Ngaphambilini sibike ukuthi amazinga e-serum e-tryptophan metabolite indole-3-propionic acid (IPA) etholakala emathunjini aphansi ezigulini ezine-fibrosis yesibindi. Kulolu cwaningo, siphenye i-transcriptome kanye ne-DNA methylome esibindini esikhuluphele maqondana namazinga e-IPA e-serum, kanye nendima ye-IPA ekubangeleni ukungasebenzi kwe-phenotypic kwamaseli e-hepatic stellate (HSCs) ku-vitro.
Lolu cwaningo luhlanganise iziguli eziyi-116 ezikhuluphele ezingenaso isifo sikashukela sohlobo lwesibili (T2DM) (ezineminyaka engu-46.8 ± 9.3; i-BMI: 42.7 ± 5.0 kg/m²) ezahlinzwa nge-bariatric e-Kuopio Bariatric Surgery Center (KOBS). Amazinga e-IPA ajikelezayo alinganiswa nge-liquid chromatography-mass spectrometry (LC-MS), ukuhlaziywa kwe-liver transcriptome kwenziwa nge-total RNA sequencing, kanti ukuhlaziywa kwe-DNA methylation kwenziwa kusetshenziswa i-Infinium HumanMethylation450 BeadChip. Amaseli e-hepatic stellate abantu (LX-2) asetshenziselwa ukuhlolwa kwe-in vitro.
Amazinga e-IPA e-serum ahlobene nokuvezwa kwezakhi zofuzo ezihilelekile ezindleleni ze-apoptotic, mitophagic, kanye ne-longitude esibindini. Izakhi zofuzo ze-AKT serine/threonine kinase 1 (AKT1) kwakuyizakhi zofuzo ezisebenzisana kakhulu nezinamandla kakhulu kuphrofayili yesibindi kanye ne-DNA methylation. Ukwelashwa kwe-IPA kwabangela i-apoptosis, ukuncipha kokuphefumula kwe-mitochondrial, kanye nokushintsha kwesimo seseli kanye ne-mitochondrial dynamics ngokushintsha ukuvezwa kwezakhi zofuzo ezaziwa ngokulawula i-fibrosis, i-apoptosis, kanye nokusinda kwamaseli e-LX-2.
Uma kubhekwa konke lokhu, lolu lwazi lusekela ukuthi i-IPA inemiphumela yokwelapha engaba khona futhi ingadala i-apoptosis futhi ishintshe i-phenotype ye-HSC iye esimweni esingasebenzi, ngaleyo ndlela yandisa amathuba okuvimbela i-fibrosis yesibindi ngokuphazamisa ukusebenza kwe-HSC kanye ne-metabolism ye-mitochondrial.
Ukusabalala kokukhuluphala kanye nesifo se-metabolic syndrome kuye kwahlotshaniswa nokwanda kwesifo sesibindi esinamafutha esihlotshaniswa ne-metabolically associated (MASLD); lesi sifo sithinta u-25% kuya ku-30% wabantu abaningi [1]. Umphumela oyinhloko we-MASLD etiology yi-fibrosis yesibindi, inqubo eguquguqukayo ebonakala ngokuqongelela okuqhubekayo kwe-fibrous extracellular matrix (ECM) [2]. Amaseli amakhulu ahilelekile ku-fibrosis yesibindi amaseli e-hepatic stellate (HSCs), abonisa izinhlobo ezine ezaziwayo: i-quiescent, i-activated, i-inactivated, kanye ne-senescent [3, 4]. Ama-HSC angasetshenziswa futhi ahlukaniswe kusuka efomini ye-quiescent abe amaseli afana ne-fibroblast akhula ngokushesha anezidingo zamandla aphezulu, ngokuvezwa okwandisiwe kwe-α-smooth muscle actin (α-SMA) kanye ne-type I collagen (Col-I) [5, 6]. Ngesikhathi sokuguqulwa kwe-fibrosis yesibindi, ama-HSC asebenzayo asuswa nge-apoptosis noma i-inactivation. Lezi zinqubo zifaka phakathi ukwehla kwezakhi zofuzo ze-fibrogenic kanye nokuguqulwa kwezakhi zofuzo ze-prosurvival (njengezindlela ze-NF-κB kanye ne-PI3K/Akt signaling) [7, 8], kanye nezinguquko ku-dynamics kanye nomsebenzi we-mitochondrial [9].
Amazinga e-serum ye-tryptophan metabolite indole-3-propionic acid (IPA), ekhiqizwa emathunjini, atholakale enciphile ezifweni ze-metabolic zabantu okuhlanganisa ne-MASLD [10-13]. I-IPA ihlotshaniswa nokudla i-fiber yokudla, yaziwa ngemiphumela yayo yokulwa ne-antioxidant kanye ne-anti-inflammatory, futhi inciphisa i-phenotype ye-steatohepatitis (NASH) engeyona eyotshwala ebangelwa ukudla emzimbeni kanye ne-in vitro [11-14]. Ubufakazi obuthile buvela ocwaningweni lwethu lwangaphambilini, olubonise ukuthi amazinga e-IPA e-serum ayephansi ezigulini ezine-fibrosis yesibindi kunasezigulini ezikhuluphele ezingenayo i-fibrosis yesibindi ku-Kuopio Bariatric Surgery Study (KOBS). Ngaphezu kwalokho, sibonise ukuthi ukwelashwa kwe-IPA kunganciphisa ukubonakaliswa kwezakhi zofuzo eziyizimpawu zakudala zokunamathela kweseli, ukufuduka kweseli kanye nokusebenza kwe-hematopoietic stem cell kumodeli ye-human hepatic stellate cell (LX-2) futhi iyi-metabolite engaba khona ye-hepatoprotective [15]. Kodwa-ke, akukacaci ukuthi i-IPA ibangela kanjani ukuhlehla kwe-fibrosis yesibindi ngokusebenzisa i-HSC apoptosis kanye ne-mitochondrial bioenergetics.
Lapha, sibonisa ukuthi i-serum IPA ihlotshaniswa nokuvezwa kwezakhi zofuzo ezicebile ku-apoptosis, i-mitophagy, kanye nezindlela zokuphila isikhathi eside esibindini sabantu abanesifo sikashukela esikhuluphele kodwa esingesona uhlobo lwesibili (i-KOBS). Ngaphezu kwalokho, sithole ukuthi i-IPA ingadala ukuqedwa kanye nokuwohloka kwamaseli e-hematopoietic stem cell (ama-HSC) asebenzayo ngendlela yokungasebenzi. Le miphumela yembula indima entsha ye-IPA, okwenza kube yinto engaba umgomo wokwelapha ukukhuthaza ukuhlehla kwe-fibrosis yesibindi.
Ucwaningo lwangaphambilini eqenjini le-KOBS lubonise ukuthi iziguli ezine-fibrosis yesibindi zazinamazinga aphansi e-IPA ajikelezayo uma kuqhathaniswa neziguli ezingenayo i-fibrosis yesibindi [15]. Ukuze sikhiphe umphumela odidayo ongaba khona wesifo sikashukela sohlobo 2, siqashe iziguli eziyi-116 ezikhuluphele ezingenaso isifo sikashukela sohlobo 2 (iminyaka ephakathi ± SD: 46.8 ± 9.3 iminyaka; BMI: 42.7 ± 5.0 kg/m2) (Ithebula 1) ocwaningweni lwe-KOBS oluqhubekayo njengabantu abacwaningwayo [16]. Bonke abahlanganyeli banikeze imvume ebhaliwe futhi inqubo yocwaningo yavunywa yiKomidi Lokuziphatha leSibhedlela saseNorth Savo County ngokuhambisana neSimemezelo saseHelsinki (54/2005, 104/2008 kanye no-27/2010).
Amasampula e-biopsy yesibindi atholakale ngesikhathi sokuhlinzwa kwe-bariatric futhi ahlolwa ngokwe-histological ngodokotela bezifo abanolwazi ngokwemigomo echazwe ngaphambilini [17, 18]. Imigomo yokuhlola ifingqiwe kuThebula Elingeziwe S1 futhi ichazwe ngaphambilini [19].
Amasampula e-fasting serum ahlaziywe nge-untargeted liquid chromatography-mass spectrometry (LC-MS) yokuhlaziywa kwe-metabolomics (n = 116). Amasampula ahlaziywe kusetshenziswa uhlelo lwe-UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) njengoba kuchaziwe ngaphambilini19. Ukuhlonza i-isopropyl alcohol (IPA) kwakusekelwe esikhathini sokugcina kanye nokuqhathaniswa kwe-MS/MS spectrum nezindinganiso ezimsulwa. Ukuqina kwesignali ye-IPA (indawo ephakeme) kucatshangelwe kuzo zonke ezinye izihlaziyi [20].
Ukulandelana kwe-RNA yesibindi sonke kwenziwa kusetshenziswa i-Illumina HiSeq 2500 futhi idatha yacutshungulwa kusengaphambili njengoba kuchaziwe ngaphambilini [19, 21, 22]. Senze ukuhlaziywa kokubonakaliswa kokwehluka okuqondiwe kokulotshiweyo okuthinta umsebenzi/i-biogenesis ye-mitochondrial sisebenzisa izakhi zofuzo zango-1957 ezikhethwe kusizindalwazi se-MitoMiner 4.0 [23]. Ukuhlaziywa kwe-methylation ye-DNA yesibindi kwenziwa kusetshenziswa i-Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) kusetshenziswa indlela efanayo njengoba kuchaziwe ngaphambilini [24, 25].
Amaseli e-hepatic stellate kubantu (LX-2) ahlinzekwe ngomusa nguSolwazi Stefano Romeo futhi akhuliswa futhi agcinwa endaweni ye-DMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Ukuze kukhethwe umthamo osebenzayo we-IPA, amaseli e-LX-2 aphathwe ngamanani ahlukene e-IPA (10 μM, 100 μM kanye ne-1 mM; Sigma, 220027) endaweni ye-DMEM/F12 amahora angama-24. Ngaphezu kwalokho, ukuze kuhlolwe ikhono le-IPA lokuvimbela ama-HSC, amaseli e-LX-2 aphathwe kanye ne-5 ng/ml TGF-β1 (izinhlelo ze-R&D, 240-B-002/CF) kanye ne-1 mM IPA endaweni ye-serum engenawo amahora angama-24. Kuzilawuli zezimoto ezifanele, i-4 nM HCL equkethe i-0.1% BSA yasetshenziswa ekwelashweni kwe-TGF-β1 kanti i-0.05% DMSO yasetshenziswa ekwelashweni kwe-IPA, futhi zombili zasetshenziswa ndawonye ekwelashweni okuhlanganisiwe.
I-Apoptosis ihlolwe kusetshenziswa i-FITC Annexin V Apoptosis Detection Kit ene-7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) ngokwemiyalelo yomenzi. Kafushane, i-LX-2 (1 × 105 cells/well) yakhuliswa ubusuku bonke emapuletini angu-12-well bese iphathwa ngemithamo eminingi ye-IPA noma i-IPA kanye ne-TGF-β1. Ngosuku olulandelayo, amaseli antantayo nanamathelayo aqoqwa, afakwa i-trypsin, agezwa nge-PBS, aphinde axhunywe ku-Annexin V binding buffer, futhi afakwa ku-FITC-Annexin V kanye ne-7-AAD imizuzu eyi-15.
Ama-Mitochondria kumaseli aphilayo afakwe umbala ngenxa yomsebenzi we-oxidative kusetshenziswa i-Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Ekuhlolweni kwe-MTR, amaseli e-LX-2 afakwa ngobuningi obufanayo ne-IPA kanye ne-TGF-β1. Ngemva kwamahora angu-24, amaseli aphilayo afakwa i-trypsin, agezwa nge-PBS, bese efakwa i-100 μM MTR endaweni engenawo ama-serum ku-37 °C imizuzu engu-20 njengoba kuchaziwe ngaphambilini [26]. Ekuhlaziyweni kwesimo seseli eliphilayo, usayizi weseli kanye nobunzima be-cytoplasmic kwahlaziywa kusetshenziswa amapharamitha e-forward scatter (FSC) kanye ne-side scatter (SSC), ngokulandelana.
Yonke idatha (imicimbi engu-30,000) itholakale kusetshenziswa i-NovoCyte Quanteon (Agilent) futhi yahlaziywa kusetshenziswa i-NovoExpress® 1.4.1 noma isofthiwe ye-FlowJo V.10.
Izinga lokusetshenziswa kwe-oxygen (OCR) kanye nezinga le-extracellular acidification (ECAR) kwalinganiswa ngesikhathi sangempela kusetshenziswa i-Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) ehlonyiswe nge-Seahorse XF Cell Mito Stress ngokwemiyalelo yomenzi. Kafushane, amaseli angu-2 × 104 LX-2/umthofu atshalwe kuma-plate e-XF96 cell culture. Ngemva kokufukamela ubusuku bonke, amaseli aphathwa nge-isopropanol (IPA) kanye ne-TGF-β1 (Izindlela Ezingeziwe 1). Ukuhlaziywa kwedatha kwenziwa kusetshenziswa isofthiwe ye-Seahorse XF Wave, ehlanganisa i-Seahorse XF Cell Energy Phenotype Test Report Generator. Kusukela kulokhu, kwabalwa i-Bioenergetic Health Index (BHI) [27].
I-RNA iyonke ihunyushwe yaba yi-cDNA. Ukuze uthole izindlela ezithile, bheka ireferensi [15]. Amazinga e-mRNA e-ribosomal acidic protein P0 (RPLP0) yomuntu kanye namazinga e-cyclophilin A1 (PPIA) asetshenziswa njengezilawuli zezakhi zofuzo ezihlanganisiwe. I-QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, The Netherlands) isetshenziswe ne-TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) noma i-Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), kanye ne-relative gene expression fold kubalwe kusetshenziswa amapharamitha okujikeleza inani le-Ct eliqhathaniswayo (ΔΔCt) kanye nendlela ye-∆∆Ct. Imininingwane yama-primers inikezwe kumaThebula Angeziwe S2 kanye ne-S3.
I-Nuclear DNA (ncDNA) kanye ne-mitochondrial DNA (mtDNA) zikhishwe kusetshenziswa i-DNeasy blood and tissue kit (Qiagen) njengoba kuchaziwe ngaphambilini [28]. Inani elilinganiselwe le-mtDNA libalwe ngokubala isilinganiso sesifunda ngasinye se-mtDNA esiqondiwe nesilinganiso sejiyometri sezifunda ezintathu ze-nuclear DNA (mtDNA/ncDNA), njengoba kuchaziwe ku-Supplementary Methods 2. Imininingwane yama-primer e-mtDNA kanye ne-ncDNA inikezwe ku-Supplementary Table S4.
Amaseli aphilayo afakwe i-Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) ukuze abone ngeso lengqondo amanethiwekhi e-mitochondrial aphakathi kwamangqamuzana nangaphakathi kwamangqamuzana. Amaseli e-LX-2 (amaseli angu-1 × 104/umthofu) akhuliswa kuma-slide engilazi kuma-culture plate ahambisanayo ane-glass-bottom (Ibidi GmbH, Martinsried, Germany). Ngemva kwamahora angu-24, amaseli e-LX-2 aphilayo afakwa i-100 μM MTR imizuzu engama-20 ku-37 °C kanti ama-cell nuclei afakwe i-DAPI (1 μg/ml, Sigma-Aldrich) njengoba kuchaziwe ngaphambilini [29]. Amanethiwekhi e-Mitochondrial afakwe ngeso lengqondo kusetshenziswa i-microscope eguquliwe ye-Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, Germany) efakwe i-confocal module ye-Zeiss LSM 800 ku-37 °C endaweni enomswakama ene-5% CO2 kusetshenziswa i-63×NA 1.3 objective. Sithole izithombe eziyishumi zochungechunge lwe-Z zohlobo ngalunye lwesampula. Uchungechunge ngalunye lwe-Z luqukethe izingxenye ezingu-30, ngasinye sinobukhulu obungu-9.86 μm. Kusampula ngayinye, izithombe zemikhakha yokubuka eyishumi ehlukene zatholakala kusetshenziswa isofthiwe ye-ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany), futhi ukuhlaziywa kwesimo se-mitochondrial kwenziwa kusetshenziswa isofthiwe ye-ImageJ (v1.54d) [30, 31] ngokwemingcele echazwe ku-Supplementary Methods 3.
Amaseli aqiniswa nge-2% glutaraldehyde ku-0.1 M phosphate buffer, kwalandela ukufakwa ngesisombululo se-osmium tetroxide esingu-1% (Sigma Aldrich, MO, USA), kancane kancane ancishiswa ngamanzi nge-acetone (Merck, Darmstadt, Germany), futhi ekugcineni afakwa ku-epoxy resin. Izingxenye ezincane kakhulu zalungiswa futhi zapendwa nge-1% uranyl acetate (Merck, Darmstadt, Germany) kanye ne-1% lead citrate (Merck, Darmstadt, Germany). Izithombe ze-Ultrastructural zatholakala kusetshenziswa i-JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) nge-voltage esheshisa engu-80 kV.
Ukwakheka kwamaseli e-LX-2 aphathwe nge-IPA amahora angama-24 kwahlaziywa nge-phase-contrast microscopy ekukhulisweni okungu-50x kusetshenziswa i-Zeiss inverted light microscope (Zeiss Axio Vert.A1 kanye ne-AxioCam MRm, eJena, eJalimane).
Idatha yezokwelapha ivezwe njenge-mean ± standard deviation noma i-median (ibanga le-interquartile: IQR). Ukuhlaziywa kwe-one-way kokwehluka (continuous variables) noma ukuhlolwa kwe-χ² (categorical variables) kusetshenziswe ukuqhathanisa umehluko phakathi kwamaqembu amathathu ocwaningo. Isilinganiso se-false positive (FDR) sisetshenziswe ukulungisa ukuhlolwa okuningi, kanti izakhi zofuzo ezine-FDR <0.05 zibhekwe njengezibalulekile ngokwezibalo. Ukuhlaziywa kwe-Spearman correlation kusetshenziswe ukuhlanganisa i-CpG DNA methylation namandla esignali ye-IPA, ngamanani e-nominal p ​​(p <0.05) abikiwe.
Ukuhlaziywa kwendlela kwenziwe kusetshenziswa ithuluzi lokuhlaziya isethi yezakhi zofuzo elisekelwe kuwebhu (i-WebGestalt) lama-transcript angu-268 (i-nominal p < 0.01), ama-transcript ahlobene ne-mitochondria angu-119 (i-nominal p < 0.05), kanye nama-CpG angu-4350 kuma-transcript esibindi angu-3093 ahlotshaniswa namazinga e-IPA e-serum ajikelezayo. Ithuluzi le-Venny DB (version 2.1.0) elitholakala mahhala lasetshenziswa ukuthola izakhi zofuzo ezihambisanayo, kanti i-StringDB (version 11.5) yasetshenziswa ukubona ngeso lengqondo ukusebenzisana kwamaprotheni namaphrotheni.
Kokuhlolwa kwe-LX-2, amasampula ahlolwe ukuthi ajwayelekile yini kusetshenziswa ukuhlolwa kwe-D'Agostino-Pearson. Idatha itholakale okungenani kumakhophi amathathu ebhayoloji futhi yafakwa ku-ANOVA yendlela eyodwa ngokuhlolwa kwe-Bonferroni post hoc. Inani le-p elingaphansi kuka-0.05 libhekwa njengelibalulekile ngokwezibalo. Idatha yethulwa njenge-± SD ephakathi, futhi inani lokuhlolwa likhonjisiwe kusibalo ngasinye. Konke ukuhlaziya namagrafu kwenziwe kusetshenziswa isofthiwe yezibalo ye-GraphPad Prism 8 ye-Windows (GraphPad Software Inc., inguqulo 8.4.3, eSan Diego, e-USA).
Okokuqala, sihlole ukuhlangana kwamazinga e-IPA egazini nokubhalwa kwesibindi, umzimba wonke, kanye ne-mitochondrial. Kuphrofayili yokubhalwa okuphelele, i-gene enamandla kakhulu ehlotshaniswa namazinga e-IPA egazini kwakuyi-MAPKAPK3 (FDR = 0.0077; i-protein kinase-activated protein kinase-activated protein kinase 3); kuphrofayili yokubhalwa okuhlobene ne-mitochondria, i-gene enamandla kakhulu ehlotshaniswa nayo kwakuyi-AKT1 (FDR = 0.7621; i-AKT serine/threonine kinase 1) (Ifayela elingeziwe 1 kanye nefayela elingeziwe 2).
Sabe sesihlaziya imibhalo yomhlaba wonke (n = 268; p < 0.01) kanye nemibhalo ehlobene ne-mitochondria (n = 119; p < 0.05), ekugcineni sathola i-apoptosis njengendlela ebaluleke kakhulu ye-canonical (p = 0.0089). Ngemibhalo ye-mitochondrial ehlotshaniswa namazinga e-IPA e-serum, sigxile ku-apoptosis (FDR = 0.00001), i-mitophagy (FDR = 0.00029), kanye nezindlela zokubonisa i-TNF (FDR = 0.000006) (Isithombe 1A, Ithebula 2, kanye Nezithombe Ezingeziwe 1A-B).
Ukuhlaziywa okuphindaphindekayo kwemibhalo yomhlaba wonke, ehlobene ne-mitochondria, kanye ne-methylation ye-DNA esibindini somuntu ngokuhambisana namazinga e-IPA esegazini. I-A imele imibhalo yomhlaba wonke engu-268, imibhalo ehlobene ne-mitochondria engu-119, kanye nemibhalo ehlobene ne-DNA ehlanganiswe ne-methyl ehlanganiswe nezindawo ze-CpG ezingu-3092 ezihlotshaniswa namazinga e-IPA esegazini (amanani e-p < 0.01 yemibhalo yomhlaba wonke kanye ne-DNA ehlanganiswe ne-methyl, kanye namanani e-p < 0.05 yemibhalo ehlobene ne-mitochondrial). Imibhalo ephindaphindekayo emikhulu iboniswe phakathi (i-AKT1 kanye ne-YKT6). B Imephu yokusebenzisana kwezakhi zofuzo ezingu-13 ezinesilinganiso sokusebenzisana esiphezulu (0.900) nezinye izakhi zofuzo yakhiwe ngamazakhi zofuzo angu-56 aphindaphindekayo (isifunda somugqa omnyama) ahlotshaniswa kakhulu namazinga e-IPA esegazini kusetshenziswa ithuluzi eliku-inthanethi i-StringDB. Okuluhlaza: Izakhi zofuzo ezihlanganiswe nengxenye yeselula ye-Gene Ontology (GO): mitochondria (GO:0005739). I-AKT1 yiprotheni enesilinganiso esiphezulu (0.900) sokusebenzisana namanye amaprotheni ngokusekelwe kudatha (ngokusekelwe ekumeni umbhalo, ukuhlolwa, izizindalwazi, kanye nokubonakaliswa ngokubambisana). Ama-node enethiwekhi amelela amaprotheni, kanti imiphetho imelela ukuxhumana phakathi kwamaprotheni.
Njengoba ama-metabolites e-gut microbiota angalawula ukwakheka kwe-epigenetic nge-methylation ye-DNA [32], siphenye ukuthi amazinga e-serum IPA ahlotshaniswa yini ne-methylation ye-DNA yesibindi. Sithole ukuthi izindawo ezimbili ezinkulu ze-methylation ezihlotshaniswa namazinga e-serum IPA zaziseduze nesifunda 3 esicebile nge-proline-serine (C19orf55) kanye nomndeni we-heat shock protein B (small) ilungu 6 (HSPB6) (Ifayela elengeziwe 3). I-DNA methylation ye-4350 CpG (p < 0.01) yayihlotshaniswa namazinga e-serum IPA futhi yacebiswa ezindleleni zokulawula isikhathi eside (p = 0.006) (Isithombe 1A, Ithebula 2, kanye nesithombe esengeziwe 1C).
Ukuze siqonde izindlela eziphilayo ezisekela ubudlelwano phakathi kwamazinga e-IPA egazini, imibhalo yomhlaba wonke, imibhalo ehlobene ne-mitochondria, kanye ne-methylation ye-DNA esibindini somuntu, senze ukuhlaziywa kokugqagqana kwezakhi zofuzo ezikhonjwe ekuhlaziyweni kwendlela kwangaphambilini (Isithombe 1A). Imiphumela yokuhlaziywa kokunongwa kwendlela yezakhi zofuzo ezingu-56 ezigqagqanayo (ngaphakathi komugqa omnyama ku-Isithombe 1A) ibonise ukuthi indlela ye-apoptosis (p = 0.00029) iqokomise izakhi zofuzo ezimbili ezivamile kulezi zihlaziyo ezintathu: i-AKT1 kanye ne-YKT6 (YKT6 v-SNARE homolog), njengoba kuboniswe kumdwebo we-Venn (Isithombe Esingeziwe 2 kanye nesithombe 1A). Ngokuthakazelisayo, sithole ukuthi i-AKT1 (cg19831386) kanye ne-YKT6 (cg24161647) zazihlobene kahle namazinga e-IPA egazini (Ifayela elingeziwe 3). Ukuze sithole ukusebenzisana okungenzeka kwamaprotheni phakathi kwemikhiqizo yezakhi zofuzo, sikhethe izakhi zofuzo ezingu-13 ezinesilinganiso sesifunda esivamile esiphezulu (0.900) phakathi kwezakhi zofuzo ezingu-56 ezigqagqanayo njengokufaka futhi sakha imephu yokusebenzisana. Ngokwezinga lokuzethemba (ukuzethemba okusemaphethelweni), i-AKT1 gene ene-score ephezulu kakhulu (0.900) yayiphezulu (Isithombe 1B).
Ngokusekelwe ekuhlaziyweni kwendlela, sithole ukuthi i-apoptosis yiyona ndlela eyinhloko, ngakho-ke siphenye ukuthi ukwelashwa kwe-IPA kuzothinta yini i-apoptosis yama-HSCs ku-vitro. Ngaphambilini sibonise ukuthi imithamo ehlukene ye-IPA (10 μM, 100 μM, kanye ne-1 mM) yayingewona ubuthi kumaseli e-LX-2 [15]. Lolu cwaningo lubonise ukuthi ukwelashwa kwe-IPA ku-10 μM kanye ne-100 μM kwandisa inani lamaseli asebenzayo nane-necrotic. Kodwa-ke, uma kuqhathaniswa neqembu lokulawula, ukuphila kwamaseli kwehle ekugxilweni kwe-IPA okungu-1 mM, kuyilapho izinga le-necrosis yamaseli lingashintshi (Isithombe 2A, B). Okulandelayo, ukuthola ukugxilwa okufanele kakhulu kokubangela i-apoptosis kumaseli e-LX-2, sihlole i-IPA engu-10 μM, 100 μM, kanye ne-1 mM amahora angama-24 (Isithombe 2A-E kanye nesithombe esengeziwe 3A-B). Ngokuthakazelisayo, i-IPA 10 μM kanye ne-100 μM zehlise izinga le-apoptosis (%), noma kunjalo, i-IPA 1 mM yandise izinga le-apoptosis kanye nezinga le-apoptosis (%) uma kuqhathaniswa nokulawula futhi ngenxa yalokho yakhethwa ukuze kwenziwe ezinye izivivinyo (Izibalo 2A–D).
I-IPA ibangela i-apoptosis yamaseli e-LX-2. Indlela ye-Annexin V kanye ne-7-AAD yokudaya kabili isetshenziswe ukulinganisa izinga le-apoptotic kanye nokwakheka kwamaseli nge-flow cytometry. Amaseli e-BA afakwe i-10 μM, 100 μM kanye ne-1 mM IPA amahora angama-24 noma nge-F–H TGF-β1 (5 ng/ml) kanye ne-1 mM IPA endaweni engena-serum amahora angama-24. A: amaseli aphilayo (i-Annexin V -/ 7AAD-); B: amaseli e-necrotic (i-Annexin V -/ 7AAD+); C, F: ekuqaleni (i-Annexin V +/ 7AAD-); D, G: ngasekupheleni (i-Annexin V+/7AAD+.); E, H: iphesenti lamaseli e-apoptotic asekuqaleni nasekupheleni kwesikhathi ngesilinganiso se-apoptotic (%). Idatha ivezwa njengesilinganiso ± SD, n = izivivinyo ezizimele ezi-3. Ukuqhathaniswa kwezibalo kwenziwe kusetshenziswa i-ANOVA yendlela eyodwa nokuhlolwa kwe-Bonferroni post hoc. *p < 0.05; ****p < 0.0001
Njengoba sesibonisile ngaphambilini, i-5 ng/ml TGF-β1 ingadala ukusebenza kwe-HSC ngokwandisa ukubonakaliswa kwezakhi zofuzo ze-classical marker [15]. Amaseli e-LX-2 aphathwe nge-5 ng/ml TGF-β1 kanye ne-1 mM IPA ngokuhlanganiswa (Isithombe 2E–H). Ukwelashwa kwe-TGF-β1 akuzange kushintshe izinga le-apoptosis, nokho, ukwelashwa ngokubambisana kwe-IPA kwandise izinga le-apoptosis kanye ne-apoptosis (%) uma kuqhathaniswa nokwelashwa kwe-TGF-β1 (Isithombe 2E–H). Le miphumela ikhombisa ukuthi i-1 mM IPA ingakhuthaza i-apoptosis kumaseli e-LX-2 ngaphandle kokungeniswa kwe-TGF-β1.
Siqhubekile sahlola umphumela we-IPA ekuphefumuleni kwe-mitochondrial kumaseli e-LX-2. Imiphumela ibonise ukuthi i-1 mM IPA yehlise imingcele yesilinganiso sokusebenzisa umoya-mpilo (OCR): ukuphefumula okungeyona i-mitochondrial, ukuphefumula okuyisisekelo kanye nokuphezulu, ukuvuza kweproton kanye nokukhiqizwa kwe-ATP uma kuqhathaniswa neqembu lokulawula (Isithombe 3A, B), kuyilapho i-bioenergetic health index (BHI) ingashintshi.
I-IPA yehlisa ukuphefumula kwe-mitochondrial kumaseli e-LX-2. Ijika lokuphefumula le-mitochondrial (i-OCR) livezwa njengemingcele yokuphefumula kwe-mitochondrial (ukuphefumula okungeyona i-mitochondrial, ukuphefumula okuyisisekelo, ukuphefumula okuphezulu, ukuvuza kwe-proton, ukukhiqizwa kwe-ATP, i-SRC ne-BHI). Amaseli A no-B afakwe i-10 μM, i-100 μM kanye ne-1 mM IPA amahora angama-24, ngokulandelana. Amaseli C no-D afakwe i-TGF-β1 (5 ng/ml) kanye ne-1 mM IPA endaweni engena-serum amahora angama-24, ngokulandelana. Zonke izilinganiso zalungiswa ngokokuqukethwe kwe-DNA kusetshenziswa i-CyQuant kit. I-BHI: inkomba yezempilo ye-bioenergetic; I-SRC: umthamo wokugcina wokuphefumula; I-OCR: izinga lokusetshenziswa komoya-mpilo. Idatha ivezwa njengokuphambuka okujwayelekile ± okujwayelekile (SD), n = izivivinyo ezizimele ezi-5. Ukuqhathaniswa kwezibalo kwenziwe kusetshenziswa ukuhlolwa kwe-ANOVA kanye ne-Bonferroni post hoc eyodwa. *p < 0.05; **p < 0.01; kanye ***p < 0.001
Ukuze sithole ukuqonda okubanzi ngomphumela we-IPA kuphrofayili ye-bioenergetic yamaseli e-LX-2 asebenze yi-TGF-β1, sihlaziye i-phosphorylation ye-mitochondrial oxidative yi-OCR (Isithombe 3C, D). Imiphumela ibonise ukuthi ukwelashwa kwe-TGF-β1 kunganciphisa ukuphefumula okuphezulu, amandla okugcina ukuphefumula (i-SRC) kanye ne-BHI uma kuqhathaniswa neqembu lokulawula (Isithombe 3C, D). Ngaphezu kwalokho, ukwelashwa okuhlanganisiwe kunciphisa ukuphefumula okuyisisekelo, ukuvuza kwe-proton kanye nokukhiqizwa kwe-ATP, kodwa i-SRC ne-BHI zaziphakeme kakhulu kunalezo eziphathwe nge-TGF-β1 (Isithombe 3C, D).
Senze futhi "Ukuhlolwa Kwamandla Eselula" okuhlinzekwe yisoftware ye-Seahorse (Isithombe Esingeziwe 4A–D). Njengoba kuboniswe ku-Supplementary Fig. 3B, amandla okusebenza kwe-metabolic e-OCR kanye ne-ECAR ancishisiwe ngemuva kokwelashwa kwe-TGF-β1, nokho, akukho mehluko owabonwa emaqenjini okwelashwa okuhlanganiswayo kanye ne-IPA uma kuqhathaniswa neqembu lokulawula. Ngaphezu kwalokho, amazinga omabili e-basal kanye nokucindezeleka kwe-OCR ancishisiwe ngemuva kokwelashwa okuhlanganiswayo kanye ne-IPA uma kuqhathaniswa neqembu lokulawula (Isithombe Esingeziwe 4C). Ngokuthakazelisayo, iphethini efanayo yabonwa ngokwelashwa okuhlanganiswayo, lapho kungekho shintsho emazingeni e-basal kanye nokucindezeleka kwe-ECAR olubonwe uma kuqhathaniswa nokwelashwa kwe-TGF-β1 (Isithombe Esingeziwe 4C). Kuma-HSC, ukwehla kwe-phosphorylation ye-mitochondrial oxidative kanye nekhono lokwelashwa okuhlanganiswayo ukubuyisela i-SCR kanye ne-BHI ngemuva kokuchayeka ekwelashweni kwe-TGF-β1 akuzange kushintshe amandla okusebenza kwe-metabolic (OCR kanye ne-ECAR). Uma kuqhathaniswa, le miphumela ikhombisa ukuthi i-IPA inganciphisa i-bioenergetics kuma-HSC, okuphakamisa ukuthi i-IPA ingadala iphrofayili yamandla aphansi eshintsha i-phenotype ye-HSC ibe yi-inactivation (Isithombe Esingeziwe 4D).
Umphumela we-IPA ku-mitochondrial dynamics uhlolwe kusetshenziswa ukulinganisa okunezinhlangothi ezintathu kwesimo se-mitochondrial kanye nokuxhumeka kwenethiwekhi kanye nokufaka imibala kwe-MTR (Isithombe 4 kanye nesithombe esengeziwe 5). Isithombe 4 sibonisa ukuthi, uma kuqhathaniswa neqembu lokulawula, ukwelashwa kwe-TGF-β1 kwehlise indawo ephakathi nendawo, inombolo yegatsha, ubude begatsha lonke, kanye nenombolo yokuhlangana kwegatsha (Isithombe 4A kanye no-B) futhi kwashintsha isilinganiso se-mitochondria kusuka kusimo esiyindilinga kuya kusimo esiphakathi (Isithombe 4C). Ukwelashwa kwe-IPA kuphela kwehlise ivolumu ephakathi ye-mitochondrial futhi kwashintsha isilinganiso se-mitochondria kusuka kusimo esiyindilinga kuya kusimo esiphakathi uma kuqhathaniswa neqembu lokulawula (Isithombe 4A). Ngokuphambene nalokho, i-sphericity, ubude begatsha eliphakathi, kanye nomsebenzi we-mitochondrial ohlolwe yi-mitochondrial membrane potential-dependent MTR (Isithombe 4A kanye no-E) kwahlala kungashintshi futhi lawa mapharamitha awazange ahluke phakathi kwamaqembu. Uma kuqhathaniswa, le miphumela iphakamisa ukuthi ukwelashwa kwe-TGF-β1 kanye ne-IPA kubonakala kuguqula ukuma nosayizi we-mitochondrial kanye nobunzima benethiwekhi kumaseli e-LX-2 aphilayo.
I-IPA ishintsha amandla e-mitochondrial kanye nobuningi be-DNA ye-mitochondrial kumaseli e-LX-2. A. Izithombe ezimele eziyimfihlo zamaseli e-LX-2 aphilayo afakwe i-TGF-β1 (5 ng/ml) kanye ne-1 mM IPA amahora angama-24 endaweni engenaso i-serum ekhombisa amanethiwekhi e-mitochondrial afakwe i-Mitotracker™ Red CMXRos kanye nama-nuclei afakwe i-DAPI eluhlaza okwesibhakabhaka. Yonke idatha iqukethe okungenani izithombe eziyi-15 eqenjini ngalinye. Sithole izithombe eziyi-10 ze-Z-stack zohlobo ngalunye lwesampula. Uchungechunge ngalunye lwe-Z-axis lwaluqukethe izingcezu ezingama-30, ngasinye sinobukhulu obungu-9.86 μm. Ibha yesikali: 10 μm. B. Izinto ezimele (i-mitochondria kuphela) ezitholwe ngokusebenzisa umkhawulo oguquguqukayo esithombeni. Ukuhlaziywa kwenani kanye nokuqhathaniswa kokuxhumeka kwenethiwekhi ye-mitochondrial morphological kwenziwa kuwo wonke amaseli eqenjini ngalinye. C. Ukuvama kwezilinganiso zesimo se-mitochondrial. Amanani aseduze no-0 abonisa izimo eziyindilinga, kanye namanani aseduze no-1 abonisa izimo ze-filamentous. D Okuqukethwe kwe-Mitochondrial DNA (mtDNA) kunqunywe njengoba kuchaziwe ku-Materials and Methods. Ukuhlaziywa kwe-E Mitotracker™ Red CMXRos kwenziwe nge-flow cytometry (imicimbi engu-30,000) njengoba kuchaziwe ku-Materials and Methods. Idatha yethulwa njenge-mean ± SD, n = izivivinyo ezizimele ezi-3. Ukuqhathaniswa kwezibalo kwenziwe kusetshenziswa ukuhlolwa kwe-ANOVA kanye ne-Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Sabe sesihlaziya okuqukethwe kwe-mtDNA kumaseli e-LX-2 njengesibonakaliso senombolo ye-mitochondrial. Uma kuqhathaniswa neqembu lokulawula, okuqukethwe kwe-mtDNA kwandisiwe eqenjini eliphathwe nge-TGF-β1 (Isithombe 4D). Uma kuqhathaniswa neqembu eliphathwe nge-TGF-β1, okuqukethwe kwe-mtDNA kwancishiswa eqenjini lokwelashwa okuhlanganisiwe (Isithombe 4D), okuphakamisa ukuthi i-IPA inganciphisa okuqukethwe kwe-mtDNA futhi mhlawumbe inombolo ye-mitochondrial kanye nokuphefumula kwe-mitochondrial (Isithombe 3C). Ngaphezu kwalokho, i-IPA ibonakala sengathi inciphisa okuqukethwe kwe-mtDNA ekwelashweni okuhlanganisiwe kodwa ayizange ithinte umsebenzi we-mitochondrial oqhutshwa yi-MTR (Izithombe 4A–C).
Sihlole ukuhlangana kwe-IPA namazinga e-mRNA ezakhi zofuzo ezihlotshaniswa ne-fibrosis, i-apoptosis, ukusinda, kanye ne-mitochondrial dynamics kumaseli e-LX-2 (Isithombe 5A–D). Uma kuqhathaniswa neqembu lokulawula, iqembu eliphathwe nge-TGF-β1 libonise ukwanda kokubonakaliswa kwezakhi zofuzo ezifana ne-collagen type I α2 chain (COL1A2), i-α-smooth muscle actin (αSMA), i-matrix metalloproteinase 2 (MMP2), i-tissue inhibitor ye-metalloproteinase 1 (TIMP1), kanye ne-dynamin 1-like gene (DRP1), okubonisa ukwanda kwe-fibrosis kanye nokusebenza. Ngaphezu kwalokho, uma kuqhathaniswa neqembu lokulawula, ukwelashwa kwe-TGF-β1 kunciphisa amazinga e-mRNA e-nuclear pregnane X receptor (PXR), i-caspase 8 (CASP8), i-MAPKAPK3, i-inhibitor ye-B-cell α, i-enhancer ye-nuclear factor κ gene light peptide (NFκB1A), kanye ne-inhibitor ye-nuclear factor κB kinase subunit β (IKBKB) (Isithombe 5A–D). Uma kuqhathaniswa nokwelashwa kwe-TGF-β1, ukwelashwa okuhlangene ne-TGF-β1 kanye ne-IPA kunciphisa ukuvezwa kwe-COL1A2 kanye ne-MMP2, kodwa kwandisa amazinga e-mRNA e-PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, kanye ne-IKBKB. Ukwelashwa kwe-IPA kunciphisa kakhulu ukuvezwa kwe-MMP2, i-Bcl-2-associated protein X (BAX), AKT1, i-optic atrophy protein 1 (OPA1), kanye ne-mitochondrial fusion protein 2 (MFN2), kanti ukuvezwa kwe-CASP8, NFκB1A, NFκB1B, kanye ne-IKBKB kwanda uma kuqhathaniswa neqembu lokulawula. Kodwa-ke, akukho mehluko otholakale ekubonakalisweni kwe-caspase-3 (CASP3), i-apoptotic peptidase activating factor 1 (APAF1), i-mitochondrial fusion protein 1 (MFN1), kanye ne-fission protein 1 (FIS1). Ngokubambisana, le miphumela iphakamisa ukuthi ukwelashwa kwe-IPA kuguqula ukubonakaliswa kwezakhi zofuzo ezihlotshaniswa ne-fibrosis, i-apoptosis, ukusinda, kanye ne-mitochondrial dynamics. Idatha yethu iphakamisa ukuthi ukwelashwa kwe-IPA kunciphisa i-fibrosis kumaseli e-LX-2; ngesikhathi esifanayo, kukhuthaza ukusinda ngokushintsha i-phenotype iye ekungasebenzini.
I-IPA ilawula ukuvezwa kwezakhi zofuzo ze-fibroblast, i-apoptotic, i-viability, kanye ne-mitochondrial dynamics kumaseli e-LX-2. Ama-histogram abonisa ukuvezwa kwe-mRNA maqondana nokulawulwa kwe-endogenous (RPLP0 noma i-PPIA) ngemuva kokuthi amaseli e-LX-2 eqanjwe nge-TGF-β1 kanye ne-IPA endaweni engena-serum amahora angama-24. I-A ikhombisa ama-fibroblast, i-B ikhombisa amaseli e-apoptotic, i-C ikhombisa amaseli asindayo, kanti i-D ikhombisa ukuvezwa kwezakhi zofuzo ze-mitochondrial dynamics. Idatha yethulwa njenge-mean ± standard deviation (SD), n = izivivinyo ezizimele ezi-3. Ukuqhathaniswa kwezibalo kwenziwe kusetshenziswa ukuhlolwa kwe-ANOVA kanye ne-Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Ngemuva kwalokho, izinguquko kusayizi weseli (FSC-H) kanye nobunzima be-cytoplasmic (SSC-H) zahlolwa nge-flow cytometry (Isithombe 6A,B), kanti izinguquko ku-morphology yeseli ngemuva kokwelashwa kwe-IPA zahlolwa nge-transmission electron microscopy (TEM) kanye ne-phase contrast microscopy (Isithombe Esingeziwe 6A-B). Njengoba bekulindelekile, amaseli eqenjini eliphathwe nge-TGF-β1 anda ngobukhulu uma kuqhathaniswa neqembu lokulawula (Isithombe 6A,B), okubonisa ukwanda okuvamile kwe-rough endoplasmic reticulum (ER*) kanye ne-phagolysosomes (P), okubonisa ukusebenza kwe-hematopoietic stem cell (HSC) (Isithombe Esingeziwe 6A). Kodwa-ke, uma kuqhathaniswa neqembu eliphathwe nge-TGF-β1, usayizi weseli, ubunzima be-cytoplasmic (Isithombe 6A,B), kanye nokuqukethwe kwe-ER* kwehlisiwe eqenjini lokwelashwa okuhlanganisiwe kwe-TGF-β1 kanye ne-IPA (Isithombe Esingeziwe 6A). Ngaphezu kwalokho, ukwelashwa kwe-IPA kwehlise usayizi weseli, ubunzima be-cytoplasmic (Izithombe 6A,B), okuqukethwe kwe-P kanye ne-ER* (Isithombe Esingeziwe 6A) uma kuqhathaniswa neqembu lokulawula. Ngaphezu kwalokho, okuqukethwe kwamaseli e-apoptotic kukhuphuke ngemuva kwamahora angama-24 okwelashwa kwe-IPA uma kuqhathaniswa neqembu lokulawula (imicibisholo emhlophe, Isithombe Esingeziwe 6B). Ngokubambisana, le miphumela isikisela ukuthi i-1 mM IPA ingakhuthaza i-HSC apoptosis futhi iguqule izinguquko kumapharamitha e-morphological eseli abangelwa yi-TGF-β1, ngaleyo ndlela ilawule usayizi weseli kanye nobunzima, okungase kuhlotshaniswe nokungasebenzi kwe-HSC.
I-IPA ishintsha usayizi weseli kanye nobunzima be-cytoplasmic kumaseli e-LX-2. Izithombe ezimele zokuhlaziywa kwe-flow cytometry. Ukuhlaziywa kusebenzise isu lokugaya eliqondene namaseli e-LX-2: i-SSC-A/FSC-A ukuchaza inani lamaseli, i-FSC-H/FSC-A ukuhlonza ama-doublet, kanye ne-SSC-H/FSC-H yobukhulu beseli kanye nokuhlaziywa kobunzima. Amaseli afakwe i-TGF-β1 (5 ng/ml) kanye ne-1 mM IPA endaweni engena-serum amahora angama-24. Amaseli e-LX-2 asatshalaliswa kwi-quadrant engezansi kwesobunxele (SSC-H-/FSC-H-), i-quadrant ephezulu kwesobunxele (SSC-H+/FSC-H-), i-quadrant engezansi kwesokudla (SSC-H-/FSC-H+), kanye ne-quadrant ephezulu kwesokudla (SSC-H+/FSC-H+) yobukhulu beseli kanye nokuhlaziywa kobunzima be-cytoplasmic. B. Ukwakheka kweseli kuhlaziywe nge-flow cytometry kusetshenziswa i-FSC-H (i-forward scatter, usayizi weseli) kanye ne-SSC-H (i-side scatter, i-cytoplasmic complexity) (imicimbi engu-30,000). Idatha yethulwa njenge-mean ± SD, n = izivivinyo ezizimele ezi-3. Ukuqhathaniswa kwezibalo kwenziwe kusetshenziswa i-one-way ANOVA kanye ne-Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 kanye ne-***p < 0.0001
Ama-metabolite amathumbu afana ne-IPA asephenduke isihloko esishisayo socwaningo, okuphakamisa ukuthi kungase kutholakale izinhloso ezintsha ku-gut microbiota. Ngakho-ke kuyathakazelisa ukuthi i-IPA, i-metabolite esiyixhumanise ne-fibrosis yesibindi kubantu [15], iboniswe ukuthi iyinto engaba khona yokulwa ne-fibrotic kumamodeli ezilwane [13, 14]. Lapha, sibonisa okokuqala ubudlelwano phakathi kwe-IPA ye-serum kanye ne-transcriptomics yesibindi yomhlaba wonke kanye ne-methylation ye-DNA kubantu abakhuluphele abangenaso isifo sikashukela sohlobo 2 (T2D), siqokomisa i-apoptosis, i-mitophagy kanye nokuphila isikhathi eside, kanye ne-gene engaba khona ye-candidate AKT1 elawula i-homeostasis yesibindi. Enye into entsha ocwaningweni lwethu ukuthi sibonise ukusebenzisana kokwelashwa kwe-IPA ne-apoptosis, i-cell morphology, i-mitochondrial bioenergetics kanye ne-dynamics kumaseli e-LX-2, okubonisa i-spectrum yamandla ephansi eshintsha i-phenotype ye-HSC iye ekuqalisweni kokusebenza, okwenza i-IPA ibe yinto engaba khona yokuthuthukisa i-fibrosis yesibindi.
Sithole ukuthi i-apoptosis, i-mitophagy kanye nokuphila isikhathi eside kwakuyizindlela ezibaluleke kakhulu ze-canonical ezicebile kuma-gene esibindi ahlotshaniswa ne-IPA ejikelezayo ye-serum. Ukuphazamiseka kohlelo lokulawula ikhwalithi ye-mitochondrial (MQC) kungaholela ekungasebenzi kahle kwe-mitochondrial, i-mitophagy kanye ne-apoptosis, ngaleyo ndlela kukhuthaze ukwenzeka kwe-MASLD[33, 34]. Ngakho-ke, singacabanga ukuthi i-IPA ingase ihileleke ekugcineni amandla eseli kanye nobuqotho be-mitochondrial ngokusebenzisa i-apoptosis, i-mitophagy kanye nokuphila isikhathi eside esibindini. Idatha yethu ibonise ukuthi ama-gene amabili ayevamile kuzo zonke izivivinyo ezintathu: i-YKT6 kanye ne-AKT1. Kubalulekile ukuqaphela ukuthi i-YKT6 iyiprotheni ye-SNARE ehilelekile enkambisweni yokuhlanganiswa kwe-membrane yeseli. Idlala indima ku-autophagy kanye ne-mitophagy ngokwenza i-initiation complex ene-STX17 kanye ne-SNAP29 ku-autophagosome, ngaleyo ndlela ikhuthaze ukuhlanganiswa kwama-autophagosome kanye nama-lysosome[35]. Ngaphezu kwalokho, ukulahlekelwa umsebenzi we-YKT6 kuholela ekuphazamisekeni kwe-mitophagy[36], kuyilapho ukwanda kwe-YKT6 kuhlotshaniswa nokuqhubeka kwe-hepatocellular carcinoma (HCC), okubonisa ukwanda kokusinda kwamaseli[37]. Ngakolunye uhlangothi, i-AKT1 iyi-gene ebaluleke kakhulu esebenzisana futhi idlala indima ebalulekile ezifweni zesibindi, okuhlanganisa indlela yokubonisana ye-PI3K/AKT, umjikelezo weseli, ukufuduka kwamaseli, ukwanda, ukunamathela okugxile, umsebenzi we-mitochondrial, kanye nokukhiqizwa kwe-collagen[38-40]. Indlela yokubonisana ye-PI3K/AKT esebenzayo ingasebenzisa amaseli e-hematopoietic stem (ama-HSC), angamaseli anesibopho sokukhiqizwa kwe-extracellular matrix (ECM), futhi ukungasebenzi kwayo kungabangela ukwenzeka nokuqhubeka kwe-liver fibrosis[40]. Ngaphezu kwalokho, i-AKT ingenye yezinto ezibalulekile zokusinda kwamaseli ezivimbela i-p53-dependent cell apoptosis, futhi ukusebenza kwe-AKT kungahlotshaniswa nokuvinjelwa kwe-liver cell apoptosis[41, 42]. Imiphumela etholakele iphakamisa ukuthi i-IPA ingase ihileleke ku-apoptosis ehlobene ne-mitochondria yesibindi ngokuthinta isinqumo sama-hepatocytes phakathi kokungena ku-apoptosis noma ukusinda. Le miphumela ingalawulwa yi-AKT kanye/noma izakhi zofuzo ze-YKT6, ezibalulekile kwi-homeostasis yesibindi.
Imiphumela yethu ikhombisile ukuthi i-1 mM IPA ibangele i-apoptosis futhi yehlisa ukuphefumula kwe-mitochondrial kumaseli e-LX-2 ngaphandle kokwelashwa kwe-TGF-β1. Kuyaphawuleka ukuthi i-apoptosis iyindlela enkulu yokuxazulula i-fibrosis kanye nokusebenza kwe-hematopoietic stem cell (HSC), futhi iyisenzakalo esibalulekile ekuphenduleni okuguqukayo kwe-fibrosis yesibindi [4, 43]. Ngaphezu kwalokho, ukubuyiselwa kwe-BHI kumaseli e-LX-2 ngemuva kokwelashwa okuhlanganisiwe kunikeze ukuqonda okusha ngendima engaba khona ye-IPA ekulawulweni kwe-mitochondrial bioenergetics. Ngaphansi kwezimo zokuphumula nezingasebenzi, amaseli e-hematopoietic avame ukusebenzisa i-mitochondrial oxidative phosphorylation ukukhiqiza i-ATP futhi abe nomsebenzi ophansi we-metabolic. Ngakolunye uhlangothi, ukusebenza kwe-HSC kuthuthukisa ukuphefumula kwe-mitochondrial kanye ne-biosynthesis ukuze kuhlangatshezwane nezidingo zamandla zokungena esimweni se-glycolytic [44]. Iqiniso lokuthi i-IPA ayizange ithinte amandla e-metabolic kanye ne-ECAR lisikisela ukuthi indlela ye-glycolytic ayibekwanga phambili kangako. Ngokufanayo, olunye ucwaningo lubonise ukuthi i-1 mM IPA ikwazile ukulawula umsebenzi we-mitochondrial respiratory chain kuma-cardiomyocytes, umugqa weseli le-hepatocyte lomuntu (i-Huh7) kanye namaseli e-endothelial e-umbilical vein yomuntu (i-HUVEC); Kodwa-ke, akukho mphumela we-IPA otholakale ku-glycolysis kuma-cardiomyocytes, okuphakamisa ukuthi i-IPA ingathinta ama-bioenergetics ezinye izinhlobo zamaseli [45]. Ngakho-ke, sicabanga ukuthi i-1 mM IPA ingasebenza njenge-uncoupler yamakhemikhali emnene, ngoba inganciphisa kakhulu ukubonakaliswa kwezakhi zofuzo ze-fibrogenic, i-morphology yeseli kanye ne-mitochondrial bioenergetics ngaphandle kokushintsha inani le-mtDNA [46]. Ama-uncouplers e-Mitochondrial angavimbela i-culture-induced fibrosis kanye nokusebenza kwe-HSC [47] futhi anciphise ukukhiqizwa kwe-mitochondrial ATP okulawulwa noma okubangelwa amaprotheni athile njenge-uncoupling proteins (UCP) noma i-adenine nucleotide translocase (ANT). Kuye ngohlobo lweseli, lesi simo singavikela amaseli ku-apoptosis kanye/noma sikhuthaze i-apoptosis [46]. Kodwa-ke, kudingeka izifundo ezengeziwe ukuze kucaciswe indima ye-IPA njenge-mitochondrial uncoupler ekuqalisweni kokusebenza kweseli le-hematopoietic stem.
Sabe sesihlola ukuthi izinguquko ekuphefumuleni kwe-mitochondrial zibonakala yini ekubunjweni kwe-mitochondrial kumaseli e-LX-2 aphilayo. Ngokuthakazelisayo, ukwelashwa kwe-TGF-β1 kushintsha isilinganiso se-mitochondrial kusuka ku-spherical kuya ku-medium, ngokuncipha kwamagatsha e-mitochondrial kanye nokwanda kokubonakaliswa kwe-DRP1, okuyisici esibalulekile ekuqhekekeni kwe-mitochondrial [48]. Ngaphezu kwalokho, ukuqhekeka kwe-mitochondrial kuhlotshaniswa nobunzima benethiwekhi iyonke, futhi ukuguquka kusuka ekuhlanganisweni kuya ekuqhekekeni kubalulekile ekusebenzeni kwe-hematopoietic stem cell (HSC), kanti ukuvinjelwa kokuqhekeka kwe-mitochondrial kuholela ku-HSC apoptosis [49]. Ngakho-ke, imiphumela yethu ikhombisa ukuthi ukwelashwa kwe-TGF-β1 kungabangela ukwehla kobunzima benethiwekhi ye-mitochondrial ngokuncipha kwamagatsha, okuyinto evame kakhulu ekuqhekekeni kwe-mitochondrial okuhlotshaniswa namaseli e-hematopoietic stem cell (HSCs) asebenzayo. Ngaphezu kwalokho, idatha yethu ibonise ukuthi i-IPA ingashintsha isilinganiso se-mitochondria kusuka ku-spherical kuya ku-medium shape, ngaleyo ndlela kuncishiswe ukubonakaliswa kwe-OPA1 kanye ne-MFN2. Izifundo zibonise ukuthi ukwehla kwe-OPA1 kungabangela ukwehla kwamandla e-mitochondrial membrane kanye nokubangela i-apoptosis yamaseli [50]. I-MFN2 yaziwa ngokuxhumanisa ukuhlanganiswa kwe-mitochondrial kanye ne-apoptosis [51]. Imiphumela etholakele isikisela ukuthi ukungeniswa kwamaseli e-LX-2 yi-TGF-β1 kanye/noma i-IPA kubonakala kushintsha ukuma nosayizi we-mitochondrial, kanye nesimo sokusebenza kanye nobunzima benethiwekhi.
Imiphumela yethu ikhombisa ukuthi ukwelashwa okuhlanganisiwe kwe-TGFβ-1 kanye ne-IPA kunganciphisa i-mtDNA kanye nemingcele yesimo seseli ngokulawula ukubonakaliswa kwe-mRNA kwe-fibrosis, i-apoptosis kanye nezakhi zofuzo ezihlobene nokusinda kumaseli agwema i-apoptosis. Ngempela, i-IPA yehlise izinga lokubonakaliswa kwe-mRNA kwezakhi zofuzo ze-AKT1 kanye nezakhi zofuzo ezibalulekile ze-fibrosis njenge-COL1A2 kanye ne-MMP2, kodwa yandisa izinga lokubonakaliswa kwe-CASP8, elihlotshaniswa ne-apoptosis. Imiphumela yethu ibonise ukuthi ngemva kokwelashwa kwe-IPA, ukubonakaliswa kwe-BAX kwehlile kanye nokubonakaliswa kwe-mRNA kwama-subunit omndeni we-TIMP1, i-BCL-2 kanye ne-NF-κB kwanda, okuphakamisa ukuthi i-IPA ingavuselela izimpawu zokusinda kumaseli e-hematopoietic stem (ama-HSC) agwema i-apoptosis. Lawa ma-molecule angasebenza njengezibonakaliso zokusinda kumaseli e-hematopoietic stem asebenzayo, okungase kuhlotshaniswe nokubonakaliswa okwandisiwe kwamaprotheni alwa ne-apoptotic (njenge-Bcl-2), ukubonakaliswa okwehla kwe-pro-apoptotic BAX, kanye nokuxhumana okuyinkimbinkimbi phakathi kwe-TIMP ne-NF-κB [5, 7]. I-IPA isebenzisa imiphumela yayo nge-PXR, futhi sithole ukuthi ukwelashwa okuhlangene ne-TGF-β1 kanye ne-IPA kwandise amazinga okuvezwa kwe-PXR mRNA, okubonisa ukucindezelwa kokusebenza kwe-HSC. Ukusayinwa kwe-PXR okusebenzayo kwaziwa ukuthi kuvimbela ukusebenza kwe-HSC kokubili ku-vivo kanye naku-vitro [52, 53]. Imiphumela yethu ikhombisa ukuthi i-IPA ingabamba iqhaza ekususweni kwama-HSC asebenzayo ngokukhuthaza i-apoptosis, ukunciphisa i-fibrosis kanye ne-metabolism ye-mitochondrial, kanye nokuthuthukisa izimpawu zokusinda, okuyizinqubo ezijwayelekile eziguqula i-phenotype ye-HSC esebenze ibe yileyo engasebenzi. Enye incazelo engaba khona yendlela engaba khona kanye nendima ye-IPA ku-apoptosis ukuthi isusa i-mitochondria engasebenzi kahle ikakhulukazi nge-mitophagy (indlela yangaphakathi) kanye nendlela yokusayinwa ye-TNF yangaphandle (Ithebula 1), exhunywe ngqo nendlela yokusayinwa kokusinda ye-NF-κB (Isithombe Esingeziwe 7). Ngokuthakazelisayo, izakhi zofuzo ezicebile ezihlobene ne-IPA ziyakwazi ukuvusa izimpawu ze-pro-apoptotic kanye ne-pro-survival endleleni ye-apoptotic [54], okuphakamisa ukuthi i-IPA ingase ivuse indlela ye-apoptotic noma ukusinda ngokusebenzisana nalezi zakhi zofuzo. Kodwa-ke, ukuthi i-IPA ivuse kanjani i-apoptosis noma ukusinda ngesikhathi sokusebenza kwe-HSC kanye nezindlela zayo zokusebenza azikacaci.
I-IPA iyi-metabolite yama-microbial eyakhiwe nge-tryptophan yokudla nge-gut microbiota. Izifundo zikhombisile ukuthi inezakhiwo zokulawula ezilwa nokuvuvukala, i-antioxidant, kanye ne-epigenetic endaweni yamathumbu.[55] Izifundo zikhombisile ukuthi i-IPA ingalawula ukusebenza kwesithiyo samathumbu futhi inciphise ukucindezeleka kwe-oxidative, okungaba nomthelela emiphumeleni yayo yemvelo yendawo.[56] Eqinisweni, i-IPA ithuthwa ezithweni eziqondiwe ngokujikeleza kwegazi, futhi njengoba i-IPA inesakhiwo esifanayo se-metabolite esikhulu ne-tryptophan, i-serotonin, kanye ne-indole derivatives, i-IPA inezenzo ze-metabolic eziholela eziphethweni ze-metabolic ezincintisanayo.[52] I-IPA ingase incintisane nama-metabolite atholakala nge-tryptophan ukuze ibophe izindawo kuma-enzyme noma kuma-receptor, okungase kuphazamise izindlela ezivamile ze-metabolic. Lokhu kugcizelela isidingo socwaningo olwengeziwe nge-pharmacokinetics yayo kanye ne-pharmacodynamics ukuze kuqondwe kangcono ifasitela layo lokwelapha.[57] Kusazobonakala ukuthi lokhu kungenzeka yini nakuma-hematopoietic stem cells (HSCs).
Siyavuma ukuthi ucwaningo lwethu lunemikhawulo ethile. Ukuze sihlole ngqo ukuhlobana okuhlobene ne-IPA, sikhiphe iziguli ezinesifo sikashukela sohlobo lwesibili (i-T2DM). Siyavuma ukuthi lokhu kunciphisa ukusebenza okubanzi kokutholakele kwethu ezigulini ezinesifo sikashukela sohlobo lwesibili kanye nesifo sesibindi esithuthukile. Nakuba ukuhlushwa kwe-IPA emzimbeni womuntu kungu-1–10 μM [11, 20], ukuhlushwa kwe-IPA engu-1 mM kukhethwe ngokusekelwe ekuhlushweni okuphezulu okungenabo ubuthi [15] kanye nesilinganiso esiphezulu se-apoptosis, kungekho mehluko ephesenti labantu abasele be-necrotic. Nakuba amazinga e-IPA angaphezulu kwe-physiological asetshenziswe kulolu cwaningo, okwamanje akukho ukuvumelana mayelana nomthamo osebenzayo we-IPA [52]. Nakuba imiphumela yethu ibalulekile, isiphetho esibanzi se-metabolic se-IPA sisalokhu siyindawo esebenzayo yocwaningo. Ngaphezu kwalokho, okutholakele kwethu ekuhlotsheni phakathi kwamazinga e-IPA e-serum kanye ne-methylation ye-DNA ye-liver transcripts atholakalanga kuphela kuma-hematopoietic stem cells (HSCs) kodwa nasezicutshini zesibindi. Sikhethe ukusebenzisa amaseli e-LX-2 omuntu ngokusekelwe kokutholakele kwethu kwangaphambilini okuvela ekuhlaziyweni kwe-transcriptome ukuthi i-IPA ihlotshaniswa nokusebenza kwe-hematopoietic stem cell (HSC) [15], kanti ama-HSC angamaseli amakhulu ahilelekile ekuqhubekeni kwe-fibrosis yesibindi. Isibindi sakhiwe ngezinhlobo eziningi zamaseli, ngakho-ke amanye amamodeli amaseli afana nesistimu yokukhulisa amaseli e-hepatocyte-HSC-immune ehlanganiswe nokusebenza kwe-caspase kanye nokuqhekeka kwe-DNA kanye nendlela yokusebenza okuhlanganisa nezinga leprotheni kufanele kucatshangelwe ukuze kufundwe indima ye-IPA kanye nokusebenzisana kwayo nezinye izinhlobo zamaseli esibindi.