Twaba twagambire enyima ngu ebiriisa by‘omubiri ebirikuruga omu mara ebirikwetwa indole-3-propionic acid (IPA) biri ahansi omu barwaire abaine obuhuta bw‘ebihaha. Omu kucondooza oku, tukacondooza aha transcriptome na DNA methylome omu bihaha ebiine omubiri muhango omu nkoragana n‘embeera za IPA omu shagama, hamwe n‘omurimo gwa IPA omu kureetaho okutakora kw‘obutafaari bw‘ebihaha (HSCs) omu nkora ya vitro.
Okucondooza oku kukaba kurimu abarwaire 116 abaine omubiri muhango abatarikutunga endwara ya shukaari ey‘omuringo gwa 2 (T2DM) (emyaka 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m2) abaakozirweho okushemezibwa kw‘okukyebera omubiri omu kitongore ky‘okukyebera omubiri ekya Kuopio (KOBS). Emiringo ya IPA erikwetoorora ekapimwa omu muringo gw’okukyebera amaizi (LC-MS), okucondooza aha bihandiiko by’ebihaha kukakorwa omu kucondooza kwa RNA yoona, kandi okucondooza kwa DNA kukakorwa barikukoresa Infinium HumanMethylation450 BeadChip. Obutafaari bw’ekibumba ky’omuntu (LX-2) bukakoresibwa omu kucondooza okwa in vitro.
Emiringo ya IPA omu shagama neekwatagana n‘okureebeka kw‘ebicweka by‘omubiri ebirikwejumba omu mihanda ya apoptotic, mitophagic, n‘okumara obwire buraingwa omu kifuba. Ekirungo kya AKT serine/threonine kinase 1 (AKT1) kikaba kiri ekika ky‘obuzaarwa ekirikukirayo kandi ekirikukirayo kukoragana omu nkora y‘okuhandiika ekibumba n‘enkora ya DNA. Obujanjabi bwa IPA bukareetaho okufa kw‘obutafaari, okukyendeeza aha kuhuuta kw‘obutafaari bw‘omubiri, n‘okuhindura aha nkora y‘obutafaari n‘entunguuka y‘obutafaari bw‘omubiri obwo burikuhindura okureebeka kw‘ebicweka by‘omubiri ebirikumanywa ngu nibitegyeka fibrosis, apoptosis, n‘okugumaho kw‘obutafaari bwa LX-2.
Kutwarwa hamwe, ebihandiiko ebi nibishagika ngu IPA eine obushoboorozi bw‘okujanjaba kandi neebaasa kureetera apoptosis n‘okuhindura HSC phenotype kuza omu mbeera etarikukora, obwo n‘okwongyera aha kubaasa kw‘okuzibira liver fibrosis obwo erikuteganisa HSC okukora n‘okuhindura eby‘okurya omu mitochondrial.
Okujanjaara kw‘okugira omubiri muhango munonga n‘oburwaire bw‘okuhindura eby‘okurya omu mubiri (metabolic syndrome) kubaire nikukwatagana n‘okwongyera aha ndwara y‘ebishaju by‘ebihaha erikureetwaho okuhindura eby‘okurya omu mubiri (MASLD); endwara egi neekwata 25% kuhika 30% y‘abantu boona [1]. Ekikuru ekirikuruga omu MASLD etiology n‘okusharara kw‘ebihaha, omuringo gw‘amaani ogurikumanyirwa aha kuguma nigurundaana kw‘ebihaha ebiri aheeru y‘obutafaari (ECM) [2]. Obutafaari obukuru oburikwejumba omu kusheeshasheesha kw‘ekibumba n‘obutafaari bw‘ekibumba oburikwetwa hepatic stellate cells (HSCs), oburikworeka emiringo ena emanyirwe: obutafaari oburikuhuumura, oburikukora, obutarikukora, n‘oburikukura [3, 4]. HSCs nizibaasa kukora kandi zihindurwe kuruga omu muringo gw‘okuhuumuraho kuza omu butafaari oburikushushana n‘obw‘ebihimba oburikweyongyera oburikwetaaga amaani maingi, n‘okwongyera aha kworeka α-smooth muscle actin (α-SMA) n‘omuringo I collagen (Col-I) [5, 6]. Omu bwire bw‘okugaruka enyima kw‘ekibumba, HSCs ezikozirwe nizihwaho kurabira omu apoptosis nari obutakora. Enkora ezi nizitwariramu okukyendeeza aha nkora y‘obutafaari bw‘omubiri n‘okuhindura obutafaari bw‘omubiri oburikureetaho okugumaho (nk‘emihanda y‘okumanyisa NF-κB na PI3K/Akt) [7, 8], hamwe n‘empindahinduka omu nkora y‘obutafaari bw‘omubiri n‘emirimo [9].
Omubaro gw‘eshagama y‘omubiri (IPA), ogurikuruga omu mara, gushangirwe ngu nigukyendeera omu ndwara z‘omubiri z‘abantu ezirimu MASLD [10–13]. IPA neekwatagana n‘okurya ebiriisa by‘omubiri, neemanyirwa aha nkora yaayo ey‘okurwanisa obukooko oburikureeta endwara n‘okurwanisa okuzimba, kandi neekyendeeza aha nkora y‘okurya erikureetwa steatohepatitis etari y‘okunywa amaarwa (NASH) omu nkora y‘amagara n‘omu nkora y‘amagara [11–14]. Obuhame obumwe niburuga omu kucondooza kwaitu okwabandize, okwayorekire ngu omubaro gwa IPA omu shagama gukaba guri ahansi omu barwaire abaine obuzibu bw‘okusharara kw‘ebihaha kukira omu barwaire abaine omubiri muhango abatarikusharara kw‘ebihaha omu kucondooza kwa Kuopio Bariatric Surgery Study (KOBS). Ekindi, tukooreka ngu obujanjabi bwa IPA nibubaasa kukyendeeza aha kumanyisa kw‘ebicweka by‘omubiri ebirikuba biri obumanyiso bw‘okuhamira aha butafaari, okufuruka kw‘obutafaari n‘okukora kw‘obutafaari bw‘omubiri oburikureeta eshagama omu nkora y‘obutafaari bw‘omubiri oburikwetwa hepatic stellate cell (LX-2) model kandi nibubaasa kuba buri obumanyiso bw‘okurinda obutafaari bw‘omubiri [15]. Kwonka, tikikamanyirwe gye oku IPA erikureetaho okugaruka enyima kw‘ebihaha obwo erikukora HSC apoptosis n‘eby‘amagara by‘omubiri.
Aha, nitworeka ngu serum IPA neekwatagana n‘okworeka ebiriisa ebiine obugaiga omu apoptosis, mitophagy, n‘emihanda y‘okumara obwire buraingwa omu kifuba ky‘abantu abaine omubiri muhango kwonka abatari ba shukaari ey‘omuringo 2 (KOBS). Ekindi, tukashanga ngu IPA neebaasa kureetera okusheenya n‘okusheenya obutafaari oburikureeta eshagama (HSCs) kurabira omu muhanda gw‘okutakora. Ebirugiremu ebi nibyoreka omurimo musya gwa IPA, ekirikugihindura ekigyendererwa ky‘obujanjabi ekirikubaasa kutunguura okugaruka enyima kw‘ebihaha.
Okucondooza okwabandize omu guruupu ya KOBS kukooreka ngu abarwaire abaine obuzibu bw‘okusharara kw‘ebihaha bakaba baine omubaro gwa IPA ogurikutambura ahansi waagyeragyeranisa n‘abarwaire abatarikusharara kw‘ebihaha [15]. Kubingaho ebirikubaasa kureetaho okushoberwa ahabwa shukaari ey‘omuringo 2, tukahandiika abarwaire 116 abaine omubiri muhango abatarikutunga shukaari ey‘omuringo 2 (emyaka y‘ahaiguru ± SD: 46.8 ± 9.3 emyaka; BMI: 42.7 ± 5.0 kg/m2) (Ekipande kya KOBS) kuruga omu kucondooza okurikugyenda omu maisho]. Abeetabiremu boona bakaha okwikiriza kwabo omu buhandiikye kandi enkora y‘okwega ekaikirizibwa akakiiko k‘emitwarize y‘eirwariro rya North Savo County kurugiirira aha kihandiiko kya Helsinki (54/2005, 104/2008 na 27/2010).
Ebishushani by‘okukyebera ebihaha bikatunga omu bwire bw‘okushemezibwa kwa bariatric kandi byakyeberwa abacondoozi b‘endwara abaine obumanyirizi kurugiirira aha nkora ezaabaire zishobooroirwe enyima [17, 18]. Enkora y‘okushwijuma neefunzibwa omu nkora y‘okwongyeraho S1 kandi ekashoboororwa enyima [19].
Sampuli z‘eshagama y‘okusiiba zikeeberwa omu nkora y‘okukyebera ebiri omu mubiri (LC-MS) ahabw‘okukyebera eby‘okurya (n = 116). Sampuli zikakyeberwa barikukoresa enkora ya UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Bugirimaani) nk‘oku kyashoboororwa enyima19. Okumanya isopropyl alcohol (IPA) kukaba kuri aha bwire bw‘okurinda n‘okugyeragyeranisa ebiri omu nkora ya MS/MS n‘omutindo ogushemeire. Obuhango bw‘akamanyiso ka IPA (omwanya gw‘ahaiguru) bukateekateekwaho omu kucondooza kwona okwakuratsireho [20].
Okukuratana kwa RNA y’ekibumba kyona kukakorwa barikukoresa Illumina HiSeq 2500 kandi ebihandiiko bikabanza byakorwa nk’oku byashoboororwa enyima [19, 21, 22]. Twakora okucondooza okugyendereire okurikutaana aha bihandiiko ebirikukwata aha mirimo y‘emibiri/ebihangirwe ebiine amagara turikukoresa ebiriisa 1957 ebyatoorainwe kuruga omu nkora ya MitoMiner 4.0 [ 23 ]. Okucondooza aha DNA y‘ekibumba kukakorwa barikukoresa ekikwato ky‘okucondooza eky‘omubiri (Illumina, San Diego, CA, USA) barikukoresa omuringo nigwo gumwe nk‘oku gwashoboororwa enyima [24, 25].
Obutafaari bw‘omuntu obw‘omubiri oburikwetwa stellate stellate (LX-2) bukaheebwa omu muringo gw‘embabazi na Prof. Okutoorana omubaro gw‘okukora gwa IPA, obutafaari bwa LX-2 bukajanjabibwa n‘ebicweka bya IPA ebitarikushushana (10 μM, 100 μM na 1 mM; Sigma, 220027) omu nkora ya DMEM/F12 kumara eshaaha 24 h. Ekindi, okucondooza aha busobozi bwa IPA obw‘okuremesa HSCs kukora, obutafaari bwa LX-2 bukajanjabibwa hamwe na 5 ng/ml TGF-β1 (enkora za R&D, 240-B-002/CF) na 1 mM IPA omu mwanya ogutarimu serum kumara eshaaha 24 h. Aha nkora z‘okukyebera emotoka ezirikuhikaana, 4 nM HCL erimu 0.1% BSA ekakoresibwa omu kujanjaba TGF-β1 kandi 0.05% DMSO ekakoresibwa omu kujanjaba IPA, kandi byombi bikakoresibwa hamwe omu kujanjaba kw‘okuteerana.
Okusheeshasheesha kw‘eshagama kukakyeberwa barikukoresa ekikwato ky‘okukyebera okusheeshasheesha kw‘eshagama ekya FITC Annexin V ekirimu 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) kurugiirira aha biragiro by‘omukozi. Omu bugufu, LX-2 (1 × 105 cells/well) zikaba zirimu ekiro kyona omu bipande by’ebiina 12 reero bwanyima zijanjabwa n’ebicweka bingi bya IPA nari IPA na TGF-β1. Ekiro ekyakuratsireho, obutafaari oburikugyendera aha maizi n’oburikuhamira aha maizi bukatorotoorwamu, bwateebwamu trypsin, bwayozibwa na PBS, bwagaruka bwateebwa omu buffer y’okukwatiraho Annexin V, kandi bwateebwa omu FITC-Annexin V na 7-AAD kumara edakiika 15.
Mitochondria omu butafaari obuhuriire zikateebwaho erangi ahabw‘emirimo y‘okuhindura ebiri omu mubiri barikukoresa MitotrackerTM Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Aha kukyebera kwa MTR, obutafaari bwa LX-2 bukateebwa omu nkora aha nkora erikwingana na IPA na TGF-β1. Bwanyima y’eshaaha 24, obutafaari obuhuriire bukateebwa omu trypsin, bwayozibwa na PBS, reero bwateebwa omu 100 μM MTR omu mwanya ogutarimu serum aha 37 °C kumara edakiika 20 nk’oku kyashoboororwa enyima [ 26 ]. Aha kucondooza aha nkora y‘obutafaari obuhuriire, obuhango bw‘obutafaari n‘oburemeezi bw‘obutafaari bw‘omubiri bikakyeberwa barikukoresa okujanjaara kw‘omu maisho (FSC) n‘okujanjaara kw‘aha rubaju (SSC), nk‘oku biri.
Ebihandiiko byona (ebintu 30,000) bikatunga barikukoresa puroguraamu ya NovoCyte Quanteon (Agilent) kandi byashwijumwa barikukoresa puroguraamu ya NovoExpress® 1.4.1 nari FlowJo V.10.
Omubaro gw‘okukoresa oxygen (OCR) n‘omubaro gw‘okuhindura asidi omu butafaari bw‘omubiri (ECAR) bikapimwa omu bwire obwabuzima barikukoresa ekikwato ky‘okukyebera okusheeshasheesha kw‘omubiri (Agilent Technologies, Santa Clara, CA) ekiine ekikwato ky‘okukyebera obutafaari bw‘omubiri (Seahorse XF Cell Mito Stress) kurugiirira aha biragiro by‘omukozi. Omu bugufu, obutafaari bwa LX-2 2 × 104/ekiina bukabyarwa aha bipande by’okuhinga obutafaari bwa XF96. Bwanyima y’okumara ekiro kyona, obutafaari bukajanjabwa na isopropanol (IPA) na TGF-β1 (Emiringo y’okwongyeraho 1). Okushwijuma amakuru kukakorwa barikukoresa puroguraamu ya Seahorse XF Wave, erimu ekikwato ky‘okukyebera ripooti y‘okukyebera amaani g‘obutafaari bwa Seahorse XF. Kuruga ahari eki, omubaro gw‘amagara g‘abantu (BHI) gukabarwa [27].
RNA yoona ekahandiikwa omu cDNA. Okumanya emiringo emwe, reeba ebihandiiko [15]. Omubaro gw‘abantu 60S ebiriisa by‘omubiri ebirimu asidi P0 (RPLP0) na cyclophilin A1 (PPIA) mRNA bikakoresibwa nk‘okutegyeka ebiri omu nkora y‘obuzaarwa. Enkora ya QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, Netherlands) ekakoresibwa n‘ekikwato kya TaqManTM Fast Advanced Master Mix Kit (Ebikwato by‘okujanjaaza eby‘obuhangwa ebirikukoresibwa) nari ekikwato kya Sensifast SYBR Lo-ROX (omuhendo gw‘okugyeragyeranisa eby‘obuhangwa, BIO 94050), kandi ebipimo (ΔΔCt) n‘omuringo gwa ∆∆Ct. Ebirikukwata aha bikwatiraine n‘okubanza nibiheebwa omu bipande by‘okwongyeraho S2 na S3.
DNA y’omubiri (ncDNA) na DNA y’omubiri (mtDNA) zikaihwaho barikukoresa ekikwato kya DNeasy eky’eshagama n’ebicweka by’omubiri (Qiagen) nk’oku kyashoboororwa enyima [28]. Omuhendo gw‘okugyeragyeranisa ogwa mtDNA gukabarwa barikubara omubaro gwa buri kicweka kya mtDNA ekigyendereirwe n‘omubaro gwa geometric gw‘ebicweka bishatu bya DNA eby‘omubiri (mtDNA/ncDNA), nk‘oku birikworekwa omu nkora z‘okwongyeraho 2. Ebirikukwata aha ntandikwa za mtDNA na ncDNA nibiheebwa omu kipande ky‘okwongyeraho S4.
Obutafaari obuhuriire bukateebwaho erangi ya MitotrackerTM Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) kureeba omu maisho obutafaari oburi ahagati y’obutafaari n’omunda y’obutafaari bw’omubiri. Obutafaari bwa LX-2 (obutafaari 1 × 104/eiziba) bukakurwa aha bipande by’endabirwamu omu bipande by’okuhinga ebirikushushana n’eby’ahansi y’endabirwamu (Ibidi GmbH, Martinsried, Germany). Bwanyima y’eshaaha 24, obutafaari bwa LX-2 obuhuriire bukateebwa omu 100 μM MTR kumara edakiika 20 ahari 37 °C kandi obutafaari bw’omubiri bukateebwamu DAPI (1 μg/ml, Sigma-Aldrich) nk’oku kyashoboororwa enyima [29]. Emikutu y‘omubiri ekareebeka barikukoresa ekikwato ky‘okureeba ebiri omu nkora (Carl Zeiss Microimaging GmbH, Jena, Germany) ekiine ekikwato ky‘okureeba ebiri omu nkora ya Zeiss LSM 800 aha 37 °C omu mwanya ogurimu omuyaga ogurimu 5% CO2 barikukoresa ekikwato kya 1NA×3je. Twatunga ebishushani ikumi ebya Z-series ahari buri muringo gw‘eky‘okureeberaho. Buri Z-series erimu ebicweka 30, buri kimwe kiine oburaingwa bwa 9.86 μm. Aha buri sampo, ebishushani by‘ebicweka ikumi eby‘okureeberaho eby‘okureeberaho bikatunga barikukoresa puroguraamu ya ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany), kandi okucondooza aha nkora y‘ebicweka by‘omubiri eby‘omubiri kukakorwa barikukoresa puroguraamu ya ImageJ (v1.54d) [30, 31] eshobooroirwe gye kurugiirira aha nkora ya Metho3.
Obutafaari bukahamibwa n’ekiriisa kya 2% omu 0.1 M phosphate buffer, bwakuratirwa okuhamibwa n’ekiriisa kya osmium tetroxide 1% (Sigma Aldrich, MO, USA), mporampora bukakyeenwa amaizi n’ekiriisa kya asetoni (Merck, Darmstadt, Germany), kandi bwanyima bwateebwa omu nshaho y’epokisi. Ebicweka ebikye munonga bikatebeekanisibwa kandi byateebwaho erangi 1% y’ekiriisa kya uranyl (Merck, Darmstadt, Bugirimaani) na 1% y’ekiriisa kya lead citrate (Merck, Darmstadt, Bugirimaani). Ebishushani by‘ahaiguru munonga bikatunga okukoresa ekikwato ky‘okukyebera ebiri omu nkora ya JEM 2100F EXII (JEOL Ltd, Tokyo, Japan) aha nkora y‘okutunguura 80 kV.
Enkora y‘obutafaari bwa LX-2 obujanjabirwe na IPA kumara eshaaha 24 ekakyeberwa omu nkora y‘okukyebera ebiri omu nkora y‘okugyeragyeranisa ebiri omu nkora aha kutunguuka kwa 50x barikukoresa enkora ya Zeiss ey‘omushana oguhindukire (Zeiss Axio Vert.A1 na AxioCam MRm, Jena, Germany).
Ebihandiiko by‘obushaho bikooreka nk‘ebiri ahagati ± okutaana kw‘omutindo nari ebiri ahagati (omubaro gw‘ebicweka bina: IQR). Okushwijuma kw‘omuringo gumwe kw‘okuhinduka (ebintu ebirikuhinduka ebitarikuhwaho) nari okukyebera χ2 (ebintu ebirikuhinduka omu miringo) bikakoresibwa kugyeragyeranisa entaaniso ahagati y‘ebicweka bishatu by‘okwega. Omubaro gw‘ebishuba (FDR) gukakoresibwa kushoboorora ahabw‘okukyeberwa kwingi, kandi ebiriisa ebiine FDR < 0.05 bikatwarwa nk‘eby‘omugasho omu by‘embariirira. Okushwijuma kwa Spearman kukakoresibwa kukwatiraine n‘okuhindura CpG DNA methylation n‘amaani g‘akamanyiso ka IPA, n‘emihendo ya p ey‘eiziina (p < 0.05) eyaagambirweho.
Okushwijuma omuhanda kukakorwa barikukoresa ekikwato ky‘okushwijuma ebiri aha mukutu (WebGestalt) ahabw‘ebihandiiko 268 (eiziina p < 0.01), ebihandiiko 119 ebiine akakwate na mitochondria (eiziina p < 0.05), na 4350 CpGs ezirikutambura zikaba ziri aha rurengo rwa IPAs eziine akakwate na liver3. Ekikwato kya Venny DB (omuringo 2.1.0) ekirikushangwa busha kikakoresibwa kusherura ebiriisa ebirikuteerana, kandi StringDB (omuringo 11.5) kikakoresibwa kureeba omu maisho oku puroteyini zirikukoragana.
Aha kugyezesa kwa LX-2, eshampu zikakyeberwa kureeba yaaba ziri ez‘obutoosha barikukoresa okugyezesa kwa D‘Agostino-Pearson. Ebihandiiko bikatunga kuruga omu bishushani bishatu eby‘ebihangirwe ebiine amagara kandi byateebwa omu nkora ya ANOVA ey‘omuringo gumwe n‘okukyebera kwa Bonferroni post hoc. Omuhendo gwa p-guri ahansi ya 0.05 gukatwarwa nk‘ogw‘omugasho omu by‘embariirira. Ebihandiiko nibiheebwayo nk‘ebiri ahagati ± SD, kandi omubaro gw‘okucondooza nigworeka omu buri kishushani. Okushwijuma kwona n‘okuhandiika ebishushani bikakorwa barikukoresa puroguraamu y‘embariirira eya GraphPad Prism 8 eya Windows (enkora ya GraphPad Inc., omuringo 8.4.3, San Diego, USA).
Eky‘okubanza, tukacondooza aha nkoragana eri ahagati y‘embeera za IPA omu shagama n‘ebihandiiko by‘ekibumba, omubiri gwona, n‘eby‘omubiri. Omu nkora y‘okuhandiika yoona, oruzaaro orurikukirayo amaani orurikukwatagana n‘embeera za IPA omu shagama rukaba ruri MAPKAPK3 (FDR = 0.0077; puroteyini erikukoresibwa mitogen 3); omu nkora y‘okuhandiika ebirikukwata aha mitochondria, ebiriisa ebiine akakwate n‘omubiri eby‘amaani bikaba biri AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Fayiro y‘okwongeraho 1 na Fayiro y‘okwongeraho 2).
Bwanyima tukashwijuma ebihandiiko by‘ensi yoona (n = 268; p < 0.01) n‘ebihandiiko ebiine akakwate n‘emibiri y‘omubiri (n = 119; p < 0.05), aha muheru twamanya ngu okufa kw‘omubiri n‘omuhanda gw‘ebiragiro ogurikukirayo obukuru (p = 0.0089). Aha bihandiiko by‘omubiri ebiine akakwate n‘embeera za IPA omu shagama, tukata omutima aha kufa kw‘omubiri (FDR = 0.00001), okucwekyerera kw‘omubiri (FDR = 0.00029), n‘emihanda y‘okumanyisa TNF (FDR = 0.000006) (ekishushani 1A, ekipande 2, n‘ebishushani by‘okwongyeraho1).
Okushwijuma okurikurenga aha nkora y‘ensi yoona, ebihandiiko ebiine akakwate na mitochondria, n‘okuhindura DNA omu kihaha ky‘omuntu omu kukwatiraine n‘embeera za IPA omu shagama. A neejwekyera ebihandiiko 268 eby‘ensi yoona, ebihandiiko 119 ebiine akakwate na mitochondria, n‘ebihandiiko bya DNA ebiine akakwate na DNA ebiri aha maapu aha myanya 3092 eya CpG erikukwatagana n‘embeera za IPA omu shagama (emihendo ya p < 0.01 ahabw‘ebihandiiko by‘ensi yoona n‘ebihandiiko bya DNA methylated, n‘ebihandiiko bya p dri5on <0). Ebihandiiko ebikuru ebirikuteerana nibyorekwa ahagati (AKT1 na YKT6). B Maapu y‘okukoragana kw‘ebicweka 13 eby‘omubiri ebiine obubonero bw‘okukoragana oburikukirayo (0.900) n‘ebicweka ebindi eby‘omubiri ekakorwa kuruga omu bicweka 56 ebyabaire nibikwatagana (ekicweka ky‘omurongooti ogurikwiragura) ebyabaire biine akakwate kakuru n‘embeera za IPA omu shagama y‘omubiri barikukoresa ekikwato ky‘aha Intaneeti StringDB. Ekibira: Ebicweka by‘omubiri ebihandiikirwe aha maapu y‘ebicweka by‘omubiri eby‘omubiri (GO): obutafaari bw‘omubiri (GO:0005739). AKT1 ni puroteyini erimu obubonero bw‘ahaiguru (0.900) ahabw‘okukoragana na puroteyini ezindi kurugiirira aha makuru (okurugiirira aha kucondooza ebihandiiko, okugyezesa, ebihandiiko, n‘okushoboorora hamwe). Enyuguta z’omutimbagano nizijwekyera puroteyini, kandi emikono neejwekyera enkwatanisa ahagati ya puroteyini.
Ahabw‘okugira ngu ebiriisa by‘omunda nibibaasa kutebeekanisa ebiri omu nkora y‘epigenetic kurabira omu kuhindura DNA [32], tukacondooza yaaba emihendo ya IPA omu shagama y‘omubiri ekaba neekwatagana n‘okuhindura DNA y‘ekibumba. Twashanga ngu emyanya ebiri emikuru ey‘okukoreramu methylation erikukwatagana n‘embeera za IPA omu shagama ekaba eri haihi n‘ekicweka ky‘obugaiga bwa proline-serine 3 (C19orf55) n‘ekyanga kya puroteyini y‘okushoberwa ahabw‘okwotsya B (ekikye) memba 6 (HSPB6) (Fairo y‘okwongeraho 3). DNA methylation ya 4350 CpG (p < 0.01) ekaba neekwatagana n‘embeera za IPA omu shagama kandi ekaba etungire omu mihanda y‘okutegyeka amagara marungi (p = 0.006) (ekishushani 1A, ekipande 2, n‘ekishushani ky‘okwongyeraho 1C).
Kubaasa kwetegyereza enkora z‘ebihangwa eziri ahansi y‘enkoragana ahagati y‘embeera za IPA omu shagama, ebihandiiko by‘ensi yoona, ebihandiiko ebiine akakwate na mitochondria, n‘okuhindura DNA omu kihaha ky‘omuntu, tukakora okucondooza okurikuraba aha nkora y‘obuzaarwa eyamanyirwe omu kucondooza kw‘omuhanda okwabandize (ekishushani 1A). Ebirugire omu kucondooza kw‘okugaita omuhanda gw‘ebicweka by‘omubiri 56 ebirikuteerana (omunda y‘omurongooti gw‘erangi erikutukura omu kishushani 1A) bikooreka ngu omuhanda gw‘okucwekyerera (p = 0.00029) gukashoboorora ebicweka bibiri ebirikushushana omu kucondooza okushatu: AKT1 na YKT6 (YKT6 v-SNARE homolog omu kishushani Ven2), nk‘oku kirikworekwa. 1A). Ekirikushemeza, tukashanga ngu AKT1 (cg19831386) na YKT6 (cg24161647) zikaba ziine akakwate karungi n‘embeera za IPA omu shagama (Fayiro y‘okwongeraho 3). Kubaasa kumanya enkoragana erikubaasa kubaho ahagati y‘ebintu by‘obuzaarwa, tukatoorana obuzaarwa 13 obuine obubonero bw‘ekicweka ky‘omutaano oburikukirayo (0.900) omu butafaari 56 oburikuteerana nk‘ebirikuteebwamu kandi twakora maapu y‘enkoragana. Okurugirira aha rurengo rw‘okwesiga (okwesiga kw‘aha rubaju), oruzaaro rwa AKT1 oruine obubonero bw‘ahaiguru (0.900) rukaba ruri ahaiguru (ekishushani 1B).
Okurugirira aha kucondooza kw‘omuhanda, tukashanga ngu apoptosis nigwo muhanda mukuru, n‘ahabw‘ekyo tukacondooza yaaba obujanjabi bwa IPA nibubaasa kukwata aha apoptosis ya HSCs omu vitro. Twabanza kworeka ngu emibazi etari emwe n‘emwe eya IPA (10 μM, 100 μM, na 1 mM) ekaba etari y‘obutwa aha butafaari bwa LX-2 [15]. Okucondooza oku kukooreka ngu okujanjaba IPA ahari 10 μM na 100 μM kukongyera aha mubaro gw‘obutafaari oburikubaasa kugumaho n‘oburikufa. Kwonka, waagyeragyeranisa n‘ekibiina ky‘okukyebera, obutafaari bw‘okugumaho bukakyendeera aha 1 mM IPA concentration, obwo omubaro gw‘okusheeshasheesha kw‘obutafaari gukaguma gutahindukire (ekishushani 2A, B). Ekindi, kutunga ekicweka ekihikire eky‘okureetaho apoptosis omu butafaari bwa LX-2, tukagyezaho 10 μM, 100 μM, na 1 mM IPA kumara 24 h (ekishushani 2A-E n‘ekishushani ky‘okwongyeraho 3A-B). Ekirikushemeza, IPA 10 μM na 100 μM zikakyendeeza aha mubaro gw‘okucwekyerera (%), kwonka, IPA 1 mM ekeyongyera aha kukyerererwa kw‘okusheeshasheesha n‘omubaro gw‘okusheeshasheesha (%) kugyeragyeranisa n‘okukyebera n‘ahabw‘ekyo ekatooranwa kukora okucondooza okundi (ebishushani 2A–D).
IPA neereetaho okufa kw’obutafaari bwa LX-2. Enkora ya Annexin V na 7-AAD ey’okusiiga emirundi ebiri ekakoresibwa kugyeragyeranisa omubaro gw’okufa kw’obutafaari n’omuringo gw’obutafaari kurabira omu kucondooza okurikukoresibwa flow cytometry. Obutafaari bwa BA bukateebwa omu nkora ya 10 μM, 100 μM na 1 mM IPA kumara eshaaha 24 nari na F–H TGF-β1 (5 ng/ml) na 1 mM IPA omu mwanya ogutarimu serum kumara eshaaha 24. A: obutafaari obuhuriire (Ekicweka V -/ 7AAD-); B: obutafaari oburikusheeshasheesha (Ekicweka V -/ 7AAD+); C, F: kare (Ekihandiiko ky’okureeberaho V +/ 7AAD-); D, G: okukyerererwa (Ekihandiiko ky’okureeberaho V+/7AAD.+); E, H: ebicweka 10 ahari 100 eby‘obutafaari bwona obw‘okubanza n‘obw‘aha muheru omu mubaro gw‘okufa (%). Ebihandiiko nibyorekwa nk‘ebiri ahagati ± SD, n = 3 okucondooza okwetongoire. Okugyeragyeranisa kw‘embariirira kukakorwa barikukoresa ANOVA y‘omuringo gumwe n‘ekigyezo kya Bonferroni post hoc. * p < 0.05; p<0.0001
Nkoku twayorekire enyima, 5 ng/ml TGF-β1 neebaasa kureetera HSC kukora obwo erikweyongyera aha kumanyisa ebiri omu nkora y‘obumanyiso bw‘omubiri [15]. Obutafaari bwa LX-2 bukajanjabibwa na 5 ng/ml TGF-β1 na 1 mM IPA omu kugaita (ekishushani 2E–H). Obujanjabi bwa TGF-β1 tiburahinduire omubaro gw‘okufa kw‘eshagama, kwonka, obujanjabi bwa IPA hamwe bukayongyera aha kufa kw‘eshagama okukyerereirwe n‘omubaro gw‘okufa kw‘eshagama (%) kugyeragyeranisa n‘obujanjabi bwa TGF-β1 (Fig. 2E–H). Ebirugire omu kucondooza oku nibyoreka ngu 1 mM IPA neebaasa kutunguura apoptosis omu butafaari bwa LX-2 hatariho kureetaho TGF-β1.
Twongyeire twacondooza aha nkora ya IPA aha kuhuuta kwa mitochondrial omu butafaari bwa LX-2. Ebirugiremu byorekire ngu 1 mM IPA ekakyendeeza aha nkora y‘okukoresa oxygen (OCR): okuhuuta okutari kwa mitochondrial, okuhuuta kw‘ahansi n‘okw‘ahaiguru, okusheeshasheesha kwa purotoni n‘okukora ATP kugyeragyeranisa n‘ekibiina ky‘okukyebera (ekishushani 3A, B), obwo omubaro gw‘amagara g‘ebihangwa (BHI) gutahindukire.
IPA neekyendeeza aha kuhuuta kw‘omubiri omu butafaari bwa LX-2. Omubaro gw‘okuhuumura kw‘omubiri (OCR) niguheebwayo nk‘ebicweka by‘okuhuumura kw‘omubiri (okuhuumura okutari kw‘omubiri, okuhuumura kw‘ahansi, okuhuumura okurikukirayo, okusheeshasheesha kwa purotoni, okureetaho ATP, SRC na BHI). Obutafaari A na B bukateebwa omu nkora ya 10 μM, 100 μM na 1 mM IPA kumara eshaaha 24, nk’oku biri. Obutafaari C na D bukateebwa omu nkora ya TGF-β1 (5 ng/ml) na 1 mM IPA omu mwanya ogutarimu serum kumara eshaaha 24, nk’oku biri. Ebipimo byona bikahindurwa bya buriijo kuhika aha biri omu DNA barikukoresa ekikwato kya CyQuant. BHI: omubaro gw‘amagara g‘amaani g‘ebihangirwe; SRC: obushoboorozi bw‘okubiika eby‘okuhuumura; OCR: omubaro gw‘okukoresa oxygen. Ebihandiiko nibiheebwayo nk‘ebiri ahagati ± okutaana kw‘omutindo (SD), n = 5 okucondooza okwetongoire. Okugyeragyeranisa kw‘embariirira kukakorwa barikukoresa ANOVA y‘omuringo gumwe n‘okukyebera kwa Bonferroni post hoc. * p < 0.05; ** p < 0.01; na ***p <0.001
Kubaasa kutunga okwetegyereza okuhikire aha nkora ya IPA aha nkora y‘amagara g‘obutafaari bwa LX-2 oburikukoresibwa TGF-β1, tukashwijuma okuhinduka kw‘omubiri okurikukoresibwa OCR (Fig. 3C,D). Ebirugiremu byorekire ngu obujanjabi bwa TGF-β1 nibubaasa kukyendeeza aha kuhuuta okurikukirayo, obushoboorozi bw‘okuhuuta (SRC) na BHI kugyeragyeranisa n‘ekibiina ky‘okukyebera (Fig. 3C,D). Okwongyera ahari ekyo, obujanjabi bw‘okuteerana bukakyendeeza aha kuhuuta kw‘ahansi, okusheeshasheesha kwa purotoni n‘okukora ATP, kwonka SRC na BHI bikaba biri ahaiguru munonga kukira ebyabaire bijanjabirwe na TGF-β1 (Fig. 3C,D).
Kandi tukakora “okukyebera kw‘amaani g‘obutafaari” okwaheebwa puroguraamu ya Seahorse (Ekishushani ky‘okwongyeraho 4A–D). Nk‘oku kirikworekwa omu kishushani ky‘okwongyeraho 3B, obushoboorozi bw‘okuhindura eby‘okurya bwa OCR na ECAR bwombi bukakyendeera bwanyima y‘okujanjaba TGF-β1, kwonka, tihaine ntaaniso eyaareebekire omu guruupu z‘okujanjaba z‘okuteerana n‘ez‘okujanjaba IPA kugyeragyeranisa n‘ekibiina ky‘okukyebera. Ekindi, ebiri aha rurengo rwa OCR byombi eby‘ahansi n‘eby‘okushoberwa bikakyeera bwanyima y‘okugaita n‘okujanjaba IPA kugyeragyeranisa n‘ekibiina ky‘okukyebera (Ekishushani ky‘okwongyeraho 4C). Ekirikushemeza, enkora nk‘egi ekareebeka n‘obujanjabi bw‘okuteerana, ahu hatariho mpindahinduka omu rurengo rwa basal n‘orw‘okushoberwa rwa ECAR eyaareebekire kugyeragyeranisa n‘obujanjabi bwa TGF-β1 (Ekishushani ky‘okwongyeraho 4C). Omu HSCs, okukyendeera omu mitochondrial oxidative phosphorylation n‘obushoboorozi bw‘okujanjaba okurikugaita kugaruraho SCR na BHI bwanyima y‘okukwatwaho obujanjabi bwa TGF-β1 tikuhinduire aha bushoboorozi bw‘okuhindura eby‘okurya (OCR na ECAR). Ebirugiremu ebi byatwarwa hamwe, nibyoreka ngu IPA neebaasa kukyendeeza aha bioenergetics omu HSCs, ekirikumanyisa ngu IPA neebaasa kureetaho enkora y‘amaani makye erikuhindura HSC phenotype kuza omu kutakora (Ekishushani ky‘okwongyeraho 4D).
Eki IPA erikukora aha nkora y‘emibiri y‘omubiri kikacondoozibwa barikukoresa okupima kw‘ebicweka bishatu eby‘omubiri n‘enkwatanisa z‘emikutu hamwe n‘okutaho erangi ya MTR (ekishushani 4 n‘ekishushani ky‘okwongyeraho 5). Ekishushani 4 nikyoreka ngu, waagyeragyeranisa n‘ekibiina ky‘okukyebera, obujanjabi bwa TGF-β1 bukakyendeeza aha mwanya gw‘ahaiguru oguri ahagati, omubaro gw‘amataagi, oburaingwa bw‘amataagi goona, n‘omubaro gw‘okuteerana kw‘amataagi (ekishushani 4A na B) kandi bwahindura omubaro gw‘ebicweka by‘omubiri ebirikwetwa mitochondria kuruga aha nkora y‘okwetoorora kuza aha nkora y‘ahagati (ekishushani 4C). Obujanjabi bwa IPA bwonka bukakyendeeza aha bwingi bw‘ebicweka by‘omubiri ebiri ahagati y‘ebicweka by‘omubiri kandi bwahindura omubaro gw‘ebicweka by‘omubiri kuruga aha nkora y‘enziga kuza aha nkora y‘ahagati waagyeragyeranisa n‘ekibiina ky‘okukyebera (ekishushani 4A). Okutaana n‘ekyo, obuhango bw‘amataagi, oburaingwa bw‘amataagi oburikwingana, n‘emirimo y‘emibiri y‘omubiri eyakyeberwa MTR erikwegamira aha bushoboorozi bw‘omubiri (ekishushani 4A na E) bikaguma bitahindukire kandi ebipimo ebi tibiine ntaaniso ahagati y‘ebicweka. Ebirugire omu kucondooza oku byatwarwa hamwe, nibyoreka ngu obujanjabi bwa TGF-β1 na IPA nibushusha ngu nibuhindura omu nkora n‘obuhango bwa mitochondrial hamwe n‘okugumizamu kw‘omutimbagano omu butafaari bwa LX-2 obuhuriire.
IPA neehindura enkora y‘obutafaari bw‘omubiri n‘obungi bwa DNA y‘omubiri omu butafaari bwa LX-2. A. Ebishushani by‘okujwekyera eby‘obutafaari bwa LX-2 obuhuriire obuteirwe omu nkora ya TGF-β1 (5 ng/ml) na 1 mM IPA kumara eshaaha 24 omu mwanya ogutarimu serum ebirikworeka emikutu y‘obutafaari oburimu erangi ya MitotrackerTM Red CMXRos n‘obutafaari oburimu erangi ya bururu na DAPI. Ebihandiiko byona bikaba birimu ebishushani haakiri 15 ahari buri guruupu. Twatunga ebishushani 10 bya Z-stack ahari buri muringo gw‘eky‘okureeberaho. Buri mubaro gwa Z-axis gukaba gurimu ebicweka 30, buri kimwe kiine oburaingwa bwa 9.86 μm. Omubaro gw’okupima: 10 μm. B. Ebintu ebirikujwekyera (mitochondria byonka) ebirikumanyirwa aha kukozesa okutaho omutindo gw‘okuhindura aha kishushani. Okushwijuma kw‘omubaro n‘okugyeragyeranisa enkwatanisa z‘omutindo gw‘omubiri gw‘omubiri (mitochondrial morphological network) bikakorwa aha butafaari bwona omu buri guruupu. C. Emirundi ei emibaro y‘obubonero bw‘emibiri erikubaho. Emihendo eri haihi na 0 neeyoreka ebishushani by’enziga, kandi emihendo eri haihi na 1 neeyoreka ebishushani by’ebihimba. D Ebirimu omu mitochondria DNA (mtDNA) bikamanyirwa nk‘oku byashoboororwa omu bikoreso n‘emiringo y‘okukora. E MitotrackerTM Red CMXRos okucondooza kukakorwa omu kucondooza okurikukoresibwa okukyebera okurikugyendera aha nkora y‘okusheeshasheesha (ebintu 30,000) nk‘oku byashoboororwa omu bikoreso n‘emiringo y‘okukora. Ebihandiiko nibiheebwayo nk‘ebiri ahagati ± SD, n = 3 okucondooza okwetongoire. Okugyeragyeranisa kw‘embariirira kukakorwa barikukoresa ANOVA y‘omuringo gumwe n‘okukyebera kwa Bonferroni post hoc. * p < 0.05; ** p < 0.01; ***p < 0,001; p<0.0001
Bwanyima tukashwijuma ebiri omu mtDNA omu butafaari bwa LX-2 nk’eky’okureeberaho ky’omubaro gw’ebicweka by’omubiri ebirikwetwa mitochondrial. Kugyeragyeranisa n‘ekibiina ky‘okukyebera, ebiri omu mtDNA bikeyongyera omu guruupu eyaabaire neejanjabwa TGF-β1 (ekishushani 4D). Kugyeragyeranisa n‘ekibiina ekyajanjabirwe TGF-β1, ebiri omu mtDNA bikakyendeera omu guruupu eyaabaire neejanjabwa omu nkora y‘okuteerana (ekishushani 4D), ekirikumanyisa ngu IPA neebaasa kukyendeeza aha biri omu mtDNA kandi nikibaasika n‘omubaro gw‘emibiri y‘omubiri hamwe n‘okuhuuta kw‘emibiri y‘omubiri (ekishushani 3C). Ekindi, IPA ekashusha nk‘erikukyendeeza aha mtDNA omu kujanjaba kw‘okuteerana kwonka ekaba etaine kakwate n‘emirimo y‘emibiri erikuteebwaho MTR (ebishushani 4A–C).
Twacondooza aha nkoragana ya IPA n‘embeera za mRNA z‘ebicweka by‘omubiri ebiine akakwate n‘okusheeshasheesha, apoptosis, okugumaho, n‘okuhinduka kw‘ebicweka by‘omubiri omu butafaari bwa LX-2 (Ekishushani 5A–D). Kugyeragyeranisa n‘ekibiina ky‘okukyebera, ekibiina ekyabaire kiine obujanjabi bwa TGF-β1 kikayoreka okweyongyera kw‘okureebeka kw‘ebicweka by‘omubiri nk‘omurunga gwa kolajeni ogw‘omuringo I α2 (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), ekirikuzibira obutafaari bw‘omubiri oburikworeka obutafaari bw‘omubiri (dy-RPD1), na dyli-RP1). okweyongyera kw‘okusharara n‘okukora. Ekindi, kugyeragyeranisa n‘ekibiina ky‘okukyebera, obujanjabi bwa TGF-β1 bukakyendeeza aha rurengo rwa mRNA rw‘omubazi gw‘okutwara enda X (PXR), caspase 8 (CASP8), MAPKAPK3, omuzibizi w‘obutafaari bwa B α, omuhi w‘obuhwezi bw‘omubiri κ omubazi gw‘omushana gw‘omubiri (NFκbiB1A), . β (IKBKB) (Ekishushani 5A–D). Kugyeragyeranisa n‘obujanjabi bwa TGF-β1, obujanjabi oburimu TGF-β1 na IPA bukakyendeeza aha kureebeka kwa COL1A2 na MMP2, kwonka bwayongyera aha rurengo rwa mRNA rwa PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκ,βKB1-. Obujanjabi bwa IPA bukakyendeeza munonga aha kureebeka kwa MMP2, Bcl-2-eine akakwate na puroteyini X (BAX), AKT1, puroteyini y‘okubura amaisho 1 (OPA1), na puroteyini y‘okuteerana kw‘emibiri (MFN2), obwo okureebeka kwa CASP8, NFκB1A, NFκBB1B kwabaire nikweyongyera kugyeragyeranisibwa n‘ekibiina ky‘okutegyeka. Kwonka tihaine ntaaniso eyashangirwe omu kumanyisa kw‘ekiriisa kya caspase-3 (CASP3), ekirikureetaho okusheeshasheesha kw‘omubiri (APAF1), puroteyini y‘okuteerana kw‘eshagama (MFN1), n‘ekiriisa ky‘okuteerana kw‘eshagama (FIS1). Omu kugaita, ebyarugamu ebi nibyoreka ngu obujanjabi bwa IPA nibuhindura okureebeka kw‘ebicweka by‘omubiri ebiine akakwate n‘okusheeshasheesha, okufa kw‘ebicweka by‘omubiri, okugumaho, n‘okuhinduka kw‘ebicweka by‘omubiri. Ebihandiiko byaitu nibyoreka ngu obujanjabi bwa IPA nibukyendeeza aha fibrosis omu butafaari bwa LX-2; omu bwire nibwo bumwe, nikireetaho okugumaho obwo kirikuhindura ebiri omu nkora (phenotype) kuza omu kutakora.
IPA neehindura okureebeka kw’ebicweka by’omubiri ebirikwetwa fibroblast, apoptotic, okubaasa kugumaho, n’ebiri omu butafaari bwa LX-2. Histograms nizooreka mRNA okureebeka okurikuhikaana n‘okutegyeka kw‘omubiri (RPLP0 nari PPIA) bwanyima y‘obutafaari bwa LX-2 kureetaho TGF-β1 na IPA omu mwanya ogutarimu serum kumara eshaaha 24 h. A neeyoreka obutafaari oburikwetwa fibroblasts, B neeyoreka obutafaari oburikufa, C neeyoreka obutafaari oburikugumaho, kandi D neeyoreka oku obutafaari bw’omubiri burikukora nk’obutafaari oburikwetwa mitochondrial dynamics. Ebihandiiko nibiheebwayo nk‘ebiri ahagati ± okutaana kw‘omutindo (SD), n = 3 okucondooza okwetongoire. Okugyeragyeranisa kw‘embariirira kukakorwa barikukoresa ANOVA y‘omuringo gumwe n‘okukyebera kwa Bonferroni post hoc. * p < 0.05; ** p < 0.01; ***p < 0,001; p<0.0001
Bwanyima, empindahinduka omu bugabe bw‘obutafaari (FSC-H) n‘oburemeezi bw‘obutafaari (SSC-H) bikakyeberwa omu kucondooza kwa flow cytometry (ekishushani 6A,B), kandi empindahinduka omu nkora y‘obutafaari bwanyima y‘okujanjaba IPA zikakyeberwa omu kucondooza kwa transmission electron microscopy (TEM) n‘okucondooza kwa phase contrast microscopy (ekishushani ky‘okwongyeraho-B). Nk‘oku kyabaire nikiteekateekwaho, obutafaari omu guruupu eyaabaire neejanjabwa TGF-β1 bukayeyongyera omu buhango waagyeragyeranisa n‘ekibiina ekyabaire nikikyebera (ekishushani 6A,B), kworeka okutunguuka kwa kiraasi kw‘obutafaari bw‘omubiri obugumire (ER*) na phagolysosomes (P), kworeka okutandika kw‘obutafaari bw‘omubiri oburikureeta eshagama (HSC) (ekishushani ky‘okwongyeraho 6A). Kwonka, waagyeragyeranisa n‘ekibiina ekyajanjabirwe TGF-β1, obuhango bw‘obutafaari, oburemeezi bw‘obutafaari (Fig. 6A,B), n‘ebiri omu ER* bikakyeera omu guruupu eyaabaire neejanjabwa TGF-β1 na IPA (Ekishushani ky‘okwongyeraho 6A). Ekindi, obujanjabi bwa IPA bukakyendeeza aha buhango bw‘obutafaari, oburemeezi bw‘obutafaari bw‘omubiri (ebishushani 6A,B), P na ER* ebiri omu (ekishushani ky‘okwongyeraho 6A) kugyeragyeranisa n‘ekibiina ky‘okukyebera. Okwongyera ahari ekyo, ebiri omu butafaari bwa apoptotic bikeyongyera bwanyima y‘eshaha 24 z‘okujanjaba IPA kugyeragyeranisa n‘ekibiina ky‘okukyebera (obushaarizi oburikwera, Supplementary Fig. 6B). Omu kugaita, ebyarugamu ebi nibyoreka ngu 1 mM IPA neebaasa kureetaho HSC apoptosis kandi egarure enyima empindahinduka omu nkora y‘obutafaari oburikureetwaho TGF-β1, obwo erikutebeekanisa obuhango bw‘obutafaari n‘okugumizamu, ekirikubaasa kuba nikikwatagana n‘okutakora kwa HSC.
IPA neehindura obuhango bw‘obutafaari n‘okugumizamu kw‘obutafaari bw‘omubiri omu butafaari bwa LX-2. Ebishushani ebirikujwekyera okushwijuma kwa flow cytometry. Okweshwijuma kukakoresa enkora y‘okutaho omuryango (gating strategy) ey‘omutaano aha butafaari bwa LX-2: SSC-A/FSC-A kushoboorora omubaro gw‘obutafaari, FSC-H/FSC-A kumanyisa obutafaari bubiri, na SSC-H/FSC-H ahabw‘okushwijuma obuhango bw‘obutafaari n‘oburemeezi. Obutafaari bukateebwa omu nkora ya TGF-β1 (5 ng/ml) na 1 mM IPA omu mwanya ogutarimu serum kumara eshaaha 24 h. Obutafaari bwa LX-2 bukagabwa omu kicweka kya hansi aha rubaju rwa bumosho (SSC-H-/FSC-H-), ahaiguru aha rubaju rwa bumosho (SSC-H+/FSC-H-), ekicweka kya ahansi ekya buryo (SSC-H-/FSC-H+), n'ekicweka kya ahaiguru ekya buryo (SSC-H+/FSC-H+) ahabw'obuhango bw'obutafaari n'obuhango bw'obutafaari. B. Enkora y‘obutafaari ekakyeberwa omu nkora ya flow cytometry barikukoresa FSC-H (okujanjaara omu maisho, obuhango bw‘obutafaari) na SSC-H (okujanjaara aha rubaju, okugumizamu kw‘obutafaari) (ebintu 30,000 ebyabaireho). Ebihandiiko nibiheebwayo nk‘ebiri ahagati ± SD, n = 3 okucondooza okwetongoire. Okugyeragyeranisa kw‘embariirira kukakorwa barikukoresa ANOVA y‘omuringo gumwe n‘okukyebera kwa Bonferroni post hoc. * p < 0.05; ** p < 0.01; ***p <0.001 na ****p <0.0001
Ebirungo by‘omunda nka IPA bihindukire omutwe gw‘okucondooza ogurikwotsya, ekirikumanyisa ngu ebigyendererwa bisya nibibaasa kuzoorwaho omu bihingwa by‘omunda. Nahabwekyo nikishemeza ngu IPA, ekirungo eki twakwatanisa n‘okusharara kw‘ebihaha omu bantu [15], kyorekwa kuba ekirungo ekirikubaasa kurwanisa okusharara omu bishushani by‘enyamaishwa [13, 14]. Aha, nitworeka omurundi gw‘okubanza akakwate akari ahagati ya serum IPA n‘okuhandiika kw‘ekibumba omu nsi yoona n‘okuhindura DNA omu bantu abaine omubiri muhango abatarikutunga endwara ya shukaari ey‘omuringo 2 (T2D), turikworeka apoptosis, mitophagy n‘okumara obwire buraingwa, hamwe n‘ekirikubaasa kuba eky‘omubazi gw‘okutoorana AKT1 ogurikutegyeka okuguma kw‘ekibumba. Ekindi ekisya omu kucondooza kwaitu n‘okugira ngu tukooreka okujanjaba kwa IPA kurikukoragana n‘okufa kw‘obutafaari, enkora y‘obutafaari, mitochondrial bioenergetics n‘entunguuka omu butafaari bwa LX-2, ekirikworeka omubaro gw‘amaani oguri ahansi ogurikuhindura HSC phenotype kuza omu kutakora, ekirikuhindura IPA kuba orikubaasa kutunguura li fibrosis.
Twashanga ngu apoptosis, mitophagy n‘okumara obwire buraingwa bikaba biri emihanda y‘ebiragiro erikukirayo obukuru erimu obugaiga omu nkora y‘ebihaha erikukwatagana n‘okutambura kw‘eshagama IPA. Okuteganisibwa kw‘enkora y‘okurinda omutindo gw‘ebicweka by‘omubiri (MQC) nikubaasa kureetera okuremwa kukora gye kw‘ebicweka by‘omubiri, mitophagy na apoptosis, obwo nikireetaho okubaho kwa MASLD [33, 34]. Nahabwekyo, nitubaasa kuteebereza ngu IPA neebaasa kwejumba omu kuguma n‘obutafaari bw‘omubiri n‘obuhame bw‘obutafaari bw‘omubiri kurabira omu apoptosis, mitophagy n‘okumara obwire buraingwa omu kihaha. Ebihandiiko byaitu nibyoreka ngu ebiriisa bibiri bikaba biri bingi omu kucondooza okushatu: YKT6 na AKT1. Nikirungi kumanya ngu YKT6 ni puroteyini ya SNARE erikwejumba omu nkora y‘okuteerana kw‘obutafaari bw‘omubiri. Nikikora omurimo omu kurya eby‘okurya n‘okurya eby‘okurya by‘omubiri (mitophagy) obwo kirikukora ekicweka ky‘okutandika na STX17 na SNAP29 aha by‘okurya by‘omubiri, obwo nikitunguura okuteerana kw‘eby‘okurya by‘omubiri n‘eby‘okurya by‘omubiri [35]. Ekindi, okufeerwa emirimo ya YKT6 nikirugamu okuremwa kukora mitophagy [36], obwo okutunguuka kwa YKT6 kurikukwatagana n‘okutunguuka kw‘oburwaire bwa hepatocellular carcinoma (HCC), kworeka okweyongyera kw‘obutafaari bw‘okugumaho [37]. Aha rubaju orundi, AKT1 niyo nkora y‘okukoragana erikukirayo obukuru kandi neekora omurimo muhango omu ndwara z‘ebihaha, harimu omuhanda gw‘okumanyisa PI3K/AKT, enzindo y‘obutafaari, okufuruka kw‘obutafaari, okukanyira, okuhamira aha nshonga, okukora kwa mitochondrial, n‘okushohoza collagen [38–40]. Omuhanda gw‘okumanyisa ogwa PI3K/AKT ogukozirwe nigubaasa kukora obutafaari bw‘omubiri oburikukora eshagama (HSCs), obutafaari oburikukora aha kureetaho obutafaari oburikuruga omu mubiri (ECM), kandi okutakora gye kwabwo nikubaasa kureetaho okubaho n‘okutunguuka kw‘oburwaire bw‘ekibumba [40]. Okwongyera ahari ekyo, AKT n‘emwe aha nshonga nkuru z‘okugumaho kw‘obutafaari oburikuzibira p53-dependent cell apoptosis, kandi AKT okukora nikubaasa kuba nikukwatagana n‘okuzibira obutafaari bw‘ekibumba apoptosis [41, 42]. Ebirugire omu kucondooza oku bitungirwe nibyoreka ngu IPA neebaasa kwejumba omu kusheeshasheesha kw‘ebihaha okurikukwatagana n‘okucwekyerera kw‘ebihaha (hepatocytes) ahagati y‘okutaaha omu kusheeshasheesha nari okugumaho. Ebirugiremu ebi nibibaasa kutegyekwa AKT na/nari YKT6 candidate genes, ebiri bikuru munonga aha kuguma kw‘ekibumba.
Ebirugire omu kucondooza kwaitu nibyoreka ngu 1 mM IPA ekareetaho apoptosis kandi ekakyendeeza aha kuhuuta kwa mitochondrial omu butafaari bwa LX-2 obutarikwegamira aha bujanjabi bwa TGF-β1. Nikimanyisa ngu apoptosis n‘omuhanda mukuru gw‘okushoboorora fibrosis n‘okukora kw‘obutafaari bw‘eshagama (HSC), kandi n‘ekintu kikuru omu kugarukamu kw‘omubiri okurikugaruka enyima kw‘okusharara kw‘ekibumba [4, 43]. Ekindi, okugaruraho BHI omu butafaari bwa LX-2 bwanyima y‘okujanjaba okurikugaita kikaha okumanya okusya aha murimo ogurikubaasa kukorwa IPA omu kuhabura aha nkora y‘amagara g‘omubiri. Omu mbeera z‘okuhuumura n‘okutakora, obutafaari oburikureeta eshagama nibukira kukoresa obutafaari bw‘omubiri oburikwetwa mitochondrial oxidative phosphorylation kureetaho ATP kandi bwine emirimo mikye y‘okuhindura eby‘okurya. Aha rubaju orundi, okukora kwa HSC nikutunguura okuhuumura kw‘emibiri n‘okukora eby‘okurya by‘omubiri kugaruzaho ebyetaago by‘amaani eby‘okutaaha omu mbeera ya glycolytic [44]. Okugira ngu IPA teine ​​kakwate n‘obushoboorozi bw‘okuhindura eby‘okurya hamwe na ECAR nikimanyisa ngu omuhanda gwa glycolytic tigurikwebembezibwa munonga. Omu muringo nigwo gumwe, okucondooza okundi kukooreka ngu 1 mM IPA ekaba neebaasa kuhindura emirimo y‘omurunga gw‘okuhuumura ogwa mitochondrial omu butafaari bw‘omutima, omu nkora y‘obutafaari bw‘ekibumba bw‘omuntu (Huh7) n‘obutafaari bw‘omutsi gw‘omubiri gw‘omuntu (HUVEC); Kwonka, tihaine ekirikubaho kya IPA ekyashangirwe aha glycolysis omu cardiomyocytes, ekirikumanyisa ngu IPA neebaasa kukwata aha bioenergetics z‘ebindi bika by‘obutafaari [45]. Nahabwekyo, nituteekateeka ngu 1 mM IPA neebaasa kukora nk’omubazi gw’okutaho emibazi y’omutaano, ahabw’okuba neebaasa kukyendeeza munonga aha kumanyisa kw’obuzaarwa bw’omubiri, enkora y’obutafaari n’amaani g’omubiri g’omubiri hatariho kuhindura omuhendo gwa mtDNA [46]. Ebirikureetaho okutaikirizana kw‘emibiri nibibaasa kuzibira okusheeshasheesha okurikureetwaho obuhangwa n‘okukora kwa HSC [47] kandi bikyendeeze aha kureetaho ATP omu mitochondria erikutegyekwa nari erikureetwa puroteyini ezimwe nk‘ebiriisa ebitarikuteerana (UCP) nari adenine nucleotide translocase (ANT). Okurugirira aha kika ky‘obutafaari, eki nikibaasa kurinda obutafaari obutafa kandi/nari kutunguura okufa [46]. Kwonka, okucondooza okurikukiraho nikwetengyesa kushoboorora omurimo gwa IPA nk‘ekintu ekirikutaho obutafaari bw‘omubiri oburikukora eshagama omu kutakora.
Bwanyima tukacondooza yaaba empindahinduka omu kuhuumura kw‘ebicweka by‘omubiri nizireebeka omu nkora y‘ebicweka by‘omubiri omu butafaari bwa LX-2 obuhuriire. Ekirikushemeza, obujanjabi bwa TGF-β1 nibuhindura omubaro gw‘emibiri kuruga aha nkora y‘okwetoorora kuza aha nkora y‘ahagati, n‘okukyendeeza aha kusharaho kw‘emibiri n‘okwongyera aha kumanyisa kwa DRP1, enshonga nkuru omu kuhendagura kw‘emibiri [48]. Ekindi, okucwanyagura kwa mitochondrial nikukwatagana n’okugumizamu kw’omutimbagano gwona, kandi okuhinduka kuruga omu kugaita kuza omu kucwanyagura ni kikuru munonga omu kukora kw’obutafaari bw’eshagama (HSC), obwo okuzibira okucwanyagura kwa mitochondrial kurikureetaho HSC apoptosis [49]. Nahabwekyo, ebyarugamu byaitu nibyoreka ngu obujanjabi bwa TGF-β1 nibubaasa kureetera okukyendeera omu kugumizamu kw‘omutimbagano gw‘emibiri n‘okukyendeera kw‘okutaho amataagi, ekirikukirayo omu kusheesheka kw‘emibiri okurikukwatagana n‘obutafaari bw‘omubiri oburikukora (HSCs). Ekindi, ebihandiiko byaitu nibyoreka ngu IPA neebaasa kuhindura omubaro gwa mitochondria kuruga omu nkora y’enziga kuza omu nkora y’ahagati, obwo erikukyendeeza aha kureebeka kwa OPA1 na MFN2. Okucondooza kworekire ngu okukyendeeza aha nkora ya OPA1 nikubaasa kureetera okukyendeera omu bushoboorozi bw’omubiri gw’omubiri kandi kireetaho okufa kw’obutafaari [50]. MFN2 neemanyirwa aha kureetaho okuteerana kw’ebicweka by’omubiri n’okufa kw’ebicweka by’omubiri [51]. Ebirugire omu kucondooza oku nibyoreka ngu okutaho obutafaari bwa LX-2 na TGF-β1 na/nari IPA nikireebeka ngu nikihindura omu nkora n‘obuhango bwa mitochondrial, hamwe n‘embeera y‘okukora n‘okugumizamu kw‘omutimbagano.
Ebirugire omu kucondooza kwaitu nibyoreka ngu okujanjaba okurikugaita TGFβ-1 na IPA nikubaasa kukyendeeza mtDNA n’ebicweka by’obutafaari bw’omubiri obwo birikutebeekanisa oku mRNA erikworeka fibrosis, apoptosis n’ebicweka by’omubiri ebiine akakwate n’okugumaho omu butafaari oburikwetantara apoptosis. Buzima, IPA ekakyendeeza aha rurengo rw‘okumanyisa mRNA rwa AKT1 n‘ebicweka by‘omubiri ebikuru eby‘omubiri nka COL1A2 na MMP2, kwonka ekayongyera aha rurengo rw‘okumanyisa CASP8, orurikukwatagana n‘okucwekyerera kw‘omubiri. Ebirugire omu kucondooza kwaitu nibyoreka ngu bwanyima y‘okujanjaba IPA, okureebeka kwa BAX kukakyeera kandi okureebeka kwa mRNA kw‘ebicweka by‘eka ya TIMP1, BCL-2 na NF-κB kukeeyongyera, ekirikumanyisa ngu IPA neebaasa kureetaho obumanyiso bw‘okugumaho omu butafaari bw‘eshagama (HSCs) oburikwetantara apoptosis. Ebicweka ebi nibibaasa kukora nk‘obumanyiso bw‘okugumaho omu butafaari bw‘eshagama oburikukora, oburikubaasa kuba nibukwatagana n‘okwongyera aha kumanyisa kw‘ebiriisa ebirikurwanisa okufa (nka Bcl-2), okukyendeera omu kumanyisa kwa pro-apoptotic BAX, n‘okuzaana okugumire ahagati ya TIMP na NF-κB [5, 7]. IPA neekora emirimo yaayo kurabira omu PXR, kandi tukashanga ngu okujanjaba okurikugaita TGF-β1 na IPA kukaba kwongyera aha rurengo rw‘okumanyisa PXR mRNA, ekirikworeka okuzibira okukora kwa HSC. Okumanyisa kwa PXR okukozirwe nikumanyirwa aha kuzibira HSC kukora omu nkora y‘amagara n‘omu nkora y‘amagara [52, 53]. Ebirugire omu kucondooza kwaitu nibyoreka ngu IPA neebaasa kwejumba omu kukyebera HSCs ezikozirweho obwo erikutunguura apoptosis, okukyendeeza aha fibrosis na mitochondrial metabolism, n‘okutunguura obumanyiso bw‘okugumaho, oburi enkora z‘omutaano ezirikuhindura HSC ezikozirweho phenotype kuhinduka ezitarikukora. Ekindi ekirikubaasa kushoboorora aha nkora erikubaasa kubaho n‘omurimo gwa IPA omu apoptosis n‘okugira ngu neesheenya mitochondria etarikukora gye okukira munonga kurabira omu mitophagy (omuhanda gw‘omunda) n‘omuhanda gw‘okumanyisa TNF ogw‘aheeru (Ekipande 1), ogurikukwatagana butunu n‘omuhanda gw‘okumanyisa ogw‘okugumaho ogwa NF-κB Figure7 (omuhanda gw‘okumanyisa ogw‘okugumaho Figure7). Ekirikushemeza, ebiriisa ebitunguukire ebiine akakwate na IPA nibibaasa kureetaho obumanyiso bwa pro-apoptotic n‘obw‘okugumaho omu muhanda gwa apoptotic [54], ekirikumanyisa ngu IPA neebaasa kureetaho omuhanda gw‘okufa nari okugumaho ahabw‘okukoragana n‘ebiriisa ebi. Kwonka oku IPA erikureetaho apoptosis nari okugumaho omu bwire bw‘okukora kwa HSC n‘emihanda yaayo ey‘obukoryo tibirikwetegyerezibwa gye.
IPA n‘ekintu ekirikuhindura ebiriisa by‘omubiri kuruga omu tryptophan y‘omu by‘okurya kurabira omu bihingwa by‘omunda. Okucondooza kworekire ngu kiine obushoboorozi bw’okurwanisa okuzimba, okuzibira okusheeshasheesha, n’okutegyeka epigenetic omu mbeera y’omu mara.[55] Okucondooza kworekire ngu IPA neebaasa kuhindura omu nkora y‘okuzibira amaizi g‘omu mara n‘okukyendeeza aha kushoberwa kw‘okwosya, ekirikubaasa kureetaho ebirikurugamu omu mubiri gwayo omu mwanya.[56] Omu mazima, IPA neetwarwa omu bicweka by‘omubiri ebigyendereirwe kurabira omu kugyendera omu mubiri, kandi ahabw‘okugira ngu IPA neegaaniira aha nkora y‘okuhindura eby‘okurya ebikuru erikushushana na tryptophan, serotonin, n‘ebirikuruga omu indole, IPA neekora ebikorwa by‘okuhindura eby‘okurya ebirikurugamu okuhakanisa kw‘eby‘okurya.[52] IPA neebaasa kuhakanisa n‘ebintu ebirikuruga omu tryptophan ahabw‘emyanya y‘okukwatiraho aha nkora z‘omubiri nari aha bikwatiraine n‘okutunga, ekirikubaasa kuteganisa emihanda y‘okuhindura eby‘okurya eya butoosha. Eki nikyoreka ekyetengo ky’okweyongyera kwega aha nkora yaayo ey’okukoresa emibazi n’okukoresa emibazi yaayo kubaasa kushoboorora gye edirisa yaayo ey’okujanjaba.[57] Nikigumizamu kureeba yaaba eki nikibaasa kubaho n’omu butafaari bw’eshagama (HSCs).
Nitwikiriza ngu okwega kwaitu kwine obugarukiro. Kukyebera buzima enkoragana eziine akakwate na IPA, tukaihaho abarwaire abaine endwara ya shukaari ey‘omuringo 2 (T2DM). Nitwikiriza ngu eki nikizibira okukoresa okuhango kw‘ebi twashangire aha barwaire abaine endwara ya shukaari ey‘omuringo 2 n‘endwara y‘ebihaha erikutunguuka. N‘obu omubaro gw‘omubiri gwa IPA omu shagama y‘omuntu guri 1–10 μM [11, 20], omubaro gwa 1 mM IPA gukatooranwa kurugiirira aha mubaro gw‘ahaiguru ogutarimu obutwa [15] n‘omubaro gw‘ahaiguru gw‘okucwekyerera kw‘omubiri, hatariho ntaaniso omu bicweka 10 ahari 100 by‘obutafaari oburikufa. N‘obu emiringo ya supraphysiological ya IPA yaakozesiibwe omu kucondooza oku, hati tihaine okuhikirizana aha bikwatiraine n‘omubaro murungi gwa IPA [52]. N‘obu ebyarugamu byaitu biraabe biri bikuru, enkora y‘okuhindura eby‘okurya omu mubiri (metabolic fate) eya IPA neeguma eri omwanya gw‘okucondooza ogurikukora. Ekindi, ebi twashangire aha nkoragana eri ahagati y‘embeera za IPA omu shagama n‘okuhindura DNA omu bihandiiko by‘ebihaha tibikatungirwe kuruga omu butafaari bw‘eshagama (HSCs) bwonka kureka n‘omu bicweka by‘ebihaha. Twatoorana kukoresa obutafaari bw‘omuntu LX-2 kurugiirira aha bi twashangire enyima kuruga omu kucondooza kwa transcriptome ngu IPA neekwatagana n‘okukora kw‘obutafaari bw‘eshagama (HSC) [15], kandi HSCs nizo butafaari obukuru oburikwejumba omu kutunguuka kw‘okusharara kw‘ekibumba. Ekibumba kirimu ebika by‘obutafaari bingi, n‘ahabw‘ekyo ebindi bishushani by‘obutafaari nka hepatocyte-HSC-immune cell co-culture system hamwe n‘okukora kwa caspase n‘okucwanyagura DNA hamwe n‘omuringo gw‘okukora obariiremu omutindo gwa puroteyini biine kuteekateekwaho kwega omurimo gwa IPA n‘enkoragana yaayo n‘ebindi bishushani by‘obutafaari bw‘ekibumba.