Mbere twatangaje ko urugero rw'amaraso rwa aside indole-3-propionic (IPA) ikomoka mu mara ari hasi mu barwayi bafite fibrosis y'umwijima. Muri ubu bushakashatsi, twakoze ubushakashatsi ku bijyanye na transcriptome na methylome ya ADN mu mwijima w'umubyibuho ukabije ugereranyije n'urugero rwa IPA mu mara, ndetse n'uruhare rwa IPA mu gutuma uturemangingo tw'umwijima (HSCs) tudakora neza mu buryo bwa vitro.
Ubushakashatsi bwakorewe ku barwayi 116 bafite umubyibuho ukabije badafite diyabete yo mu bwoko bwa 2 (T2DM) (bafite imyaka 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m²) babazwe bariatric mu kigo cy’ubuvuzi cya Kuopio Bariatric Surgery Center (KOBS). Ingano ya IPA yapimwe hakoreshejwe liquid chromatography-mass spectrometry (LC-MS), isesengura rya transcriptome y’umwijima ryakozwe hakoreshejwe sequencing ya RNA yose, naho isesengura rya ADN methylation ryakozwe hakoreshejwe Infinium HumanMethylation450 BeadChip. Uturemangingo twa stellate y’umwijima w’umuntu (LX-2) twakoreshejwe mu bushakashatsi bwakozwe muri vitro.
Ingano ya IPA mu maraso ya serum yafitanye isano no kugaragaza imiterere y’uturemangingo twibasira inzira zo kuzura kw’uturemangingo, kuzura kw’uturemangingo, no kuramba kw’uturemangingo mu mwijima. Ingirabuzimafatizo ya AKT serine/threonine kinase 1 (AKT1) niyo yari ngirabuzimafatizo nyinshi kandi zikora cyane mu nyandiko y’umwijima na ADN methylation. Ubuvuzi bwa IPA bwatumye habaho kuzura kw’uturemangingo, kugabanya guhumeka kwa mitochondrial, no guhindura imiterere y’uturemangingo n’imikorere ya mitochondrial binyuze mu guhindura imiterere y’uturemangingo tuzwiho kugenga fibrosis, kuzura kw’uturemangingo, no kubaho kw’uturemangingo twa LX-2.
Iyo mibare yose hamwe, igaragaza ko IPA ishobora kugira ingaruka ku buvuzi kandi ko ishobora gutera apoptosis no guhindura imiterere ya HSC ikajya mu gihe idakora, bityo bikarushaho kwagura amahirwe yo kubuza umwijima gutera fibrosis binyuze mu kubangamira ibikorwa bya HSC no gukora mitochondrial metabolism.
Ubukana bw'umubyibuho ukabije n'indwara ya metabolic syndrome byagiye bijyana no kwiyongera kw'indwara y'umwijima w'ibinure bifitanye isano na metabolically associated thickens (MASLD); iyi ndwara yibasira 25% kugeza 30% by'abaturage muri rusange [1]. Ingaruka nyamukuru za MASLD ni fibrosis y'umwijima, inzira ihinduka irangwa no kwibumbira hamwe kw'uturemangingo tw'ingirabuzimafatizo (ECM) [2]. Uturemangingo tw'ingenzi tugira uruhare mu gutera fibrosis y'umwijima ni uturemangingo tw'umwijima (HSCs), tugaragaza ubwoko bune buzwi: quiescent, activated, inactivated, na senescent [3, 4]. HSCs zishobora gukoreshwa no gutandukana kuva ku buryo butinze zikajya mu turemangingo tumeze nka fibroblast dukenera ingufu nyinshi, hamwe no kwiyongera k'imikorere ya α-smooth muscle actin (α-SMA) na type I collagen (Col-I) [5, 6]. Mu gihe cy'ihinduka rya fibrosis y'umwijima, HSCs zikoreshwa zikurwaho binyuze mu kuzura cyangwa kudakora. Izi nzira zirimo kugabanya imiterere y’uturemangingo tw’umubiri (fibrogen génétique) no guhindura imiterere y’uturemangingo tw’umubiri (prosurvival genes) (nk’inzira za NF-κB na PI3K/Akt signaling pathways) [7, 8], ndetse n’impinduka mu mikorere n’imikorere ya mitochondrial [9].
Urugero rwa serum rwa tryptophan metabolite indole-3-propionic aside (IPA) ikorerwa mu mara, byagaragaye ko rugabanuka mu ndwara za metabolite mu bantu harimo na MASLD [10-13]. IPA ifitanye isano no kurya fibre, izwiho ingaruka zayo zo kurwanya antioxidant no kurwanya ububyimbirwe, kandi ikagabanya uburyo steatohepatitis idakoresheje alcoholic (NASH) iterwa n'indyo mu mubiri no mu mubiri [11-14]. Hari ibimenyetso byavuye mu bushakashatsi bwacu bwa mbere, bwagaragaje ko urugero rwa IPA mu mara rwari ruri hasi mu barwayi bafite fibrosis y'umwijima kurusha mu barwayi bafite umubyibuho ukabije badafite fibrosis y'umwijima mu Inyigo yo Kubaga Kuopio Bariatric (KOBS). Byongeye kandi, twerekanye ko ubuvuzi bwa IPA bushobora kugabanya igaragara rya genes ari ikimenyetso cya kera cyo gufatana kw'uturemangingo, kwimuka kw'uturemangingo no gukorana kw'uturemangingo tw'amaraso mu icyitegererezo cya selile y'umwijima (LX-2) kandi ni metabolite ishobora kurinda umwijima [15]. Ariko, ntibirasobanuka neza uburyo IPA itera gusubira inyuma kw'urugingo rw'umwijima binyuze mu gukora HSC apoptosis na mitochondrial bioenergetics.
Aha, tugaragaza ko IPA mu maraso ifitanye isano no kugaragaza imiterere y’uturemangingo twinshi mu mikorere y’uturemangingo, imitofagitire, n’inzira zo kuramba mu mwijima w’abantu bafite diyabete y’umubyibuho ukabije ariko itari iya diyabete yo mu bwoko bwa 2 (KOBS). Byongeye kandi, twasanze IPA ishobora gutuma uturemangingo tw’amaraso dukoreshwa mu gusohora no kwangirika kw’uturemangingo tw’amaraso (HSCs) binyuze mu nzira yo kudakora. Ibi bisubizo bigaragaza uruhare rushya rwa IPA, bigatuma iba intego ishobora kuvura yo guteza imbere umwijima.
Ubushakashatsi bwakozwe mbere mu itsinda rya KOBS bwagaragaje ko abarwayi bafite umwijima bafite urugero rwa IPA ruri hasi ugereranije n’abarwayi badafite umwijima [15]. Kugira ngo dukureho ingaruka zishobora guterwa na diyabete yo mu bwoko bwa 2, twashyize mu matsinda abarwayi 116 bafite umubyibuho ukabije badafite diyabete yo mu bwoko bwa 2 (imyaka ± SD: 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m2) (Imbonerahamwe ya 1) mu bushakashatsi bwa KOBS buri gukorwa nk'umubare w'abantu bakoreweho ubushakashatsi [16]. Abitabiriye bose batanze icyemezo cyanditse kandi amabwiriza y'ubushakashatsi yemejwe na Komite Ishinzwe Imyitwarire y'Ibitaro bya North Savo County hakurikijwe Itangazo rya Helsinki (54/2005, 104/2008 na 27/2010).
Ibipimo bya biopsy y'umwijima byabonetse mu gihe cyo kubaga bariatric kandi bisuzumwa histological n'abahanga mu ndwara hakurikijwe ibipimo byasobanuwe mbere [17, 18]. Ibipimo by'isuzuma bikubiye mu mbonerahamwe y'inyongera S1 kandi byasobanuwe mbere [19].
Ingero za serum yo kwiyiriza ubusa zasesenguwe hakoreshejwe uburyo bwa chromatography-mass spectrometry (LC-MS) butagenewe gukoreshwa mu gusesengura metabolomics (n = 116). Ingero zasesenguwe hakoreshejwe uburyo bwa UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Ubudage) nk'uko byasobanuwe mbere19. Kumenya isopropyl alcohol (IPA) byashingiye ku gihe cyo kubika no kugereranya MS/MS spectrum n'ibipimo bisukuye. Ubukana bw'ikimenyetso cya IPA (agace k'inyongera) bwasuzumwe mu isesengura ryose [20].
Gusesengura RNA y'umwijima wose byakozwe hakoreshejwe Illumina HiSeq 2500 kandi amakuru yaratunganyijwe mbere nkuko byasobanuwe mbere [19, 21, 22]. Twakoze isesengura ry’itandukaniro ry’imvugo ry’inyandiko rigira ingaruka ku mikorere ya mitochondrial/biogenesis dukoresheje gene zo mu 1957 zatoranijwe muri database ya MitoMiner 4.0 [23]. Isesengura rya methylation ya ADN y'umwijima ryakozwe hakoreshejwe Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) dukoresheje uburyo bumwe nk’ubwo byasobanuwe mbere [24, 25].
Uturemangingo tw’umwijima w’umuntu (LX-2) twatanzwe n’ubuntu na Porofeseri Stefano Romeo maze dukorerwa ubworozi kandi tubungabungwa mu buryo bwa DMEM/F12 (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Kugira ngo hatoranywe urugero rw’imikorere rwa IPA, uturemangingo twa LX-2 twavuwe hakoreshejwe intera zitandukanye za IPA (10 μM, 100 μM na 1 mM; Sigma, 220027) mu buryo bwa DMEM/F12 mu gihe cy’amasaha 24. Byongeye kandi, kugira ngo harebwe ubushobozi bwa IPA bwo guhagarika HSCs, uturemangingo twa LX-2 twavuwe hamwe na 5 ng/ml TGF-β1 (systems za R&D, 240-B-002/CF) na 1 mM IPA mu buryo budafite serum mu gihe cy’amasaha 24. Ku bijyanye n'uburyo bwo kugenzura imodoka, 4 nM HCL irimo 0.1% BSA yakoreshejwe mu kuvura TGF-β1 naho 0.05% DMSO ikoreshwa mu kuvura IPA, kandi byombi byakoreshejwe hamwe mu kuvura bivanze.
Apoptosis yapimwe hakoreshejwe FITC Annexin V Apoptosis Detection Kit ifite 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) hakurikijwe amabwiriza y'uwakoze. Muri make, LX-2 (1 × 105 selile/isupu) yashyizwe mu masahani 12 y'amasupu hanyuma ivurwa hakoreshejwe doze nyinshi za IPA cyangwa IPA na TGF-β1. Umunsi wakurikiyeho, selile zireremba n'izifata zakusanyijwe, zishyirwamo trypsini, zozwa na PBS, zongerwa mu gikoresho cyo gufata Annexin V, hanyuma zishyirwamo FITC-Annexin V na 7-AAD mu gihe cy'iminota 15.
Mitochondria mu turemangingo tuzima yashyizwemo ibara ry’imikorere ya oxidative hakoreshejwe Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Mu isuzuma rya MTR, uturemangingo twa LX-2 twashyizwemo ubucucike bungana na IPA na TGF-β1. Nyuma y'amasaha 24, uturemangingo tuzima twashyizwemo trypsinize, twozwa na PBS, hanyuma dushyirwamo 100 μM MTR mu buryo budafite serum kuri 37 °C mu gihe cy'iminota 20 nk'uko byasobanuwe mbere [26]. Mu isesengura ry'imiterere y'uturemangingo tuzima, ingano y'uturemangingo n'ubuhanga bwa cytoplasmic byasesenguwe hakoreshejwe ibipimo bya forward scatter (FSC) na side scatter (SSC).
Amakuru yose (ibintu 30.000) yabonetse hakoreshejwe NovoCyte Quanteon (Agilent) kandi yasesenguwe hakoreshejwe NovoExpress® 1.4.1 cyangwa porogaramu ya FlowJo V.10.
Igipimo cyo gukoresha umwuka wa ogisijeni (OCR) n'igipimo cyo gushyira aside hanze y'ingirabuzimafatizo (ECAR) byapimwe mu gihe nyacyo hakoreshejwe Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) ifite Seahorse XF Cell Mito Stress hakurikijwe amabwiriza y'uwakoze. Muri make, uturemangingo 2 × 104 LX-2/ikiraro byatewe ku dupande tw'uturemangingo twa XF96. Nyuma yo gukubitwa nijoro, uturemangingo twavuwe na isopropanol (IPA) na TGF-β1 (Uburyo bw'inyongera 1). Isesengura ry'amakuru ryakozwe hakoreshejwe porogaramu ya Seahorse XF Wave, irimo Seahorse XF Cell Energy Phenotype Test Report Generator. Hashingiwe kuri ibi, hakozwe Bioenergetic Health Index (BHI) [27].
RNA yose yahinduwe muri cDNA. Ku buryo bwihariye, reba referensi [15]. Ibipimo bya poroteyine ya aside ya ribosomal ya 60S ribosomal P0 (RPLP0) na cyclophilin A1 (PPIA) mRNA byakoreshejwe nk'igenzura rya gene. Sisitemu ya QuantStudio 6 pro Real-Time PCR (Thermo Fisher, Landsmeer, Ubuholandi) yakoreshejwe hamwe na TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) cyangwa Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), kandi gene expression fold yarabazwe hakoreshejwe ibipimo bya Ct value cycling (ΔΔCt) hamwe n'uburyo bwa ∆∆Ct. Ibisobanuro birambuye kuri primers bitangwa mu mbonerahamwe z'inyongera S2 na S3.
ADN y'ingirabuzimafatizo (ncDNA) na ADN ya mitochondrial (mtDNA) byakuwemo hakoreshejwe DNeasy blood and tissue kit (Qiagen) nk'uko byasobanuwe mbere [28]. Ingano ya mtDNA yabazwe hashingiwe ku kubara igipimo cya buri gace ka mtDNA kigenewe ugereranije n'ikigereranyo cy'imiterere y'uturere dutatu twa ADN y'ingirabuzimafatizo (mtDNA / ncDNA), nk'uko bisobanuwe mu buryo bw'inyongera bwa 2. Ibisobanuro birambuye ku byakozwe kuri mtDNA na ncDNA bitangwa mu mbonerahamwe y'inyongera S4.
Uturemangingo tuzima twashyizwemo ibara rya Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) kugira ngo tugaragaze urusobe rw’uturemangingo n’urw’uturemangingo. Uturemangingo twa LX-2 (uturemangingo 1 × 104 / well) twashyizwe ku birahuri mu mpande z’ibirahuri zihuye n’ikirahuri (Ibidi GmbH, Martinsried, Ubudage). Nyuma y’amasaha 24, uturemangingo twa LX-2 twashyizwemo 100 μM MTR mu gihe cy’iminota 20 kuri 37 °C naho uturemangingo twashyizwemo ibara rya DAPI (1 μg / ml, Sigma-Aldrich) nk’uko byasobanuwe mbere [29]. Uturemangingo twa Mitochondrial twashyizwemo ibara rya mikorosikopi ya Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, Ubudage) ifite module ya Zeiss LSM 800 confocal kuri 37 °C mu kirere gifite ubushuhe bwa 5% CO2 hakoreshejwe intego ya 63 × NA 1.3. Twabonye amashusho icumi ya Z-series kuri buri bwoko bw'icyitegererezo. Buri Z-series irimo ibice 30, buri kimwe gifite ubugari bwa 9.86 μm. Kuri buri cyitegererezo, amashusho y'ibice icumi bitandukanye byabonetse hakoreshejwe porogaramu ya ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Ubudage), kandi isesengura ry'imiterere ya mitochondrial ryakozwe hakoreshejwe porogaramu ya ImageJ (v1.54d) [30, 31] hakurikijwe ibipimo byasobanuwe mu buryo bw'inyongera bwa 3.
Uturemangingo twashyizwemo glutaraldehyde ya 2% muri 0.1 M phosphate buffer, hagakurikiraho gushingwamo umuti wa 1% osmium tetroxide (Sigma Aldrich, MO, USA), buhoro buhoro hagakurwamo acetone (Merck, Darmstadt, Ubudage), hanyuma bishyirwamo epoxy resin. Ibice bito cyane byarateguwe kandi bisiga irangi rya 1% uranyl acetate (Merck, Darmstadt, Ubudage) na 1% citrate y'icyitegererezo (Merck, Darmstadt, Ubudage). Amashusho y'imiterere yabonetse hakoreshejwe mikorosikopi ya JEM 2100F EXII transmission electron (JEOL Ltd, Tokyo, Ubudage) ku muvuduko wihuta wa 80 kV.
Imiterere y'uturemangingo twa LX-2 twavuwe na IPA mu gihe cy'amasaha 24 yasesenguwe hakoreshejwe mikorosikopi ya phase-contrast kuri magnification ya 50x hakoreshejwe mikorosikopi y'urumuri rwa Zeiss inverted (Zeiss Axio Vert.A1 na AxioCam MRm, Jena, Ubudage).
Amakuru y’ubuvuzi yagaragajwe nk’ikigereranyo ± kunyuranya gusanzwe cyangwa hagati (interquartile range: IQR). Isesengura ry’uburyo bumwe bw’impinduka (continuous variables) cyangwa ikizamini cya χ² (categorical variables) ryakoreshejwe mu kugereranya itandukaniro riri hagati y’amatsinda atatu y’ubushakashatsi. Igipimo cy’ubwiza bw’ikinyoma (FDR) cyakoreshejwe mu gukosora ibizamini byinshi, naho gene zifite FDR <0.05 zafashwe nk’izifite agaciro mu mibare. Isesengura rya Spearman ry’imibanire ryakoreshejwe mu guhuza methylation ya CpG ADN n’imbaraga z’ibimenyetso bya IPA, hamwe n’agaciro k’amazina ya p (p <0.05) kagaragajwe.
Isesengura ry’inzira ryakozwe hakoreshejwe igikoresho cyo gusesengura imiterere y’uturemangingo (WebGestalt) gishingiye ku rubuga (transcripts 268) (nominal p < 0.01), transcripts 119 zijyanye na mitochondria (nominal p < 0.05), na CpGs 4350 muri transcripts 3093 z’umwijima zari zifitanye isano n’urugero rwa IPA mu maraso. Igikoresho cya Venny DB (version 2.1.0) cyakoreshejwe ku buntu mu gushaka imiterere y’uturemangingo, naho StringDB (version 11.5) ikoreshwa mu kwerekana imikoranire ya poroteyine na poroteyine.
Ku igerageza rya LX-2, ingero zapimwe kugira ngo zirebe ko zisanzwe hakoreshejwe ikizamini cya D'Agostino-Pearson. Amakuru yabonetse muri kopi eshatu z’ibinyabuzima nibura maze akorerwa ANOVA y’inzira imwe hamwe na Bonferroni post hoc test. Agaciro ka p kari munsi ya 0.05 kafatwaga nk'akamaro mu mibare. Amakuru agaragazwa nk'ikigereranyo ± SD, kandi umubare w'ibizamini ugaragazwa muri buri gishushanyo. Isesengura ryose n'imbonerahamwe byakozwe hakoreshejwe porogaramu ya GraphPad Prism 8 statistical ya Windows (GraphPad Software Inc., verisiyo 8.4.3, San Diego, USA).
Ubwa mbere, twakoze ubushakashatsi ku isano iri hagati y’urugero rwa IPA mu maraso n’inyandiko z’umwijima, umubiri wose, n’iz’imitochondrial. Mu nyandiko zose, gene ikomeye cyane ifitanye isano n’urugero rwa IPA mu maraso ni MAPKAPK3 (FDR = 0.0077; protein kinase-activated protein kinase-activated protein 3); mu nyandiko zifitanye isano na mitochondria, gene ikomeye cyane ifitanye isano ni AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Dosiye y’inyongera 1 na Dosiye y’inyongera 2).
Hanyuma twasesenguye inyandiko z’isi yose (n = 268; p < 0.01) n’inyandiko zijyanye na mitochondria (n = 119; p < 0.05), amaherezo tumenya ko apoptosis ari yo nzira ikomeye cyane (p = 0.0089). Ku nyandiko za mitochondria zijyanye n’urugero rwa IPA mu maraso, twibanze kuri apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), n’inzira z’ibimenyetso bya TNF (FDR = 0.000006) (Ishusho ya 1A, Imbonerahamwe ya 2, n’Ishusho z’inyongera 1A-B).
Isesengura ry’inyandiko zijyanye na mitochondria ku isi, na methylation ya ADN mu mwijima w’umuntu bifitanye isano n’urugero rwa IPA mu maraso. A ihagarariye inyandiko 268 ku isi, inyandiko 119 zijyanye na mitochondria, n’inyandiko za methyl za ADN zishushanyije ku duce 3092 twa CpG dufitanye isano n’urugero rwa IPA mu maraso (agaciro ka p < 0.01 ku nyandiko zijyanye na ADN na methyl ya ADN, n’agaciro ka p < 0.05 ku nyandiko zijyanye na mitochondria). Inyandiko zikomeye zifatanye zigaragara hagati (AKT1 na YKT6). B Ikarita y’imikoranire ya genes 13 zifite amanota menshi yo gukorana (0.900) n’izindi genes yakozwe mu magene 56 afatanye (akarere k’umurongo w’umukara) yahujwe cyane n’urugero rwa IPA mu maraso hakoreshejwe igikoresho cyo kuri interineti StringDB. Icyatsi kibisi: Genes zishushanyije ku gice cy’uturemangingo cya Gene Ontology (GO): mitochondria (GO: 0005739). AKT1 ni poroteyine ifite amanota menshi (0.900) mu mikoranire n'izindi poroteyine hashingiwe ku makuru (hashingiwe ku gusesengura inyandiko, igerageza, ububiko bw'amakuru, no guhuza). Uduce tw'urusobe rw'amakuru tugaragaza poroteyine, naho impande zigaragaza isano iri hagati ya poroteyine.
Kubera ko metabolite za microbiota mu mara zishobora kugenzura imiterere ya epigenetic binyuze muri methylation ya ADN [32], twakoze ubushakashatsi ku birebana n'uko urugero rwa IPA mu mara rufitanye isano na methylation ya ADN mu mwijima. Twasanze ahantu habiri hanini ha methylation ifitanye isano n'urugero rwa IPA mu mara hari hafi y'agace ka 3 gakungahaye kuri proline-serine (C19orf55) n'umuryango wa poroteyine ya B (nto) mu gihe cy'ubushyuhe (HSPB6) (Inyandiko y'inyongera ya 3). methylation ya ADN ya 4350 CpG (p < 0.01) yahujwe n'urugero rwa IPA mu mara kandi yakungahajwe n'inzira zo kugenzura igihe kirekire (p = 0.006) (Ishusho ya 1A, Imbonerahamwe ya 2, n'Ishusho y'inyongera ya 1C).
Kugira ngo dusobanukirwe uburyo bw’ibinyabuzima bushingiye ku isano iri hagati y’urugero rwa IPA mu maraso, inyandiko z’isi yose, inyandiko zifitanye isano na mitochondria, na methylation ya ADN mu mwijima w’umuntu, twakoze isesengura ry’imiterere y’uturemangingo twagaragaye mu isesengura ry’inzira ryabanje (Ishusho ya 1A). Ibisubizo by’isesengura ry’uburyo bwo kongera uburyo bw’uturemangingo 56 duhurirana (muri umurongo w’umukara mu Ishusho ya 1A) byagaragaje ko inzira ya apoptosis (p = 0.00029) yagaragaje uturemangingo tubiri duhuriyeho mu isesengura ritatu: AKT1 na YKT6 (YKT6 v-SNARE homolog), nk’uko bigaragara mu ishusho ya Venn (Ishusho y’inyongera ya 2 n’Ishusho ya 1A). Igishimishije ni uko twasanze AKT1 (cg19831386) na YKT6 (cg24161647) bifitanye isano ryiza n’urugero rwa IPA mu maraso (Idosiye y’inyongera ya 3). Kugira ngo tumenye imikoranire ya poroteyine hagati y’ibikomoka kuri gene, twahisemo gene 13 zifite amanota menshi y’akarere (0.900) muri gene 56 zihurirana nk’inyongera maze twubaka ikarita y’imikoranire. Dukurikije urwego rw'icyizere (icyizere cyo ku rwego rwo hejuru), gene ya AKT1 ifite amanota menshi (0.900) yari ku isonga (Ishusho ya 1B).
Dukurikije isesengura ry’inzira, twasanze apoptosis ari yo nzira nyamukuru, bityo twakoze ubushakashatsi ku bijyanye no kuvura IPA byagira ingaruka ku kuvura HSCs muri vitro. Mbere twagaragaje ko dose zitandukanye za IPA (10 μM, 100 μM, na 1 mM) zitari uburozi ku turemangingo twa LX-2 [15]. Ubu bushakashatsi bwagaragaje ko kuvura IPA kuri 10 μM na 100 μM byongera umubare w’uturemangingo dushobora kubaho kandi dufite necrotic. Ariko, ugereranije n’itsinda ry’abagenzura, ubushobozi bwo kubaho bw’uturemangingo bwagabanutse kuri 1 mM IPA, mu gihe igipimo cya necrosis y’uturemangingo kitahindutse (Ishusho ya 2A, B). Hanyuma, kugira ngo tubone igipimo cyiza cyo gutera apoptosis mu turemangingo twa LX-2, twapimye 10 μM, 100 μM, na 1 mM IPA mu gihe cy’amasaha 24 (Ishusho ya 2A-E n’Ishusho y’inyongera 3A-B). Igishimishije ni uko IPA 10 μM na 100 μM byagabanyije igipimo cya apoptosis (%), icyakora, IPA 1 mM yongereye igipimo cya apoptosis ya nyuma n'igipimo cya apoptosis (%) ugereranije n'uburyo bwo kuyigenzura, bityo ikaba yaratoranyijwe kugira ngo ikorerwe andi magerageza (Ishusho ya 2A–D).
IPA itera apoptosis y'uturemangingo twa LX-2. Uburyo bwa Annexin V na 7-AAD bwo gusiga irangi inshuro ebyiri bwakoreshejwe kugira ngo harebwe igipimo cya apoptotic n'imiterere y'uturemangingo hakoreshejwe flow cytometry. Uturemangingo twa BA twashyizwemo 10 μM, 100 μM na 1 mM IPA mu gihe cy'amasaha 24 cyangwa hamwe na F–H TGF-β1 (5 ng/ml) na 1 mM IPA mu buryo budafite serum mu gihe cy'amasaha 24. A: uturemangingo tuzima (Annexin V -/ 7AAD-); B: uturemangingo twa necrotic (Annexin V -/ 7AAD+); C, F: kare (Annexin V +/ 7AAD-); D, G: nyuma (Annexin V+/7AAD+.); E, H: ijanisha ry'uturemangingo twose twa apoptotic twa mbere n'igihe cya nyuma mu gipimo cya apoptotic (%). Amakuru agaragazwa nk'ikigereranyo ± SD, n = igerageza 3 ryigenga. Igereranya ry'imibare ryakozwe hakoreshejwe uburyo bumwe bwa ANOVA na Bonferroni post hoc test. *p < 0.05; ****p < 0.0001
Nkuko twabigaragaje mbere, 5 ng/ml TGF-β1 ishobora gutuma HSC ikora binyuze mu kongera uburyo gene za kera zikora [15]. Uturemangingo twa LX-2 twavuwe na 5 ng/ml TGF-β1 na 1 mM IPA hamwe (Ishusho ya 2E–H). Ubuvuzi bwa TGF-β1 ntibwahinduye igipimo cya apoptosis, icyakora, IPA hamwe na IPA byongera igipimo cya apoptosis cyatinze na apoptosis (%) ugereranije n'ubuvuzi bwa TGF-β1 (Ishusho ya 2E–H). Ibi bisubizo bigaragaza ko 1 mM IPA ishobora guteza apoptosis mu turemangingo twa LX-2 hatitawe ku gukurura TGF-β1.
Twakomeje gukora ubushakashatsi ku ngaruka za IPA ku guhumeka kwa mitochondrial mu turemangingo twa LX-2. Ibisubizo byagaragaje ko 1 mM IPA yagabanije ibipimo byo gukoresha ogisijeni (OCR): guhumeka bitari mitochondrial, guhumeka kw'ibanze n'ukw'ikirenga, kuvunika kwa protoni no gukora ATP ugereranije n'itsinda ry'abagenzura (Ishusho ya 3A, B), mu gihe igipimo cy'ubuzima bw'umubiri (BHI) kitahindutse.
IPA igabanya guhumeka kwa mitochondrial mu turemangingo twa LX-2. Umuyoboro w'ubuhumekero wa mitochondrial (OCR) ugaragazwa nk'ibipimo by'ubuhumekero bwa mitochondrial (ubuhumekero butari bwa mitochondrial, ubuhumekero bw'ibanze, ubuhumekero bwa maximal, kuvuja kwa proton, gusohora ATP, SRC na BHI). Uturemangingo A na B twashyizwemo 10 μM, 100 μM na 1 mM IPA mu gihe cy'amasaha 24, uko bikurikirana. Uturemangingo C na D twashyizwemo TGF-β1 (5 ng/ml) na 1 mM IPA mu buryo budafite serum mu gihe cy'amasaha 24, uko bikurikirana. Ibipimo byose byagenwe ku gipimo cya ADN hakoreshejwe CyQuant kit. BHI: igipimo cy'ubuzima bw'umubiri; SRC: ubushobozi bwo guhumeka; OCR: igipimo cyo gukoresha ogisijeni. Amakuru agaragazwa nk'ikigereranyo ± kunyuranaho gusanzwe (SD), n = igerageza 5 ryigenga. Igereranya ry'imibare ryakozwe hakoreshejwe ikizamini cya ANOVA na Bonferroni post hoc. *p < 0.05; **p < 0.01; na ***p < 0.001
Kugira ngo turusheho gusobanukirwa neza ingaruka za IPA ku miterere y’uturemangingo twa LX-2 twa TGF-β1, twasesenguye phosphorylation ya oxidative ya mitochondrial hakoreshejwe OCR (Ishusho ya 3C, D). Ibisubizo byagaragaje ko uburyo bwo kuvura TGF-β1 bushobora kugabanya ubushobozi bwo guhumeka, ubushobozi bwo guhumeka (SRC) na BHI ugereranije n’itsinda rigenzura (Ishusho ya 3C, D). Byongeye kandi, uburyo bwo kuvura buvanze bwagabanyije uburyo bwo guhumeka bw’ibanze, kuvunika kwa protoni no gukora ATP, ariko SRC na BHI byari hejuru cyane ugereranyije n’ibyavuwe na TGF-β1 (Ishusho ya 3C, D).
Twanakoze "Ikizamini cya Cellular Energy Phenotype Test" cyatanzwe na porogaramu ya Seahorse (Ishusho y'inyongera 4A–D). Nkuko bigaragara ku Ishusho y'inyongera 3B, ubushobozi bwa OCR na ECAR bwo kuvura bwaragabanutse nyuma yo kuvurwa na TGF-β1, ariko nta tandukaniro ryagaragaye mu matsinda yo kuvurwa na IPA ugereranije n'itsinda ryo kugenzura. Byongeye kandi, urugero rwa OCR rwo hasi n'urw'ihungabana rwagabanutse nyuma yo kuvurwa na IPA ugereranije n'itsinda ryo kugenzura (Ishusho y'inyongera 4C). Igishimishije ni uko hari imiterere isa n'iyo yagaragaye mu kuvura bivanze, aho nta mpinduka mu rugero rwa ECAR rwo hasi n'urw'ihungabana rwagaragaye ugereranije n'ubuvuzi bwa TGF-β1 (Ishusho y'inyongera 4C). Muri HSCs, kugabanuka kwa phosphorylation ya oxidative ya mitochondrial hamwe n'ubushobozi bwo kuvura bivanze mu gusubiza SCR na BHI nyuma yo kuvurwa na TGF-β1 ntabwo byahinduye ubushobozi bwa metabolic (OCR na ECAR). Ibyo bisubizo byose hamwe bigaragaza ko IPA ishobora kugabanya ingufu zikomoka ku binyabuzima muri HSC, bigaragaza ko IPA ishobora gutera imbaraga nke zihindura imiterere ya HSC ikagera ku kudakora (Ishusho y'inyongera ya 4D).
Ingaruka za IPA ku mihindagurikire ya mitochondrial zasuzumwe hakoreshejwe uburyo butatu bwo gupima imiterere ya mitochondrial n'imiyoboro y'itumanaho ndetse no gusiga irangi rya MTR (Ishusho ya 4 n'Ishusho y'inyongera ya 5). Ishusho ya 4 igaragaza ko, ugereranije n'itsinda ry'abagenzura, uburyo bwo kuvura TGF-β1 bwagabanije ubuso bw'impuzandengo, umubare w'ishami, uburebure bw'ishami ryose, n'umubare w'ihuriro ry'amashami (Ishusho ya 4A na B) kandi bwahinduye igipimo cya mitochondrial kuva ku mpande kugeza ku mpande (Ishusho ya 4C). Uburyo bwo kuvura IPA gusa ni bwo bwagabanije ingano ya mitochondrial kandi bwahinduye igipimo cya mitochondrial kuva ku mpande kugeza ku mpande ugereranije n'itsinda ry'abagenzura (Ishusho ya 4A). Mu buryo bunyuranye, imiterere y'ubugari, uburebure bw'ishami, n'imikorere ya mitochondrial yapimwe na MTR ishingiye ku ruhu rw'imitochondrial (Ishusho ya 4A na E) ntibyahindutse kandi ibi bipimo ntibyatandukanye hagati y'amatsinda. Ibyo bisubizo bigaragaza ko uburyo bwo kuvura TGF-β1 na IPA busa n'aho buhindura imiterere n'ingano ya mitochondrial ndetse n'ubukana bw'urusobe rw'amatsinda mu turemangingo tuzima twa LX-2.
IPA ihindura imiterere ya mitochondrial na ADN ya mitochondrial mu turemangingo twa LX-2. A. Amashusho y’inyuma y’uturemangingo tuzima twa LX-2 twashyizwemo TGF-β1 (5 ng/ml) na 1 mM IPA mu gihe cy’amasaha 24 mu buryo budafite serum yerekana imiyoboro ya mitochondrial yasizwe na Mitotracker™ Red CMXRos na nuclei yasizwe ubururu na DAPI. Amakuru yose yari afite nibura amashusho 15 kuri buri tsinda. Twabonye amashusho 10 ya Z-stack kuri buri bwoko bw’icyitegererezo. Buri gice cya Z-axis cyari gifite ibice 30, buri kimwe gifite ubunini bwa 9.86 μm. Umugozi w’ingero: 10 μm. B. Ibintu by’inyuma (mitochondria gusa) byagaragajwe hakoreshejwe uburyo bwo guhuza ishusho. Isesengura ry’ingano n’igereranya ry’imiyoboro ya mitochondrial byakozwe ku turemangingo twose muri buri tsinda. C. Inshuro ikigereranyo cy’imiterere ya mitochondrial. Agaciro kari hafi ya 0 kerekana imiterere y’uruziga, naho agaciro kari hafi ya 1 kerekana imiterere y’urukiramende. D Ibikubiye muri ADN ya Mitochondrial (mtDNA) byagenwe nk'uko byasobanuwe muri Ibikoresho n'Uburyo. Isesengura rya E Mitotracker™ Red CMXRos ryakozwe hakoreshejwe flow cytometry (ibintu 30.000) nk'uko byasobanuwe muri Ibikoresho n'Uburyo. Amakuru agaragazwa nk'ikigereranyo ± SD, n = igerageza 3 ryigenga. Igereranya ry'imibare ryakozwe hakoreshejwe ikizamini cya ANOVA na Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Hanyuma twasesenguye ingano ya mtDNA mu turemangingo twa LX-2 nk'ikimenyetso cy'umubare wa mitochondrial. Ugereranije n'itsinda ry'abagenzuye, ingano ya mtDNA yiyongereye mu itsinda ryavuwe na TGF-β1 (Ishusho ya 4D). Ugereranyije n'itsinda ryavuwe na TGF-β1, ingano ya mtDNA yagabanutse mu itsinda ry'abavuwe na combination (Ishusho ya 4D), bigaragaza ko IPA ishobora kugabanya ingano ya mtDNA ndetse wenda n'umubare wa mitochondrial ndetse n'ubuhumekero bwa mitochondrial (Ishusho ya 3C). Byongeye kandi, IPA yasaga nkaho igabanya ingano ya mtDNA mu buvuzi buvanze ariko ntabwo yagize ingaruka ku mikorere ya mitochondrial iterwa na MTR (Ishusho ya 4A–C).
Twakoze ubushakashatsi ku isano iri hagati ya IPA n’urugero rwa mRNA rw’uturemangingo dufitanye isano na fibrosis, apoptosis, kubaho, na mitochondrial dynamics mu turemangingo twa LX-2 (Ishusho ya 5A–D). Ugereranije n’itsinda ry’abagenzuye, itsinda ryavuwe na TGF-β1 ryagaragaje kwiyongera k’imikorere ya jeni nka collagen type I α2 chain (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), tissue inhibitor ya metalloproteinase 1 (TIMP1), na dynamin 1-like gene (DRP1), bigaragaza kwiyongera kwa fibrosis no gukora. Byongeye kandi, ugereranije n’itsinda ry’abagenzuye, ubuvuzi bwa TGF-β1 bwagabanyije urugero rwa mRNA rwa nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, inhibitor ya B-cell α, enhancer ya nuclear factor κ gene light peptide (NFκB1A), na inhibitor ya nuclear factor κB kinase subunit β (IKBKB) (Ishusho ya 5A–D). Ugereranyije n'ubuvuzi bwa TGF-β1, uburyo bwo kuvura buvanze na TGF-β1 na IPA bwagabanije igaragara rya COL1A2 na MMP2, ariko byongera urugero rwa mRNA rwa PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, na IKBKB. Uburyo bwo kuvura bwa IPA bwagabanije cyane igaragara rya MMP2, Bcl-2-affiliated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), na mitochondrial fusion protein 2 (MFN2), mu gihe uburyo bwa CASP8, NFκB1A, NFκB1B, na IKBKB bwongerewe ugereranije n'itsinda ry'abagenzura. Ariko, nta tandukaniro ryabonetse mu igaragara rya caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), na fission protein 1 (FIS1). Muri rusange, ibi byavuye mu bushakashatsi bigaragaza ko uburyo bwo kuvura bwa IPA buhindura uburyo imiterere y’uturemangingo dufitanye isano na fibrosis, apoptosis, kubaho, na mitochondrial igaragara. Amakuru yacu agaragaza ko uburyo bwo kuvura bwa IPA bugabanya fibrosis mu turemangingo twa LX-2; icyarimwe, butuma habaho kubaho binyuze mu guhindura uburyo bwo kuvura bugana ku kudakora.
IPA ihindura uburyo imiterere ya fibroblast, apoptotike, viability, na mitochondrial dynamics genes mu turemangingo twa LX-2 igaragara. Histograms zigaragaza imiterere ya mRNA ugereranije n'uburyo endogenous control (RPLP0 cyangwa PPIA) nyuma y'uko uturemangingo twa LX-2 dukoreshejwe TGF-β1 na IPA mu buryo budafite serum mu gihe cy'amasaha 24. A igaragaza fibroblasts, B igaragaza uturemangingo twa apoptotike, C igaragaza uturemangingo tukiriho, naho D igaragaza imiterere ya gene ya mitochondrial dynamics. Amakuru agaragazwa nk'ikigereranyo ± deviation standard (SD), n = igerageza 3 ryigenga. Igereranya ry'imibare ryakozwe hakoreshejwe ikizamini cya ANOVA na Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Hanyuma, impinduka mu bunini bw'uturemangingo (FSC-H) na complexity ya cytoplasmic (SSC-H) byapimwe hakoreshejwe flow cytometry (Ishusho ya 6A, B), kandi impinduka mu miterere y'uturemangingo nyuma yo kuvurwa na IPA zapimwe hakoreshejwe microscopy ya transmission electron (TEM) na phase contrast microscopy (Ishusho y'inyongera ya 6A-B). Nkuko byari byitezwe, uturemangingo two mu itsinda ryavuwe na TGF-β1 twiyongereye mu bunini ugereranije n'itsinda rigenzura (Ishusho ya 6A, B), bigaragaza kwaguka kwa kera kwa rough endoplasmic reticulum (ER*) na phagolysosomes (P), bigaragaza gukora kwa hematopoietic stem cell (HSC) (Ishusho y'inyongera ya 6A). Ariko, ugereranije n'itsinda ryavuwe na TGF-β1, ingano y'uturemangingo, complexity ya cytoplasmic (Ishusho ya 6A, B), n'ingano ya ER* byagabanutse mu itsinda ryavuwe na TGF-β1 na IPA (Ishusho y'inyongera ya 6A). Byongeye kandi, uburyo bwo kuvura bwa IPA bwagabanije ingano y'uturemangingo, imiterere ya cytoplasm (Ishusho ya 6A, B), P na ER* (Ishusho y'inyongera ya 6A) ugereranije n'itsinda rigenzura. Byongeye kandi, ingano y'uturemangingo twa apoptotic yiyongereye nyuma y'amasaha 24 yo kuvurwa na IPA ugereranije n'itsinda rigenzura (imyambi yera, Ishusho y'inyongera ya 6B). Muri rusange, ibi bisubizo bigaragaza ko 1 mM IPA ishobora gutera HSC apoptosis no gusubiza inyuma impinduka mu miterere y'uturemangingo ziterwa na TGF-β1, bityo ikagenzura ingano n'imiterere y'uturemangingo, bishobora kuba bifitanye isano no kudakora kwa HSC.
IPA ihindura ingano y'uturemangingo n'ubuso bw'uturemangingo mu turemangingo twa LX-2. Amashusho agaragaza isesengura rya cytometry y'imiyoboro y'amaraso. Isesengura ryakoresheje ingamba zihariye zo gupima uturemangingo twa LX-2: SSC-A/FSC-A kugira ngo hamenyekane umubare w'uturemangingo, FSC-H/FSC-A kugira ngo hamenyekane doublets, na SSC-H/FSC-H kugira ngo hamenyekane ingano y'uturemangingo n'ubuso bw'uturemangingo. Uturemangingo twashyizwemo TGF-β1 (5 ng/ml) na 1 mM IPA mu buryo budafite serum mu gihe cy'amasaha 24. Uturemangingo twa LX-2 twagabanyijwe mu gice cyo hasi cy'ibumoso (SSC-H-/FSC-H-), igice cyo hejuru cy'ibumoso (SSC-H+/FSC-H-), igice cyo hasi cy'iburyo (SSC-H-/FSC-H+), n'igice cyo hejuru cy'iburyo (SSC-H+/FSC-H+) kugira ngo hamenyekane ingano y'uturemangingo n'ubuso bw'uturemangingo. B. Imiterere y'uturemangingo yasesenguwe hakoreshejwe uburyo bwa "flow cytometry" hakoreshejwe FSC-H (foward scatter, cell size) na SSC-H (side scatter, cytoplasmic complexity) (ibintu 30.000). Amakuru agaragazwa nk'ikigereranyo ± SD, n = igerageza 3 ryigenga. Igereranya ry'imibare ryakozwe hakoreshejwe ikizamini cya "one-way ANOVA" na "Bonferroni post hoc test". *p < 0.05; **p < 0.01; ***p < 0.001 na ****p < 0.0001
Ibyuma bihindura imiterere y’amara nka IPA byabaye ingingo ikomeye mu bushakashatsi, bigaragaza ko intego nshya zishobora kuvumburwa muri mikorobe y’amara. Niyo mpamvu bishimishije kuba IPA, ikinyabuzima twahuje na fibrosis y’umwijima mu bantu [15], byagaragaye ko ari ikintu gishobora kurwanya fibrosis mu nyamaswa [13, 14]. Aha, tugaragaje bwa mbere isano iri hagati ya IPA yo mu mara n’iy’umwijima ku isi hamwe na methylation ya ADN ku bantu babyibushye cyane badafite diyabete yo mu bwoko bwa 2 (T2D), bigaragaza apoptosis, mitophagy no kuramba, ndetse n’ingirabuzimafatizo ishobora kuba AKT1 igenga homeostasis y’umwijima. Ikindi kintu gishya mu bushakashatsi bwacu ni uko twerekanye imikoranire y’ubuvuzi bwa IPA na apoptosis, imiterere y’uturemangingo, bioenergetics ya mitochondrial na dynamics mu turemangingo twa LX-2, bigaragaza imbaraga nke zihindura imiterere ya HSC igana ku kudakora, bigatuma IPA iba umukandida ushobora kunoza fibrosis y’umwijima.
Twasanze apoptosis, mitophagy no kuramba ari byo nzira z'ingenzi cyane zikungahaye ku ngirabuzimafatizo z'umwijima zijyanye na IPA yo mu maraso izenguruka. Guhungabana kwa sisitemu yo kugenzura ubuziranenge bwa mitochondrial (MQC) bishobora gutera imikorere mibi ya mitochondrial, mitophagy na apoptosis, bityo bigatera imbere kugaragara kwa MASLD [33, 34]. Kubwibyo, dushobora gukeka ko IPA ishobora kugira uruhare mu kubungabunga imikorere y'uturemangingo n'ubusugire bwa mitochondrial binyuze mu apoptosis, mitophagy no kuramba mu mwijima. Amakuru yacu yagaragaje ko ngirabuzimafatizo ebyiri zari zisanzwe muri ibi bipimo bitatu: YKT6 na AKT1. Ni ngombwa kumenya ko YKT6 ari poroteyine ya SNARE igira uruhare mu guhuza uturemangingo. Igira uruhare mu guhuza uturemanfatizo na mitophagy binyuze mu gukora complex yo gutangira hamwe na STX17 na SNAP29 kuri autophagosome, bityo bigateza imbere guhuza uturemanfatizo na lysosomes [35]. Byongeye kandi, kubura imikorere ya YKT6 bitera kugabanuka kwa mitophagy [36], mu gihe kwiyongera kwa YKT6 bifitanye isano no kwiyongera kwa kanseri y'umwijima (HCC), bigaragaza ko ubuzima bw'uturemangingo bwiyongera [37]. Ku rundi ruhande, AKT1 ni gene ikomeye cyane ikorana kandi igira uruhare runini mu ndwara z'umwijima, harimo inzira y'ibimenyetso ya PI3K/AKT, imikorere y'uturemangingo, kwimuka kw'uturemangingo, kwiyongera, gufatana kw'ingingo, imikorere ya mitochondrial, no gusohora kolajeni [38-40]. Inzira y'ibimenyetso ya PI3K/AKT ishobora gukora uturemangingo tw'amaraso (HSCs), ari two turemangingo dufasha gukora matrix ya extracellular matrix (ECM), kandi kudakora kwayo bishobora kugira uruhare mu kubaho no kwiyongera kwa fibrosis y'umwijima [40]. Byongeye kandi, AKT ni imwe mu ngingo z'ingenzi zituma uturemangingo tudashobora kubaho zigabanya p53-dependent cell apoptosis, kandi gukora kwa AKT bishobora kuba bifitanye isano no gukumira apoptosis y'uturemangingo tw'umwijima [41, 42]. Ibisubizo byabonetse bigaragaza ko IPA ishobora kugira uruhare mu kugabanuka kw'uturemangingo tw'umwijima bitewe n'uko umwijima uhagaze, bigatuma habaho ingaruka ku ifatwa ry'uturemangingo tw'umwijima hagati yo kwinjira mu kugabanuka kw'uturemangingo cyangwa kubaho kwatwo. Izi ngaruka zishobora kugenzurwa na AKT na/cyangwa YKT6, ari byo by'ingenzi mu gutuma umwijima ukomeza gukora neza.
Ibisubizo byacu byagaragaje ko 1 mM IPA yateje apoptosis kandi ikagabanya guhumeka kwa mitochondrial mu turemangingo twa LX-2 hatitawe ku buvuzi bwa TGF-β1. Ni ngombwa kumenya ko apoptosis ari inzira ikomeye yo gukemura fibrosis no gukora kwa hematopoietic stem cell (HSC), kandi ni ikintu cy'ingenzi mu gusubiza imikorere ya fibrosis y'umwijima [4, 43]. Byongeye kandi, kugarura BHI mu turemangingo twa LX-2 nyuma yo kuvurwa hamwe byatanze ubumenyi bushya ku ruhare rwa IPA mu kugenzura bioenergetics za mitochondrial. Mu bihe byo kuruhuka no kudakora, uturemangingo twa hematopoietic dusanzwe dukoresha phosphorylation ya mitochondrial oxidative kugira ngo dukore ATP kandi tugire imikorere mike ya metabolic. Ku rundi ruhande, gukora kwa HSC byongera guhumeka kwa mitochondrial na biosynthesis kugira ngo bibe bihuye n'ingufu zisabwa mu kwinjira muri glycolytic state [44]. Kuba IPA itaragize ingaruka ku bushobozi bwa metabolic na ECAR bigaragaza ko inzira ya glycolytic idashyirwa imbere cyane. Mu buryo nk'ubwo, ubundi bushakashatsi bwagaragaje ko 1 mM IPA yashoboye guhindura imikorere y'uruhererekane rw'ubuhumekero rwa mitochondrial mu miyoboro y'umutima, umurongo w'uturemangingo tw'amaraso y'umuntu (Huh7) n'uturemangingo tw'imitsi y'amaraso y'umuntu (HUVEC); Ariko, nta ngaruka za IPA zabonetse kuri glycolysis mu miyoboro y'umutima, bigaragaza ko IPA ishobora kugira ingaruka ku binyabuzima by'izindi selile [45]. Kubwibyo, twibwira ko 1 mM IPA ishobora gukora nk'ikintu cyoroshye kigabanya ubuhumekero, kuko ishobora kugabanya cyane imiterere ya fibrogenic gene, imiterere y'uturemangingo na miyochondrial bioenergetics idahinduye ingano ya mtDNA [46]. Uncouplers za Mitochondrial zishobora kubuza culture-induced fibrosis na HSC activation [47] no kugabanya umusaruro wa ATP ya mitochondrial igenzurwa cyangwa iterwa na poroteyine zimwe na zimwe nka poroteyine zo gukuraho (UCP) cyangwa adenine nucleotide translocase (ANT). Bitewe n'ubwoko bw'uturemangingo, iki kintu gishobora kurinda uturemangingo apoptosis cyangwa/cyangwa guteza imbere apoptosis [46]. Ariko, hakenewe ubundi bushakashatsi kugira ngo hamenyekane uruhare rwa IPA nk'ikintu kigabanya utwo dukoko mu gutuma uturemangingo tw'amaraso tudakora neza.
Hanyuma twakoze ubushakashatsi niba impinduka mu guhumeka kwa mitochondrial zigaragarira mu miterere ya mitochondrial mu turemangingo twa LX-2 tuzima. Igishimishije ni uko uburyo bwo kuvura TGF-β1 buhindura igipimo cya mitochondrial kuva ku gice cy’umurambararo kugera ku gice cy’umurambararo, hamwe no kugabanuka kw’amashami ya mitochondrial no kwiyongera k’imikorere ya DRP1, ikintu cy’ingenzi mu gucikamo ibice bya mitochondrial [48]. Byongeye kandi, gucikamo ibice bya mitochondrial bifitanye isano n’uburyo urusobe rw’imiyoboro muri rusange ruhagaze, kandi kuva ku guhuza no gucikamo ibice ni ingenzi cyane mu gukora kwa hematopoietic stem cell (HSC), mu gihe kubuza gucikamo ibice bya mitochondrial bitera HSC apoptosis [49]. Bityo, ibisubizo byacu bigaragaza ko uburyo bwo kuvura TGF-β1 bushobora gutuma urusobe rw’imiyoboro rwa mitochondrial rugabanuka bitewe n’uko amashami yagabanutse, ibi bikaba bikunze kugaragara cyane mu gucikamo ibice bya mitochondrial bifitanye isano n’uturemangingo twa hematopoietic stem cells (HSCs). Byongeye kandi, amakuru yacu yagaragaje ko IPA ishobora guhindura igipimo cya mitochondrial kuva ku gice cy’umurambararo kugera ku gice cy’umurambararo, bityo bigabanyiriza OPA1 na MFN2. Ubushakashatsi bwagaragaje ko kugabanya OPA1 bishobora gutuma ubushobozi bwa mitochondrial membrane bugabanuka kandi bigatera apoptosis y’uturemangingo [50]. MFN2 izwiho kuba ihuza mitochondrial fusion na apoptosis [51]. Ibisubizo byabonetse bigaragaza ko kwinjizwa kwa selile za LX-2 na TGF-β1 na/cyangwa IPA bisa nkaho bihindura imiterere n'ingano ya mitochondrial, ndetse n'imiterere y'imikorere n'ubukana bw'urusobe rw'amakuru.
Ibisubizo byacu bigaragaza ko uburyo bwo kuvura TGFβ-1 na IPA bushobora kugabanya ibipimo bya mtDNA n'imiterere y'uturemangingo binyuze mu kugenzura uburyo mRNA igaragaramo fibrosis, apoptosis n'uturemangingo dufitanye isano no kubaho mu turemangingo twirinda apoptosis. Mu by'ukuri, IPA yagabanyije urugero rwa mRNA rw'uturemangingo twa AKT1 n'uturemangingo tw'ingenzi twa fibrosis nka COL1A2 na MMP2, ariko yongera urugero rwa CASP8, rufitanye isano no apoptosis. Ibisubizo byacu byagaragaje ko nyuma yo kuvurwa na IPA, imiterere ya BAX yagabanutse kandi imiterere ya mRNA y'uturemangingo tw'umuryango wa TIMP1, BCL-2 na NF-κB yiyongera, bigaragaza ko IPA ishobora gukurura ibimenyetso byo kubaho mu turemangingo tw'amaraso (HSCs) twirinda apoptosis. Izi molekyuli zishobora gukora nk'ibimenyetso bishyigikira kubaho mu turemangingo tw'amaraso twifashishwa mu gukora, bishobora kujyana no kwiyongera kwa poroteyine zirwanya apoptosis (nka Bcl-2), kugabanuka kwa pro-apoptosis BAX, n'imikoranire ikomeye hagati ya TIMP na NF-κB [5, 7]. IPA ikora neza binyuze muri PXR, kandi twasanze uburyo bwo kuvura buvanze na TGF-β1 na IPA byongera urugero rw’imikorere ya PXR mRNA, bigaragaza ko HSC igabanya gukora. Gukoresha PXR mu buryo bwa "activated signaling" bizwiho kubuza HSC gukora haba mu mubiri no mu mubiri [52, 53]. Ibisubizo byacu bigaragaza ko IPA ishobora kugira uruhare mu gukuraho HSC zikora mu buryo bwa "activated HSCs" binyuze mu guteza imbere apoptosis, kugabanya fibrosis na mitochondrial metabolism, no kongera ibimenyetso byo kubaho, ari byo bikorwa bisanzwe bihindura "activated HSC phenotype" mu buryo bwa "inactivated". Ikindi gisobanuro gishoboka ku buryo IPA ishobora gukora n'uruhare rwayo muri apoptosis ni uko ikuraho mitochondria idakora neza cyane cyane binyuze muri mitophagy (inzira y'imbere) n'inzira y'itangazo rya TNF (Imbonerahamwe ya 1), ifitanye isano itaziguye n'inzira y'itangazo rya NF-κB (Ishusho y'inyongera ya 7). Igishimishije ni uko imiterere y’uturemangingo twinshi dufitanye isano na IPA ishobora gutuma habaho ibimenyetso bishyigikira apoptosis n’ibishyigikira ubuzima mu nzira ya apoptotic [54], bigaragaza ko IPA ishobora gutuma habaho inzira ya apoptotic cyangwa ubuzima binyuze mu gukorana n’utu turemangingo. Ariko, uburyo IPA itera apoptosis cyangwa ubuzima mu gihe cyo gukora HSC n’inzira zayo za tekiniki ntibirasobanuka neza.
IPA ni metabolite ikomoka kuri tryptophan ikomoka ku mirire ikoresheje microbiota y'igifu. Ubushakashatsi bwagaragaje ko ifite ubushobozi bwo kurwanya ububyimbirwe, antioxidant, na epigenetic mu mara. [55] Ubushakashatsi bwagaragaje ko IPA ishobora guhindura imikorere y'imyororokere y'amara no kugabanya stress ya oxidative, ishobora kugira uruhare mu ngaruka zayo za oxidative mu mubiri. [56] Mu by'ukuri, IPA ijyanwa mu ngingo zigomba kwitabwaho binyuze mu nzira z'amaraso, kandi kubera ko IPA isangiye imiterere y'ingufu nini na tryptophan, serotonin, na indole derivatives, IPA igira ingaruka ku mikorere ya metabolite igatuma habaho guhangana kwa metabolite. [52] IPA ishobora guhangana na metabolite ikomoka kuri tryptophan kugira ngo ifatanye n'aho enzymes cyangwa receptors zihurira, bishobora kubangamira inzira zisanzwe za metabolite. Ibi bigaragaza ko hakenewe ubushakashatsi bwimbitse ku bijyanye na pharmacokinetics yayo na pharmacodynamics kugira ngo isobanukirwe neza uburyo bwo kuvura. [57] Haracyari kureba niba ibi bishobora no kubaho mu turemangingo tw'amaraso (HSCs).
Twemera ko ubushakashatsi bwacu bufite imbogamizi zimwe na zimwe. Kugira ngo dusuzume by'umwihariko isano ifitanye isano na IPA, twakuyemo abarwayi ba diyabete yo mu bwoko bwa 2 (T2DM). Twemera ko ibi bigabanya ikoreshwa ryacu ku barwayi ba diyabete yo mu bwoko bwa 2 n'indwara z'umwijima zikomeye. Nubwo ingano ya IPA mu maraso y'umuntu ari hagati ya 1–10 μM [11, 20], ingano ya 1 mM IPA yatoranyijwe hashingiwe ku ngano iri hejuru cyane idahumanya [15] n'igipimo kinini cya apoptosis, nta tandukaniro riri mu kigero cy'uturemangingo twangiritse. Nubwo ingano ya IPA yakoreshejwe muri ubu bushakashatsi, nta mpungenge ihari ku bijyanye n'ingano ya IPA [52]. Nubwo ibisubizo byacu ari ingenzi, iherezo ryagutse rya IPA riracyari agace k'ubushakashatsi. Byongeye kandi, ibyavuye mu bushakashatsi bwacu ku isano iri hagati y'ingano ya IPA mu maraso n'imiti ya ADN mu maraso y'umwijima byabonetse atari mu turemangingo fatizo tw'amaraso (HSCs) gusa ahubwo byabonetse no mu turemangingo tw'umwijima. Twahisemo gukoresha uturemangingo twa LX-2 tw’abantu dukurikije ibyavuye mu bushakashatsi bwacu bwa transcriptome bwerekana ko IPA ifitanye isano no gukorana kwa hematopoietic stem cell (HSC) [15], kandi HSC ni zo turemangingo ingenzi zigira uruhare mu iterambere rya fibrosis y’umwijima. Umwijima ugizwe n’ubwoko bwinshi bw’uturemangingo, bityo izindi ngero z’uturemangingo nka sisitemu yo gukusanya uturemangingo twa hepatocyte-HSC hamwe no gukorana kwa caspase no gucikamo kwa ADN ndetse n’uburyo bw’imikorere harimo n’urwego rwa poroteyine bigomba kuzirikanwa kugira ngo higwe uruhare rwa IPA n’imikoranire yayo n’izindi turemangingo tw’umwijima.