Poyamba tidanena kuti kuchuluka kwa tryptophan metabolite indole-3-propionic acid (IPA) m'magazi ndi kotsika kwa odwala omwe ali ndi fibrosis ya chiwindi. Mu kafukufukuyu, tidafufuza transcriptome ndi DNA methylome m'machiwindi onenepa poyerekeza ndi kuchuluka kwa IPA m'magazi, komanso ntchito ya IPA pakupangitsa kuti maselo a hepatic stellate cells (HSCs) asamagwire ntchito m'thupi.
Kafukufukuyu adaphatikizapo odwala onenepa 116 omwe alibe matenda a shuga amtundu wachiwiri (T2DM) (zaka 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m²) omwe adachitidwa opaleshoni ya bariatric ku Kuopio Bariatric Surgery Center (KOBS). Kuchuluka kwa IPA komwe kumazungulira kunayesedwa pogwiritsa ntchito madzi a chromatography-mass spectrometry (LC-MS), kusanthula kwa chiwindi kunachitika pogwiritsa ntchito total RNA sequencing, ndipo kusanthula kwa DNA methylation kunachitika pogwiritsa ntchito Infinium HumanMethylation450 BeadChip. Maselo a hepatic stellate a anthu (LX-2) adagwiritsidwa ntchito poyesa mu vitro.
Miyezo ya IPA m'magazi imagwirizana ndi kufotokozera kwa majini omwe amagwira ntchito mu njira za apoptotic, mitophagic, ndi moyo wautali m'chiwindi. Jini ya AKT serine/threonine kinase 1 (AKT1) inali jini yochuluka kwambiri komanso yolankhulirana kwambiri mu mbiri ya chiwindi ndi DNA methylation. Chithandizo cha IPA chinayambitsa apoptosis, kuchepetsa kupuma kwa mitochondrial, komanso kusintha mawonekedwe a maselo ndi mitochondrial mwa kusintha kufotokozera kwa majini omwe amadziwika kuti amalamulira fibrosis, apoptosis, ndi kupulumuka kwa maselo a LX-2.
Poganizira zonsezi, deta iyi ikutsimikizira kuti IPA ili ndi zotsatirapo zochiritsira ndipo ikhoza kuyambitsa apoptosis ndikusuntha mawonekedwe a HSC kukhala osagwira ntchito, motero kukulitsa kuthekera koletsa chiwindi fibrosis mwa kusokoneza kuyambika kwa HSC ndi kagayidwe ka mitochondrial.
Kuchuluka kwa kunenepa kwambiri ndi matenda a metabolic syndrome kwagwirizanitsidwa ndi kuwonjezeka kwa matenda a chiwindi amafuta ogwirizana ndi kagayidwe kachakudya (MASLD); matendawa amakhudza 25% mpaka 30% ya anthu onse [1]. Chotsatira chachikulu cha MASLD ndi fibrosis ya chiwindi, njira yosinthika yomwe imadziwika ndi kusonkhana kosalekeza kwa fibrous extracellular matrix (ECM) [2]. Maselo akuluakulu omwe amakhudzidwa ndi fibrosis ya chiwindi ndi maselo a hepatic stellate (HSCs), omwe amasonyeza mitundu inayi yodziwika: quiescent, activated, inactivated, and senescent [3, 4]. Ma HSC amatha kuyatsidwa ndikusinthidwa kuchoka ku mawonekedwe a quiescent kupita ku maselo ofanana ndi fibroblast omwe amafunikira mphamvu zambiri, ndi kuwonjezeka kwa α-smooth muscle actin (α-SMA) ndi type I collagen (Col-I) [5, 6]. Panthawi yosintha fibrosis ya chiwindi, ma HSC omwe amayatsidwa amachotsedwa kudzera mu apoptosis kapena inactivation. Njirazi zikuphatikizapo kuchepetsa majini a fibrogenic ndi kusintha kwa majini a prosurvival (monga NF-κB ndi PI3K/Akt signaling pathways) [7, 8], komanso kusintha kwa mitochondrial dynamics ndi ntchito [9].
Kuchuluka kwa tryptophan metabolite indole-3-propionic acid (IPA) m'magazi, komwe kumapangidwa m'matumbo, kwapezeka kuti kumachepa m'matenda a kagayidwe kachakudya m'thupi la munthu kuphatikizapo MASLD [10-13]. IPA imagwirizanitsidwa ndi kudya ulusi wazakudya, imadziwika ndi mphamvu zake zotsutsana ndi antioxidant komanso zotsutsana ndi kutupa, ndipo imachepetsa mawonekedwe a steatohepatitis (NASH) omwe amayambitsidwa ndi zakudya m'thupi komanso m'thupi [11-14]. Umboni wina umachokera ku kafukufuku wathu wakale, womwe udawonetsa kuti kuchuluka kwa IPA m'magazi kunali kotsika mwa odwala omwe ali ndi fibrosis ya chiwindi kuposa odwala onenepa kwambiri omwe alibe fibrosis ya chiwindi mu Kuopio Bariatric Surgery Study (KOBS). Kuphatikiza apo, tidawonetsa kuti chithandizo cha IPA chingachepetse kuwonetsedwa kwa majini omwe ndi zizindikiro zakale za kumamatira kwa maselo, kusamuka kwa maselo ndi kuyamwa kwa maselo oyambira m'magazi mu chitsanzo cha hepatic stellate cell (LX-2) ndipo ndi metabolite yoteteza chiwindi [15]. Komabe, sizikudziwika bwino momwe IPA imathandizira kutsika kwa fibrosis ya chiwindi poyambitsa HSC apoptosis ndi mitochondrial bioenergetics.
Apa, tikuwonetsa kuti IPA ya m'magazi imagwirizana ndi kufotokozera kwa majini olemera mu apoptosis, mitophagy, ndi njira zokhalitsa m'chiwindi cha anthu onenepa koma osadwala matenda a shuga amtundu wa 2 (KOBS). Kuphatikiza apo, tapeza kuti IPA ikhoza kuyambitsa kuchotsedwa ndi kuwonongeka kwa maselo oyambira a hematopoietic (HSCs) kudzera mu njira yoletsa kugwira ntchito. Zotsatirazi zikuwonetsa ntchito yatsopano ya IPA, zomwe zimapangitsa kuti ikhale cholinga chochiritsira kubwezeretsanso kwa fibrosis ya chiwindi.
Kafukufuku wakale mu gulu la KOBS adawonetsa kuti odwala omwe ali ndi fibrosis ya chiwindi anali ndi milingo yochepa ya IPA poyerekeza ndi odwala omwe alibe fibrosis ya chiwindi [15]. Pofuna kuchotsa zotsatira zosokoneza za matenda a shuga amtundu wa 2, tidalemba odwala onenepa 116 omwe alibe matenda a shuga amtundu wa 2 (zaka zapakati ± SD: 46.8 ± 9.3 zaka; BMI: 42.7 ± 5.0 kg/m2) (Table 1) kuchokera ku kafukufuku wa KOBS womwe ukupitilira ngati gulu la kafukufuku [16]. Ophunzira onse adapereka chilolezo cholembedwa ndipo njira yophunzirirayo idavomerezedwa ndi Komiti ya Makhalidwe Abwino ya Chipatala cha North Savo County motsatira Chilengezo cha Helsinki (54/2005, 104/2008 ndi 27/2010).
Zitsanzo za biopsy ya chiwindi zinapezeka panthawi ya opaleshoni ya bariatric ndipo zinawunikidwa ndi akatswiri odziwa bwino matenda malinga ndi zomwe zafotokozedwa kale [17, 18]. Zofunikira zowunikira zafotokozedwa mwachidule mu Supplementary Table S1 ndipo zafotokozedwa kale [19].
Zitsanzo za seramu yosala kudya zinasanthulidwa pogwiritsa ntchito untargeted liquid chromatography-mass spectrometry (LC-MS) kuti ziwonetse metabolomics analysis (n = 116). Zitsanzo zinasanthulidwa pogwiritsa ntchito UHPLC-qTOF-MS system (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) monga tafotokozera kale19. Kuzindikira isopropyl alcohol (IPA) kunachokera pa nthawi yosungira ndi kuyerekeza kwa MS/MS spectrum ndi miyezo yoyera. Mphamvu ya chizindikiro cha IPA (malo okwera kwambiri) inaganiziridwa mu kusanthula kwina konse [20].
Kusanthula kwa RNA ya chiwindi chonse kunachitika pogwiritsa ntchito Illumina HiSeq 2500 ndipo deta idakonzedwa kale monga momwe tafotokozera kale [19, 21, 22]. Tinachita kusanthula kosiyana kwa ma transcripts okhudzana ndi ntchito ya mitochondrial/biogenesis pogwiritsa ntchito majini a 1957 osankhidwa kuchokera ku database ya MitoMiner 4.0 [23]. Kusanthula kwa methylation ya DNA ya chiwindi kunachitika pogwiritsa ntchito Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) pogwiritsa ntchito njira yomweyi monga momwe tafotokozera kale [24, 25].
Maselo a chiwindi cha anthu (LX-2) adaperekedwa mokoma mtima ndi Pulofesa Stefano Romeo ndipo adakulitsidwa ndikusungidwa mu DMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Kuti asankhe mlingo wogwirira ntchito wa IPA, maselo a LX-2 adachiritsidwa ndi kuchuluka kosiyanasiyana kwa IPA (10 μM, 100 μM ndi 1 mM; Sigma, 220027) mu DMEM/F12 medium kwa maola 24. Kuphatikiza apo, kuti afufuze kuthekera kwa IPA kuletsa ma HSC, maselo a LX-2 adathandizidwa ndi 5 ng/ml TGF-β1 (machitidwe a R&D, 240-B-002/CF) ndi 1 mM IPA mu serum-free medium kwa maola 24. Pa zowongolera zamagalimoto zomwe zikugwirizana, 4 nM HCL yokhala ndi 0.1% BSA idagwiritsidwa ntchito pochiza TGF-β1 ndipo 0.05% DMSO idagwiritsidwa ntchito pochiza IPA, ndipo zonse ziwiri zidagwiritsidwa ntchito pamodzi pochiza kuphatikiza.
Apoptosis inayesedwa pogwiritsa ntchito FITC Annexin V Apoptosis Detection Kit yokhala ndi 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) malinga ndi malangizo a wopanga. Mwachidule, LX-2 (maselo 1 × 105/chitsime) inaleredwa usiku wonse m'mabolo 12-zitsime kenako n’kupatsidwa mankhwala osiyanasiyana a IPA kapena IPA ndi TGF-β1. Tsiku lotsatira, maselo oyandama ndi omatira anasonkhanitsidwa, kutsukidwa, kutsukidwa ndi PBS, kubwezeretsedwa mu Annexin V binding buffer, ndikuyikidwa mu FITC-Annexin V ndi 7-AAD kwa mphindi 15.
Mitochondria m'maselo amoyo adapakidwa utoto chifukwa cha ntchito ya okosijeni pogwiritsa ntchito Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Pa mayeso a MTR, maselo a LX-2 adayikidwa mu kachulukidwe kofanana ndi IPA ndi TGF-β1. Pambuyo pa maola 24, maselo amoyo adayikidwa mu trypsinize, kutsukidwa ndi PBS, kenako adayikidwa mu 100 μM MTR mu seramu yopanda seramu pa 37 °C kwa mphindi 20 monga tafotokozera kale [26]. Pa kusanthula kwa mawonekedwe a maselo amoyo, kukula kwa maselo ndi kusinthasintha kwa cytoplasmic zidasanthulidwa pogwiritsa ntchito magawo a forward scatter (FSC) ndi side scatter (SSC), motsatana.
Deta yonse (zochitika 30,000) idapezedwa pogwiritsa ntchito NovoCyte Quanteon (Agilent) ndipo idasanthulidwa pogwiritsa ntchito NovoExpress® 1.4.1 kapena pulogalamu ya FlowJo V.10.
Kuchuluka kwa okosijeni (OCR) ndi kuchuluka kwa acidification yakunja (ECAR) zinayesedwa nthawi yeniyeni pogwiritsa ntchito Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) yokhala ndi Seahorse XF Cell Mito Stress malinga ndi malangizo a wopanga. Mwachidule, maselo 2 × 104 LX-2/chitsime anabzalidwa pa mbale zokulira maselo a XF96. Pambuyo poikamo usiku wonse, maselo anachiritsidwa ndi isopropanol (IPA) ndi TGF-β1 (Njira Zowonjezera 1). Kusanthula deta kunachitika pogwiritsa ntchito pulogalamu ya Seahorse XF Wave, yomwe imaphatikizapo Seahorse XF Cell Energy Phenotype Test Report Generator. Kuchokera apa, Bioenergetic Health Index (BHI) inawerengedwa [27].
RNA yonse idasinthidwa kukhala cDNA. Kuti mudziwe njira zinazake, onani buku [15]. Ma RNA a anthu a 60S ribosomal acidic protein P0 (RPLP0) ndi cyclophilin A1 (PPIA) adagwiritsidwa ntchito ngati ma constitutive gene controls. QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, The Netherlands) idagwiritsidwa ntchito ndi TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) kapena Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), ndipo relative gene expression fold idawerengedwa pogwiritsa ntchito ma comparative Ct value cycling parameters (ΔΔCt) ndi njira ya ∆∆Ct. Tsatanetsatane wa ma primers aperekedwa mu Supplementary Tables S2 ndi S3.
DNA ya nyukiliya (ncDNA) ndi DNA ya mitochondrial (mtDNA) zinachotsedwa pogwiritsa ntchito DNeasy blood and tissue kit (Qiagen) monga tafotokozera kale [28]. Kuchuluka kwa mtDNA kunawerengedwa powerengera chiŵerengero cha dera lililonse la mtDNA lomwe likufunidwa ku mean ya geometric ya madera atatu a nyukiliya (mtDNA/ncDNA), monga momwe zafotokozedwera mu Supplementary Methods 2. Tsatanetsatane wa ma primers a mtDNA ndi ncDNA aperekedwa mu Supplementary Table S4.
Maselo amoyo adapakidwa utoto ndi Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) kuti awonetse maukonde a mitochondrial pakati pa maselo ndi amkati mwa maselo. Maselo a LX-2 (maselo 1 × 104/chitsime) adakulitsidwa pa magalasi m'mabolo omera omwe ali pansi pa galasi (Ibidi GmbH, Martinsried, Germany). Pambuyo pa maola 24, maselo amoyo a LX-2 adapakidwa utoto ndi 100 μM MTR kwa mphindi 20 pa 37 °C ndipo ma nuclei a maselo adapakidwa utoto ndi DAPI (1 μg/ml, Sigma-Aldrich) monga tafotokozera kale [29]. Maukonde a Mitochondrial adawonetsedwa pogwiritsa ntchito maikulosikopu yozungulira ya Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, Germany) yokhala ndi Zeiss LSM 800 confocal module pa 37 °C mumlengalenga wonyowa ndi 5% CO2 pogwiritsa ntchito cholinga cha 63×NA 1.3. Tinapeza zithunzi khumi za mndandanda wa Z pa mtundu uliwonse wa chitsanzo. Mndandanda uliwonse wa Z uli ndi magawo 30, chilichonse chokhala ndi makulidwe a 9.86 μm. Pa chitsanzo chilichonse, zithunzi za magawo khumi osiyanasiyana a mawonekedwe zidapezedwa pogwiritsa ntchito pulogalamu ya ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany), ndipo kusanthula kwa mitochondrial morphology kunachitika pogwiritsa ntchito pulogalamu ya ImageJ (v1.54d) [30, 31] malinga ndi magawo omwe afotokozedwa mu Supplementary Methods 3.
Maselowo anakhazikika ndi 2% glutaraldehyde mu 0.1 M phosphate buffer, kutsatiridwa ndi kukhazikika ndi 1% osmium tetroxide solution (Sigma Aldrich, MO, USA), pang'onopang'ono anauma ndi acetone (Merck, Darmstadt, Germany), ndipo potsiriza anaikidwa mu epoxy resin. Zigawo zoonda kwambiri zinakonzedwa ndikupaka utoto ndi 1% uranyl acetate (Merck, Darmstadt, Germany) ndi 1% lead citrate (Merck, Darmstadt, Germany). Zithunzi za ultrastructural zinapezeka pogwiritsa ntchito JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) pa voltage yothamanga ya 80 kV.
Kapangidwe ka maselo a LX-2 omwe adathandizidwa ndi IPA kwa maola 24 adawunikidwa pogwiritsa ntchito microscopy ya phase-contrast pa kukula kwa 50x pogwiritsa ntchito microscope ya Zeiss inverted light (Zeiss Axio Vert.A1 ndi AxioCam MRm, Jena, Germany).
Deta yachipatala inafotokozedwa ngati mean ± standard deviation kapena median (interquartile range: IQR). Kusanthula kwa njira imodzi kwa kusiyana (continuous variables) kapena χ² test (categorical variables) kunagwiritsidwa ntchito poyerekeza kusiyana pakati pa magulu atatu ophunzirira. False positive rate (FDR) inagwiritsidwa ntchito kukonza mayeso angapo, ndipo majini omwe ali ndi FDR <0.05 amaonedwa kuti ndi ofunika kwambiri. Kusanthula kwa Spearman correlation kunagwiritsidwa ntchito kulumikiza CpG DNA methylation ndi mphamvu ya chizindikiro cha IPA, ndi ma nominal p values ​​​​(p <0.05) omwe anenedwa.
Kusanthula njira kunachitika pogwiritsa ntchito chida chowunikira majini pa intaneti (WebGestalt) cha ma transcript 268 (nominal p < 0.01), ma transcript 119 okhudzana ndi mitochondria (nominal p < 0.05), ndi ma CpG 4350 mwa ma transcript 3093 a chiwindi omwe anali okhudzana ndi kuchuluka kwa IPA m'magazi. Chida cha Venny DB (version 2.1.0) chomwe chimapezeka kwaulere chinagwiritsidwa ntchito kupeza majini ogwirizana, ndipo StringDB (version 11.5) inagwiritsidwa ntchito kuwona momwe mapuloteni ndi mapuloteni zimagwirira ntchito.
Pa kuyesa kwa LX-2, zitsanzo zinayesedwa kuti zitsimikizire kuti zili bwino pogwiritsa ntchito mayeso a D'Agostino-Pearson. Deta inapezedwa kuchokera ku ma replica atatu achilengedwe ndipo inayesedwa njira imodzi ya ANOVA ndi mayeso a Bonferroni post hoc. P-value yochepera 0.05 inkaonedwa kuti ndi yofunika kwambiri pa ziwerengero. Deta imaperekedwa ngati mean ± SD, ndipo chiwerengero cha mayeserocho chikuwonetsedwa pachithunzi chilichonse. Kusanthula konse ndi ma graph kunachitika pogwiritsa ntchito pulogalamu ya ziwerengero ya GraphPad Prism 8 ya Windows (GraphPad Software Inc., mtundu 8.4.3, San Diego, USA).
Choyamba, tinafufuza mgwirizano wa milingo ya IPA m'magazi ndi zolemba za chiwindi, thupi lonse, ndi mitochondrial. Mu mbiri yonse ya zolemba, jini yamphamvu kwambiri yogwirizana ndi milingo ya IPA m'magazi inali MAPKAPK3 (FDR = 0.0077; mitogen-activated protein kinase-activated protein kinase 3); mu mbiri ya zolemba zokhudzana ndi mitochondria, jini yamphamvu kwambiri yogwirizana inali AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Fayilo yowonjezera 1 ndi Fayilo yowonjezera 2).
Kenako tinasanthula zolemba zapadziko lonse lapansi (n = 268; p < 0.01) ndi zolemba zokhudzana ndi mitochondria (n = 119; p < 0.05), pomaliza pake tinazindikira apoptosis ngati njira yofunika kwambiri yodziwira (p = 0.0089). Pa zolemba za mitochondrial zokhudzana ndi kuchuluka kwa IPA m'magazi, tinayang'ana kwambiri pa apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), ndi njira zolumikizirana za TNF (FDR = 0.000006) (Chithunzi 1A, Table 2, ndi Zithunzi Zowonjezera 1A-B).
Kusanthula kophatikizana kwa zolemba zapadziko lonse lapansi, zokhudzana ndi mitochondria, ndi methylation ya DNA m'chiwindi cha munthu mogwirizana ndi milingo ya IPA m'magazi. A ikuyimira zolemba zapadziko lonse lapansi 268, zolemba 119 zokhudzana ndi mitochondria, ndi zolemba za methylated za DNA zomwe zalembedwa ku malo 3092 a CpG okhudzana ndi milingo ya IPA m'magazi (p values ​​​​< 0.01 ya zolemba zapadziko lonse lapansi ndi DNA methylated, ndi p values ​​​​< 0.05 ya zolemba za mitochondrial). Zolemba zazikulu zolumikizana zikuwonetsedwa pakati (AKT1 ndi YKT6). B Mapu olumikizirana a majini 13 omwe ali ndi chiŵerengero chapamwamba kwambiri cholumikizirana (0.900) ndi majini ena adapangidwa kuchokera ku majini 56 ophatikizana (dera la mzere wakuda) omwe adalumikizidwa kwambiri ndi milingo ya IPA m'magazi pogwiritsa ntchito chida cha pa intaneti cha StringDB. Chobiriwira: Majini olumikizidwa ku gawo la maselo a Gene Ontology (GO): mitochondria (GO:0005739). AKT1 ndi puloteni yokhala ndi chigoli chapamwamba kwambiri (0.900) pa kuyanjana ndi mapuloteni ena kutengera deta (kutengera kusanthula zolemba, zoyeserera, ma database, ndi ma co-expression). Ma netiweki amayimira mapuloteni, ndipo m'mphepete mwake mumayimira kulumikizana pakati pa mapuloteni.
Popeza ma metabolites a m'matumbo amatha kulamulira kapangidwe ka epigenetic kudzera mu methylation ya DNA [32], tidafufuza ngati milingo ya IPA m'magazi inali yogwirizana ndi methylation ya DNA ya chiwindi. Tapeza kuti malo awiri akuluakulu a methylation okhudzana ndi milingo ya IPA m'magazi anali pafupi ndi dera la 3 lolemera proline-serine (C19orf55) ndi heat shock protein family B (small) member 6 (HSPB6) (Fayilo yowonjezera 3). DNA methylation ya 4350 CpG (p < 0.01) idagwirizana ndi milingo ya IPA m'magazi ndipo idakulitsidwa mu njira zowongolera moyo wautali (p = 0.006) (Chithunzi 1A, Table 2, ndi Chithunzi Chowonjezera 1C).
Kuti timvetse njira zamoyo zomwe zimagwirizana pakati pa milingo ya IPA m'magazi, zolemba zapadziko lonse lapansi, zolemba zokhudzana ndi mitochondria, ndi methylation ya DNA m'chiwindi cha munthu, tinachita kusanthula kofanana kwa majini omwe adapezeka mu kusanthula kwakale kwa njira (Chithunzi 1A). Zotsatira za kusanthula kwa njira yowonjezerera majini 56 ophatikizana (mkati mwa mzere wakuda mu Chithunzi 1A) zinawonetsa kuti njira ya apoptosis (p = 0.00029) idawonetsa majini awiri ofanana ndi kusanthula katatu: AKT1 ndi YKT6 (YKT6 v-SNARE homolog), monga momwe zasonyezedwera mu chithunzi cha Venn (Chithunzi Chowonjezera 2 ndi Chithunzi 1A). Chosangalatsa n'chakuti, tapeza kuti AKT1 (cg19831386) ndi YKT6 (cg24161647) zinali zogwirizana bwino ndi milingo ya IPA m'magazi (Fayilo Yowonjezera 3). Kuti tidziwe momwe mapuloteni angagwirizanire pakati pa zinthu za majini, tinasankha majini 13 omwe ali ndi chiwerengero chapamwamba kwambiri cha dera (0.900) pakati pa majini 56 ophatikizana ngati cholowera ndipo tinapanga mapu olumikizirana. Malinga ndi mulingo wodzidalira (kudzidalira kwa m'mphepete), jini ya AKT1 yokhala ndi chigoli chapamwamba kwambiri (0.900) inali pamwamba (Chithunzi 1B).
Kutengera ndi kusanthula kwa njira, tinapeza kuti apoptosis ndiyo njira yayikulu, kotero tinafufuza ngati chithandizo cha IPA chingakhudze apoptosis ya HSCs mu vitro. Poyamba tinawonetsa kuti milingo yosiyanasiyana ya IPA (10 μM, 100 μM, ndi 1 mM) sinali yoopsa kwa maselo a LX-2 [15]. Kafukufukuyu adawonetsa kuti chithandizo cha IPA pa 10 μM ndi 100 μM chinawonjezera kuchuluka kwa maselo okhazikika komanso otupa. Komabe, poyerekeza ndi gulu lolamulira, kuthekera kwa maselo kukhala ndi moyo kunachepa pa 1 mM IPA, pomwe kuchuluka kwa necrosis ya maselo sikunasinthe (Chithunzi 2A, B). Kenako, kuti tipeze kuchuluka koyenera koyambitsa apoptosis m'maselo a LX-2, tinayesa 10 μM, 100 μM, ndi 1 mM IPA kwa maola 24 (Chithunzi 2A-E ndi Chithunzi Chowonjezera 3A-B). Chochititsa chidwi n'chakuti, IPA 10 μM ndi 100 μM zinachepetsa kuchuluka kwa apoptosis (%), komabe, IPA 1 mM inawonjezera kuchuluka kwa apoptosis ndi apoptosis (%) poyerekeza ndi kulamulira ndipo motero inasankhidwa kuti igwiritsidwe ntchito poyesa kwina (Zithunzi 2A–D).
IPA imayambitsa apoptosis ya maselo a LX-2. Njira ya Annexin V ndi 7-AAD yopaka utoto kawiri idagwiritsidwa ntchito poyesa kuchuluka kwa apoptotic rate ndi mawonekedwe a maselo pogwiritsa ntchito flow cytometry. Maselo a BA adayikidwa mu incubation ndi 10 μM, 100 μM ndi 1 mM IPA kwa maola 24 kapena ndi F–H TGF-β1 (5 ng/ml) ndi 1 mM IPA mu serum-free medium kwa maola 24. A: maselo amoyo (Annexin V -/ 7AAD-); B: maselo otupa (Annexin V -/ 7AAD+); C, F: oyambirira (Annexin V +/ 7AAD-); D, G: mochedwa (Annexin V+/7AAD+.); E, H: peresenti ya maselo onse oyambirira ndi ochedwa apoptotic mu apoptotic rate (%). Deta imafotokozedwa ngati apakati ± SD, n = 3 zoyesera zodziyimira pawokha. Kuyerekeza ziwerengero kunachitika pogwiritsa ntchito njira imodzi ya ANOVA ndi mayeso a Bonferroni post hoc. *p < 0.05; ****p < 0.0001
Monga tawonetsera kale, 5 ng/ml TGF-β1 ikhoza kuyambitsa HSC powonjezera kufotokozedwa kwa majini akale [15]. Maselo a LX-2 adathandizidwa ndi 5 ng/ml TGF-β1 ndi 1 mM IPA pamodzi (Chithunzi 2E–H). Chithandizo cha TGF-β1 sichinasinthe kuchuluka kwa apoptosis, komabe, chithandizo cha IPA chinawonjezera kuchuluka kwa apoptosis ndi apoptosis mochedwa (%) poyerekeza ndi chithandizo cha TGF-β1 (Chithunzi 2E–H). Zotsatirazi zikusonyeza kuti 1 mM IPA ikhoza kulimbikitsa apoptosis m'maselo a LX-2 mosasamala kanthu za kulowetsedwa kwa TGF-β1.
Tinafufuzanso momwe IPA imakhudzira kupuma kwa mitochondrial m'maselo a LX-2. Zotsatira zake zinasonyeza kuti 1 mM IPA inachepetsa kuchuluka kwa okosijeni m'thupi (OCR): kupuma kosakhala kwa mitochondrial, kupuma kwa basal ndi maximal, kutayikira kwa proton ndi kupanga kwa ATP poyerekeza ndi gulu lolamulira (Chithunzi 3A, B), pomwe index ya thanzi la bioenergetic (BHI) sinasinthe.
IPA imachepetsa kupuma kwa mitochondrial m'maselo a LX-2. Mzere wozungulira wa kupuma kwa mitochondrial (OCR) umawonetsedwa ngati magawo a kupuma kwa mitochondrial (kupuma kosagwira ntchito kwa mitochondrial, kupuma kwa basal, kupuma kwakukulu, kutayikira kwa proton, kupanga ATP, SRC ndi BHI). Maselo A ndi B adayikidwa mu incubation ndi 10 μM, 100 μM ndi 1 mM IPA kwa maola 24, motsatana. Maselo C ndi D adayikidwa mu incubation ndi TGF-β1 (5 ng/ml) ndi 1 mM IPA mu serum-free medium kwa maola 24, motsatana. Miyeso yonse idasinthidwa kukhala yachibadwa ku DNA pogwiritsa ntchito CyQuant kit. BHI: bioenergetic health index; SRC: respiratory reserve capacity; OCR: oxygen consumption rate. Deta imawonetsedwa ngati mean ± standard deviation (SD), n = 5 zoyeserera zodziyimira pawokha. Kuyerekeza kwa ziwerengero kunachitika pogwiritsa ntchito mayeso a ANOVA ndi Bonferroni post hoc. *p < 0.05; **p < 0.01; ndi ***p < 0.001
Kuti timvetse bwino momwe IPA imakhudzira bioenergetic profile ya TGF-β1-activated LX-2 cells, tinasanthula mitochondrial oxidative phosphorylation ndi OCR (Chithunzi 3C,D). Zotsatira zake zinasonyeza kuti chithandizo cha TGF-β1 chingachepetse kupuma kwakukulu, mphamvu yosungira mpweya (SRC) ndi BHI poyerekeza ndi gulu lolamulira (Chithunzi 3C,D). Kuphatikiza apo, chithandizo chophatikizana chinachepetsa kupuma kwa basal, proton leak ndi kupanga ATP, koma SRC ndi BHI zinali zapamwamba kwambiri kuposa zomwe zinathandizidwa ndi TGF-β1 (Chithunzi 3C,D).
Tinachitanso "Cellular Energy Phenotype Test" yoperekedwa ndi pulogalamu ya Seahorse (Chithunzi Chowonjezera 4A–D). Monga momwe zasonyezedwera mu Chithunzi Chowonjezera 3B, mphamvu zonse za kagayidwe kachakudya za OCR ndi ECAR zinachepa pambuyo pa chithandizo cha TGF-β1, komabe, palibe kusiyana komwe kunawonedwa m'magulu ophatikizana ndi ochizira IPA poyerekeza ndi gulu lowongolera. Kuphatikiza apo, milingo yonse ya basal ndi stress ya OCR inachepa pambuyo pa chithandizo chophatikizana ndi IPA poyerekeza ndi gulu lowongolera (Chithunzi Chowonjezera 4C). Chosangalatsa n'chakuti, njira yofananayo idawonedwa ndi chithandizo chophatikizana, komwe palibe kusintha kwa milingo ya basal ndi stress ya ECAR komwe kudawonedwa poyerekeza ndi chithandizo cha TGF-β1 (Chithunzi Chowonjezera 4C). Mu HSCs, kuchepa kwa phosphorylation ya mitochondrial oxidative ndi kuthekera kwa chithandizo chophatikizana kubwezeretsa SCR ndi BHI pambuyo pokumana ndi chithandizo cha TGF-β1 sikunasinthe mphamvu ya kagayidwe kachakudya (OCR ndi ECAR). Zotsatirazi zikusonyeza kuti IPA ingachepetse mphamvu zamagetsi mu ma HSC, zomwe zikusonyeza kuti IPA ikhoza kuyambitsa mphamvu zochepa zomwe zimasintha mawonekedwe a HSC kukhala osagwira ntchito (Chithunzi Chowonjezera 4D).
Zotsatira za IPA pa mitochondrial dynamics zinafufuzidwa pogwiritsa ntchito kuchuluka kwa magawo atatu a mawonekedwe a mitochondrial ndi maukonde olumikizirana komanso utoto wa MTR (Chithunzi 4 ndi Chithunzi Chowonjezera 5). Chithunzi 4 chikuwonetsa kuti, poyerekeza ndi gulu lolamulira, chithandizo cha TGF-β1 chinachepetsa dera lapakati, chiwerengero cha nthambi, kutalika kwa nthambi yonse, ndi chiwerengero cha nthambi yolumikizana (Chithunzi 4A ndi B) ndipo chinasintha kuchuluka kwa mitochondria kuchokera ku mawonekedwe ozungulira kupita ku mawonekedwe apakati (Chithunzi 4C). Chithandizo cha IPA chokha chinachepetsa kuchuluka kwa mitochondrial ndipo chinasintha kuchuluka kwa mitochondria kuchokera ku mawonekedwe ozungulira kupita ku mawonekedwe apakati poyerekeza ndi gulu lolamulira (Chithunzi 4A). Mosiyana ndi zimenezi, sphericity, kutalika kwa nthambi yapakati, ndi ntchito ya mitochondrial yomwe imayesedwa ndi mitochondrial membrane potential-dependent MTR (Chithunzi 4A ndi E) sizinasinthe ndipo magawo awa sanasiyane pakati pa magulu. Ponseponse, zotsatirazi zikusonyeza kuti chithandizo cha TGF-β1 ndi IPA chikuwoneka kuti chimasintha mawonekedwe ndi kukula kwa mitochondrial komanso zovuta za netiweki m'maselo amoyo a LX-2.
IPA imasintha mphamvu za mitochondrial ndi kuchuluka kwa DNA ya mitochondrial m'maselo a LX-2. A. Zithunzi zoyimira za confocal za maselo amoyo a LX-2 omwe ali ndi TGF-β1 (5 ng/ml) ndi 1 mM IPA kwa maola 24 mu serum-free medium zomwe zikuwonetsa maukonde a mitochondrial okhala ndi Mitotracker™ Red CMXRos ndi nuclei zokhala ndi buluu wokhala ndi DAPI. Deta yonse inali ndi zithunzi zosachepera 15 pagulu lililonse. Tinapeza zithunzi 10 za Z-stack pa mtundu uliwonse wa chitsanzo. Mndandanda uliwonse wa Z-axis unali ndi magawo 30, chilichonse chokhala ndi makulidwe a 9.86 μm. Scale bar: 10 μm. B. Zinthu zoyimira (mitochondria yokha) zinadziwika pogwiritsa ntchito adaptive thresholding pachithunzichi. Kusanthula kwa kuchuluka ndi kufananiza kulumikizana kwa mitochondrial morphological network kunachitika pa maselo onse m'gulu lililonse. C. Kuchuluka kwa ma ratios a mawonekedwe a mitochondrial. Mitengo yoyandikira 0 imasonyeza mawonekedwe ozungulira, ndipo mitengo yoyandikira 1 imasonyeza mawonekedwe a filamentous. D Kuchuluka kwa DNA ya Mitochondrial (mtDNA) kunapezeka monga momwe kwafotokozedwera mu Zipangizo ndi Njira. Kusanthula kwa E Mitotracker™ Red CMXRos kunachitika pogwiritsa ntchito flow cytometry (zochitika 30,000) monga momwe kwafotokozedwera mu Zipangizo ndi Njira. Deta imaperekedwa ngati mean ± SD, n = mayesero atatu odziyimira pawokha. Kuyerekeza ziwerengero kunachitika pogwiritsa ntchito mayeso a ANOVA ndi Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Kenako tinasanthula kuchuluka kwa mtDNA m'maselo a LX-2 ngati chizindikiro cha kuchuluka kwa mitochondrial. Poyerekeza ndi gulu lolamulira, kuchuluka kwa mtDNA kunawonjezeka m'gulu lothandizidwa ndi TGF-β1 (Chithunzi 4D). Poyerekeza ndi gulu lothandizidwa ndi TGF-β1, kuchuluka kwa mtDNA kunachepetsedwa m'gulu lothandizira kuphatikiza (Chithunzi 4D), zomwe zikusonyeza kuti IPA ikhoza kuchepetsa kuchuluka kwa mtDNA ndipo mwina kuchuluka kwa mitochondrial komanso kupuma kwa mitochondrial (Chithunzi 3C). Kuphatikiza apo, IPA imawoneka kuti imachepetsa kuchuluka kwa mtDNA mu chithandizo chophatikiza koma sinakhudze ntchito ya mitochondrial yomwe imayendetsedwa ndi MTR (Chithunzi 4A–C).
Tinafufuza mgwirizano wa IPA ndi milingo ya mRNA ya majini okhudzana ndi fibrosis, apoptosis, kupulumuka, ndi mitochondrial dynamics mu maselo a LX-2 (Chithunzi 5A–D). Poyerekeza ndi gulu lolamulira, gulu lothandizidwa ndi TGF-β1 linawonetsa kuwonjezeka kwa ma gene monga collagen type I α2 chain (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), tissue inhibitor ya metalloproteinase 1 (TIMP1), ndi dynamin 1-like gene (DRP1), zomwe zikusonyeza kuwonjezeka kwa fibrosis ndi activation. Kuphatikiza apo, poyerekeza ndi gulu lolamulira, chithandizo cha TGF-β1 chinachepetsa milingo ya mRNA ya nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, inhibitor ya B-cell α, enhancer ya nuclear factor κ gene light peptide (NFκB1A), ndi inhibitor ya nuclear factor κB kinase subunit β (IKBKB) (Chithunzi 5A–D). Poyerekeza ndi chithandizo cha TGF-β1, chithandizo chophatikizana ndi TGF-β1 ndi IPA chinachepetsa kufalikira kwa COL1A2 ndi MMP2, koma chinawonjezera kuchuluka kwa mRNA kwa PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, ndi IKBKB. Chithandizo cha IPA chinachepetsa kwambiri kufalikira kwa MMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), ndi mitochondrial fusion protein 2 (MFN2), pomwe kufalikira kwa CASP8, NFκB1A, NFκB1B, ndi IKBKB kunawonjezeka poyerekeza ndi gulu lolamulira. Komabe, palibe kusiyana komwe kunapezeka pa kufalikira kwa caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), ndi fission protein 1 (FIS1). Zotsatirazi zikusonyeza kuti chithandizo cha IPA chimasintha momwe majini amagwirizanirana ndi fibrosis, apoptosis, kupulumuka, ndi mitochondrial dynamics zimagwirira ntchito. Deta yathu ikusonyeza kuti chithandizo cha IPA chimachepetsa fibrosis m'maselo a LX-2; nthawi yomweyo, chimalimbikitsa kupulumuka mwa kusintha mawonekedwe ake kukhala osagwira ntchito.
IPA imasintha momwe majini a fibroblast, apoptotic, viability, ndi mitochondrial dynamics amaonekera m'maselo a LX-2. Ma histogram amasonyeza mRNA expression poyerekeza ndi endogenous control (RPLP0 kapena PPIA) pambuyo poti maselo a LX-2 ayambitsidwa ndi TGF-β1 ndi IPA mu serum-free medium kwa maola 24. A imasonyeza ma fibroblast, B imasonyeza maselo a apoptotic, C imasonyeza maselo opulumuka, ndipo D imasonyeza momwe majini a mitochondrial dynamics amaonekera. Deta imaperekedwa ngati mean ± standard deviation (SD), n = 3 zoyeserera zodziyimira pawokha. Kuyerekeza ziwerengero kunachitika pogwiritsa ntchito mayeso a ANOVA ndi Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Kenako, kusintha kwa kukula kwa maselo (FSC-H) ndi cytoplasmic complexity (SSC-H) kunayesedwa pogwiritsa ntchito flow cytometry (Chithunzi 6A,B), ndipo kusintha kwa mawonekedwe a maselo pambuyo pa chithandizo cha IPA kunayesedwa pogwiritsa ntchito transmission electron microscopy (TEM) ndi phase contrast microscopy (Chithunzi Chowonjezera 6A-B). Monga momwe zinalili, maselo omwe ali mu gulu lothandizidwa ndi TGF-β1 adakula kukula poyerekeza ndi gulu lolamulira (Chithunzi 6A,B), kuwonetsa kukula kwa classical kwa rough endoplasmic reticulum (ER*) ndi phagolysosomes (P), kusonyeza hematopoietic stem cell (HSC) activation (Chithunzi Chowonjezera 6A). Komabe, poyerekeza ndi gulu lothandizidwa ndi TGF-β1, kukula kwa maselo, cytoplasmic complexity (Chithunzi 6A,B), ndi ER* zinachepa mu gulu lothandizira la TGF-β1 ndi IPA (Chithunzi Chowonjezera 6A). Kuphatikiza apo, chithandizo cha IPA chinachepetsa kukula kwa maselo, kusinthasintha kwa cytoplasmic (Zithunzi 6A,B), kuchuluka kwa P ndi ER* (Chithunzi Chowonjezera cha 6A) poyerekeza ndi gulu lolamulira. Kuphatikiza apo, kuchuluka kwa maselo a apoptotic kunawonjezeka pambuyo pa maola 24 a chithandizo cha IPA poyerekeza ndi gulu lolamulira (mivi yoyera, Chithunzi Chowonjezera cha 6B). Zotsatirazi zikusonyeza kuti 1 mM IPA ikhoza kuyambitsa apoptosis ya HSC ndikubweza kusintha kwa magawo a morphological a maselo omwe ayambitsidwa ndi TGF-β1, potero kuwongolera kukula kwa maselo ndi kusinthasintha kwawo, komwe kungagwirizane ndi kuletsa kwa HSC.
IPA imasintha kukula kwa maselo ndi kusinthasintha kwa maselo m'maselo a LX-2. Zithunzi zoyimira kusanthula kwa kayendedwe ka madzi. Kusanthulaku kunagwiritsa ntchito njira yolumikizira maselo a LX-2: SSC-A/FSC-A kuti afotokoze kuchuluka kwa maselo, FSC-H/FSC-A kuti azindikire ma doublet, ndi SSC-H/FSC-H kuti awonetse kukula kwa maselo ndi kusanthula kusinthasintha. Maselo anaikidwa mu TGF-β1 (5 ng/ml) ndi 1 mM IPA mu seramu yopanda seramu kwa maola 24. Maselo a LX-2 anagawidwa mu gawo la kumanzere lakumunsi (SSC-H-/FSC-H-), gawo lakumanzere lakumtunda (SSC-H+/FSC-H-), gawo lakumanja lakumunsi (SSC-H-/FSC-H+), ndi gawo lakumanja lakumtunda lakumtunda (SSC-H+/FSC-H+) kuti awonetse kukula kwa maselo ndi kusanthula kusinthasintha kwa maselo. B. Kapangidwe ka maselo kanasanthulidwa pogwiritsa ntchito njira yoyezera kuchuluka kwa maselo pogwiritsa ntchito FSC-H (forward scatter, cell size) ndi SSC-H (side scatter, cytoplasmic complexity) (zochitika 30,000). Deta imaperekedwa ngati mean ± SD, n = 3 zoyeserera zodziyimira pawokha. Kuyerekeza ziwerengero kunachitika pogwiritsa ntchito njira imodzi ya ANOVA ndi Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 ndi ****p < 0.0001
Ma metabolites a m'matumbo monga IPA akhala nkhani yofunika kwambiri yofufuza, zomwe zikusonyeza kuti zolinga zatsopano zitha kupezeka mu gut microbiota. Chifukwa chake n'zosangalatsa kuti IPA, metabolite yomwe tagwirizanitsa ndi fibrosis ya chiwindi mwa anthu [15], yawonetsedwa kuti ndi mankhwala oletsa fibrosis m'zinyama [13, 14]. Pano, tikuwonetsa koyamba mgwirizano pakati pa serum IPA ndi global liver transcriptomics ndi DNA methylation mwa anthu onenepa omwe alibe matenda a shuga a mtundu wa 2 (T2D), kuwonetsa apoptosis, mitophagy ndi moyo wautali, komanso jini lomwe lingakhale loyenera AKT1 lolamulira homeostasis ya chiwindi. Chinthu china chatsopano cha kafukufuku wathu ndikuti tawonetsa kuyanjana kwa chithandizo cha IPA ndi apoptosis, mawonekedwe a maselo, bioenergetics ya mitochondrial ndi mphamvu m'maselo a LX-2, kusonyeza mphamvu yochepa yomwe imasintha phenotype ya HSC kupita ku inactivation, zomwe zimapangitsa IPA kukhala yoyenera kukonza fibrosis ya chiwindi.
Tinapeza kuti apoptosis, mitophagy ndi moyo wautali zinali njira zofunika kwambiri zodziwika bwino zomwe zimaphatikizidwa mu majini a chiwindi omwe amagwirizanitsidwa ndi IPA yozungulira seramu. Kusokonezeka kwa dongosolo lowongolera khalidwe la mitochondrial (MQC) kungayambitse kusagwira bwino ntchito kwa mitochondrial, mitophagy ndi apoptosis, motero kumalimbikitsa kuchitika kwa MASLD[33, 34]. Chifukwa chake, titha kuganiza kuti IPA ikhoza kukhala ndi gawo pakusunga mphamvu za maselo ndi umphumphu wa mitochondrial kudzera mu apoptosis, mitophagy ndi moyo wautali m'chiwindi. Deta yathu yawonetsa kuti majini awiri anali ofala pa mayeso atatuwa: YKT6 ndi AKT1. Ndikofunikira kudziwa kuti YKT6 ndi puloteni ya SNARE yomwe imakhudzidwa ndi njira yolumikizirana kwa nembanemba ya maselo. Imagwira ntchito mu autophagy ndi mitophagy popanga complex yoyambitsa ndi STX17 ndi SNAP29 pa autophagosome, motero imalimbikitsa kuphatikizana kwa autophagosomes ndi lysosomes[35]. Kuphatikiza apo, kutayika kwa ntchito ya YKT6 kumabweretsa vuto la mitophagy [36], pomwe kukwera kwa YKT6 kumalumikizidwa ndi kupita patsogolo kwa hepatocellular carcinoma (HCC), kusonyeza kuwonjezeka kwa kupulumuka kwa maselo [37]. Kumbali inayi, AKT1 ndiye jini yofunika kwambiri yolumikizana ndipo imagwira ntchito yofunika kwambiri pa matenda a chiwindi, kuphatikizapo njira yolumikizirana ya PI3K/AKT, kuzungulira kwa maselo, kusamuka kwa maselo, kuchulukana, kulumikizidwa kwa focal, ntchito ya mitochondrial, ndi kutulutsa kwa collagen [38-40]. Njira yolumikizirana ya PI3K/AKT yoyambitsidwa imatha kuyambitsa maselo oyambira a hematopoietic (HSCs), omwe ndi maselo omwe amachititsa kupanga matrix akunja (ECM), ndipo kusakhazikika kwake kungathandize kuchitika ndi kupita patsogolo kwa fibrosis ya chiwindi [40]. Kuphatikiza apo, AKT ndi chimodzi mwazinthu zofunika kwambiri zopulumuka maselo zomwe zimaletsa apoptosis ya maselo odalira p53, ndipo kuyambika kwa AKT kungagwirizane ndi kuletsa apoptosis ya maselo a chiwindi [41, 42]. Zotsatira zomwe zapezeka zikusonyeza kuti IPA ikhoza kukhala ndi gawo mu apoptosis yokhudzana ndi chiwindi pogwiritsa ntchito mitochondria pokhudza chisankho cha ma hepatocytes pakati pa kulowa mu apoptosis kapena kupulumuka. Zotsatirazi zitha kulamulidwa ndi majini a AKT ndi/kapena YKT6, omwe ndi ofunikira kwambiri pa homeostasis ya chiwindi.
Zotsatira zathu zasonyeza kuti 1 mM IPA inayambitsa apoptosis ndi kuchepetsa kupuma kwa mitochondrial m'maselo a LX-2 popanda chithandizo cha TGF-β1. N'zodziwikiratu kuti apoptosis ndi njira yayikulu yothetsera fibrosis ndi kuyambitsa maselo oyambira a hematopoietic (HSC), komanso ndi chochitika chofunikira kwambiri pakuyankha kwa thupi kwa fibrosis ya chiwindi [4, 43]. Komanso, kubwezeretsedwa kwa BHI m'maselo a LX-2 pambuyo pa chithandizo chophatikizana kunapereka chidziwitso chatsopano pa ntchito yomwe IPA ingakhale nayo pakulamulira bioenergetics ya mitochondrial. Pansi pa mikhalidwe yopumula komanso yosagwira ntchito, maselo a hematopoietic nthawi zambiri amagwiritsa ntchito phosphorylation ya mitochondrial oxidative kuti apange ATP ndipo amakhala ndi ntchito yochepa ya kagayidwe kachakudya. Kumbali ina, kuyambitsa HSC kumawonjezera kupuma kwa mitochondrial ndi biosynthesis kuti kulipirire kufunikira kwa mphamvu yolowera mu glycolytic state [44]. Chowonadi chakuti IPA sinakhudze mphamvu ya kagayidwe kachakudya ndipo ECAR ikusonyeza kuti njira ya glycolytic siiyang'aniridwa kwambiri. Mofananamo, kafukufuku wina adawonetsa kuti 1 mM IPA inatha kusintha ntchito ya unyolo wa kupuma kwa mitochondrial mu cardiomyocytes, mzere wa maselo a hepatocyte a munthu (Huh7) ndi maselo a endothelial a m'mitsempha ya munthu (HUVEC); Komabe, palibe chomwe chidapezeka pa glycolysis mu cardiomyocytes, zomwe zikusonyeza kuti IPA ingakhudze bioenergetics ya mitundu ina ya maselo [45]. Chifukwa chake, tikuganiza kuti 1 mM IPA ikhoza kugwira ntchito ngati uncoupler wofatsa wa mankhwala, popeza imatha kuchepetsa kwambiri mawonekedwe a majini a fibrogen, mawonekedwe a maselo ndi mitochondrial bioenergetics popanda kusintha kuchuluka kwa mtDNA [46]. Mitochondrial uncouplers imatha kuletsa culture-induced fibrosis ndi HSC activation [47] ndikuchepetsa kupanga kwa mitochondrial ATP komwe kumayendetsedwa kapena kuyambitsidwa ndi mapuloteni ena monga ma uncoupling proteins (UCP) kapena adenine nucleotide translocase (ANT). Kutengera mtundu wa maselo, izi zitha kuteteza maselo ku apoptosis ndi/kapena kulimbikitsa apoptosis [46]. Komabe, maphunziro ena akufunika kuti afotokoze bwino ntchito ya IPA monga cholumikizira cha mitochondrial mu kuletsa maselo oyambira a hematopoietic.
Kenako tinafufuza ngati kusintha kwa kupuma kwa mitochondrial kumawonekera mu mawonekedwe a mitochondrial m'maselo amoyo a LX-2. Chochititsa chidwi n'chakuti, chithandizo cha TGF-β1 chimasintha kuchuluka kwa mitochondrial kuchokera ku spherical kupita ku intermediate, ndi kuchepa kwa nthambi za mitochondrial ndi kuwonjezeka kwa DRP1, chinthu chofunikira kwambiri pakugawanika kwa mitochondrial [48]. Kuphatikiza apo, kugawanika kwa mitochondrial kumalumikizidwa ndi zovuta zonse za netiweki, ndipo kusintha kuchokera ku fusion kupita ku fission ndikofunikira kwambiri pa kuyambitsa hematopoietic stem cell (HSC), pomwe kuletsa kugawanika kwa mitochondrial kumabweretsa HSC apoptosis [49]. Chifukwa chake, zotsatira zathu zikuwonetsa kuti chithandizo cha TGF-β1 chingayambitse kuchepa kwa zovuta za netiweki ya mitochondrial ndi kuchepa kwa nthambi, zomwe zimachitika kwambiri pakugawanika kwa mitochondrial komwe kumalumikizidwa ndi maselo oyambira a hematopoietic (HSCs). Kuphatikiza apo, deta yathu idawonetsa kuti IPA ikhoza kusintha kuchuluka kwa mitochondria kuchokera ku mawonekedwe ozungulira kupita ku apakati, potero kuchepetsa kuwonetsedwa kwa OPA1 ndi MFN2. Kafukufuku wasonyeza kuti kuchepetsa OPA1 kungayambitse kuchepa kwa mphamvu ya netiweki ya mitochondrial ndikuyambitsa apoptosis ya maselo [50]. MFN2 imadziwika kuti imayambitsa kusakanikirana kwa mitochondrial ndi apoptosis [51]. Zotsatira zomwe zapezeka zikusonyeza kuti kuyambitsa maselo a LX-2 ndi TGF-β1 ndi/kapena IPA kumawoneka kuti kumasintha mawonekedwe ndi kukula kwa mitochondrial, komanso momwe activation imagwirira ntchito komanso zovuta za netiweki.
Zotsatira zathu zikusonyeza kuti kuphatikiza chithandizo cha TGFβ-1 ndi IPA kungachepetse magawo a mtDNA ndi mawonekedwe a maselo mwa kuwongolera kufotokozedwa kwa mRNA kwa fibrosis, apoptosis ndi majini okhudzana ndi kupulumuka m'maselo omwe amapewa apoptosis. Zoonadi, IPA idachepetsa kuchuluka kwa mRNA kwa majini a AKT1 ndi majini ofunikira a fibrosis monga COL1A2 ndi MMP2, koma idakulitsa kuchuluka kwa CASP8, komwe kumagwirizanitsidwa ndi apoptosis. Zotsatira zathu zidawonetsa kuti pambuyo pa chithandizo cha IPA, kufotokozedwa kwa BAX kudachepa ndipo kufotokozedwa kwa mRNA kwa ma subunits a banja la TIMP1, BCL-2 ndi NF-κB kudakwera, zomwe zikusonyeza kuti IPA ikhoza kuyambitsa zizindikiro zopulumuka m'maselo oyambira a hematopoietic (HSCs) omwe amathawa apoptosis. Mamolekyu awa amatha kugwira ntchito ngati zizindikiro zoteteza kupulumuka m'maselo oyambira a hematopoietic, zomwe zitha kugwirizanitsidwa ndi kufotokozedwa kwakukulu kwa mapuloteni otsutsana ndi apoptosis (monga Bcl-2), kuchepa kwa kufotokozedwa kwa pro-apoptotic BAX, komanso mgwirizano wovuta pakati pa TIMP ndi NF-κB [5, 7]. IPA imagwira ntchito yake kudzera mu PXR, ndipo tapeza kuti kuphatikiza chithandizo ndi TGF-β1 ndi IPA kunawonjezera kuchuluka kwa PXR mRNA expression, zomwe zikusonyeza kuletsa HSC activation. Kuwonetsa kwa PXR komwe kumagwira ntchito kumadziwika kuti kumaletsa HSC activation mu vivo komanso mu vitro [52, 53]. Zotsatira zathu zikusonyeza kuti IPA ikhoza kutenga nawo mbali pakuchotsa ma HSC ogwiritsidwa ntchito polimbikitsa apoptosis, kuchepetsa fibrosis ndi kagayidwe ka mitochondrial, ndikuwonjezera zizindikiro zopulumuka, zomwe ndi njira zomwe zimasintha mtundu wa HSC wogwiritsidwa ntchito kukhala wosagwira ntchito. Kufotokozera kwina komwe kungachitike pa njira ndi ntchito ya IPA mu apoptosis ndikuti imachotsa mitochondria yosagwira ntchito makamaka kudzera mu mitophagy (njira yamkati) ndi njira yolumikizira ya TNF yakunja (Table 1), yomwe imalumikizidwa mwachindunji ndi njira yolumikizira ya NF-κB (Chithunzi Chowonjezera 7). Chochititsa chidwi n'chakuti majini olemera okhudzana ndi IPA amatha kuyambitsa zizindikiro zoyambitsa apoptotic ndi pro-suvival mu apoptotic pathway [54], zomwe zikusonyeza kuti IPA ikhoza kuyambitsa apoptotic pathway kapena kupulumuka mwa kuyanjana ndi majini awa. Komabe, momwe IPA imayambitsira apoptosis kapena kupulumuka panthawi ya HSC activation ndi njira zake sizikudziwika bwino.
IPA ndi metabolite ya tizilombo toyambitsa matenda yomwe imapangidwa kuchokera ku tryptophan yazakudya kudzera mu microbiota ya m'mimba. Kafukufuku wasonyeza kuti ili ndi mphamvu zoletsa kutupa, antioxidant, komanso epigenetic m'matumbo.[55] Kafukufuku wasonyeza kuti IPA imatha kusintha magwiridwe antchito a zotchinga m'matumbo ndikuchepetsa kupsinjika kwa okosijeni, zomwe zingathandize pa zotsatira zake za thupi.[56] Ndipotu, IPA imatumizidwa ku ziwalo zomwe zimayang'aniridwa kudzera mu kayendedwe ka magazi, ndipo popeza IPA ili ndi kapangidwe kofanana ka metabolite ndi tryptophan, serotonin, ndi indole derivatives, IPA imagwira ntchito za metabolic zomwe zimapangitsa kuti metabolic ipambane.[52] IPA ikhoza kupikisana ndi metabolites zochokera ku tryptophan kuti zigwirizane ndi ma enzymes kapena ma receptors, zomwe zingasokoneze njira zachibadwa za metabolic. Izi zikuwonetsa kufunikira kopitiliza maphunziro pa pharmacokinetics yake ndi pharmacodynamics kuti amvetse bwino njira yake yothandizira.[57] Zikuonekabe ngati izi zitha kuchitikanso m'maselo oyambira a hematopoietic (HSCs).
Tikuvomereza kuti kafukufuku wathu uli ndi zofooka zina. Pofuna kuwona bwino mgwirizano wokhudzana ndi IPA, sitinaphatikizepo odwala matenda a shuga amtundu wa 2 (T2DM). Tikuvomereza kuti izi zimachepetsa kugwiritsidwa ntchito kwa zomwe tapeza kwa odwala omwe ali ndi matenda a shuga amtundu wa 2 komanso matenda a chiwindi opitilira. Ngakhale kuti kuchuluka kwa IPA m'magazi a anthu ndi 1–10 μM [11, 20], kuchuluka kwa 1 mM IPA kunasankhidwa kutengera kuchuluka kwakukulu kosakhala ndi poizoni [15] komanso kuchuluka kwakukulu kwa apoptosis, popanda kusiyana kwa kuchuluka kwa maselo otupa. Ngakhale kuchuluka kwa IPA kwagwiritsidwa ntchito mu kafukufukuyu, pakadali pano palibe mgwirizano wokhudza mlingo wogwira ntchito wa IPA [52]. Ngakhale kuti zotsatira zathu ndizofunikira, tsogolo lalikulu la kagayidwe kachakudya ka IPA likadali gawo lofufuza. Kuphatikiza apo, zomwe tapeza pa mgwirizano pakati pa kuchuluka kwa IPA m'magazi ndi methylation ya DNA ya zolemba za chiwindi sizinapezeke kokha kuchokera ku maselo oyambira a hematopoietic (HSCs) komanso kuchokera ku minofu ya chiwindi. Tinasankha kugwiritsa ntchito maselo a LX-2 a anthu kutengera zomwe tapeza kale kuchokera ku kusanthula kwa transcriptome kuti IPA imagwirizanitsidwa ndi kuyambika kwa maselo oyambira a hematopoietic (HSC) [15], ndipo ma HSC ndi maselo akuluakulu omwe amakhudzidwa ndi kupita patsogolo kwa fibrosis ya chiwindi. Chiwindi chimapangidwa ndi mitundu yambiri ya maselo, kotero mitundu ina ya maselo monga hepatocyte-HSC-immune cell co-culture system kuphatikiza ndi kuyambika kwa caspase ndi kugawikana kwa DNA komanso momwe ntchito ikuyendera kuphatikiza kuchuluka kwa mapuloteni ziyenera kuganiziridwa kuti ziphunzire ntchito ya IPA ndi momwe imagwirira ntchito ndi mitundu ina ya maselo a chiwindi.