Ngaphambilini sibike bona amazinga we-serum we-gut-derived tryptophan metabolite indole-3-propionic acid (IPA) aphasi eengulini ezine-liver fibrosis. Kulesi sifundo, siphenye nge-transcriptome ne-DNA methylome esibindini esikhulupheleko malungana namazinga we-serum IPA, kanye nendima ye-IPA ekukhuthazeni ukungasebenzi kwamaseli we-hepatic stellate (HSCs) ku-vitro.
Irhubhululo lifaka hlangana iingulani ezikhulupheleko ezili-116 ezinganasifo seswekile (T2DM) (iminyaka ema-46.8 ± 9.3; i-BMI: 42.7 ± 5.0 kg/m2) ezahlinzwa nge-bariatric e-Kuopio Bariatric Surgery Center (KOBS). Amazinga we-IPA ajikelezako alinganiswa nge-liquid chromatography-mass spectrometry (LC-MS), ukuhlaziywa kwe-liver transcriptome kwenziwa ngokulandelana kwe-RNA epheleleko, begodu ukuhlaziywa kwe-DNA methylation kwenziwa kusetjenziswa i-Infinium HumanMethylation450 BeadChip. Amaseli we-hepatic stellate womuntu (LX-2) asetjenziselwa ukuhlolwa kwe-in vitro.
Amazinga we-IPA we-serum ahlotjaniswa nokuvezwa kwamajini abandakanyeka eendleleni ze-apoptotic, ze-mitophagic, nezokuphila isikhathi eside esibindini. Ijini le-AKT serine/threonine kinase 1 (AKT1) bekulijini elinengi khulu nelisebenzisana khulu emtlolweni wesibindi kanye namaphrofayili we-DNA methylation. Ukwelatjhwa nge-IPA kwabangela i-apoptosis, ukwehlisa ukuphefumula kwe-mitochondrial, begodu kwatjhugulula ukwakheka kwamaseli kanye nokusebenza kwe-mitochondrial ngokutjhugulula ukuvezwa kwamajini aziwako bona alawula i-fibrosis, i-apoptosis, nokuphila kwamaseli we-LX-2.
Nakuthathwa ndawonye, ​​idatha le isekela bona i-IPA inemiphumela yokwelapha begodu ingabangela i-apoptosis begodu itjhugulule i-HSC phenotype ibe sebujameni obungasebenziko, ngaleyo ndlela kwandise amathuba wokuvimbela i-liver fibrosis ngokuphazamisa ukusebenza kwe-HSC kanye nokugaya ukudla kwe-mitochondrial.
Ukusabalala kokukhuluphala khulu nesifo sokugaya ukudla kuhlotjaniswa nokwanda kwezigameko zesifo sesibindi esinamafutha esihlobene nokugaya ukudla (MASLD); ubulwele buthinta ama-25% ukuya kwama-30% wabantu [1]. Umphumela omkhulu we-MASLD etiology yi-liver fibrosis, ikambiso enamandla ebonakala ngokubuthelela okuragela phambili kwe-fibrous extracellular matrix (ECM) [2]. Amaseli amakhulu abandakanyeka ekuvuvukeni kwesibindi ngamaseli we-hepatic stellate (ama-HSC), atjengisa ama-phenotypes amane aziwako: athulileko, asebenzako, angasebenziko, kanye nama-senescent [3, 4]. Ama-HSC angasebenza begodu atjhuguluke ukusuka esimweni esithulileko abe ngamaseli afana ne-fibroblast aneemfuno eziphezulu zamandla, ngokuvezwa okukhulu kwe-α-smooth muscle actin (α-SMA) kanye ne-type I collagen (Col-I) [5, 6]. Ngesikhathi sokubuyiselwa emuva kwe-fibrosis yesibindi, ama-HSC asebenzako aqedwa nge-apoptosis nofana ukungasebenzi. Iinqubo lezi zifaka hlangana ukwehla kwamajini we-fibrogenic nokuguqulwa kwamajini we-prosurvival (njenge-NF-κB kanye ne-PI3K/Akt iindlela zokubonisa) [7, 8], kanye nokutjhuguluka kwamandla we-mitochondrial nomsebenzi [9].
Amazinga we-serum we-tryptophan metabolite i-indole-3-propionic acid (IPA), ekhiqizwa emathunjini, atholakele bona ehlile emalwelweni womuntu afaka hlangana i-MASLD [10–13]. I-IPA ihlotjaniswa nokuthatha ifayibha yokudla, yaziwa ngemiphumela yayo yokulwisana nokuvuvuka, begodu yehlisa i-phenotype ye-non-alcoholic steatohepatitis (NASH) ebangelwa kukudla ku-vivo naku-vitro [11-14]. Ubufakazi obuthileko buvela esifundweni sethu sangaphambilini, esitjengise bona amazinga we-serum IPA bekaphasi eengulini ezine-liver fibrosis kunasezigulini ezikhulupheleko ezingana-liver fibrosis ku-Kuopio Bariatric Surgery Study (KOBS). Ukudlula lapho, sitjengise bona ukwelatjhwa nge-IPA kungehlisa ukuvezwa kwamajini angamatshwayo weklasikhi wokunamathelana kwamaseli, ukufuduka kwamaseli kanye nokusebenza kwamaseli we-hematopoietic stem kumodeli ye-hepatic stellate cell (LX-2) begodu kungaba yi-metabolite yokuvikela isibindi [15]. Kodwana, akukacaci bona i-IPA yenza njani ukubuyela emuva kwe-liver fibrosis ngokusebenzisa i-HSC apoptosis kanye ne-mitochondrial bioenergetics.
Lapha, sitjengisa bona i-IPA ye-serum ihlotjaniswa nokuvezwa kwamajini anothileko nge-apoptosis, i-mitophagy, kanye neendlela zokuphila isikhathi eside esibindini sabantu abakhulupheleko kodwana abangekho ngohlobo lwe-2 lwesifo seswekileko (KOBS). Ukudlula lapho, sithole bona i-IPA ingabanga ukuhlanzwa nokuwohloka kwamaseli weengazi (ama-HSC) ngendlela yokungasebenzi. Imiphumela le iveza indima etja ye-IPA, okwenza bona ibe lithagethi yokwelapha ukukhuthaza ukubuyela emuva kwe-fibrosis yesibindi.
Irhubhululo langaphambilini esiqhemeni se-KOBS litjengise bona iingulani ezine-fibrosis yesibindi bezinamazinga aphasi we-IPA nazimadaniswa nezigulani ezingana-liver fibrosis [15]. Ukukhipha umphumela ophazamisako womhlobo we-2 wobulwelwe betjhukela, saqasha iingulani ezikhulupheleko eziyi-116 ezinganawo umhlobo we-2 wobulwelwe betjhukela (iminyaka ephakathi ± SD: 46.8 ± 9.3 iminyaka; BMI: 42.7 ± 5.0 kg/m2) (Ithebula 1) ukusuka esifundweni esiragela phambili se-KOBS njengesibalo sokufunda [16]. Boke abahlanganyeli banikela imvumo etloliweko begodu iphrothokholi yokufunda yavunywa yiKomidi yokuZiphatha ye-North Savo County Hospital ngokuya kweSimemezelo se-Helsinki (54/2005, 104/2008 kanye no-27/2010).
Iimbonelo ze-biopsy yesibindi zatholwa ngesikhathi sokuhlinzwa kwe-bariatric begodu zahlolwa ngokwe-histologically ziingcwethi ezinolwazi ngokuya kwemibandela echazwe ngaphambilini [17, 18]. Iindlela zokuhlola zifingqwe kuThebuli lokuNgezelelako S1 begodu sele zichazwe ngaphambilini [19].
Amasampula we-serum yokuzila ukudla ahlaziywa nge-untargeted liquid chromatography-mass spectrometry (LC-MS) ukuhlaziywa kwe-metabolomics (n = 116). Amasampula ahlaziywa kusetjenziswa ihlelo le-UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) njengombana kuchaziwe ngaphambilini19. Ukukhonjwa kwe-isopropyl alcohol (IPA) bekusekelwe esikhathini sokugcinwa nokumadanisa i-MS/MS spectrum namazinga ahlanzekileko. Ukuqina kwesiginali ye-IPA (indawo ephezulu) kwaqalwa kikho koke ukuhlaziywa [20].
Ukulandelana kwe-RNA yesibindi soke kwenziwa kusetjenziswa i-Illumina HiSeq 2500 begodu idatha yalungiswa ngaphambilini njengombana kuchaziwe ngaphambilini [19, 21, 22]. Senze ukuhlaziywa kokuvezwa okuhlukileko okuqothelweko kwemitlolo ethinta umsebenzi we-mitochondrial/i-biogenesis sisebenzisa amajini ayi-1957 akhethwe ku-MitoMiner 4.0 database [23]. Ukuhlaziywa kwe-DNA yesibindi kwenziwa kusetjenziswa i-Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) kusetjenziswa indlela efanako naleyo echazwe ngaphambilini [24, 25].
Amaseli we-hepatic stellate womuntu (LX-2) anikelwa ngomusa nguProf. Stefano Romeo begodu akhuliswa begodu agcinwa ku-DMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 107-1601). Ukukhetha umthamo wokusebenza we-IPA, amaseli we-LX-2 alatjhwa ngamazinga ahlukileko we-IPA (10 μM, 100 μM kanye ne-1 mM; Sigma, 220027) ku-DMEM/F12 medium ama-awa ama-24. Ukudlula lapho, ukuphenya ikghono le-IPA lokuqeda ama-HSC, amaseli we-LX-2 alatjhwa ngokubambisana nge-5 ng/ml TGF-β1 (R&D systems, 240-B-002/CF) kanye ne-1 mM IPA endaweni engena-serum ama-awa ama-24. Ukulawulwa kwekoloyi okuhambelanako, i-4 nM HCL ene-0.1% BSA yasetjenziselwa ukwelapha i-TGF-β1 begodu i-0.05% DMSO yasetjenziselwa ukwelapha i-IPA, begodu zombili zasetjenziswa ndawonye ekwelapheni okuhlanganisiweko.
I-apoptosis yahlolwa kusetjenziswa i-FITC Annexin V Apoptosis Detection Kit ene-7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) ngokuya kwemilayo yomkhiqizi. Kafitjhani, i-LX-2 (1 × 105 amaseli/umgodi) yakhuliswa ubusuku boke emapuletini we-12-well bese yaphathwa ngemithamo eminengi ye-IPA nofana i-IPA kanye ne-TGF-β1. Ngelanga elilandelako, amaseli antantako nanamathelako abuthelelwa, afakwa i-trypsin, ahlanzwa nge-PBS, afakwa kabutjha ku-Annexin V binding buffer, begodu afakwa nge-FITC-Annexin V kanye ne-7-AAD imizuzu eli-15.
Ama-mitochondria emaseli aphilako ahlanjululwa ukusebenza kwe-oxidative kusetjenziswa i-Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Ukuhlolwa kwe-MTR, amaseli we-LX-2 afukanywa nge-density elinganako ne-IPA ne-TGF-β1. Ngemva kwama-awa ama-24, amaseli aphilako ahlanjululwa, ahlanzwa nge-PBS, bese afakwa nge-100 μM MTR ngaphakathi kwe-serum-free medium ku-37 °C imizuzu ema-20 njengombana kuchaziwe ngaphambilini [26]. Ukuhlaziywa kwesakhiwo samaseli aphilako, ubukhulu bamaseli nobunzima be-cytoplasmic kwahlaziywa kusetjenziswa amapharamitha wokusabalalisa phambili (FSC) kanye nokusabalalisa eceleni (SSC), ngokulandelana.
Yonke idatha (izehlakalo ezingama-30,000) yatholwa kusetjenziswa i-NovoCyte Quanteon (Agilent) begodu yahlaziywa kusetjenziswa isofthiwe ye-NovoExpress® 1.4.1 nofana i-FlowJo V.10.
Izinga lokusetjenziswa kwe-oksijini (i-OCR) kanye nezinga lokukhiqizwa kwe-extracellular (ECAR) kwalinganiswa ngesikhathi sangempela kusetjenziswa i-Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) ene-Seahorse XF Cell Mito Stress ngokuya kwemilayo yomenzi. Kafitjhani, amaseli we-LX-2 ama-2 × 104/umthombo atjalwa emapuletini wesiko lamaseli we-XF96. Ngemva kokufukamela ubusuku boke, amaseli alatjhwa nge-isopropanol (IPA) kanye ne-TGF-β1 (Iindlela ezingezelelweko 1). Ukuhlaziywa kwedatha kwenziwa kusetjenziswa isofthiwe ye-Seahorse XF Wave, efaka hlangana i-Seahorse XF Cell Energy Phenotype Test Report Generator. Ukusuka kilokhu, i-Bioenergetic Health Index (BHI) yabalwa [27].
I-RNA epheleleko yatlolwa yaba yi-cDNA. Ukuze uthole iindlela ezithileko, qala ireferensi [15]. Amazinga we-Human 60S ribosomal acidic protein P0 (RPLP0) kanye ne-cyclophilin A1 (PPIA) mRNA asetjenziswa njengezilawuli zamajini. I-QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, The Netherlands) yasetjenziswa ne-TaqManTM Fast Advanced Master Mix Kit (Applied Biosystems) nofana i-Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), begodu ukuvezwa kwezakhi zofuzo okuhlobeneko kwabalwa kusetjenziswa i-cyc amapharamitha (ΔΔCt) kanye nendlela ye-∆∆Ct. Imininingwana yama-primers inikelwe kuThebuli yokuNgezelela i-S2 ne-S3.
I-DNA yenyukliya (ncDNA) kanye ne-mitochondrial DNA (mtDNA) zakhishwa kusetjenziswa i-DNeasy blood and tissue kit (Qiagen) njengombana kuchaziwe ngaphambilini [28]. Inani elihlobeneko le-mtDNA labalwa ngokubala isilinganiso sendawo ngayinye ye-mtDNA ehlosiweko ukuya esilinganisweni sejometri sezifunda ezintathu ze-DNA yenyukliya (mtDNA/ncDNA), njengombana kuvezwe ku-Supplementary Methods 2. Imininingwana yama-primers we-mtDNA ne-ncDNA inikelwe ku-Supplementary Table S4.
Amaseli aphilako ahlanjululwe nge-MitotrackerTM Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) ukubona ngeso lengqondo amanethiwekhi we-mitochondrial aphakathi kwamaseli nangaphakathi kwamaseli. Amaseli we-LX-2 (amaseli ayi-1 × 104/umthombo) akhuliswa emaslayidini weglasi emapuletini wokukhulisa ane-glass-bottomed (Ibidi GmbH, Martinsried, Germany). Ngemva kwama-awa ama-24, amaseli we-LX-2 aphilako afukanywa nge-100 μM MTR imizuzu ema-20 ku-37 °C begodu ama-nuclei wamaseli ahlanjululwe nge-DAPI (1 μg/ml, Sigma-Aldrich) njengombana kuchaziwe ngaphambilini [29]. Amanethiwekhi we-mitochondrial abonwa ngokusebenzisa i-Zeiss Axio Observer inverted microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) ene-Zeiss LSM 800 confocal module ku-37 °C emmoyeni onomswakama one-5% CO2 kusetjenziswa i-63×NA 1.3 objective. Sithole iinthombe ezilitjhumi ze-Z-series zomhlobo ngamunye wesampula. I-Z-series ngayinye ineengaba ezingama-30, enye nenye enobukhulu obungu-9.86 μm. Isampula ngayinye, iinthombe zemikhakha elitjhumi ehlukileko yokubuka zitholwe kusetjenziswa isofthiwe ye-ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany), begodu ukuhlaziywa kwe-mitochondrial morphology kwenziwa kusetjenziswa isofthiwe ye-ImageJ (v1.54d) [30, 31] ngokuya ngamapharamitha achazwe ku-Supplementary Methods 3.
Amaseli alungiswa nge-2% glutaraldehyde ku-0.1 M phosphate buffer, kwalandela ukulungiswa nge-1% osmium tetroxide solution (Sigma Aldrich, MO, USA), kancanikancani aphelelwa mamanzi nge-acetone (Merck, Darmstadt, Germany), begodu ekugcineni afakwa nge-epoxy resin. Iingcenye ezincani khulu zalungiswa begodu zafakwa ibala nge-1% ye-uranyl acetate (Merck, Darmstadt, Germany) kanye ne-1% ye-lead citrate (Merck, Darmstadt, Germany). Iinthombe ze-ultrastructural zitholwe kusetjenziswa i-JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) nge-accelerating voltage ye-80 kV.
I-morphology yamaseli we-LX-2 aphathwe nge-IPA ama-awa ama-24 yahlaziywa nge-phase-contrast microscopy nge-50x magnification kusetjenziswa i-Zeiss inverted light microscope (Zeiss Axio Vert.A1 kanye ne-AxioCam MRm, Jena, Germany).
Idatha yezokwelapha yavezwa njengesilinganiso ± ukuphambuka okujayelekileko nofana okuphakathi (ububanzi be-interquartile: IQR). Ukuhlaziywa kwendlela eyodwa yokuhlukahluka (amatjhuguluko aragela phambili) nofana ukuhlolwa kwe-χ2 (amatjhuguluko wesigaba) kwasetjenziselwa ukumadanisa umehluko hlangana namaqembu amathathu wokufunda. Izinga lokungasiqiniso (i-FDR) lasetjenziselwa ukulungisa ukuhlolwa okunengi, begodu amajini ane-FDR < 0.05 athathwa njengabalulekileko. Ukuhlaziywa kokuhlobana kwe-Spearman kwasetjenziselwa ukuhlobanisa i-CpG DNA methylation namandla wesignali ye-IPA, ngamanani we-p (p <0.05) abikwe.
Ukuhlaziywa kwendlela kwenziwa kusetjenziswa ithulusi lokuhlaziywa kwamajini asekelwe kuwebhu (i-WebGestalt) yemitlolo ema-268 (nominal p <0.01), imitlolo ema-119 ehlobene ne-mitochondria (nominal p <0.05), kanye nama-CpG ama-4350 ngaphandle kwama-3093 wemitlolo yesibindi ebegade ihlotjaniswa namazinga we-IPA we-serum. Ithuluzi le-Venny DB (inguqulo 2.1.0) elitholakala simahla lasetjenziselwa ukuthola amajini ahlangana, begodu i-StringDB (inguqulo 11.5) yasetjenziselwa ukubona ngelihlo lengqondo ukusebenzisana kwamaphrotheyini namaphrotheyini.
Ekuhlolweni kwe-LX-2, amasampula ahlolwa ukujayelekile kusetjenziswa i-D'Agostino-Pearson test. Idatha yatholwa okungenani ekuphindaphindweni okuthathu kwezinto eziphilako begodu yafakwa ngaphasi kwe-ANOVA yendlela eyodwa nge-Bonferroni post hoc test. Inani le-p elingaphasi kwe-0.05 belithathwa njengeliqakathekileko. Idatha ivezwa njenge-mean ± SD, begodu inani lokuhlolwa litjengiswe emfanekisweni ngamunye. Konke ukuhlaziywa namagrafu kwenziwa kusetjenziswa isofthiwe ye-GraphPad Prism 8 ye-Windows (I-GraphPad Software Inc., version 8.4.3, San Diego, USA).
Kokuthoma, sihlole ukuhlobana kwamazinga we-IPA we-serum nesibindi, umzimba woke, kanye nemibhalo ye-mitochondrial. Kuphrofayili epheleleko, ijini enamandla khulu ehlotjaniswa namazinga we-IPA ye-serum bekuyi-MAPKAPK3 (FDR = 0.0077; i-mitogen-activated protein kinase-activated protein kinase 3); ku-mitochondria-related transcript profile, isakhi esinamandla khulu esihlotjaniswako bekuyi-AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Ifayela elengeziweko 1 kanye nefayela elengeziweko 2).
Bese sihlaziya imitlolo yephasi loke (n = 268; p < 0.01) kanye nemitlolo ehlobene ne-mitochondria (n = 119; p < 0.05), ekugcineni sikhombe i-apoptosis njengendlela eqakathekileko khulu (p = 0.0089). Malungana nama-mitochondrial transcripts ahlotjaniswa namazinga we-serum IPA, sitjheje khulu i-apoptosis (FDR = 0.00001), i-mitophagy (FDR = 0.00029), kanye neendlela zokubonisa i-TNF (FDR = 0.000006) (Umfanekiso 1A, Ithebula 2, kanye nama-Supplementary Figures 1-B).
Ukuhlaziywa okudlulako kwephasi loke, imibhalo ehlobene ne-mitochondria, kanye ne-DNA methylation esibindini somuntu ngokuhlobana namazinga we-serum IPA. I-A ijamele imitlolo yephasi loke ema-268, imitlolo ema-119 ehlotjaniswa nama-mitochondria, kanye nama-transkripthi we-DNA methylated ahlanganiswe kumasayithi we-3092 we-CpG ahlobene namazinga we-serum IPA (amanani we-p < 0.01 wemitlolo yephasi loke kanye ne-DNA methylated, kanye namanani we-p < 0.00 we-mitochondrial transcript). Imitlolo emikhulu edlulako itjengiswa phakathi (i-AKT1 ne-YKT6). B Imephu yokusebenzisana yamajini ali-13 anezinga eliphezulu lokusebenzisana (0.900) namanye amajini yakhiwa ukusuka kumajini ama-56 ahlangana (isifunda somgqa omnyama) ebekahlotjaniswa khulu namazinga we-IPA we-serum kusetjenziswa ithulusi le-inthanethi i-StringDB. Okuluhlaza: Amajini ahlelwe ku-Gene Ontology (GO) ingcenye yamaseli: i-mitochondria (GO:0005739). I-AKT1 yiphrotheyini enesikoro esiphezulu (0.900) sokusebenzisana namanye amaphrotheyini ngokuya ngedatha (ngokuya ngokumba umtlolo, ukuhlolwa, ama-database, nokuvezwa ngokubambisana). Amanothi wenethiwekhi ajamele amaphrotheyini, begodu imiphetho ijamele ukuhlobana hlangana namaphrotheyini.
Njengombana ama-gut microbiota metabolites angalawula ukwakheka kwe-epigenetic ngokusebenzisa i-DNA methylation [32], siphenye bona amazinga we-serum IPA ahlotjaniswa na ne-liver DNA methylation. Sithole bona iindawo ezimbili ezikulu ze-methylation ezihlobene namazinga we-serum IPA beziseduze ne-proline-serine-rich region 3 (C19orf55) kanye ne-heat shock protein family B (small) member 6 (HSPB6) (Ifayela elengeziweko 3). I-DNA methylation ye-4350 CpG (p < 0.01) beyihlotjaniswa namazinga we-serum IPA begodu beyinothiswe eendleleni zokulawula isikhathi eside (p = 0.006) (Isithombe 1A, Ithebula 2, kanye nesithombe esingezelelweko 1C).
Ukuzwisisa iindlela zebhayoloji ezisekela ukuhlobana hlangana namazinga we-serum IPA, imitlolo yephasi loke, imitlolo ehlotjaniswa ne-mitochondria, kanye ne-DNA methylation esibindini somuntu, senze ukuhlaziywa kokuhlangana kwamajini akhonjwe ekuhlaziyweni kwendlela yangaphambilini (Umfanekiso 1A). Imiphumela yokuhlaziywa kokunothisa kwendlela yamajini ama-56 ahlangana (ngaphakathi komgqa omnyama kuMfanekiso 1A) itjengise bona indlela ye-apoptosis (p = 0.00029) iveze amajini amabili avamileko ekuhlaziyweni okuthathu: i-AKT1 ne-YKT6 (YKT6 v-SNARE homolog), njengombana kutjengisiwe emfanekisweni we-Ven (Umfanekiso 2 1A). Ngokukarisako, sithole bona i-AKT1 (cg19831386) kanye ne-YKT6 (cg24161647) bezihlobene kuhle namazinga we-serum IPA (Ifayela elengeziweko 3). Ukukhomba ukusebenzisana kwamaphrotheyini hlangana nemikhiqizo yamajini, sakhetha amajini ali-13 anesikoro esiphezulu sesifunda esijayelekileko (0.900) hlangana namajini ali-56 ahlangana njengesifakelo begodu sakha imephu yokusebenzisana. Ngokuya ngezinga lokuzithemba (ukuzithemba okuncani), ijini le-AKT1 elinamaphuzu aphezulu (0.900) beliphezulu (Umfanekiso 1B).
Ngokuya ngokuhlaziywa kwendlela, sithole bona i-apoptosis bekuyindlela ekulu, yeke saphenya bona ukwelatjhwa nge-IPA kuzokuthinta na i-apoptosis yama-HSCs ku-vitro. Sitjengise ngaphambilini bona imithamo ehlukileko ye-IPA (10 μM, 100 μM, kanye ne-1 mM) beyinganabuthi kumaseli we-LX-2 [15]. Irhubhululo leli litjengise bona ukwelatjhwa nge-IPA nge-10 μM kanye ne-100 μM kwandisa inani lamaseli aphilako nawokufa. Kodwana, nayiqathaniswa nesiqhema sokulawula, ukuphila kwamaseli kwehla nge-1 mM IPA, lokha izinga lokubhubha kwamaseli lingatjhuguluki (Umfanekiso 2A, B). Okulandelako, ukuthola ukugxila okuhle ukubangela i-apoptosis kumaseli we-LX-2, sihlole i-10 μM, i-100 μM, kanye ne-1 mM IPA ama-awa ama-24 (Umfanekiso 2A-E kanye noMfanekiso ongeziweko 3A-B). Ngokuthakazelisako, i-IPA 10 μM ne-100 μM yehlisa izinga lokufa (%), nanyana kunjalo, i-IPA 1 mM yakhuphula izinga lokufa ngemva kwesikhathi kanye nezinga lokufa (%) nayiqathaniswa nokulawula begodu ngalokho yakhethelwa ukuhlolwa okuragela phambili (Imifanekiso 2A-D).
I-IPA ibanga ukubulawa kwamaseli we-LX-2. I-Annexin V kanye ne-7-AAD indlela yokufaka amabala kabili yasetjenziselwa ukulinganisa izinga lokufa kanye nokubunjwa kwamaseli nge-flow cytometry. Amaseli we-BA afukanywa nge-10 μM, 100 μM kanye ne-1 mM IPA ama-awa ama-24 nofana nge-F-H TGF-β1 (5 ng/ml) kanye ne-1 mM IPA endaweni engana-serum ama-awa ama-24. A: amaseli aphilako (I-Annexin V -/ 7AAD-); B: amaseli we-necrotic (Annexin V -/ 7AAD+); C, F: ekuthomeni (I-Annexin V +/ 7AAD-); D, G: late (Annexin V+/7AAD.+); E, H: iphesenteji yamaseli we-apoptotic yokuthoma newemuva ngezinga lokufa (%). Idatha ivezwa njengesilinganiso ± SD, n = 3 ukuhlolwa okuzijameleko. Ukumadanisa iimbalo kwenziwa kusetjenziswa i-ANOVA yendlela eyodwa nge-Bonferroni post hoc test. *p < 0.05; ****p < 0.0001
Njengombana sitjengise ngaphambilini, i-5 ng/ml ye-TGF-β1 ingabanga ukusebenza kwe-HSC ngokukhuphula ukuvezwa kwamajini we-classic marker [15]. Amaseli we-LX-2 alatjhwa nge-5 ng/ml TGF-β1 kanye ne-1 mM IPA ngokuhlanganiswa (Umfanekiso 2E–H). Ukwelatjhwa nge-TGF-β1 azange kutjhugulule izinga lokufa, nanyana kunjalo, ukwelatjhwa ngokubambisana kwe-IPA kwandisa izinga lokufa kwe-apoptosis ne-apoptosis (%) nayiqathaniswa nokwelatjhwa nge-TGF-β1 (Umfanekiso 2E–H). Imiphumela le itjengisa bona i-1 mM IPA ingakhuthaza i-apoptosis kumaseli we-LX-2 ngaphandle kokungeniswa kwe-TGF-β1.
Siragele phambili nokuphenya umphumela we-IPA ekuphefumuleni kwe-mitochondrial kumaseli we-LX-2. Imiphumela itjengise bona i-1 mM IPA yehlisa amapharamitha wezinga lokusetjenziswa kwe-oksijini (OCR): ukuphefumula okungekho kwe-mitochondrial, ukuphefumula okusezingeni eliphasi nokukhulu, ukuvuza kwephrothoni nokukhiqizwa kwe-ATP nayiqathaniswa nesiqhema sokulawula (Umfanekiso 3A, B), ngesikhathi i-bioenergetic health index (BHI) ingatjhugulukanga.
I-IPA yehlisa ukuphefumula kwe-mitochondrial kumaseli we-LX-2. I-mitochondrial respiration curve (OCR) yethulwa njengamapharamitha wokuphefumula kwe-mitochondrial (ukuphefumula okungekho kwe-mitochondrial, ukuphefumula okusezingeni eliphezulu, ukuphefumula okukhulu, ukuvuza kwephrothoni, ukukhiqizwa kwe-ATP, i-SRC kanye ne-BHI). Amaseli A no-B afukanywa nge-10 μM, 100 μM kanye ne-1 mM IPA ama-awa ama-24, ngokulandelana. Amaseli C no-D afakwa nge-TGF-β1 (5 ng/ml) kanye ne-1 mM IPA ngaphakathi kwe-serum-free medium ama-awa ama-24, ngokulandelana. Zoke iinlinganiso zalungiswa ngokuqukethwe kwe-DNA kusetjenziswa ikhithi ye-CyQuant. I-BHI: inkomba yezamaphilo ye-bioenergetic; I-SRC: umthamo wokuphefumula; I-OCR: izinga lokusetjenziswa kwe-oksijini. Idatha ivezwa njengesilinganiso ± ukuphambuka okujayelekileko (SD), n = 5 ukuhlolwa okuzijameleko. Ukumadanisa iimbalo kwenziwa kusetjenziswa i-ANOVA ye-one-way kanye ne-Bonferroni post hoc test. *p < 0.05; **p < 0.01; begodu ***p < 0.001
Ukuthola ukuzwisisa okupheleleko komphumela we-IPA kuphrofayili ye-bioenergetic yamaseli we-TGF-β1-activated LX-2, sihlaziye i-mitochondrial oxidative phosphorylation nge-OCR (Fig. 3C,D). Imiphumela itjengise bona ukwelatjhwa nge-TGF-β1 kunganciphisa ukuphefumula okukhulu, amandla wokuphefumula (SRC) kanye ne-BHI nayiqathaniswa nesiqhema sokulawula (Umfanekiso 3C,D). Ukungezelela, ukwelatjhwa okuhlanganisiweko kwehlise ukuphefumula kwe-basal, ukuvuza kwephrothoni nokukhiqizwa kwe-ATP, kodwana i-SRC ne-BHI beziphezulu khulu kunalezo eziphathwe nge-TGF-β1 (Umfanekiso 3C,D).
Siphinde senze "Ukuhlolwa Kwe-Phenotype Yamandla Amaseli" okunikelwe yi-software ye-Seahorse (Isithombe esingezelelweko 4A-D). Njengombana kutjengiswe ku-Supplementary Fig. 3B, zombili i-OCR ne-ECAR amandla we-metabolic yehla ngemva kokwelatjhwa nge-TGF-β1, nanyana kunjalo, akukho mehluko obonwako emaqenjini wokwelatjhwa okuhlanganisiweko ne-IPA nayiqathaniswa nesiqhema sokulawula. Ukudlula lapho, kokubili amazinga we-OCR aphasi nokugandeleleka kwehla ngemva kokuhlanganiswa nokwelatjhwa nge-IPA nayiqathaniswa nesiqhema sokulawula (Isithombe esingezelelweko 4C). Ngokuthakazelisako, iphetheni efanako yabonwa ngokwelapha okuhlanganisiweko, lapho kungekho ukutjhuguluka emazingeni we-ECAR aphasi nokugandeleleka okubonwako nayiqathaniswa nokwelatjhwa kwe-TGF-β1 (Isithombe esingezelelweko 4C). Ku-HSCs, ukwehla kwe-mitochondrial oxidative phosphorylation kanye nekghono lokwelatjhwa okuhlanganisiweko ukubuyisela i-SCR ne-BHI ngemva kokuvezwa ekwelatjhweni kwe-TGF-β1 azange kutjhugulule amandla wokugaya ukudla (i-OCR ne-ECAR). Nakuthathwa ndawonye, ​​imiphumela le itjengisa bona i-IPA ingehlisa i-bioenergetics kuma-HSC, okusikisela bona i-IPA ingabangela iphrofayili yamandla ephasi etjhugulula i-HSC phenotype ukuya ekungasebenzini (Isithombe esingezelelweko 4D).
Umphumela we-IPA ekuguquguqukeni kwe-mitochondrial kwaphenywa kusetjenziswa ukulinganisa okuthathu kwe-mitochondrial morphology nokuxhumana kwenethiwekhi kanye nokufakwa kwe-MTR (Umfanekiso 4 kanye noMfanekiso 5 wokungezelela). Umfanekiso 4 utjengisa bona, nayiqathaniswa nesiqhema sokulawula, ukwelatjhwa nge-TGF-β1 kwehlise indawo ephakathi, inani lamagatja, ubude begatja elipheleleko, kanye nenani lokuhlangana kwamagatja (Umfanekiso 4A no-B) begodu kwatjhugulula inani le-mitochondria ukusuka ebujameni obuyindilinga ukuya ebujamweni obuphakathi (Umfanekiso 4C). Ukwelatjhwa nge-IPA kwaphela kwehlise ivolumu ye-mitochondria begodu kwatjhugulula inani le-mitochondria ukusuka ebujameni obuyindilinga ukuya ebujamweni obuphakathi nayiqathaniswa nesiqhema sokulawula (Umfanekiso 4A). Ngokuphikisako, ubu-sphericity, ubude begatja eliphakathi, nomsebenzi we-mitochondrial ohlolwe yi-mitochondrial membrane potential-dependent MTR (Umfanekiso 4A no-E) wahlala ungatjhuguluki begodu amapharamitha la azange ahluke hlangana namaqembu. Nakuthathwa ndawonye, ​​imiphumela le iveza bona i-TGF-β1 kanye ne-IPA ukwelatjhwa kubonakala kutjhugulula ubujamo nobukhulu be-mitochondrial kanye nobunzima benethiwekhi kumaseli we-LX-2 aphilako.
I-IPA itjhugulula ama-mitochondrial dynamics kanye nobunengi be-mitochondrial DNA kumaseli we-LX-2. A. Iinthombe ezijameleko zamaseli we-LX-2 aphilako afakwe nge-TGF-β1 (5 ng/ml) kanye ne-1 mM IPA ama-awa ama-24 ngaphakathi kwe-serum-free medium atjengisa amanethiwekhi we-mitochondrial afakwe nge-MitotrackerTM Red CMXRos kanye nama-nuclei afakwe nge-DAPI. Yonke idatha ineenthombe okungenani ezili-15 ngesiqhema ngasinye. Sithole iinthombe ezili-10 ze-Z-stack zomhlobo ngamunye wesampula. Ukulandelana kwe-Z-axis ngayinye bekuneengcezu ezingama-30, enye nenye enobukhulu obungu-9.86 μm. Ibha yesikali: 10 μm. B. Izinto ezijameleko (ama-mitochondria kwaphela) ezikhonjwe ngokusebenzisa umkhawulo ovumelanako esithombeni. Ukuhlaziywa okulinganiselweko nokumadanisa ukuxhumana kwenethiwekhi ye-mitochondrial kwenziwa kiwo woke amaseli esiqhemeni ngasinye. C. Imvamisa yama-mitochondrial shape ratios. Amanani aseduze no-0 atjengisa amajamo ayindilinga, begodu amanani aseduze no-1 atjengisa amajamo anemicu. D Okuqukethwe kwe-Mitochondrial DNA (mtDNA) kwatholakala njengoba kuchaziwe ku-Materials and Methods. Ukuhlaziywa kwe-E MitotrackerTM Red CMXRos kwenziwa nge-flow cytometry (izehlakalo ezingama-30,000) njengombana kuchaziwe ku-Materials and Methods. Idatha ivezwe njengesilinganiso ± SD, n = 3 ukuhlolwa okuzijameleko. Ukumadanisa iimbalo kwenziwa kusetjenziswa i-ANOVA ye-one-way kanye ne-Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Bese sihlaziya okuqukethwe yi-mtDNA kumaseli we-LX-2 njengesibonisi senombolo ye-mitochondrial. Nawuqathaniswa nesiqhema sokulawula, okuqukethwe kwe-mtDNA kwakhuphuka esiqhemeni esiphathwe nge-TGF-β1 (Umfanekiso 4D). Nawuqathaniswa nesiqhema esiphathwe nge-TGF-β1, okuqukethwe kwe-mtDNA kwehle esiqhemeni esilatjhwa ngokuhlanganiswa (Umfanekiso 4D), okusikisela bona i-IPA inganciphisa okuqukethwe kwe-mtDNA mhlamunye inani le-mitochondrial kanye nokuphefumula kwe-mitochondrial (Umfanekiso 3C). Ngaphezu kwalokho, i-IPA beyibonakala yehlisa okuqukethwe kwe-mtDNA ekwelapheni okuhlanganisiweko kodwana azange ithinte umsebenzi we-mitochondrial ophakathi kwe-MTR (Imifanekiso 4A-C).
Sihlolisise ukuhlobana kwe-IPA namazinga we-mRNA wamajini ahlobene ne-fibrosis, i-apoptosis, ukuphila, kanye nokuguquguquka kwe-mitochondrial kumaseli we-LX-2 (Umfanekiso 5A-D). Nayiqathaniswa nesiqhema sokulawula, isiqhema esiphathwe nge-TGF-β1 sitjengisa ukuvezwa okukhulu kwamajini afana ne-collagen type I α2 chain (COL1A2), i-α-smooth muscle actin (αSMA), i-matrix metalloproteinase 2 (MMP2), i-tissue inhibitor of metalloproteinase 1 (TIMP1), kanye ne-gene-like gene (1D), okutjengisa i-gene ukwanda kwe-fibrosis nokusebenza. Ukudlula lapho, nayimadaniswa nesiqhema sokulawula, ukwelatjhwa nge-TGF-β1 kwehlise amazinga we-mRNA we-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 (Isithombe KBKB) 5A–D). Nayiqathaniswa nokwelatjhwa nge-TGF-β1, ukwelatjhwa okuhlanganisiweko nge-TGF-β1 kanye ne-IPA kwehlise ukuvezwa kwe-COL1A2 ne-MMP2, kodwana kwandisa amazinga we-mRNA we-PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, kanye ne-IKBKB. Ukwelatjhwa nge-IPA kwehlise khulu ukuvezwa kwe-MMP2, i-Bcl-2-associated protein X (BAX), i-AKT1, i-optic atrophy protein 1 (OPA1), kanye ne-mitochondrial fusion protein 2 (MFN2), lokha ukuvezwa kwe-CASP8, NFκB1A, NFκB1B, kanye ne-IKB kwakhuphuka nayiqathaniswa neqembu lokulawula. Kodwana, akukho mehluko otholakeleko ekuvezweni kwe-caspase-3 (CASP3), i-apoptotic peptidase activating factor 1 (APAF1), i-mitochondrial fusion protein 1 (MFN1), kanye ne-fission protein 1 (FIS1). Ngokuhlanganyela, imiphumela le iveza bona ukwelatjhwa nge-IPA kutjhugulula ukuvezwa kwamajini ahlobene ne-fibrosis, i-apoptosis, ukuphila, kanye nokuguquguquka kwe-mitochondrial. Idatha yethu iveza bona ukwelatjhwa nge-IPA kwehlisa i-fibrosis kumaseli we-LX-2; ngesikhathi esifanako, ikhuthaza ukuphila ngokutjhugulula i-phenotype ukuya ekungasebenzini.
I-IPA itjhugulula ukuvezwa kwe-fibroblast, i-apoptotic, i-viability, kanye namajini we-mitochondrial dynamics kumaseli we-LX-2. Ama-histogram atjengisa ukuvezwa kwe-mRNA ngokuya ngokulawula kwangaphakathi (i-RPLP0 nofana i-PPIA) ngemva kokuthi amaseli we-LX-2 afakwe nge-TGF-β1 ne-IPA endaweni engena-serum ama-awa ama-24. U-A utjengisa ama-fibroblasts, u-B utjengisa amaseli we-apoptotic, u-C utjengisa amaseli aphilako, begodu u-D utjengisa ukuvezwa kwezakhi zofuzo ze-mitochondrial dynamics. Idatha ivezwa njengesilinganiso ± ukuphambuka okujayelekileko (SD), n = 3 ukuhlolwa okuzijameleko. Ukumadanisa iimbalo kwenziwa kusetjenziswa i-ANOVA ye-one-way kanye ne-Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Bese, amatjhuguluko ngobukhulu bamaseli (FSC-H) kanye nobunzima be-cytoplasmic (SSC-H) kwahlolwa nge-flow cytometry (Umfanekiso 6A,B), begodu amatjhuguluko we-morphology yamaseli ngemva kokwelatjhwa nge-IPA kwahlolwa nge-transmission electron microscopy (TEM) kanye ne-phase contrast microscopy (Umfanekiso 6A-B). Njengombana bekulindelwe, amaseli wesiqhema esiphathwe nge-TGF-β1 akhula ngobukhulu nayiqathaniswa nesiqhema sokulawula (Umfanekiso 6A,B), okutjengisa ukwanda okujayelekileko kwe-endoplasmic reticulum (ER*) kanye nama-phagolysomes (P), okutjengisa ukusebenza kwe-hematopoietic stem cell (HSC) (Umfanekiso 6A). Kodwana, nayiqathaniswa nesiqhema esiphathwe nge-TGF-β1, ubukhulu beseli, ubudisi be-cytoplasmic (Umfanekiso 6A,B), kanye nokuqukethwe kwe-ER* kwehle esiqhemeni se-TGF-β1 ne-IPA (Isithombe esingezelelweko 6A). Ukudlula lapho, ukwelatjhwa nge-IPA kwehlise ubukhulu bamaseli, ubudisi be-cytoplasmic (Imifanekiso 6A,B), okuqukethwe kwe-P kanye ne-ER* (Imifanekiso eyengeziweko 6A) nayiqathaniswa nesiqhema sokulawula. Ukungezelela, okuqukethwe kwamaseli we-apoptotic kwanda ngemva kwama-awa ama-24 wokwelatjhwa nge-IPA nakuqathaniswa nesiqhema sokulawula (imicibisholo emhlophe, Umfanekiso ongezelelweko 6B). Ngokuhlanganyela, imiphumela le iveza bona i-1 mM IPA ingakhuthaza i-HSC apoptosis begodu ibuyisele emuva amatjhuguluko emapharamitha we-morphological yamaseli abangelwa yi-TGF-β1, ngaleyindlela ilawula ubukhulu beseli nobudisi, okungahlotjaniswa nokungasebenzi kwe-HSC.
I-IPA itjhugulula ubukhulu bamaseli nobunzima be-cytoplasmic kumaseli we-LX-2. Iinthombe ezijameleko zokuhlaziywa kwe-flow cytometry. Ukuhlaziywa kwasebenzisa iqhinga lokuvala eliqondene namaseli we-LX-2: i-SSC-A/FSC-A ukuhlathulula inani lamaseli, i-FSC-H/FSC-A ukukhomba ama-doublets, kanye ne-SSC-H/FSC-H ukuhlaziywa kobukhulu bamaseli nobunzima. Amaseli afukanywa nge-TGF-β1 (5 ng/ml) kanye ne-1 mM IPA ngaphakathi kwe-serum-free medium ama-awa ama-24. Amaseli we-LX-2 asabalaliswa abe yi-quadrant engehla kwesokunxele (SSC-H-/FSC-H-), i-quadrant ephezulu yesokunxele (SSC-H+/FSC-H-), i-quadrant engezansi kwesokudla (SSC-H-/FSC-H+), kanye ne-quadrant ephezulu kwesokudla (SSC-H+/FSC-H+) ukuhlaziywa kobukhulu bamaseli nobunzima be-cytoplasmic. B. Ukwakheka kwamaseli kwahlaziywa nge-flow cytometry kusetjenziswa i-FSC-H (ukusabalala phambili, ubukhulu bamaseli) kanye ne-SSC-H (ukusakazeka kwehlangothi, ubudisi be-cytoplasmic) (iminyanya ema-30,000). Idatha ivezwe njengesilinganiso ± SD, n = 3 ukuhlolwa okuzijameleko. Ukumadanisa iimbalo kwenziwa kusetjenziswa i-ANOVA ye-one-way kanye ne-Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 kanye ****p < 0.0001
Ama-metabolites wamathumbu afana ne-IPA sele abe yindaba etjhisako yokurhubhulula, okusikisela bona amathagethi amatjha angatholwa ema-microbiota wamathumbu. Ngalokho-ke kuyathakazelisa bona i-IPA, i-metabolite esiyihlanganise ne-liver fibrosis ebantwini [15], itjengiswe njenge-anti-fibrotic compound eenlwaneni [13, 14]. Lapha, sitjengisa kokuthoma ukuhlobana hlangana ne-serum IPA kanye ne-global liver transcriptomics kanye ne-DNA methylation ebantwini abakhulupheleko abangenayo i-type 2 yesifo seswekileko (T2D), ukuveza i-apoptosis, i-mitophagy nokuphila isikhathi eside, kanye ne-gene engenzeka i-AKT1 elawula i-liver homeostasis. Okhunye okutjha kwesifundo sethu kukuthi sitjengise ukusebenzisana kokwelatjhwa kwe-IPA ne-apoptosis, ukubunjwa kwamaseli, i-mitochondrial bioenergetics kanye nokuguquguquka kwamaseli we-LX-2, okutjengisa i-spectrum yamandla ephasi etjhugulula i-HSC phenotype ukuya ekungasebenzini, okwenza i-IPA ibe yinto engenzeka yokuthuthukisa i-liver fibrosis.
Sithole bona i-apoptosis, i-mitophagy nokuphila isikhathi eside bekuziindlela eziqakatheke khulu ezinothiswe emajini wesibindi ahlotjaniswa ne-IPA ye-serum ejikelezako. Ukuphazamiseka kwehlelo lokulawula ikhwalithi ye-mitochondrial (MQC) kungabangela ukungasebenzi kuhle kwe-mitochondrial, i-mitophagy kanye ne-apoptosis, ngaleyindlela kukhuthaze ukwenzeka kwe-MASLD[33, 34]. Ngalokho-ke, singaqagela bona i-IPA ingabandakanyeka ekugcineni amandla wamaseli nokuthembeka kwe-mitochondrial ngokusebenzisa i-apoptosis, i-mitophagy nokuphila isikhathi eside esibindini. Idatha yethu itjengise bona amajini amabili bekafana kizo zoke iinhlahlubo ezintathu: i-YKT6 ne-AKT1. Kuqakathekile ukutjheja bona i-YKT6 yiphrotheyini ye-SNARE ebandakanyeka enqubweni yokuhlanganiswa kwamaseli. Idlala indima e-autophagy ne-mitophagy ngokwakha i-initiation complex ne-STX17 ne-SNAP29 ku-autophagosome, ngaleyo ndlela ikhuthaze ukuhlanganiswa kwama-autophagosomes nama-lysosomes[35]. Ukudlula lapho, ukulahlekelwa msebenzi we-YKT6 kuphumela ekukhubazekeni kwe-mitophagy[36], ngesikhathi ukulawulwa kwe-YKT6 kuhlotjaniswa nokuragela phambili kwe-hepatocellular carcinoma (HCC), okutjengisa ukwanda kokuphila kwamaseli[37]. Ngakelinye ihlangothi, i-AKT1 lijini eliqakatheke khulu elisebenzisanako begodu lidlala indima eqakathekileko emalwelweni wesibindi, kufaka hlangana indlela yokutjengisa i-PI3K/AKT, umjikelezo wamaseli, ukufuduka kwamaseli, ukwanda, ukunamathelana, ukusebenza kwe-mitochondrial, nokukhiqizwa kwe-collagen[38–40]. Indlela yokubonisa i-PI3K/AKT esebenzako ingasebenza amaseli we-hematopoietic stem (ama-HSC), okumaseli anesibopho sokukhiqiza i-extracellular matrix (ECM), begodu ukungalawuleki kwayo kungaba nomthelela ekwenzekeni nokuthuthuka kwe-liver fibrosis[40]. Ngaphezu kwalokho, i-AKT ngenye yezinto eziqakathekileko zokuphila kwamaseli ezivimbela ukufa kwamaseli ancike ku-p53, begodu ukusebenza kwe-AKT kungahlotjaniswa nokuvimbela ukufa kwamaseli wesibindi [41, 42]. Imiphumela etholakeleko iveza bona i-IPA ingabandakanyeka ekufeni kwe-mitochondria yesibindi ngokuthinta isiqunto sama-hepatocytes hlangana nokungena ku-apoptosis nofana ukuphila. Imiphumela le ingalawulwa yi-AKT kanye/nofana amajini we-YKT6, aqakathekileko ekuhlaleni kwesibindi.
Imiphumela yethu itjengise bona i-1 mM IPA yabangela i-apoptosis begodu yehlisa ukuphefumula kwe-mitochondrial kumaseli we-LX-2 ngaphandle kokwelatjhwa nge-TGF-β1. Kuyaphawuleka bona i-apoptosis yindlela ekulu yokurarulula i-fibrosis nokuvuselelwa kwe-hematopoietic stem cell (HSC), begodu mcimbi oqakathekileko ekuphenduleni kwe-fibrosis yesibindi [4, 43]. Ngaphezu kwalokho, ukubuyiselwa kwe-BHI kumaseli we-LX-2 ngemva kokwelatjhwa okuhlanganisiweko kunikele ukuzwisisa okutjha ngendima engaba khona ye-IPA ekulawuleni i-mitochondrial bioenergetics. Ngaphasi kobujamo bokuphumula nokungasebenzi, amaseli weengazi avamise ukusebenzisa i-mitochondrial oxidative phosphorylation ukukhiqiza i-ATP begodu abe nomsebenzi omncani wokugaya ukudla. Ngakelinye ihlangothi, ukusebenza kwe-HSC kuthuthukisa ukuphefumula kwe-mitochondrial kanye ne-biosynthesis ukunxephezela iimfuno zamandla zokungena esimweni se-glycolytic [44]. Iqiniso lokobana i-IPA azange ithinte amandla wokugaya ukudla kanye ne-ECAR itjengisa bona indlela ye-glycolytic ayikaqalwa phambili. Ngokufanako, elinye irhubhululo litjengise bona i-1 mM IPA yakghona ukutjhugulula umsebenzi we-mitochondrial respiratory chain kuma-cardiomyocytes, umugqa wamaseli we-hepatocyte womuntu (Huh7) kanye namaseli we-umbilical vein endothelial womuntu (HUVEC); Kodwana, akukho mphumela we-IPA owatholakala ku-glycolysis kuma-cardiomyocytes, okusikisela bona i-IPA ingathinta i-bioenergetics yeminye imihlobo yamaseli [45]. Ngalokho-ke, siqagela bona i-1 mM IPA ingasebenza njenge-mild chemical uncoupler, ngombana ingehlisa khulu ukuvezwa kwezakhi zofuzo ze-fibrogenic, ukubunjwa kwamaseli kanye ne-mitochondrial bioenergetics ngaphandle kokutjhugulula inani le-mtDNA [46]. Ama-mitochondrial uncouplers angavimbela i-culture-induced fibrosis kanye nokusebenza kwe-HSC [47] begodu anciphise ukukhiqizwa kwe-mitochondrial ATP okulawulwa nofana okubangelwa maphrotheyini athileko afana nama-uncoupling proteins (UCP) nofana i-adenine nucleotide translocase (ANT). Ngokuya ngomhlobo wamaseli, isenzakalo lesi singavikela amaseli ekufeni begodu/nofana sikhuthaze i-apoptosis [46]. Kodwana, iimfundo ezingezelelweko ziyatlhogeka ukucacisa indima ye-IPA njenge-mitochondrial uncoupler ekunciphiseni amaseli we-hematopoietic.
Bese siphenya bona amatjhuguluko ekuphefumuleni kwe-mitochondrial abonakala na ebujameni be-mitochondrial kumaseli we-LX-2 aphilako. Ngokuthakazelisako, ukwelatjhwa nge-TGF-β1 kutjhugulula izinga le-mitochondrial ukusuka e-spherical ukuya ephakathi, ngokuncipha kokuhlukaniswa kwe-mitochondrial nokukhuphuka kokuvezwa kwe-DRP1, okuyinto eqakathekileko ekuqhekekeni kwe-mitochondrial [48]. Ukudlula lapho, ukuhlukaniswa kwe-mitochondrial kuhlotjaniswa nobudisi benethiwekhi yoke, begodu ukutjhuguluka ukusuka ekuhlanganisweni ukuya ekuhlukaneni kuqakathekile ekusebenzeni kwe-hematopoietic stem cell (HSC), lokha ukuvinjelwa kokuhlukaniswa kwe-mitochondrial kurholela ekufeni kwe-HSC [49]. Ngalokho-ke, imiphumela yethu itjengisa bona ukwelatjhwa nge-TGF-β1 kungabangela ukwehla kobunzima benethiwekhi ye-mitochondrial ngokuncipha kokuhlukana, okuvame khulu ekuqhekekeni kwe-mitochondrial okuhlobene namaseli we-hematopoietic (HSCs) asebenzako. Ukudlula lapho, idatha yethu itjengise bona i-IPA ingatjhugulula inani le-mitochondria ukusuka ebujameni obuyindilinga ukuya ebujameni obuphakathi, ngaleyo ndlela yehlise ukuvezwa kwe-OPA1 ne-MFN2. Iimfundo zitjengise bona ukwehla kwe-OPA1 kungabangela ukwehla kwamandla we-mitochondrial membrane begodu kubangele ukubulawa kwamaseli[50]. I-MFN2 yaziwa ngokuthi ilamula ukuhlanganiswa kwe-mitochondrial kanye ne-apoptosis[51]. Imiphumela etholakeleko iveza bona ukufakwa kwamaseli we-LX-2 nge-TGF-β1 kanye/nofana i-IPA kubonakala kutjhugulula ubujamo nobukhulu be-mitochondrial, kanye nobujamo bokusebenza nobudisi benethiwekhi.
Imiphumela yethu itjengisa bona ukusetjenziswa kwe-TGFβ-1 ne-IPA kungehlisa i-mtDNA namapharamitha wesakhiwo seseli ngokulawula ukuvezwa kwe-mRNA ye-fibrosis, i-apoptosis kanye namajini ahlobene nokusinda emaseli abalekela i-apoptosis. Kwamambala, i-IPA yehlise izinga lokuvezwa kwe-mRNA ye-AKT1 kanye namajini aqakathekileko we-fibrosis afana ne-COL1A2 ne-MMP2, kodwana yakhuphula izinga lokuvezwa kwe-CASP8, ehlotjaniswa ne-apoptosis. Imiphumela yethu itjengise bona ngemva kokwelatjhwa nge-IPA, ukuvezwa kwe-BAX kwehla begodu ukuvezwa kwe-mRNA yama-subunits womndeni we-TIMP1, i-BCL-2 ne-NF-κB kwanda, okusikisela bona i-IPA ingakhuthaza amatshwayo wokuphila kumaseli we-hematopoietic stem (HSCs) abalekela i-apoptosis. Amamolekhyuli la angasebenza njengeempawu zokuphila emaseli we-hematopoietic stem asebenzako, okungahlotjaniswa nokuvezwa okukhulu kwamaphrotheyini alwisana nokufa (njenge-Bcl-2), ukwehla kokuvezwa kwe-BAX, kanye nokusebenzisana okuhlukahlukeneko hlangana ne-TIMP ne-NF-κB [5, 7]. I-IPA yenza imiphumela yayo nge-PXR, begodu sithole bona ukwelatjhwa okuhlanganisiweko nge-TGF-β1 ne-IPA kwandisa amazinga wokuvezwa kwe-PXR mRNA, okutjengisa ukugandeleleka kokusebenza kwe-HSC. Ukusetjenziswa kwe-PXR kwaziwa ngokuvimbela ukusebenza kwe-HSC kokubili ku-vivo naku-vitro [52, 53]. Imiphumela yethu itjengisa bona i-IPA ingahlanganyela ekuhlanzeni ama-HSC asebenzako ngokukhuthaza i-apoptosis, ukwehlisa i-fibrosis kanye nokugaya ukudla kwe-mitochondrial, nokuthuthukisa amatshwayo wokuphila, okuziinqubo ezijayelekileko eziguqula i-HSC esebenzako ibe ngengasebenziko. Enye ihlathululo engenzeka yendlela engaba khona nendima ye-IPA ku-apoptosis kukuthi ihlanza ama-mitochondria angasebenziko khulukhulu nge-mitophagy (indlela yangaphakathi) kanye nendlela yokubonisa i-TNF yangaphandle (Ithebula 1), ehlotjaniswa ngqo nendlela yokubonisa ukusinda kwe-NF-κB (Umfanekiso wokungezelela 7). Ngokuthakazelisako, amajini anothileko ahlobene ne-IPA akghona ukuletha amatshwayo we-pro-apoptotic ne-pro-survival endleleni ye-apoptotic [54], okusikisela bona i-IPA ingabangela i-apoptotic pathway nofana ukuphila ngokusebenzisana namajini la. Kodwana, indlela i-IPA ebangela ngayo i-apoptosis nofana ukuphila ngesikhathi sokusebenza kwe-HSC kanye neendlela zayo ezisebenzako akukacaci.
I-IPA yi-microbial metabolite eyakhiwe nge-tryptophan yokudla nge-gut microbiota. Iimfundo zitjengise bona ine-anti-inflammatory, antioxidant, kanye nezakhiwo zokulawula i-epigenetic endaweni yamathumbu.[55] Iimfundo zitjengise bona i-IPA ingaguqula ukusebenza kwesithiyo samathumbu begodu yehlise ukugandeleleka kwe-oxidative, okungaba nomthelela emiphumeleni yayo yendawo.[56] Kuhlekuhle, i-IPA ithunyelwa eenthweni eziqothelweko ngokusebenzisa ukujikeleza, begodu njengoba i-IPA yabelana ngesakhiwo esifanako esikhulu se-metabolite ne-tryptophan, i-serotonin, kanye ne-indole derivatives, i-IPA yenza izenzo zokugaya ukudla eziphumela ekuncintisaneni.[52] I-IPA ingancintisana nama-metabolites avela ku-tryptophan ngeendawo zokubophelela kuma-enzyme nofana kuma-receptor, okungenzeka kuphazamise iindlela ezijayelekileko zokugaya ukudla. Lokhu kuveza itlhogeko yeemfundo ezingezelelweko nge-pharmacokinetics kanye ne-pharmacodynamics ukuzwisisa ngcono iwindi layo lokwelapha.[57] Kusazokubonakala bona lokhu kungenzeka na kumaseli we-hematopoietic stem (HSCs).
Siyavuma bona irhubhululo lethu linemikhawulo ethileko. Ukuhlola ngokukhethekileko ukuhlobana okuhlobene ne-IPA, sikhuphe ngaphandle iingulani ezine-type 2 yesifo seswekileko (T2DM). Siyavuma bona lokhu kukhawulela ukusetjenziswa okubanzi kwemiphumela yethu eengulini ezine-type 2 yesifo seswekileko nesifo sesibindi esithuthukileko. Nanyana ukugxila kwe-IPA emzimbeni womuntu kungu-1-10 μM [11, 20], ukugxila kwe-1 mM IPA kwakhethwa ngokuya ngokugxila okungekho ubuthi [15] kanye nezinga eliphezulu le-apoptosis, ngaphandle komehluko emaphesentini we-necrotic cell population. Nanyana amazinga we-IPA asetjenziswe esifundweni lesi, njenganje akukho ukuvumelana malungana nomthamo osebenzako we-IPA [52]. Nanyana imiphumela yethu iqakathekile, isiphetho esibanzi se-IPA sihlala siyindawo esebenzako yokurhubhulula. Ngaphezu kwalokho, imiphumela yethu malungana nokuhlobana hlangana namazinga we-serum IPA kanye ne-DNA methylation yama-liver transcripts ayitholwanga kwaphela emaselini we-hematopoietic stem (HSCs) kodwana nasezicutshini zesibindi. Sikhethe ukusebenzisa amaseli we-LX-2 womuntu ngokuya kokutholwe kwethu kwangaphambilini ekuhlaziyweni kwe-transcriptome ukuthi i-IPA ihlotjaniswa nokusebenza kwe-hematopoietic stem cell (HSC) [15], begodu ama-HSC ngamaseli amakhulu abandakanyeka ekurageleni phambili kwe-liver fibrosis. Isibindi sakhiwe ziinhlobo ezinengi zamaseli, njeke amanye amamodeli wamaseli afana ne-hepatocyte-HSC-immune cell co-culture system ehlanganiswe nokusebenza kwe-caspase nokuhlukaniswa kwe-DNA kanye nendlela yokusebenza efaka hlangana izinga lamaphrotheyini kufanele kuqalwe ukufunda indima ye-IPA nokusebenzisana kwayo neminye imihlobo yamaseli wesibindi.