Peb tau tshaj tawm ua ntej tias cov theem ntawm cov tshuaj tryptophan metabolite indole-3-propionic acid (IPA) hauv cov ntshav qis dua rau cov neeg mob uas muaj kab mob siab. Hauv kev tshawb fawb no, peb tau tshawb xyuas cov transcriptome thiab DNA methylome hauv cov neeg rog rog hauv kev sib raug zoo rau cov theem IPA hauv cov ntshav, nrog rau lub luag haujlwm ntawm IPA hauv kev ua kom cov phenotypic inactivation ntawm hepatic stellate cells (HSCs) hauv vitro.
Txoj kev tshawb fawb no muaj 116 tus neeg mob rog dhau uas tsis muaj mob ntshav qab zib hom 2 (T2DM) (hnub nyoog 46.8 ± 9.3 xyoo; BMI: 42.7 ± 5.0 kg/m²) uas tau phais mob bariatric ntawm Kuopio Bariatric Surgery Center (KOBS). Cov qib IPA uas nyob hauv lub cev tau ntsuas los ntawm kev siv kua chromatography-mass spectrometry (LC-MS), kev tshuaj xyuas daim siab transcriptome tau ua los ntawm kev tshuaj xyuas tag nrho RNA sequencing, thiab kev tshuaj xyuas DNA methylation tau ua los ntawm kev siv Infinium HumanMethylation450 BeadChip. Cov hlwb stellate hauv siab tib neeg (LX-2) tau siv rau kev sim hauv vitro.
Cov theem IPA hauv cov ntshav muaj feem cuam tshuam nrog kev qhia tawm ntawm cov noob caj noob ces uas koom nrog kev tuag ntawm lub cev, kev tuag ntawm lub cev, thiab txoj kev ua neej ntev hauv lub siab. Lub noob caj noob ces AKT serine/threonine kinase 1 (AKT1) yog cov noob caj noob ces uas muaj ntau tshaj plaws thiab tseem ceeb tshaj plaws hauv cov qauv ntawm daim siab thiab DNA methylation. Kev kho IPA ua rau muaj kev tuag ntawm lub cev, txo qis kev ua pa ntawm mitochondrial, thiab hloov pauv cov qauv ntawm lub hlwb thiab cov dynamics ntawm mitochondrial los ntawm kev hloov kho qhov kev qhia tawm ntawm cov noob caj noob ces uas paub tias tswj hwm fibrosis, apoptosis, thiab kev ciaj sia ntawm LX-2 hlwb.
Thaum muab cov ntaub ntawv no los sib sau ua ke, ces qhia tias IPA muaj peev xwm kho tau thiab ua rau lub cev tuag thiab hloov HSC phenotype mus rau qhov tsis ua haujlwm, yog li ua rau muaj peev xwm tiv thaiv kab mob siab los ntawm kev cuam tshuam rau HSC kev ua haujlwm thiab kev ua haujlwm ntawm mitochondrial.
Qhov kev rog dhau thiab metabolic syndrome tau cuam tshuam nrog kev nce ntxiv ntawm cov kab mob rog rog hauv daim siab (MASLD); tus kab mob cuam tshuam rau 25% txog 30% ntawm cov pej xeem [1]. Qhov tshwm sim tseem ceeb ntawm MASLD etiology yog daim siab fibrosis, ib qho txheej txheem dynamic uas tau piav qhia los ntawm kev sib sau ua ke ntawm fibrous extracellular matrix (ECM) [2]. Cov hlwb tseem ceeb uas koom nrog hauv daim siab fibrosis yog hepatic stellate cells (HSCs), uas muaj plaub hom phenotypes paub: quiescent, activated, inactivated, thiab senescent [3, 4]. HSCs tuaj yeem ua haujlwm thiab hloov pauv los ntawm daim ntawv quiescent mus rau hauv cov hlwb proliferative fibroblast-zoo li nrog kev xav tau lub zog siab, nrog rau kev qhia ntxiv ntawm α-smooth muscle actin (α-SMA) thiab hom I collagen (Col-I) [5, 6]. Thaum lub siab fibrosis rov qab, cov HSCs ua haujlwm raug tshem tawm ntawm apoptosis lossis inactivation. Cov txheej txheem no suav nrog kev txo qis ntawm cov noob fibrogenic thiab kev hloov kho ntawm cov noob prosurvival (xws li NF-κB thiab PI3K/Akt signaling pathways) [7, 8], nrog rau kev hloov pauv hauv mitochondrial dynamics thiab kev ua haujlwm [9].
Cov theem ntawm cov tryptophan metabolite indole-3-propionic acid (IPA), uas tsim tawm hauv txoj hnyuv, tau pom tias txo qis hauv cov kab mob metabolic tib neeg suav nrog MASLD [10–13]. IPA cuam tshuam nrog kev noj zaub mov fiber, paub txog nws cov teebmeem antioxidant thiab anti-inflammatory, thiab txo cov zaub mov-induced non-alcoholic steatohepatitis (NASH) phenotype hauv vivo thiab hauv vitro [11–14]. Qee cov pov thawj los ntawm peb txoj kev tshawb fawb yav dhau los, uas tau qhia tias cov theem IPA hauv cov ntshav qis dua hauv cov neeg mob uas muaj daim siab fibrosis dua li cov neeg mob rog tsis muaj daim siab fibrosis hauv Kuopio Bariatric Surgery Study (KOBS). Ntxiv mus, peb tau qhia tias kev kho mob IPA tuaj yeem txo qhov kev qhia tawm ntawm cov noob caj noob ces uas yog cov cim qhia txog kev sib txuas ntawm tes, kev tsiv teb tsaws chaw ntawm tes thiab hematopoietic stem cell activation hauv tib neeg hepatic stellate cell (LX-2) qauv thiab yog lub peev xwm hepatoprotective metabolite [15]. Txawm li cas los xij, nws tseem tsis meej tias IPA ua rau lub siab fibrosis regression los ntawm kev ua kom HSC apoptosis thiab mitochondrial bioenergetics ua haujlwm li cas.
Ntawm no, peb qhia tau hais tias serum IPA muaj feem cuam tshuam nrog kev qhia tawm ntawm cov noob caj noob ces uas nplua nuj nyob rau hauv apoptosis, mitophagy, thiab txoj kev ua neej ntev hauv lub siab ntawm cov neeg rog tab sis tsis yog hom 2 ntshav qab zib (KOBS). Ntxiv mus, peb pom tias IPA tuaj yeem ua rau muaj kev tshem tawm thiab kev puas tsuaj ntawm cov hlwb hematopoietic stem cell (HSCs) los ntawm txoj kev tsis ua haujlwm. Cov txiaj ntsig no qhia txog lub luag haujlwm tshiab rau IPA, ua rau nws yog lub hom phiaj kho mob los txhawb kev rov qab los ntawm daim siab fibrosis.
Ib txoj kev tshawb fawb yav dhau los hauv pawg neeg KOBS tau qhia tias cov neeg mob uas muaj kab mob siab fibrosis muaj cov qib IPA qis dua piv rau cov neeg mob uas tsis muaj kab mob siab fibrosis [15]. Txhawm rau kom tsis txhob muaj qhov cuam tshuam ntawm tus kab mob ntshav qab zib hom 2, peb tau nrhiav 116 tus neeg mob rog uas tsis muaj kab mob ntshav qab zib hom 2 (hnub nyoog nruab nrab ± SD: 46.8 ± 9.3 xyoo; BMI: 42.7 ± 5.0 kg/m2) (Rooj 1) los ntawm txoj kev tshawb fawb KOBS uas tab tom ua raws li cov pej xeem kawm [16]. Txhua tus neeg koom nrog tau muab kev pom zoo sau ntawv thiab cov txheej txheem tshawb fawb tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm North Savo County Tsev Kho Mob raws li Daim Ntawv Tshaj Tawm ntawm Helsinki (54/2005, 104/2008 thiab 27/2010).
Cov qauv kuaj mob siab tau txais thaum lub sijhawm phais mob bariatric thiab soj ntsuam cov ntaub ntawv los ntawm cov kws kho mob uas muaj kev paub dhau los raws li cov qauv piav qhia ua ntej [17, 18]. Cov qauv ntsuas tau muab tso ua ke hauv Supplementary Table S1 thiab tau piav qhia ua ntej [19].
Cov qauv ntshav thaum yoo mov tau raug tshuaj xyuas los ntawm kev siv cov kua chromatography-mass spectrometry (LC-MS) uas tsis tau tsom rau kev tshuaj xyuas metabolomics (n = 116). Cov qauv tau raug tshuaj xyuas siv lub kaw lus UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Lub Tebchaws Yelemees) raws li tau piav qhia ua ntej19. Kev txheeb xyuas isopropyl cawv (IPA) yog raws li lub sijhawm khaws cia thiab kev sib piv ntawm MS / MS spectrum nrog cov qauv ntshiab. Lub zog ntawm IPA teeb liab (thaj chaw siab tshaj plaws) tau raug txiav txim siab hauv txhua qhov kev tshuaj xyuas ntxiv [20].
Tag nrho daim siab RNA sequencing tau ua tiav siv Illumina HiSeq 2500 thiab cov ntaub ntawv tau ua tiav raws li tau piav qhia ua ntej [19, 21, 22]. Peb tau ua qhov kev tshuaj xyuas sib txawv ntawm cov ntawv sau uas cuam tshuam rau mitochondrial function / biogenesis siv 1957 noob xaiv los ntawm MitoMiner 4.0 database [23]. Daim siab DNA methylation kev tshuaj xyuas tau ua tiav siv Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) siv tib txoj kev raws li tau piav qhia ua ntej [24, 25].
Cov hlwb stellate hauv siab tib neeg (LX-2) tau muab los ntawm Prof. Stefano Romeo thiab tau cog qoob loo thiab tswj hwm hauv DMEM/F12 nruab nrab (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Txhawm rau xaiv cov koob tshuaj ua haujlwm ntawm IPA, LX-2 hlwb tau kho nrog ntau qhov sib txawv ntawm IPA (10 μM, 100 μM thiab 1 mM; Sigma, 220027) hauv DMEM/F12 nruab nrab rau 24 teev. Ntxiv mus, txhawm rau tshawb nrhiav lub peev xwm ntawm IPA los ua kom HSCs tsis ua haujlwm, LX-2 hlwb tau kho nrog 5 ng/ml TGF-β1 (R&D systems, 240-B-002/CF) thiab 1 mM IPA hauv nruab nrab tsis muaj serum rau 24 teev. Rau cov tsheb tswj hwm sib xws, 4 nM HCL uas muaj 0.1% BSA tau siv rau kev kho TGF-β1 thiab 0.05% DMSO tau siv rau kev kho IPA, thiab ob qho tib si tau siv ua ke rau kev kho ua ke.
Kev ntsuas Apoptosis tau raug soj ntsuam siv FITC Annexin V Apoptosis Detection Kit nrog 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) raws li cov lus qhia ntawm cov chaw tsim khoom. Hauv luv luv, LX-2 (1 × 105 hlwb/qhov dej) tau cog qoob loo thaum hmo ntuj hauv 12-qhov dej phaj thiab tom qab ntawd kho nrog ntau koob tshuaj ntawm IPA lossis IPA thiab TGF-β1. Hnub tom qab, cov hlwb ntab thiab cov hlwb lo tau raug sau, trypsinized, ntxuav nrog PBS, resuspended hauv Annexin V binding buffer, thiab incubated nrog FITC-Annexin V thiab 7-AAD rau 15 feeb.
Mitochondria hauv cov hlwb muaj sia nyob tau pleev xim rau kev ua haujlwm oxidative siv Mitotracker ™ Liab CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Rau MTR assays, LX-2 hlwb tau incubated ntawm qhov sib npaug zos nrog IPA thiab TGF-β1. Tom qab 24 teev, cov hlwb muaj sia nyob tau trypsinized, ntxuav nrog PBS, thiab tom qab ntawd incubated nrog 100 μM MTR hauv serum-free medium ntawm 37 ° C rau 20 feeb raws li tau piav qhia ua ntej [26]. Rau kev tshuaj xyuas cov hlwb muaj sia nyob, qhov loj ntawm cov hlwb thiab cytoplasmic complexity tau tshuaj xyuas siv cov kev sib txawv pem hauv ntej (FSC) thiab sab scatter (SSC), raws li.
Tag nrho cov ntaub ntawv (30,000 qhov xwm txheej) tau txais los ntawm kev siv NovoCyte Quanteon (Agilent) thiab tau tshuaj xyuas siv NovoExpress® 1.4.1 lossis FlowJo V.10 software.
Tus nqi siv oxygen (OCR) thiab tus nqi acidification sab nraud (ECAR) tau ntsuas lub sijhawm tiag tiag siv Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) nruab nrog Seahorse XF Cell Mito Stress raws li cov lus qhia ntawm cov chaw tsim khoom. Hauv luv luv, 2 × 104 LX-2 hlwb/qhov dej tau cog rau ntawm XF96 cell culture phaj. Tom qab incubation hmo ntuj, cov hlwb tau kho nrog isopropanol (IPA) thiab TGF-β1 (Supplementary Methods 1). Kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv Seahorse XF Wave software, uas suav nrog Seahorse XF Cell Energy Phenotype Test Report Generator. Los ntawm qhov no, Bioenergetic Health Index (BHI) tau suav [27].
Tag nrho RNA tau muab sau rau hauv cDNA. Rau cov txheej txheem tshwj xeeb, saib qhov siv [15]. Cov theem mRNA ntawm tib neeg 60S ribosomal acidic protein P0 (RPLP0) thiab cyclophilin A1 (PPIA) tau siv ua cov kev tswj hwm gene. Lub QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, Lub Netherlands) tau siv nrog TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) lossis Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), thiab qhov sib piv ntawm cov gene tau suav los ntawm kev siv cov kev sib piv Ct tus nqi cycling parameters (ΔΔCt) thiab txoj kev ∆∆Ct. Cov ntsiab lus ntawm cov primers tau muab rau hauv Cov Lus Qhia Ntxiv S2 thiab S3.
Nuclear DNA (ncDNA) thiab mitochondrial DNA (mtDNA) tau rho tawm siv DNeasy ntshav thiab cov ntaub so ntswg khoom siv (Qiagen) raws li tau piav qhia ua ntej [28]. Tus nqi sib piv ntawm mtDNA tau suav los ntawm kev xam qhov sib piv ntawm txhua thaj tsam mtDNA rau qhov nruab nrab geometric ntawm peb thaj tsam DNA nuclear (mtDNA / ncDNA), raws li tau piav qhia hauv Cov Txheej Txheem Ntxiv 2. Cov ntsiab lus ntawm cov primers rau mtDNA thiab ncDNA tau muab rau hauv Cov Lus Qhia Ntxiv S4.
Cov hlwb uas muaj sia nyob tau pleev xim nrog Mitotracker ™ Liab CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) kom pom tau cov tes hauj lwm intercellular thiab intracellular mitochondrial. LX-2 hlwb (1 × 104 hlwb/qhov dej) tau cog qoob loo rau ntawm cov iav swb hauv cov phaj kab lis kev cai hauv qab iav (Ibidi GmbH, Martinsried, Lub Tebchaws Yelemees). Tom qab 24 teev, cov hlwb LX-2 uas muaj sia nyob tau incubated nrog 100 μM MTR rau 20 feeb ntawm 37 ° C thiab cov hlwb nuclei tau pleev xim nrog DAPI (1 μg/ml, Sigma-Aldrich) raws li tau piav qhia ua ntej [29]. Cov tes hauj lwm Mitochondrial tau pom tau siv Zeiss Axio Observer inverted microscope (Carl Zeiss Microimaging GmbH, Jena, Lub Tebchaws Yelemees) nruab nrog Zeiss LSM 800 confocal module ntawm 37 ° C hauv huab cua humidified nrog 5% CO2 siv lub hom phiaj 63 × NA 1.3. Peb tau txais kaum daim duab Z-series rau txhua hom qauv. Txhua Z-series muaj 30 ntu, txhua daim muaj qhov tuab ntawm 9.86 μm. Rau txhua daim qauv, cov duab ntawm kaum qhov sib txawv ntawm qhov pom tau txais los ntawm kev siv ZEN 2009 software (Carl Zeiss Microimaging GmbH, Jena, Lub Tebchaws Yelemees), thiab kev tshuaj xyuas morphology mitochondrial tau ua tiav los ntawm kev siv ImageJ software (v1.54d) [30, 31] raws li cov kev ntsuas uas tau piav qhia hauv Cov Txheej Txheem Ntxiv 3.
Cov hlwb tau kho nrog 2% glutaraldehyde hauv 0.1 M phosphate buffer, ua raws li kev kho nrog 1% osmium tetroxide kua (Sigma Aldrich, MO, USA), maj mam qhuav nrog acetone (Merck, Darmstadt, Lub Tebchaws Yelemees), thiab thaum kawg muab tso rau hauv epoxy resin. Cov ntu Ultrathin tau npaj thiab pleev xim nrog 1% uranyl acetate (Merck, Darmstadt, Lub Tebchaws Yelemees) thiab 1% lead citrate (Merck, Darmstadt, Lub Tebchaws Yelemees). Cov duab Ultrastructural tau txais los ntawm kev siv JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Nyiv) ntawm qhov hluav taws xob nrawm ntawm 80 kV.
Cov morphology ntawm LX-2 hlwb kho nrog IPA rau 24 teev tau soj ntsuam los ntawm kev siv lub tshuab microscopy sib piv theem ntawm 50x magnification siv lub tshuab microscope Zeiss inverted light (Zeiss Axio Vert.A1 thiab AxioCam MRm, Jena, Lub Tebchaws Yelemees).
Cov ntaub ntawv kho mob tau qhia ua qhov nruab nrab ± tus qauv sib txawv lossis nruab nrab (interquartile range: IQR). Kev tshuaj xyuas ib txoj kev ntawm qhov sib txawv (cov hloov pauv tas mus li) lossis χ² xeem (cov hloov pauv categorical) tau siv los sib piv qhov sib txawv ntawm peb pawg kawm. Tus nqi tsis tseeb (FDR) tau siv los kho rau ntau qhov kev sim, thiab cov noob nrog FDR < 0.05 tau suav tias yog qhov tseem ceeb ntawm kev suav lej. Kev tshuaj xyuas Spearman correlation tau siv los sib raug zoo CpG DNA methylation nrog IPA lub zog teeb liab, nrog rau cov nqi nominal p ​​(p < 0.05) tau tshaj tawm.
Kev tshuaj xyuas txoj kev tau ua tiav los ntawm kev siv cov cuab yeej tshuaj xyuas cov noob caj noob ces hauv web (WebGestalt) rau 268 daim ntawv sau (nominal p < 0.01), 119 daim ntawv sau uas cuam tshuam nrog mitochondria (nominal p < 0.05), thiab 4350 CpGs ntawm 3093 daim ntawv sau siab uas cuam tshuam nrog cov qib IPA hauv cov ntshav. Lub cuab yeej Venny DB (version 2.1.0) uas muaj pub dawb tau siv los nrhiav cov noob caj noob ces sib tshooj, thiab StringDB (version 11.5) tau siv los pom kev sib cuam tshuam ntawm cov protein-protein.
Rau qhov kev sim LX-2, cov qauv tau kuaj xyuas qhov zoo li qub siv qhov kev xeem D'Agostino-Pearson. Cov ntaub ntawv tau txais los ntawm tsawg kawg peb qhov kev rov ua dua ntawm cov tsiaj txhu thiab raug rau ib txoj kev ANOVA nrog Bonferroni post hoc test. Tus nqi p tsawg dua 0.05 tau suav tias yog qhov tseem ceeb ntawm kev suav lej. Cov ntaub ntawv tau nthuav tawm ua qhov nruab nrab ± SD, thiab tus lej ntawm cov kev sim tau qhia hauv txhua daim duab. Txhua qhov kev tshuaj xyuas thiab cov duab tau ua tiav siv GraphPad Prism 8 software suav lej rau Windows (GraphPad Software Inc., version 8.4.3, San Diego, USA).
Ua ntej, peb tau tshawb nrhiav qhov kev sib raug zoo ntawm cov qib IPA hauv cov ntshav nrog daim siab, tag nrho lub cev, thiab cov ntawv sau mitochondrial. Hauv cov ntaub ntawv sau tag nrho, cov noob caj noob ces muaj zog tshaj plaws uas cuam tshuam nrog cov qib IPA hauv cov ntshav yog MAPKAPK3 (FDR = 0.0077; mitogen-activated protein kinase-activated protein kinase 3); hauv cov ntaub ntawv sau mitochondria, cov noob caj noob ces muaj zog tshaj plaws yog AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Cov ntaub ntawv ntxiv 1 thiab Cov ntaub ntawv ntxiv 2).
Tom qab ntawd peb tau tshuaj xyuas cov ntawv sau thoob ntiaj teb (n = 268; p < 0.01) thiab cov ntawv sau uas cuam tshuam nrog mitochondria (n = 119; p < 0.05), thaum kawg txheeb xyuas apoptosis ua txoj hauv kev tseem ceeb tshaj plaws (p = 0.0089). Rau cov ntawv sau mitochondrial uas cuam tshuam nrog cov theem IPA hauv cov ntshav, peb tau tsom mus rau apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), thiab TNF signaling pathways (FDR = 0.000006) (Daim Duab 1A, Rooj 2, thiab Cov Duab Ntxiv 1A-B).
Kev tshuaj xyuas sib tshooj ntawm cov ntawv sau thoob ntiaj teb, mitochondria-txuam nrog cov ntawv sau, thiab DNA methylation hauv tib neeg lub siab nrog rau cov qib IPA hauv cov ntshav. A sawv cev rau 268 cov ntawv sau thoob ntiaj teb, 119 cov ntawv sau mitochondria-txuam nrog cov ntawv sau, thiab DNA methylated transcripts uas tau mapped rau 3092 CpG qhov chaw cuam tshuam nrog cov qib IPA hauv cov ntshav (p tus nqi < 0.01 rau cov ntawv sau thoob ntiaj teb thiab DNA methylated, thiab p tus nqi < 0.05 rau cov ntawv sau mitochondrial). Cov ntawv sau sib tshooj tseem ceeb tau pom hauv nruab nrab (AKT1 thiab YKT6). B Daim ntawv qhia kev sib cuam tshuam ntawm 13 lub noob nrog qhov qhab nia sib cuam tshuam siab tshaj plaws (0.900) nrog lwm cov noob tau tsim los ntawm 56 lub noob sib tshooj (cheeb tsam kab dub) uas tau cuam tshuam nrog cov qib IPA hauv cov ntshav siv cov cuab yeej online StringDB. Ntsuab: Cov noob tau mapped rau Gene Ontology (GO) cellular Cheebtsam: mitochondria (GO: 0005739). AKT1 yog cov protein uas muaj qhov qhab nia siab tshaj plaws (0.900) rau kev sib cuam tshuam nrog lwm cov protein raws li cov ntaub ntawv (raws li kev tshawb nrhiav cov ntawv nyeem, kev sim, cov ntaub ntawv khaws cia, thiab kev sib koom ua ke). Cov nodes hauv network sawv cev rau cov protein, thiab cov npoo sawv cev rau kev sib txuas ntawm cov protein.
Vim tias cov metabolites ntawm cov kab mob hauv plab tuaj yeem tswj cov epigenetic composition los ntawm DNA methylation [32], peb tau tshawb xyuas seb cov theem IPA hauv cov ntshav puas cuam tshuam nrog lub siab DNA methylation. Peb pom tias ob qhov chaw methylation tseem ceeb cuam tshuam nrog cov theem IPA hauv cov ntshav nyob ze ntawm thaj chaw nplua nuj proline-serine 3 (C19orf55) thiab cov protein tsev neeg B (me me) 6 (HSPB6) (Cov ntaub ntawv ntxiv 3). DNA methylation ntawm 4350 CpG (p < 0.01) tau cuam tshuam nrog cov theem IPA hauv cov ntshav thiab tau nplua nuj hauv cov txheej txheem tswj kev ua neej ntev (p = 0.006) (Daim Duab 1A, Rooj 2, thiab Daim Duab Ntxiv 1C).
Yuav kom nkag siab txog cov txheej txheem ntawm lub cev uas ua rau muaj kev sib raug zoo ntawm cov qib IPA hauv cov ntshav, cov ntawv sau thoob ntiaj teb, cov ntawv sau mitochondria, thiab DNA methylation hauv tib neeg lub siab, peb tau ua qhov kev tshuaj xyuas sib tshooj ntawm cov noob caj noob ces uas tau txheeb xyuas hauv qhov kev tshuaj xyuas txoj kev dhau los (Daim Duab 1A). Cov txiaj ntsig ntawm kev tshuaj xyuas txoj kev nplua nuj ntawm 56 cov noob caj noob ces sib tshooj (sab hauv kab dub hauv Daim Duab 1A) qhia tau hais tias txoj kev apoptosis (p = 0.00029) tau hais txog ob lub noob caj noob ces zoo sib xws rau peb qhov kev tshuaj xyuas: AKT1 thiab YKT6 (YKT6 v-SNARE homolog), raws li tau pom hauv daim duab Venn (Daim Duab Ntxiv 2 thiab Daim Duab 1A). Qhov nthuav yog, peb pom tias AKT1 (cg19831386) thiab YKT6 (cg24161647) tau sib raug zoo nrog cov qib IPA hauv cov ntshav (Cov ntaub ntawv ntxiv 3). Txhawm rau txheeb xyuas cov protein sib cuam tshuam ntawm cov khoom noob caj noob ces, peb tau xaiv 13 noob caj noob ces nrog qhov qhab nia cheeb tsam siab tshaj plaws (0.900) ntawm 56 cov noob caj noob ces sib tshooj ua cov tswv yim thiab tsim daim ntawv qhia kev sib cuam tshuam. Raws li qib kev ntseeg siab (kev ntseeg siab marginal), AKT1 gene nrog cov qhab nia siab tshaj plaws (0.900) nyob rau sab saum toj (Daim Duab 1B).
Raws li kev tshuaj xyuas txoj kev, peb pom tias apoptosis yog txoj kev tseem ceeb, yog li peb tau tshawb xyuas seb kev kho mob IPA puas yuav cuam tshuam rau apoptosis ntawm HSCs hauv vitro. Peb tau ua pov thawj ua ntej tias cov koob tshuaj sib txawv ntawm IPA (10 μM, 100 μM, thiab 1 mM) tsis muaj tshuaj lom rau LX-2 hlwb [15]. Kev tshawb fawb no qhia tau tias kev kho mob IPA ntawm 10 μM thiab 100 μM ua rau muaj ntau tus lej ntawm cov hlwb muaj sia nyob thiab necrotic. Txawm li cas los xij, piv nrog pawg tswj hwm, kev muaj sia nyob ntawm cell txo qis ntawm 1 mM IPA concentration, thaum tus nqi necrosis ntawm cell tseem tsis hloov pauv (Daim Duab 2A, B). Tom ntej no, kom nrhiav tau qhov concentration zoo tshaj plaws los ua rau apoptosis hauv LX-2 hlwb, peb tau sim 10 μM, 100 μM, thiab 1 mM IPA rau 24 teev (Daim Duab 2A-E thiab Daim Duab Ntxiv 3A-B). Txaus siab kawg li, IPA 10 μM thiab 100 μM txo qhov apoptosis rate (%), txawm li cas los xij, IPA 1 mM ua rau apoptosis lig thiab apoptosis rate (%) ntau dua piv rau kev tswj hwm thiab yog li ntawd tau xaiv rau kev sim ntxiv (Daim Duab 2A-D).
IPA ua rau cov hlwb LX-2 tuag. Annexin V thiab 7-AAD ob chav staining txoj kev tau siv los ntsuas qhov apoptosis tus nqi thiab cell morphology los ntawm flow cytometry. BA hlwb tau incubated nrog 10 μM, 100 μM thiab 1 mM IPA rau 24 teev lossis nrog F-H TGF-β1 (5 ng/ml) thiab 1 mM IPA hauv serum-free medium rau 24 teev. A: cov hlwb muaj sia nyob (Annexin V -/ 7AAD-); B: cov hlwb necrotic (Annexin V -/ 7AAD+); C, F: thaum ntxov (Annexin V +/ 7AAD-); D, G: lig (Annexin V+/7AAD.+); E, H: feem pua ​​ntawm tag nrho cov hlwb apoptosis thaum ntxov thiab lig hauv apoptosis tus nqi (%). Cov ntaub ntawv tau qhia ua qhov nruab nrab ± SD, n = 3 kev sim ywj pheej. Kev sib piv cov ntaub ntawv tau ua tiav los ntawm kev siv ib txoj kev ANOVA nrog Bonferroni post hoc test. *p <0.05; ****p <0.0001
Raws li peb tau qhia ua ntej lawm, 5 ng/ml TGF-β1 tuaj yeem ua rau HSC ua haujlwm los ntawm kev nce qhov kev qhia tawm ntawm cov noob cim qub [15]. Cov hlwb LX-2 tau kho nrog 5 ng/ml TGF-β1 thiab 1 mM IPA ua ke (Daim Duab 2E-H). Kev kho TGF-β1 tsis hloov pauv tus nqi apoptosis, txawm li cas los xij, kev kho IPA ua ke ua rau apoptosis lig thiab tus nqi apoptosis nce (%) piv nrog kev kho TGF-β1 (Daim Duab 2E-H). Cov txiaj ntsig no qhia tau tias 1 mM IPA tuaj yeem txhawb nqa apoptosis hauv LX-2 hlwb ywj pheej ntawm TGF-β1 induction.
Peb tau tshawb nrhiav ntxiv txog qhov cuam tshuam ntawm IPA rau kev ua pa ntawm mitochondrial hauv LX-2 hlwb. Cov txiaj ntsig tau qhia tias 1 mM IPA txo cov kev ntsuas oxygen consumption rate (OCR): kev ua pa tsis yog mitochondrial, kev ua pa basal thiab maximal, proton leak thiab ATP ntau lawm piv rau pawg tswj (Daim Duab 3A, B), thaum lub bioenergetic health index (BHI) tsis hloov pauv.
IPA txo cov kev ua pa ntawm mitochondrial hauv LX-2 hlwb. Cov kab nkhaus mitochondrial respiration (OCR) tau nthuav tawm ua cov kev ntsuas kev ua pa ntawm mitochondrial (kev ua pa tsis yog mitochondrial, kev ua pa hauv paus, kev ua pa siab tshaj plaws, kev xau proton, ATP tiam, SRC thiab BHI). Cov hlwb A thiab B tau incubated nrog 10 μM, 100 μM thiab 1 mM IPA rau 24 teev, raws li. Cov hlwb C thiab D tau incubated nrog TGF-β1 (5 ng/ml) thiab 1 mM IPA hauv cov tshuaj tsis muaj serum rau 24 teev, raws li. Txhua qhov kev ntsuas tau normalized rau cov ntsiab lus DNA siv cov khoom siv CyQuant. BHI: bioenergetic kev noj qab haus huv index; SRC: lub peev xwm ua pa; OCR: tus nqi siv oxygen. Cov ntaub ntawv tau nthuav tawm ua qhov nruab nrab ± tus qauv sib txawv (SD), n = 5 kev sim ywj pheej. Kev sib piv cov ntaub ntawv tau ua tiav siv ib txoj kev ANOVA thiab Bonferroni post hoc test. *p < 0.05; **p < 0.01; thiab ***p < 0.001
Yuav kom nkag siab zoo dua txog qhov cuam tshuam ntawm IPA rau ntawm cov bioenergetic profile ntawm TGF-β1-activated LX-2 hlwb, peb tau tshuaj xyuas mitochondrial oxidative phosphorylation los ntawm OCR (Daim Duab 3C, D). Cov txiaj ntsig tau qhia tias kev kho TGF-β1 tuaj yeem txo qhov siab tshaj plaws ntawm kev ua pa, lub peev xwm ua pa (SRC) thiab BHI piv rau pawg tswj (Daim Duab 3C, D). Tsis tas li ntawd, kev kho ua ke txo qis kev ua pa hauv paus, proton leak thiab ATP ntau lawm, tab sis SRC thiab BHI siab dua li cov uas kho nrog TGF-β1 (Daim Duab 3C, D).
Peb kuj tau ua qhov "Cellular Energy Phenotype Test" uas Seahorse software muab (Supplementary Fig. 4A–D). Raws li pom hauv Supplementary Fig. 3B, ob qho tib si OCR thiab ECAR metabolic potentials tau txo qis tom qab TGF-β1 kev kho mob, txawm li cas los xij, tsis muaj qhov sib txawv tau pom hauv kev sib xyaw ua ke thiab IPA kev kho mob piv rau pawg tswj hwm. Ntxiv mus, ob qho tib si basal thiab stress theem ntawm OCR tau txo qis tom qab kev sib xyaw ua ke thiab IPA kev kho mob piv rau pawg tswj hwm (Supplementary Fig. 4C). Qhov nthuav yog, ib qho qauv zoo sib xws tau pom nrog kev kho mob ua ke, qhov twg tsis muaj kev hloov pauv hauv basal thiab stress theem ntawm ECAR tau pom piv rau TGF-β1 kev kho mob (Supplementary Fig. 4C). Hauv HSCs, qhov txo qis hauv mitochondrial oxidative phosphorylation thiab lub peev xwm ntawm kev kho mob ua ke los rov qab SCR thiab BHI tom qab raug rau TGF-β1 kev kho mob tsis hloov pauv metabolic potential (OCR thiab ECAR). Thaum muab cov txiaj ntsig no ua ke qhia tau tias IPA yuav txo cov bioenergetics hauv HSCs, qhia tias IPA yuav ua rau muaj qhov profile qis zog uas hloov HSC phenotype mus rau qhov tsis ua haujlwm (Daim Duab Ntxiv 4D).
Cov nyhuv ntawm IPA rau mitochondrial dynamics tau tshawb nrhiav siv peb-seem kev ntsuas ntawm mitochondrial morphology thiab kev sib txuas network nrog rau MTR staining (Daim Duab 4 thiab Daim Duab Ntxiv 5). Daim Duab 4 qhia tau tias, piv nrog pawg tswj, TGF-β1 kev kho mob txo qhov nruab nrab ntawm thaj chaw, tus lej ceg, tag nrho cov ceg ntev, thiab tus lej sib txuas ceg (Daim Duab 4A thiab B) thiab hloov qhov feem pua ​​ntawm mitochondria los ntawm spherical mus rau intermediate morphology (Daim Duab 4C). Tsuas yog IPA kev kho mob txo qhov nruab nrab ntawm mitochondrial ntim thiab hloov qhov feem pua ​​ntawm mitochondria los ntawm spherical mus rau intermediate morphology piv nrog pawg tswj (Daim Duab 4A). Qhov sib txawv, sphericity, nruab nrab ntawm ceg ntev, thiab mitochondrial kev ua ub no ntsuas los ntawm mitochondrial membrane potential-dependent MTR (Daim Duab 4A thiab E) tseem tsis hloov pauv thiab cov kev ntsuas no tsis txawv ntawm pawg. Thaum muab ua ke, cov txiaj ntsig no qhia tau tias TGF-β1 thiab IPA kev kho mob zoo li hloov kho mitochondrial duab thiab qhov loj me nrog rau qhov nyuaj ntawm network hauv cov hlwb LX-2 uas muaj sia nyob.
IPA hloov pauv mitochondrial dynamics thiab mitochondrial DNA abundance hauv LX-2 hlwb. A. Cov duab confocal sawv cev ntawm cov hlwb LX-2 nyob uas tau incubated nrog TGF-β1 (5 ng/ml) thiab 1 mM IPA rau 24 teev hauv serum-free medium uas qhia txog mitochondrial networks stained nrog Mitotracker ™ Liab CMXRos thiab nuclei stained xiav nrog DAPI. Tag nrho cov ntaub ntawv muaj tsawg kawg 15 daim duab ib pawg. Peb tau txais 10 Z-stack duab rau txhua hom qauv. Txhua qhov Z-axis sequence muaj 30 daim, txhua tus muaj qhov tuab ntawm 9.86 μm. Scale bar: 10 μm. B. Cov khoom sawv cev (mitochondria xwb) tau txheeb xyuas los ntawm kev siv adaptive thresholding rau daim duab. Kev tshuaj xyuas ntau thiab kev sib piv ntawm mitochondrial morphological network kev sib txuas tau ua rau txhua lub hlwb hauv txhua pawg. C. Zaus ntawm mitochondrial duab piv. Cov nqi ze rau 0 qhia txog cov duab kheej kheej, thiab cov nqi ze rau 1 qhia txog cov duab filamentous. D Cov ntsiab lus ntawm Mitochondrial DNA (mtDNA) tau txiav txim siab raws li tau piav qhia hauv Cov Khoom Siv thiab Cov Txheej Txheem. E Kev tshuaj xyuas Mitotracker ™ Liab CMXRos tau ua los ntawm kev ntws cytometry (30,000 qhov xwm txheej) raws li tau piav qhia hauv Cov Khoom Siv thiab Cov Txheej Txheem. Cov ntaub ntawv tau nthuav tawm ua qhov nruab nrab ± SD, n = 3 qhov kev sim ywj pheej. Kev sib piv cov ntaub ntawv tau ua tiav siv ib txoj kev ANOVA thiab Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Tom qab ntawd peb tau tshuaj xyuas cov ntsiab lus mtDNA hauv LX-2 hlwb ua qhov qhia txog tus lej mitochondrial. Piv nrog rau pawg tswj, cov ntsiab lus mtDNA tau nce ntxiv hauv pawg kho TGF-β1 (Daim Duab 4D). Piv nrog rau pawg kho TGF-β1, cov ntsiab lus mtDNA tau txo qis hauv pawg kho ua ke (Daim Duab 4D), qhia tias IPA yuav txo cov ntsiab lus mtDNA thiab tej zaum tus lej mitochondrial nrog rau kev ua pa mitochondrial (Daim Duab 3C). Ntxiv mus, IPA zoo li txo cov ntsiab lus mtDNA hauv kev kho ua ke tab sis tsis cuam tshuam rau MTR-mediated mitochondrial activity (Daim Duab 4A-C).
Peb tau tshawb nrhiav qhov kev sib txuas ntawm IPA nrog cov theem mRNA ntawm cov noob caj noob ces uas cuam tshuam nrog fibrosis, apoptosis, kev ciaj sia, thiab mitochondrial dynamics hauv LX-2 hlwb (Daim Duab 5A–D). Piv nrog pawg tswj, pawg kho TGF-β1 tau pom tias muaj kev qhia ntau ntxiv ntawm cov noob caj noob ces xws li collagen hom I α2 saw (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), cov ntaub so ntswg inhibitor ntawm metalloproteinase 1 (TIMP1), thiab dynamin 1-zoo li noob caj noob ces (DRP1), qhia tias muaj fibrosis thiab kev ua haujlwm ntau ntxiv. Ntxiv mus, piv nrog pawg tswj, kev kho mob TGF-β1 txo cov mRNA theem ntawm nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, inhibitor ntawm B-cell α, enhancer ntawm nuclear factor κ gene light peptide (NFκB1A), thiab inhibitor ntawm nuclear factor κB kinase subunit β (IKBKB) (Daim Duab 5A–D). Piv nrog kev kho mob TGF-β1, kev kho mob ua ke nrog TGF-β1 thiab IPA txo qhov kev qhia tawm ntawm COL1A2 thiab MMP2, tab sis nce cov mRNA theem ntawm PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, thiab IKBKB. Kev kho mob IPA txo qis qhov kev qhia tawm ntawm MMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), thiab mitochondrial fusion protein 2 (MFN2), thaum qhov kev qhia tawm ntawm CASP8, NFκB1A, NFκB1B, thiab IKBKB tau nce ntxiv piv rau pawg tswj. Txawm li cas los xij, tsis muaj qhov sib txawv hauv kev qhia tawm ntawm caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), thiab fission protein 1 (FIS1). Ua ke, cov txiaj ntsig no qhia tias kev kho mob IPA hloov kho qhov kev qhia tawm ntawm cov noob caj noob ces cuam tshuam nrog fibrosis, apoptosis, kev ciaj sia, thiab mitochondrial dynamics. Peb cov ntaub ntawv qhia tias kev kho mob IPA txo ​​​​cov fibrosis hauv LX-2 hlwb; tib lub sijhawm, nws txhawb kev ciaj sia los ntawm kev hloov pauv phenotype mus rau inactivation.
IPA hloov kho qhov kev qhia tawm ntawm fibroblast, apoptotic, viability, thiab mitochondrial dynamics noob hauv LX-2 hlwb. Histograms qhia mRNA kev qhia tawm piv rau endogenous tswj (RPLP0 lossis PPIA) tom qab LX-2 hlwb raug induced nrog TGF-β1 thiab IPA hauv serum-free medium rau 24 teev. A qhia txog fibroblasts, B qhia txog apoptotic hlwb, C qhia txog cov hlwb uas muaj sia nyob, thiab D qhia txog mitochondrial dynamics noob qhia tawm. Cov ntaub ntawv tau nthuav tawm ua qhov nruab nrab ± tus qauv deviation (SD), n = 3 kev sim ywj pheej. Kev sib piv cov ntaub ntawv tau ua tiav siv ib txoj kev ANOVA thiab Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Tom qab ntawd, kev hloov pauv ntawm qhov loj ntawm lub cell (FSC-H) thiab qhov nyuaj ntawm cytoplasmic (SSC-H) tau raug soj ntsuam los ntawm kev ntsuas flow cytometry (Daim Duab 6A, B), thiab kev hloov pauv ntawm cov cell morphology tom qab kev kho IPA tau raug soj ntsuam los ntawm kev siv lub tshuab hluav taws xob xa mus (TEM) thiab lub tshuab ntsuas qhov sib piv ntawm theem (Daim Duab Ntxiv 6A-B). Raws li xav tau, cov cell hauv pawg kho TGF-β1 tau nce qhov loj me piv rau pawg tswj (Daim Duab 6A, B), qhia txog kev nthuav dav ntawm cov reticulum endoplasmic rough (ER*) thiab phagolysosomes (P), qhia txog kev ua haujlwm ntawm cov hlwb hematopoietic (HSC) (Daim Duab Ntxiv 6A). Txawm li cas los xij, piv nrog pawg kho TGF-β1, qhov loj me ntawm lub cell, qhov nyuaj ntawm cytoplasmic (Daim Duab 6A, B), thiab cov ntsiab lus ER* tau txo qis hauv pawg kho TGF-β1 thiab IPA ua ke (Daim Duab Ntxiv 6A). Ntxiv mus, kev kho IPA txo qhov loj ntawm cov cell, qhov nyuaj ntawm cytoplasmic (Daim duab 6A, B), P thiab ER * cov ntsiab lus (Daim duab ntxiv 6A) piv rau pawg tswj. Tsis tas li ntawd, cov ntsiab lus ntawm cov cell apoptotic nce tom qab 24 teev ntawm kev kho IPA piv rau pawg tswj (xub dawb, Daim duab ntxiv 6B). Ua ke, cov txiaj ntsig no qhia tias 1 mM IPA tuaj yeem txhawb HSC apoptosis thiab thim rov qab cov kev hloov pauv hauv cov qauv morphological ntawm cell uas TGF-β1 ua rau, yog li tswj qhov loj ntawm cell thiab qhov nyuaj, uas tej zaum yuav cuam tshuam nrog HSC inactivation.
IPA hloov qhov loj ntawm cov cell thiab qhov nyuaj ntawm cytoplasmic hauv LX-2 cell. Cov duab sawv cev ntawm kev tshuaj xyuas flow cytometry. Kev tshuaj xyuas siv txoj kev gating tshwj xeeb rau LX-2 cell: SSC-A/FSC-A los txhais cov pejxeem cell, FSC-H/FSC-A los txheeb xyuas cov doublets, thiab SSC-H/FSC-H rau qhov loj ntawm cell thiab kev tshuaj xyuas qhov nyuaj. Cov cell tau incubated nrog TGF-β1 (5 ng/ml) thiab 1 mM IPA hauv serum-free medium rau 24 teev. LX-2 cell tau muab faib rau hauv qis sab laug quadrant (SSC-H-/FSC-H-), sab laug sab saud quadrant (SSC-H+/FSC-H-), sab xis sab qis quadrant (SSC-H-/FSC-H+), thiab sab xis sab saud quadrant (SSC-H+/FSC-H+) rau qhov loj ntawm cell thiab kev tshuaj xyuas qhov nyuaj ntawm cytoplasmic. B. Cov qauv ntawm cov hlwb tau raug tshuaj xyuas los ntawm kev siv cov txheej txheem ntws ntawm cov hlwb siv FSC-H (kev sib tsoo pem hauv ntej, qhov loj ntawm cov hlwb) thiab SSC-H (kev sib tsoo sab, qhov nyuaj ntawm cov hlwb) (30,000 qhov xwm txheej). Cov ntaub ntawv tau nthuav tawm ua qhov nruab nrab ± SD, n = 3 qhov kev sim ywj pheej. Kev sib piv cov ntaub ntawv tau ua tiav los ntawm kev siv ib txoj kev ANOVA thiab Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 thiab ****p < 0.0001
Cov metabolites hauv plab xws li IPA tau dhau los ua ib qho kev tshawb fawb kub, qhia tias cov hom phiaj tshiab yuav raug tshawb pom hauv cov kab mob hauv plab. Yog li ntawd, nws yog qhov nthuav tias IPA, ib qho metabolite uas peb tau txuas nrog rau daim siab fibrosis hauv tib neeg [15], tau pom tias yog ib qho tshuaj tiv thaiv fibrotic hauv cov qauv tsiaj [13, 14]. Ntawm no, peb qhia thawj zaug txog kev sib raug zoo ntawm serum IPA thiab kev hloov pauv thoob ntiaj teb ntawm daim siab thiab DNA methylation hauv cov neeg rog uas tsis muaj ntshav qab zib hom 2 (T2D), qhia txog apoptosis, mitophagy thiab lub neej ntev, nrog rau ib qho gene AKT1 uas tswj hwm daim siab homeostasis. Lwm qhov tshiab ntawm peb txoj kev tshawb fawb yog tias peb tau qhia txog kev sib cuam tshuam ntawm IPA kev kho mob nrog apoptosis, cell morphology, mitochondrial bioenergetics thiab dynamics hauv LX-2 hlwb, qhia txog lub zog qis dua uas hloov HSC phenotype mus rau inactivation, ua rau IPA yog tus neeg sib tw rau kev txhim kho daim siab fibrosis.
Peb pom tias apoptosis, mitophagy thiab lub neej ntev yog cov tseem ceeb tshaj plaws canonical pathways nplua nuj nyob rau hauv daim siab genes cuam tshuam nrog circulating serum IPA. Kev cuam tshuam ntawm mitochondrial zoo tswj (MQC) system tuaj yeem ua rau mitochondrial dysfunction, mitophagy thiab apoptosis, yog li txhawb nqa qhov tshwm sim ntawm MASLD [33, 34]. Yog li ntawd, peb tuaj yeem xav tias IPA yuav koom nrog kev tswj hwm cell dynamics thiab mitochondrial kev ncaj ncees los ntawm apoptosis, mitophagy thiab lub neej ntev hauv daim siab. Peb cov ntaub ntawv qhia tau hais tias ob lub noob tau pom ntau thoob plaws peb qhov kev sim: YKT6 thiab AKT1. Nws yog ib qho tsim nyog sau tseg tias YKT6 yog SNARE protein koom nrog hauv cov txheej txheem ntawm cell membrane fusion. Nws ua lub luag haujlwm hauv autophagy thiab mitophagy los ntawm kev tsim ib qho pib complex nrog STX17 thiab SNAP29 ntawm autophagosome, yog li txhawb nqa kev sib xyaw ntawm autophagosomes thiab lysosomes [35]. Ntxiv mus, kev poob ntawm YKT6 kev ua haujlwm ua rau muaj kev cuam tshuam mitophagy [36], thaum kev nce ntawm YKT6 yog txuam nrog kev nce qib ntawm hepatocellular carcinoma (HCC), qhia txog kev muaj sia nyob ntawm cov cell [37]. Ntawm qhov tod tes, AKT1 yog qhov tseem ceeb tshaj plaws interacting gene thiab ua lub luag haujlwm tseem ceeb hauv cov kab mob siab, suav nrog PI3K/AKT signaling pathway, cell cycle, cell migration, proliferation, focal adhesion, mitochondrial function, thiab collagen secretion [38–40]. Kev ua haujlwm PI3K/AKT signaling pathway tuaj yeem ua kom cov hlwb hematopoietic stem cell (HSCs), uas yog cov hlwb uas muaj lub luag haujlwm rau kev tsim cov extracellular matrix (ECM), thiab nws qhov kev ua haujlwm tsis zoo yuav pab txhawb rau qhov tshwm sim thiab kev nce qib ntawm daim siab fibrosis [40]. Ntxiv rau, AKT yog ib qho ntawm cov yam ntxwv tseem ceeb ntawm cov hlwb uas tiv thaiv p53-dependent cell apoptosis, thiab AKT activation yuav cuam tshuam nrog kev tiv thaiv ntawm daim siab cell apoptosis [41, 42]. Cov txiaj ntsig tau los qhia tias IPA yuav koom nrog rau hauv daim siab mitochondria-cuam tshuam apoptosis los ntawm kev cuam tshuam rau kev txiav txim siab ntawm hepatocytes ntawm kev nkag mus rau apoptosis lossis kev ciaj sia. Cov teebmeem no yuav raug tswj hwm los ntawm AKT thiab / lossis YKT6 cov noob sib tw, uas yog qhov tseem ceeb rau daim siab homeostasis.
Peb cov txiaj ntsig tau qhia tias 1 mM IPA ua rau apoptosis thiab txo qis mitochondrial respiration hauv LX-2 hlwb ywj pheej ntawm TGF-β1 kev kho mob. Nws yog qhov tsim nyog tias apoptosis yog ib txoj hauv kev tseem ceeb rau kev daws teeb meem fibrosis thiab hematopoietic stem cell (HSC) kev ua haujlwm, thiab kuj yog ib qho tseem ceeb hauv kev teb rov qab ntawm lub siab fibrosis [4, 43]. Ntxiv mus, kev kho dua tshiab ntawm BHI hauv LX-2 hlwb tom qab kev kho mob ua ke tau muab kev nkag siab tshiab rau hauv lub luag haujlwm ntawm IPA hauv kev tswj hwm ntawm mitochondrial bioenergetics. Hauv qab so thiab tsis ua haujlwm, hematopoietic hlwb feem ntau siv mitochondrial oxidative phosphorylation los tsim ATP thiab muaj kev ua haujlwm metabolic qis. Ntawm qhov tod tes, HSC kev ua haujlwm txhim kho mitochondrial respiration thiab biosynthesis los them rau qhov kev xav tau lub zog ntawm kev nkag mus rau hauv lub xeev glycolytic [44]. Qhov tseeb tias IPA tsis cuam tshuam rau lub peev xwm metabolic thiab ECAR qhia tias txoj hauv kev glycolytic tsis tshua muaj qhov tseem ceeb. Ib yam li ntawd, lwm txoj kev tshawb fawb tau qhia tias 1 mM IPA muaj peev xwm hloov kho cov haujlwm ntawm mitochondrial respiratory chain hauv cardiomyocytes, tib neeg lub siab hepatocyte cell kab (Huh7) thiab tib neeg lub hlab ntsha endothelial hlwb (HUVEC); Txawm li cas los xij, tsis muaj qhov cuam tshuam ntawm IPA pom ntawm glycolysis hauv cardiomyocytes, qhia tias IPA yuav cuam tshuam rau bioenergetics ntawm lwm hom cell [45]. Yog li ntawd, peb xav tias 1 mM IPA yuav ua haujlwm ua cov tshuaj tsis muaj zog, vim nws tuaj yeem txo qis fibrogenic gene expression, cell morphology thiab mitochondrial bioenergetics yam tsis hloov pauv qhov ntau ntawm mtDNA [46]. Mitochondrial uncouplers tuaj yeem tiv thaiv kab lis kev cai-induced fibrosis thiab HSC activation [47] thiab txo cov mitochondrial ATP ntau lawm tswj lossis induced los ntawm qee cov protein xws li uncoupling proteins (UCP) lossis adenine nucleotide translocase (ANT). Nyob ntawm hom cell, qhov xwm txheej no tuaj yeem tiv thaiv cov cell los ntawm apoptosis thiab / lossis txhawb nqa apoptosis [46]. Txawm li cas los xij, xav tau kev tshawb fawb ntxiv kom piav qhia txog lub luag haujlwm ntawm IPA ua tus mitochondrial uncoupler hauv hematopoietic stem cell inactivation.
Tom qab ntawd peb tau tshawb xyuas seb qhov kev hloov pauv ntawm mitochondrial respiration puas tau cuam tshuam rau hauv mitochondrial morphology hauv cov hlwb LX-2 uas muaj sia nyob. Qhov nthuav yog, kev kho mob TGF-β1 hloov pauv qhov sib piv ntawm mitochondrial los ntawm lub voj voog mus rau nruab nrab, nrog rau kev txo qis mitochondrial branching thiab kev nce ntxiv ntawm DRP1, ib qho tseem ceeb hauv mitochondrial fission [48]. Ntxiv mus, mitochondrial fragmentation yog txuam nrog tag nrho cov teeb meem ntawm network, thiab kev hloov pauv ntawm fusion mus rau fission yog qhov tseem ceeb rau hematopoietic stem cell (HSC) activation, thaum inhibition ntawm mitochondrial fission ua rau HSC apoptosis [49]. Yog li, peb cov txiaj ntsig qhia tau tias kev kho mob TGF-β1 yuav ua rau muaj kev txo qis hauv mitochondrial network complexity nrog kev txo qis branching, uas yog ntau dua hauv mitochondrial fission cuam tshuam nrog kev ua haujlwm hematopoietic stem cell (HSCs). Ntxiv mus, peb cov ntaub ntawv qhia tau tias IPA tuaj yeem hloov pauv qhov sib piv ntawm mitochondria los ntawm lub voj voog mus rau nruab nrab, yog li txo qhov kev qhia ntawm OPA1 thiab MFN2. Cov kev tshawb fawb tau qhia tias kev txo qis ntawm OPA1 tuaj yeem ua rau muaj kev txo qis hauv mitochondrial membrane potential thiab ua rau cell apoptosis [50]. MFN2 paub tias yog tus pab txhawb mitochondrial fusion thiab apoptosis [51]. Cov txiaj ntsig tau txais qhia tias kev induction ntawm LX-2 cell los ntawm TGF-β1 thiab/lossis IPA zoo li hloov kho mitochondrial duab thiab qhov loj me, nrog rau lub xeev ua haujlwm thiab qhov nyuaj ntawm network.
Peb cov txiaj ntsig qhia tau hais tias kev kho mob ua ke ntawm TGFβ-1 thiab IPA tuaj yeem txo cov mtDNA thiab cov qauv cell morphological los ntawm kev tswj hwm mRNA kev qhia ntawm fibrosis, apoptosis thiab cov noob caj noob ces uas cuam tshuam nrog kev ciaj sia nyob hauv cov hlwb apoptosis-evading. Tseeb tiag, IPA txo qis qib kev qhia mRNA ntawm AKT1 thiab cov noob caj noob ces tseem ceeb xws li COL1A2 thiab MMP2, tab sis nce qib kev qhia ntawm CASP8, uas cuam tshuam nrog apoptosis. Peb cov txiaj ntsig qhia tau hais tias tom qab kev kho mob IPA, kev qhia BAX txo qis thiab kev qhia mRNA ntawm TIMP1 tsev neeg subunits, BCL-2 thiab NF-κB nce ntxiv, qhia tias IPA tuaj yeem txhawb cov cim kev ciaj sia nyob hauv cov hlwb hematopoietic stem cell (HSCs) uas zam kev tuag. Cov molecule no yuav ua haujlwm ua cov cim qhia txog kev ciaj sia nyob hauv cov hlwb hematopoietic uas tau qhib lawm, uas tej zaum yuav cuam tshuam nrog kev nce ntxiv ntawm cov protein tiv thaiv apoptotic (xws li Bcl-2), kev txo qis ntawm kev nce ntxiv ntawm pro-apoptotic BAX, thiab kev sib cuam tshuam ntawm TIMP thiab NF-κB [5, 7]. IPA ua rau nws cov teebmeem los ntawm PXR, thiab peb pom tias kev kho mob ua ke nrog TGF-β1 thiab IPA ua rau cov qib kev qhia tawm ntawm PXR mRNA ntau ntxiv, qhia txog kev txwv tsis pub HSC ua haujlwm. Kev ua haujlwm PXR signaling paub tias inhibit HSC ua haujlwm ob qho tib si hauv vivo thiab hauv vitro [52, 53]. Peb cov txiaj ntsig qhia tau tias IPA tuaj yeem koom nrog kev tshem tawm ntawm HSCs uas tau qhib los ntawm kev txhawb nqa apoptosis, txo cov fibrosis thiab mitochondrial metabolism, thiab txhim kho cov cim qhia txog kev ciaj sia nyob, uas yog cov txheej txheem ib txwm muaj uas hloov cov phenotype HSC uas tau qhib rau ib qho uas tsis ua haujlwm. Lwm qhov piav qhia txog lub tshuab thiab lub luag haujlwm ntawm IPA hauv apoptosis yog tias nws scavenges cov mitochondria dysfunctional feem ntau los ntawm mitophagy (txoj kev intrinsic) thiab extrinsic TNF signaling pathway (Rooj 1), uas txuas ncaj qha rau NF-κB survival signaling pathway (Supplementary Figure 7). Qhov nthuav yog, IPA-related enriched genes muaj peev xwm induce pro-apoptotic thiab pro-survival signals hauv apoptotic pathway [54], qhia tias IPA yuav induce apoptotic pathway lossis survival los ntawm kev sib cuam tshuam nrog cov genes no. Txawm li cas los xij, IPA induces apoptosis lossis survival thaum lub sijhawm HSC activation thiab nws cov mechanismistic pathways tseem tsis meej.
IPA yog ib qho tshuaj lom neeg uas tsim los ntawm cov khoom noj tryptophan los ntawm cov kab mob hauv plab. Cov kev tshawb fawb tau qhia tias nws muaj cov khoom tiv thaiv kev o, antioxidant, thiab epigenetic hauv ib puag ncig plab. [55] Cov kev tshawb fawb tau qhia tias IPA tuaj yeem hloov kho txoj haujlwm ntawm txoj hnyuv thiab txo qhov kev ntxhov siab oxidative, uas yuav ua rau nws cov teebmeem hauv zos. [56] Qhov tseeb, IPA raug thauj mus rau cov kabmob ntawm lub cev los ntawm kev ncig, thiab txij li IPA sib koom ua ke cov qauv metabolite loj zoo sib xws nrog tryptophan, serotonin, thiab indole derivatives, IPA ua rau muaj kev sib tw metabolic fates. [52] IPA yuav sib tw nrog tryptophan-derived metabolites rau cov chaw khi rau ntawm cov enzymes lossis receptors, uas yuav cuam tshuam rau txoj kev metabolic ib txwm muaj. Qhov no qhia txog qhov xav tau kev tshawb fawb ntxiv txog nws cov tshuaj pharmacokinetics thiab pharmacodynamics kom nkag siab zoo dua nws lub qhov rais kho mob. [57] Nws tseem yuav tsum tau pom seb qhov no puas tuaj yeem tshwm sim hauv cov hlwb hematopoietic (HSCs).
Peb lees paub tias peb txoj kev tshawb fawb muaj qee qhov kev txwv. Txhawm rau tshuaj xyuas cov kev sib raug zoo ntsig txog IPA, peb tsis suav nrog cov neeg mob uas muaj hom 2 ntshav qab zib mellitus (T2DM). Peb lees paub tias qhov no txwv tsis pub siv dav dav ntawm peb cov kev tshawb pom rau cov neeg mob uas muaj hom 2 ntshav qab zib mellitus thiab kab mob siab heev. Txawm hais tias qhov kev sib xyaw ua ke ntawm IPA hauv tib neeg cov ntshav yog 1–10 μM [11, 20], qhov kev sib xyaw ua ke ntawm 1 mM IPA tau xaiv raws li qhov kev sib xyaw ua ke tsis muaj tshuaj lom siab tshaj plaws [15] thiab tus nqi siab tshaj plaws ntawm apoptosis, tsis muaj qhov sib txawv ntawm feem pua ​​​​​​ntawm cov pej xeem necrotic cell. Txawm hais tias cov qib supraphysiological ntawm IPA tau siv hauv txoj kev tshawb fawb no, tam sim no tsis muaj kev pom zoo txog qhov koob tshuaj zoo ntawm IPA [52]. Txawm hais tias peb cov txiaj ntsig tseem ceeb, qhov dav dav ntawm metabolic fate ntawm IPA tseem yog thaj chaw tshawb fawb nquag. Ntxiv mus, peb cov kev tshawb pom ntawm kev sib raug zoo ntawm cov qib IPA hauv cov ntshav thiab DNA methylation ntawm daim siab transcripts tau txais tsis yog los ntawm cov hlwb hematopoietic (HSCs) tab sis kuj los ntawm cov ntaub so ntswg siab. Peb tau xaiv siv tib neeg LX-2 hlwb raws li peb cov kev tshawb pom yav dhau los los ntawm kev tshuaj xyuas transcriptome uas IPA cuam tshuam nrog hematopoietic stem cell (HSC) activation [15], thiab HSCs yog cov hlwb tseem ceeb uas koom nrog kev nce qib ntawm daim siab fibrosis. Lub siab muaj ntau hom hlwb, yog li lwm cov qauv hlwb xws li hepatocyte-HSC-immune cell co-culture system ua ke nrog caspase activation thiab DNA fragmentation nrog rau cov txheej txheem ntawm kev ua haujlwm suav nrog cov protein theem yuav tsum tau txiav txim siab los kawm txog lub luag haujlwm ntawm IPA thiab nws txoj kev sib cuam tshuam nrog lwm hom hlwb siab.