lanyut 2A-D).
IPA kelo to pa cell me LX-2. Annexin V ki 7-AAD yoo me keto rangi aryo ni ki tiyo kwede me kwano rwom me apoptotic ki kit ma cell nen kwede ki flow cytometry. BA cells ki gwoko gi ki 10 μM, 100 μM ki 1 mM IPA pi cawa 24 onyo ki F-H TGF-β1 (5 ng/ml) ki 1 mM IPA i kom ma pe ki remo pi cawa 24. A: cell makwo (Annexin V -/ 7AAD-); B: cell ma otoo (Annexin V -/ 7AAD+); C, F: macon (Annexin V +/ 7AAD-); D, G: late (Annexin V+/7AAD.+); E, H: wel pa cell ma gi too con ki ma lacen i rwom me to (%). Ngec ni ki nyutu calo rwom me ± SD, n = 3 ma ki temo ma pat pat. Apoka poka me wel ki otimo kun kitiyo ki one-way ANOVA ki Bonferroni post hoc test. *p < 0.05; ****p < 0.0001
Kit ma wanyuto kwede con, 5 ng/ml TGF-β1 twero kelo tic pa HSC kun medo rwom me nyutu pa genes ma ki lwongo ni classical marker [15]. LX-2 cells ni ki tiyo kwede ki 5 ng/ml TGF-β1 ki 1 mM IPA kacel (Fig. 2E–H). Yat ma ki lwongo ni TGF-β1 pi oloko rwom me to pa dano, kadi bed kumeno, tic ki IPA omedo rwom me to pa dano ma lacen ki to pa dano (%) ka kiporo ki yat TGF-β1 (Fig. 2E–H). Adwogi eni nyutu ni 1 mM IPA twero kelo apoptosis i LX-2 cells labongo TGF-β1 induction.
Wa medo kwedo adwogi pa IPA i kom yweyo pa mitochondrial i LX-2 cells. Adwogi ni onyutu ni 1 mM IPA odwoko piny rwom me tic ki yamo (OCR): yweyo ma pe ki mitochondrial, yweyo ma lapiny ki ma malo, proton leak ki yubu ATP ka kiporo ki gurup ma ki temo (lanyut 3A, B), kun bioenergetic health index (BHI) pi oloko.
IPA dwoko piny yweyo pa mitochondrial i LX-2 cells. Kit ma mitochondrial respiration curve (OCR) ki nyutu kwede calo mitochondrial respiration parameters (non-mitochondrial respiration, basal respiration, maximum respiration, proton leak, ATP generation, SRC ki BHI). Cell A ki B ki gwoko gi ki 10 μM, 100 μM ki 1 mM IPA pi cawa 24, kit meno. Gin ma ki lwongo ni C ki D ki gwoko gi ki TGF-β1 (5 ng/ml) ki 1 mM IPA i kom ma pe ki remo pi cawa 24, kit meno. Piim weng ki dwoko gi me rwate ki DNA ma tye iye kun ki tiyo ki CyQuant kit. BHI: bioenergetic health index; SRC: kero me yweyo; OCR: rwom me tic ki yamo. Ngec ni ki nyutu calo rwom me rwom ± apoka poka (SD), n = 5 jami ma ki temo ma pat pat. Apoka poka me wel ki otimo kun kitiyo ki one-way ANOVA ki Bonferroni post hoc test. *p < 0.05; **p < 0.01; ki ***p < 0.001
Me nongo niang matut ikom adwogi pa IPA i kom kit ma TGF-β1-activated LX-2 cells tiyo kwede, wa kwano mitochondrial oxidative phosphorylation ki OCR (Fig. 3C,D). Adwogi ni onyutu ni tic ki TGF-β1 twero dwoko piny yweyo ma malo, kero me yweyo (SRC) ki BHI ka kiporo ki gurup ma ki temo (Fig. 3C,D). Medo ikom meno, yat ma ki keto kacel ni odwoko piny yamo ma ki lwongo ni basal respiration, proton leak ki ATP ma ki yubu, ento SRC ki BHI onwongo tye malo loyo ma ki miyo yat ki TGF-β1 (Fig. 3C,D).
Watimo bene "Cellular Energy Phenotype Test" ma kimiyo ki Seahorse software (Supplementary Fig. 4A–D). Kit ma ki nyutu kwede i cal ma ki medo 3B, gin aryo ni OCR ki ECAR gu dwoko piny ingee tic ki TGF-β1, kadi bed kumeno, pe ki neno apokapoka mo i kin dul ma ki keto kacel ki IPA ka kiporo ki dul ma pe ki miyo. Medo ikom meno, rwom me basal ki stress pa OCR odok piny ingee keto kacel ki IPA ka kiporo ki gurup ma ki temo (Supplementary Fig. 4C). I yo me miti, kit ma kit meno bene ki neno ki yat ma ki rubu, ma pe ki neno alokaloka mo i rwom me basal ki stress pa ECAR ka kiporo ki yat TGF-β1 (Supplementary Fig. 4C). I HSCs, dwoko piny pa mitochondrial oxidative phosphorylation ki kero me tic ki yat ma ki rubu me dwoko SCR ki BHI inge nongo yat TGF-β1 pi oloko kit ma kom tiyo kwede (OCR ki ECAR). Ka ki kwanyo ne kacel, adwogi eni nyutu ni IPA twero dwoko piny teko pa HSCs, ma nyutu ni IPA twero kelo teko ma lapiny ma loko kit ma HSC nen kwede me doko inactivation (Supplementary Figure 4D).
Adwogi pa IPA i kom kit ma mitochondrial tiyo kwede ki temo kun kitiyo ki rwom adek me kit ma mitochondrial nen kwede ki rwate pa dul kom kacel ki MTR staining (lanyut 4 ki cal ma ki medo 5). cal me 4 nyutu ni, ka kiporo ki dul ma ki temo, tic ki TGF-β1 odwoko piny rwom me rwom ma malo, wel jang yat, bor pa jang yat weny, ki wel jang yat ma gi rwate (lanyut 4A ki B) ki oloko wel pa mitochondria ki i kom ma rwate ki dul ma rwate (lanyut 4C). Yat ma ki lwongo ni IPA keken aye odwoko piny wel mitochondria ki oloko wel mitochondria ki i dul ma rwate ki dul ma ki temo (lanyut 4A). Ka i poro, sphericity, bor pa jang yat, ki tic pa mitochondrial ma ki kwano ki mitochondrial membrane potential-dependent MTR (lanyut 4A ki E) odong ma pe oloko ki jami eni pe opokke i kin gurup. Ka ki kwanyo ne kacel, adwogi eni nyutu ni TGF-β1 ki IPA ma ki tiyo kwede nen calo gi loko kit ma mitochondrial nen kwede ki dit ne kacel ki kit ma dul kom ma kwo tiyo kwede i LX-2 cells.
IPA loko kit ma mitochondrial tiyo kwede ki dwong pa DNA pa mitochondrial i LX-2 cells. A. Cal ma nyutu LX-2 ma kwo ma ki gwoko ki TGF-β1 (5 ng/ml) ki 1 mM IPA pi cawa 24 i kom ma pe ki serum ma nyutu mitochondrial networks ma ki keto ki MitotrackerTM Red CMXRos ki nuclei ma ki keto blue ki DAPI. Ngec weny tye ki cal ma romo 15 pi gurup acel acel. Wanongo cal 10 me Z-stack pi dul acel acel ma ki nyutu. Z-axis sequence acel acel tye ki dul 30, acel acel ki pek pa 9.86 μm. Lapim: 10 μm. B. Jami ma nyutu (mitochondria keken) ma ki ngeyo ki keto rwom me rwate i kom cal. Kwedo wel ki poro pa dul kom ma ki lwongo ni mitochondrial morphological network connections ki otimo pi cell weny ma tye i dul acel acel. C. Pol pa kit ma mitochondrial nen kwede. wel ma cok ki 0 nyutu jami ma rwate, ki wel macok ki 1 nyutu jami ma rwate ki dul. D Mitochondrial DNA (mtDNA) ma tye iye ki nongo kit ma ki waco kwede i jami ma ki tiyo kwede ki yoo me tic kwede. E MitotrackerTM Red CMXRos analysis ni kitimo ki flow cytometry (30,000 events) kit ma ki waco kwede i Materials and Methods. Ngec ni ki nyutu calo rwom me ± SD, n = 3 ma ki temo ma pat pat. Apoka poka me wel ki otimo kun kitiyo ki one-way ANOVA ki Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Wan dong wa kwano wel mtDNA ma tye i LX-2 cells ma calo lanyut pa namba pa mitochondrial. Ka kiporo ki dul ma ki temo, wel mtDNA omede i dul ma ki miyo TGF-β1 (lanyut 4D). Ka kiporo ki dul ma ki miyo TGF-β1, wel mtDNA odok piny i dul ma kimiyo yat kacel (lanyut 4D), ma nyutu ni IPA twero dwoko piny wel mtDNA ki twero bedo wel mitochondrial kacel ki yweyo pa mitochondrial (lanyut 3C). Medo ikom meno, IPA nen calo odwoko piny mtDNA ma tye i yat ma ki keto kacel ento pi obalo tic pa MTR-mediated mitochondrial (lanyut 4A-C).
Wa kwedo rwate pa IPA ki rwom me mRNA pa gene ma rwate ki fibrosis, apoptosis, kwo, ki mitochondrial dynamics i LX-2 cells (lanyut 5A-D). Ka kiporo ki dul ma ki temo, dul ma ki miyo TGF-β1 onyutu medo rwom me tic pa jami ma calo collagen type I α2 chain (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP1), ki 1 D medo fibrosis ki tic. Medo ikom meno, ka kiporo ki dul ma ki temo, yat TGF-β1 odwoko piny rwom me mRNA pa nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, inhibitor pa B-cell α, enhancer pa nuclear factor κ gene light peptide (NFκB1A), ki inhibitor pa nuclear factor κB kinase subunit β (KBKB) 5A–D). Ka kiporo ki yat TGF-β1, yat ma ki keto kacel ki TGF-β1 ki IPA odwoko piny nyutu pa COL1A2 ki MMP2, ento omedo rwom me mRNA pa PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, ki IKBKB. Yat me IPA odwoko piny rwom me MMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), ki mitochondrial fusion protein 2 (MFN2), kun rwom me CASP8, NFκB1A, NFκB1B, ki IKB omedde ki dul ma ki temo. Ento, pe kinongo apokapoka mo i kit ma caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), ki fission protein 1 (FIS1). Ka ki keto kacel, adwogi eni nyutu ni yat IPA loko kit ma gene ma mako fibrosis, apoptosis, kwo, ki kit ma mitochondrial loko kwede. Lok ma kinongo nyutu ni yat IPA dwoko piny fibrosis i LX-2 cells; i kare acel, en kelo kwo kun loko kit ma kom dano nen kwede me doko gin ma pe tiyo.
IPA loko kit ma fibroblast, apoptotic, viability, ki mitochondrial dynamics genes nyutu kwede i LX-2 cells. Histograms nyutu mRNA ma rwate ki endogenous control (RPLP0 onyo PPIA) inge LX-2 cells ma ki keto i TGF-β1 ki IPA i serum-free medium pi cawa 24. A nyutu fibroblasts, B nyutu apoptotic cells, C nyutu cells ma kwo, ki D nyutu mitochondrial dynamics gene expression. Ngec ni ki nyutu calo rwom me ± apoka poka (SD), n = 3 jami ma ki temo ma pat pat. Apoka poka me wel ki otimo kun kitiyo ki one-way ANOVA ki Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
I ngeye, alokaloka i dit pa cell (FSC-H) ki cytoplasmic complexity (SSC-H) ki kwano ki flow cytometry (lanyut 6A,B), ki alokaloka i kit ma cell nen kwede inge yat IPA ki kwano ki transmission electron microscopy (TEM) ki phase contrast microscopy (Supplementary Figure 6A-B). Kit ma onongo ki tamo kwede, cell ma tye i dul ma ki miyo iye TGF-β1 omede i dit ka kiporo ki dul ma ki miyo iye kony (lanyut 6A,B), ma nyutu dongo pa endoplasmic reticulum (ER*) ki phagolysosomes (P), ma nyutu tic pa cell ma ki lwongo ni hematopoietic stem cell (HSC) (lanyut ma ki medo 6A). Ento, ka kiporo ki dul ma kimiyo iye TGF-β1, dit pa cell, rwom me cytoplasmic (Fig. 6A,B), ki ER* ma tye iye odok piny i dul ma kimiyo iye TGF-β1 ki IPA (Supplementary Fig. 6A). Medo ikom meno, yat IPA odwoko piny dit pa cell, rwom me cytoplasmic (Cal 6A,B), P ki ER* ma tye iye (Cal ma ki medo 6A) ka kiporo ki dul ma ki temo. Medo ikom meno, wel cell ma otoo omede ingee cawa 24 me tic ki IPA ka kiporo ki dul ma ki temo (lanyut ma ler, cal ma ki medo 6B). Ka ki keto kacel, adwogi eni nyutu ni 1 mM IPA twero juku HSC apoptosis ki dwoko alokaloka i kit ma cell nen kwede ma TGF-β1 okelo, ma weko ki doro dit pa cell ki kit ma kome tek kwede, ma twero bedo ni kubbe ki HSC inactivation.
IPA loko dit pa cell ki kit ma cytoplasmic tek kwede i LX-2 cells. Cal ma nyutu kit ma ki kwano kwede jami ma ki lwongo ni flow cytometry. Kwedo ni otiiyo ki yoo ma ki lwongo ni gating strategy pi LX-2 cells: SSC-A/FSC-A me nyutu wel cell, FSC-H/FSC-A me nyutu doublets, ki SSC-H/FSC-H me nyutu dit pa cell ki kit ma tek kwede. Gin ma ki lwongo ni cells ni ki gwoko ki TGF-β1 (5 ng/ml) ki 1 mM IPA i kom ma pe ki serum pi cawa 24. LX-2 cells ki poko gi i dul ma piny tung lacuc (SSC-H-/FSC-H-), dul ma malo tung lacuc (SSC-H+/FSC-H-), dul ma piny tung lacuc (SSC-H-/FSC-H+), ki dul ma malo tung lacuc (SSC-H+/FSC-H+) pi dit pa cell ki rwom me cytoplasmic complexity. B. Kit ma cell nen kwede ki kwano ki flow cytometry kun kitiyo ki FSC-H (forward scatter, cell size) ki SSC-H (side scatter, cytoplasmic complexity) (30,000 events). Ngec ni ki nyutu calo rwom me ± SD, n = 3 ma ki temo ma pat pat. Apoka poka me wel ki otimo kun kitiyo ki one-way ANOVA ki Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 ki ****p < 0.0001
Gin ma kelo dongo i ic calo IPA odoko lok ma pire tek i kwan, ma nyutu ni jami manyen ma ki nongo i ic twero bedo tye. Pi meno tye me ur ni IPA, gin ma ki lwongo ni metabolite ma wa rwate ki two ma mako adunu i kom dano [15], ki nyutu ni twero bedo gin ma gengo two ma mako adunu i kom lee [13, 14]. Kany, wanyutu pi tyen mukwongo ni tye wat ma tye i kin serum IPA ki global liver transcriptomics ki DNA methylation i kom dano ma kom gi pek ma pe ki two cukari me 2 (T2D), ma nyutu apoptosis, mitophagy ki kwo mabor, kacel ki gene ma twero bedo AKT1 ma doro liver homeostasis. Gin manyen mukene i kwan wa tye ni wanyutu kit ma yat IPA rwate kwede ki apoptosis, kit ma cell nen kwede, bioenergetics me mitochondrial ki kit ma gi loko kwede i LX-2 cells, ma nyutu ni teko ma nok ma loko HSC phenotype me doko inactivation, ma miyo IPA bedo gin ma twero kelo dongo pa liver fibrosis.
Wanongo ni apoptosis, mitophagy ki kwo mabor obedo yoo ma pire tek ma ki medo i kom gene ma mako adunu ma rwate ki circulating serum IPA. Turo pa yub me gwoko rwom pa mitochondrial (MQC) twero kelo peko i tic pa mitochondrial, mitophagy ki apoptosis, ma weko MASLD dongo[33, 34]. Pi meno, waromo tamo ni IPA twero bedo tye i gwoko kit ma cell tiyo kwede ki kit ma mitochondrial tiyo kwede kun wok ki i apoptosis, mitophagy ki kwo mabor i adunu. Ngec wa onyutu ni gene aryo onongo tye marom i kin jami adek ma ki temo ni: YKT6 ki AKT1. Ber me ngeyo ni YKT6 tye SNARE protein ma tye i yub me ribbe pa cell. En tiyo tic i autophagy ki mitophagy kun yubu gin ma ki lwongo ni initiation complex ki STX17 ki SNAP29 i kom autophagosome, ma weko autophagosomes ki lysosomes rwate kacel[35]. Medo ikom meno, rwenyo tic pa YKT6 kelo rweny pa mitophagy[36], kun medo pa YKT6 rwate ki dongo pa two ma mako adunu (HCC), ma nyutu medo kwo pa cell[37]. Ki tung cing kany, AKT1 obedo gene ma pire tek ma tiyo kacel ki tiyo tic ma pire tek i two ma mako adunu, medo ki yoo me PI3K/AKT, cell cycle, cell migration, proliferation, focal adhesion, tic pa mitochondrial, ki collagen secretion[38–40]. Gin ma ki lwongo ni PI3K/AKT ma ki tiyo kwede twero tic ki hematopoietic stem cells (HSCs), ma gin aye cell ma gi tye ki twero me yubu extracellular matrix (ECM), ki kit ma gi tiiyo kwede twero konyo i dongo ki dongo pa liver fibrosis[40]. Medo ikom meno, AKT tye i kin jami ma konyo kwo pa cell ma gengo p53-dependent cell apoptosis, ki AKT activation twero bedo ki rwate ki gengo to pa cell me adunu[41, 42]. Adwogi ma kinongo nyutu ni IPA twero bedo tye i yub me to pa dul kom ma mako adunu kun weko tam pa dul kom ma i adunu i kin donyo i apoptosis onyo kwo. Adwogi eni ki twero doro gi ki AKT ki/onyo YKT6 ma ki lwongo ni genes, ma pire tek pi gwoko adunu.
Adwogi wa onyutu ni 1 mM IPA okelo apoptosis ki odwoko piny yweyo pa mitochondrial i LX-2 cells ma pe jenge i kom yat TGF-β1. Ber me poyo ni apoptosis obedo yoo madit me cobo peko me fibrosis ki tic pa hematopoietic stem cell (HSC), ki bene obedo gin ma pire tek i kit ma kom dano dwoko kwede rwom me fibrosis [4, 43]. Medo ikom meno, dwoko BHI i LX-2 cells ingee tic kacel omiyo ngec manyen ikom tic pa IPA i yub pa mitochondrial bioenergetics. I kare me yweyo ki ma pe tiyo, cell ma yubu remo pol kare tiyo ki mitochondrial oxidative phosphorylation me yubu ATP ki tye ki tic ma lapiny. Ki tung cing kany, tic pa HSC medo yweyo pa mitochondrial ki biosynthesis me culo pi teko ma mite me donyo i kit me glycolytic [44]. Lok ada ni IPA pi obalo teko pa kom ki ECAR nyutu ni yoo me glycolytic ni pe ki keto i tic. I yo acel-lu, kwan mukene onyutu ni 1 mM IPA onongo twero loko tic pa mitochondrial respiratory chain i cardiomyocytes, human hepatocyte cell line (Huh7) ki human umbilical vein endothelial cells (HUVEC); Ento, pe ki nongo adwogi pa IPA i kom glycolysis i cardiomyocytes, ma nyutu ni IPA twero gudo bioenergetics pa cell mukene [45]. Pi meno, wa tamo ni 1 mM IPA twero tic calo gin ma kwanyo jami ma ki lwongo ni mild chemical uncoupler, pien twero dwoko piny rwom me gene ma ki lwongo ni fibrogenic gene expression, cell morphology ki mitochondrial bioenergetics labongo loko wel mtDNA [46]. Mitochondrial uncouplers twero gengo culture-induced fibrosis ki HSC activation [47] ki dwoko piny mitochondrial ATP production ma ki doro onyo kelo ki jami mogo calo uncoupling proteins (UCP) onyo adenine nucleotide translocase (ANT). Malube ki dul cell, gin man twero gwoko cell ki i to pa cell ki/onyo kelo to pa cell [46]. Ento, kwan mukene mitte me gonyo tic pa IPA ma calo gin ma kwanyo dul kom ma ki lwongo ni hematopoietic stem cell inactivation.
Wan dong wa kwedo ka ce alokaloka ma tye i yweyo pa mitochondrial nen i kit ma mitochondrial nen kwede i LX-2 cells makwo. I yo me miti, yat TGF-β1 loko rwom me mitochondrial ki i kom ma rwate, ki dwoko piny rwom me mitochondrial ki medo nyutu pa DRP1, gin ma pire tek i mitochondrial fission [48]. Medo ikom meno, poko pa mitochondrial rwate ki rwom me rwate, ki loko ki i fusion me doko fission pire tek pi tic pa hematopoietic stem cell (HSC), kun gengo mitochondrial fission kelo HSC apoptosis [49]. Pi meno, adwogi wa nyutu ni tic ki TGF-β1 twero kelo dwoko piny rwom me tic pa mitochondrial ki dwoko piny branching, ma pol kare timme i mitochondrial fission ma rwate ki activated hematopoietic stem cells (HSCs). Medo ikom meno, ngec wa onyutu ni IPA twero loko rwom pa mitochondria ki ikom dul ma rwate, ma weko dwoko piny nyutu pa OPA1 ki MFN2. Kwan ma kitimo onyutu ni dwoko piny OPA1 twero kelo dwoko piny teko pa dul kom ma i kom dano ki kelo to pa cell[50]. MFN2 ki ngeyo me konyo mitochondrial fusion ki apoptosis[51]. Adwogi ma kinongo nyutu ni keto LX-2 cells ki TGF-β1 ki/onyo IPA nen calo loko kit ma mitochondrial nen kwede ki dit ne, kacel ki kit ma en tye ka tic kwede ki kit ma dul ne tye kwede.
Adwogi wa nyutu ni tic ki TGFβ-1 ki IPA twero dwoko piny mtDNA ki kit ma cell nen kwede kun ki doro kit ma mRNA nyutu kwede fibrosis, apoptosis ki genes ma mako kwo i cell ma gi kwero to. Ki adaa, IPA odwoko piny rwom me nyutu mRNA pa AKT1 ki gene ma pire tek ma calo COL1A2 ki MMP2, ento omedo rwom me nyutu pa CASP8, ma rwate ki apoptosis. Adwogi wa onyutu ni ingee tic ki IPA, nyutu pa BAX odok piny ki nyutu pa mRNA pa TIMP1, BCL-2 ki NF-κB omede, ma nyutu ni IPA twero kelo kwo i dul kom ma ki lwongo ni hematopoietic stem cells (HSCs) ma gengo apoptosis. Molecule magi twero tic calo lanyut me kwo i kom dul kom ma ki lwongo ni activated hematopoietic stem cells, ma twero bedo ki rwate ki medo rwom me tic pa anti-apoptotic proteins (ma calo Bcl-2), dwoko piny rwom me tic pa pro-apoptotic BAX, ki tic matek ma tye i kin TIMP ki NF-κB [5, 7]. IPA tiyo ki PXR, ki wanongo ni tic ki TGF-β1 ki IPA omedo rwom me nyutu PXR mRNA, ma nyutu gengo tic pa HSC. PXR ma ki keto i tic ki ngeyo ni gengo tic pa HSC i kom dano ki i kom dano [52, 53]. Adwogi wa nyutu ni IPA twero nywako i kwanyo HSCs ma ki tiyo kwede kun kelo apoptosis, dwoko piny fibrosis ki mitochondrial metabolism, ki medo rwom me kwo, ma obedo yub ma loko HSC ma ki tiyo kwede me doko gin ma pe tiyo. Gin mukene ma twero gonyo kit ma IPA tiyo kwede ki tic pa IPA i apoptosis tye ni en kwanyo mitochondria ma pe tiyo maber ma loyo ki i mitophagy (yoo ma tye iye) ki yoo ma ki lwongo ni extrinsic TNF signaling pathway (Table 1), ma rwate ki NF-κB survival signaling pathway (Supplementary Figure 7). Ma omyera, IPA-related enriched genes twero kelo pro-apoptotic ki pro-survival signals i apoptotic pathway [54], ma nyutu ni IPA twero kelo apoptotic pathway onyo kwo kun gikubbe ki genes magi. Ento, kit ma IPA kelo kwede apoptosis onyo kwo i kare me tic pa HSC ki yoo ma en tiyo kwede pwod pe tye atir.
IPA obedo gin ma ki lwongo ni microbial metabolite ma ki yubu ki i cam ma ki lwongo ni tryptophan kun wok ki i gut microbiota. Kwan ma kitimo onyutu ni tye ki jami ma gengo two, gengo two, ki jami ma gengo dongo pa kom i ic.[55] Kwan ma kitimo onyutu ni IPA twero loko tic pa dul kom ma i ic ki dwoko piny peko me oxidative, ma twero konyo i kom adwogi me kom.[56] Ki lok adaa, IPA ki tero i dul kom ma ki yubu ki remo, ki kit ma IPA tye ki dul kom ma rom ki tryptophan, serotonin, ki indole derivatives, IPA tiyo tic me metabolic ma kelo adwogi maber.[52] IPA twero pyem ki jami ma aa ki ikom tryptophan pi kabedo ma mako enzymes onyo receptors, ma twero balo yoo me tic pa jami ma mako kom. Man nyutu ni mitte ni kimed kwan i kom kit ma yat tiyo kwede ki kit ma yat tiyo kwede me niang maber kit ma yat tiyo kwede.[57] Pud tye me neno ka man twero time i kom ler ma ki lwongo ni hematopoietic stem cells (HSCs).
Wa ngeyo ni kwan wa ni tye ki goro mogo. Me temo rwate ma mako IPA, wakwanyo lutwo ma tye ki two cukari me 2 (T2DM). Wa ngeyo ni man gengo tic pa gin ma waniano i kom lutwo ma tye ki two cukari ma ki lwongo ni type 2 ki two ma mako adunu. Kadi bed ni rwom me IPA i remo pa dano tye 1-10 μM [11, 20], rwom me 1 mM IPA ki yero malube ki rwom ma malo ma pe kelo two [15] ki rwom ma malo me apoptosis, labongo apokapoka mo i wel cell ma otoo. Kadi bed ni rwom me IPA ma malo ni ki tiyo kwede i kwan man, pe tye tam mo ma ki moko i kom rwom me tic pa IPA [52]. Kadi bed ni adwogi wa tye ma pire tek, kit ma IPA tiyo kwede i kom dano pud tye kabedo ma ki tye ka kwedo iye. Medo ikom meno, gin ma wanongo ikom rwate ma tye i kin rwom me IPA ki DNA methylation pa jami ma ki coyo ikom adunu pe ki nongo ki ikom cell ma ki lwongo ni hematopoietic stem cells (HSCs) ento bene ki ikom jami ma ki lwongo ni liver tissues. Wa yero me tic ki LX-2 cell pa dano malube ki gin ma wanongo con ki ikom kwan me transcriptome ni IPA tye ki wat ki hematopoietic stem cell (HSC) activation [15], ki HSCs aye cell madongo ma tye i dongo pa liver fibrosis. Adunu tye ki dul cell mapol, ki dong dul cell mukene calo hepatocyte-HSC-immune cell co-culture system ma ki keto kacel ki caspase activation ki DNA fragmentation kacel ki kit ma en tiyo kwede ma tye ki rwom me protein omyera ki nen me kwan tic pa IPA ki kit ma en rwate kwede ki dul cell mukene me adunu.