Ua hōʻike mua mākou ua haʻahaʻa nā pae serum o ka metabolite tryptophan i loaʻa mai ka ʻōpū indole-3-propionic acid (IPA) i nā poʻe maʻi me ka fibrosis ake. Ma kēia noiʻi, ua noiʻi mākou i ka transcriptome a me ka DNA methylome i loko o nā ake momona e pili ana i nā pae serum IPA, a me ke kuleana o IPA i ka hoʻoulu ʻana i ka phenotypic inactivation o nā cell stellate hepatic (HSCs) in vitro.
Ua komo pū ka noiʻi me 116 mau maʻi momona me ka ʻole o ka maʻi diabetes mellitus type 2 (T2DM) (makahiki 46.8 ± 9.3 mau makahiki; BMI: 42.7 ± 5.0 kg/m²) i hana ʻia i ke ʻoki bariatric ma ke kikowaena ʻoki kino ʻo Kuopio Bariatric (KOBS). Ua ana ʻia nā pae IPA e kaapuni ana e ka liquid chromatography-mass spectrometry (LC-MS), ua hana ʻia ka nānā ʻana o ka transcriptome ate e ka huina RNA sequencing, a ua hana ʻia ka nānā ʻana o ka DNA methylation me ka hoʻohana ʻana i ka Infinium HumanMethylation450 BeadChip. Ua hoʻohana ʻia nā cell stellate hepatic kanaka (LX-2) no nā hoʻokolohua in vitro.
Ua pili nā pae IPA serum me ka hōʻike ʻana o nā genes i komo i nā ala apoptotic, mitophagic, a me ke ola lōʻihi i loko o ke ake. ʻO ka gene AKT serine/threonine kinase 1 (AKT1) ka gene e launa pū nui loa ana a me ke koʻikoʻi i loko o ka transcript ate a me nā ʻano methylation DNA. Ua hoʻoulu ka mālama ʻana o IPA i ka apoptosis, hoʻemi i ka hanu mitochondrial, a hoʻololi i ke ʻano o ke kelepona a me ka dynamics mitochondrial ma ka hoʻololi ʻana i ka hōʻike ʻana o nā genes i ʻike ʻia e hoʻoponopono i ka fibrosis, apoptosis, a me ke ola ʻana o nā cell LX-2.
I ka hui pū ʻia ʻana, kākoʻo kēia ʻikepili i ka loaʻa ʻana o nā hopena therapeutic i ka IPA a hiki ke hoʻoulu i ka apoptosis a hoʻololi i ka phenotype HSC i kahi kūlana hana ʻole, no laila e hoʻonui ana i ka hiki ke kāohi i ka fibrosis ate ma ke keakea ʻana i ka hoʻoulu ʻana o HSC a me ka metabolism mitochondrial.
Ua pili ka laha ʻana o ka momona a me ka metabolic syndrome me ka piʻi ʻana o ka maʻi momona o ke akepaʻa metabolically associated (MASLD); hoʻopilikia ka maʻi i 25% a 30% o ka heluna kanaka [1]. ʻO ka hopena nui o ka MASLD etiology ʻo ia ka fibrosis ake, kahi hana ikaika i hōʻike ʻia e ka hōʻiliʻili mau ʻana o ka fibrous extracellular matrix (ECM) [2]. ʻO nā cell nui i komo i ka fibrosis akepaʻa he mau cell stellate hepatic (HSCs), e hōʻike ana i ʻehā mau phenotypes i ʻike ʻia: quiescent, activated, inactivated, a senescent [3, 4]. Hiki ke hoʻāla ʻia nā HSC a transdifferentiate mai kahi ʻano quiescent i loko o nā cell fibroblast-like proliferative me nā koi ikehu kiʻekiʻe, me ka hoʻonui ʻia o ka hōʻike ʻana o ka α-smooth muscle actin (α-SMA) a me ke ʻano I collagen (Col-I) [5, 6]. I ka wā o ka hoʻohuli ʻana o ka fibrosis akepaʻa, hoʻopau ʻia nā HSC i hoʻāla ʻia ma o ka apoptosis a i ʻole ka inactivation. ʻO kēia mau kaʻina hana e komo pū ana me ka hoʻohaʻahaʻa ʻana o nā genes fibrogenic a me ka modulation o nā genes prosurvival (e like me NF-κB a me PI3K/Akt signaling pathways) [7, 8], a me nā loli i ka mitochondrial dynamics a me ka hana [9].
Ua ʻike ʻia ua emi nā pae serum o ka metabolite tryptophan indole-3-propionic acid (IPA), i hana ʻia i loko o ka ʻōpū, i nā maʻi metabolic kanaka e like me MASLD [10–13]. Pili ka IPA me ka lawe ʻana i ka fiber dietary, ua ʻike ʻia no kona hopena antioxidant a me ka anti-inflammatory, a hoʻēmi i ka phenotype non-alcoholic steatohepatitis (NASH) i hoʻoulu ʻia e ka meaʻai ma vivo a me in vitro [11–14]. Loaʻa kekahi mau hōʻike mai kā mākou noiʻi mua, kahi i hōʻike ai he haʻahaʻa nā pae serum IPA i nā maʻi me ka fibrosis akepaʻa ma mua o nā maʻi momona me ka ʻole o ka fibrosis akepaʻa ma ka Kuopio Bariatric Surgery Study (KOBS). Eia kekahi, ua hōʻike mākou e hiki i ka mālama ʻana iā IPA ke hōʻemi i ka hōʻike ʻana o nā genes he mau māka maʻamau o ka hoʻopili ʻana o nā cell, ka neʻe ʻana o nā cell a me ka hoʻāla ʻana o nā cell stem hematopoietic i loko o kahi kumu hoʻohālike cell stellate hepatic kanaka (LX-2) a he metabolite hepatoprotective hiki [15]. Eia naʻe, ʻaʻole maopopo pehea e hoʻoulu ai ʻo IPA i ka regression fibrosis akepaʻa ma ka hoʻoulu ʻana i ka apoptosis HSC a me nā bioenergetics mitochondrial.
Maanei, ke hōʻike nei mākou e pili ana ka serum IPA me ka hōʻike ʻana o nā genes i hoʻonui ʻia i ka apoptosis, mitophagy, a me nā ala lōʻihi i loko o ke akepaʻa o ka poʻe momona akā ʻaʻole type 2 diabetes (KOBS). Eia kekahi, ua ʻike mākou hiki i ka IPA ke hoʻoulu i ka hoʻomaʻemaʻe a me ka hoʻohaʻahaʻa ʻana o nā cell hematopoietic stem (HSCs) i hoʻāla ʻia ma o ke ala inactivation. Hōʻike kēia mau hopena i kahi kuleana hou no IPA, e lilo ana ia i pahuhopu therapeutic kūpono e hoʻolaha i ka regression fibrosis ate.
Ua hōʻike ʻia kahi noiʻi mua ma ka hui KOBS he haʻahaʻa nā pae IPA kahe o nā maʻi me ka fibrosis akepaʻa i hoʻohālikelike ʻia me nā maʻi me ka ʻole o ka fibrosis akepaʻa [15]. No ka hoʻokaʻawale ʻana i ka hopena hoʻohuihui o ka maʻi diabetes type 2, ua hoʻopaʻa inoa mākou i 116 mau maʻi momona me ka ʻole o ka maʻi diabetes type 2 (makahiki awelika ± SD: 46.8 ± 9.3 mau makahiki; BMI: 42.7 ± 5.0 kg/m2) (Papa 1) mai ka noiʻi KOBS e hoʻomau nei ma ke ʻano he heluna kanaka noiʻi [16]. Ua hāʻawi nā mea komo a pau i ka ʻae ʻia ma ke kākau ʻana a ua ʻae ʻia ke kaʻina hana noiʻi e ke Kōmike Ethics o ka Halemai ʻo North Savo County e like me ka Hōʻike o Helsinki (54/2005, 104/2008 a me 27/2010).
Ua loaʻa nā ʻano biopsy o ke akepaʻa i ka wā o ke ʻoki ʻana bariatric a ua loiloi ʻia e nā pathologists akamai e like me nā pae i wehewehe mua ʻia [17, 18]. Ua hōʻuluʻulu ʻia nā pae loiloi ma ka Papa Hoʻohui S1 a ua wehewehe mua ʻia [19].
Ua kālailai ʻia nā hāpana serum hoʻokē ʻai e ka untargeted liquid chromatography-mass spectrometry (LC-MS) no ka nānā ʻana i ka metabolomics (n = 116). Ua kālailai ʻia nā hāpana me ka hoʻohana ʻana i kahi ʻōnaehana UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Kelemānia) e like me ka mea i wehewehe mua ʻia19. Ua hoʻokumu ʻia ka ʻike ʻana o ka isopropyl alcohol (IPA) ma ka manawa paʻa a me ka hoʻohālikelike ʻana o ka MS/MS spectrum me nā kūlana maʻemaʻe. Ua noʻonoʻo ʻia ka ikaika hōʻailona IPA (peak area) i nā loiloi hou aʻe [20].
Ua hana ʻia ka hoʻonohonoho ʻana o ka RNA ate holoʻokoʻa me ka hoʻohana ʻana iā Illumina HiSeq 2500 a ua hana mua ʻia ka ʻikepili e like me ka mea i wehewehe mua ʻia [19, 21, 22]. Ua hana mākou i ka loiloi hōʻike ʻokoʻa i kuhikuhi ʻia o nā transcripts e pili ana i ka hana mitochondrial/biogenesis me ka hoʻohana ʻana i nā genes 1957 i koho ʻia mai ka waihona ʻikepili MitoMiner 4.0 [23]. Ua hana ʻia ka loiloi methylation DNA ate me ka hoʻohana ʻana i ka Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) me ka hoʻohana ʻana i ke ʻano hana like me ka mea i wehewehe mua ʻia [24, 25].
Ua hāʻawi lokomaikaʻi ʻia nā pūnaewele stellate hepatic kanaka (LX-2) e Polopeka Stefano Romeo a ua hoʻoulu ʻia a mālama ʻia i loko o ka medium DMEM/F12 (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). No ke koho ʻana i ka mahele hana o IPA, ua mālama ʻia nā pūnaewele LX-2 me nā ʻano like ʻole o IPA (10 μM, 100 μM a me 1 mM; Sigma, 220027) ma ka medium DMEM/F12 no 24 hola. Eia kekahi, no ka noiʻi ʻana i ka hiki o IPA ke hoʻopau i nā HSC, ua mālama pū ʻia nā pūnaewele LX-2 me 5 ng/ml TGF-β1 (nā ʻōnaehana R&D, 240-B-002/CF) a me 1 mM IPA i loko o ka medium serum-free no 24 hola. No nā kaohi kaʻa e pili ana, ua hoʻohana ʻia he 4 nM HCL nona ka 0.1% BSA no ka mālama ʻana iā TGF-β1 a ua hoʻohana ʻia ʻo 0.05% DMSO no ka mālama ʻana iā IPA, a ua hoʻohana pū ʻia nā mea ʻelua no ka mālama hui pū ʻana.
Ua loiloi ʻia ka Apoptosis me ka hoʻohana ʻana i ka FITC Annexin V Apoptosis Detection Kit me 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) e like me nā kuhikuhi a ka mea hana. I ka pōkole, ua hoʻoulu ʻia ʻo LX-2 (1 × 105 mau cell/luawai) i ka pō i loko o nā pā 12-luawai a laila mālama ʻia me nā ʻāpana he nui o IPA a i ʻole IPA a me TGF-β1. I ka lā aʻe, ua hōʻiliʻili ʻia nā cell lana a pili, trypsinized, holoi ʻia me PBS, hoʻoulu hou ʻia i loko o ka Annexin V binding buffer, a hoʻoulu ʻia me FITC-Annexin V a me 7-AAD no 15 min.
Ua hoʻoluʻu ʻia ka Mitochondria i loko o nā cell ola no ka hana oxidative me ka hoʻohana ʻana iā Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). No nā hoʻāʻo MTR, ua hoʻoulu ʻia nā cell LX-2 ma nā densities like me IPA a me TGF-β1. Ma hope o 24 hola, ua trypsinized ʻia nā cell ola, holoi ʻia me PBS, a laila hoʻoulu ʻia me 100 μM MTR i loko o ka serum-free medium ma 37 °C no 20 min e like me ka mea i wehewehe mua ʻia [26]. No ka nānā ʻana i ke ʻano o nā cell ola, ua kālailai ʻia ka nui o nā cell a me ka paʻakikī cytoplasmic me ka hoʻohana ʻana i nā parameter forward scatter (FSC) a me ka ʻaoʻao scatter (SSC).
Ua loaʻa nā ʻikepili āpau (30,000 mau hanana) me ka hoʻohana ʻana iā NovoCyte Quanteon (Agilent) a ua kālailai ʻia me ka hoʻohana ʻana iā NovoExpress® 1.4.1 a i ʻole ka polokalamu FlowJo V.10.
Ua ana ʻia ka nui o ka hoʻohana ʻana i ka oxygen (OCR) a me ka nui o ka acidification extracellular (ECAR) i ka manawa maoli me ka hoʻohana ʻana i kahi Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) i lako me kahi Seahorse XF Cell Mito Stress e like me nā kuhikuhi a ka mea hana. I ka pōkole, ua lūlū ʻia nā cell 2 × 104 LX-2/lua ma luna o nā papa moʻomeheu cell XF96. Ma hope o ka hoʻoulu ʻana i ka pō, ua mālama ʻia nā cell me isopropanol (IPA) a me TGF-β1 (Nā Hana Hoʻohui 1). Ua hana ʻia ka loiloi ʻikepili me ka hoʻohana ʻana i ka polokalamu Seahorse XF Wave, nona ka Seahorse XF Cell Energy Phenotype Test Report Generator. Mai kēia, ua helu ʻia kahi Bioenergetic Health Index (BHI) [27].
Ua hoʻololi ʻia ka RNA holoʻokoʻa i loko o cDNA. No nā ʻano hana kikoʻī, e nānā i ka kuhikuhi [15]. Ua hoʻohana ʻia nā pae mRNA ribosomal acidic protein P0 (RPLP0) a me cyclophilin A1 (PPIA) Human 60S ma ke ʻano he mau kaohi gene constitutive. Ua hoʻohana ʻia ka QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, Netherlands) me ka TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) a i ʻole ka Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), a ua helu ʻia ka fold expression gene pili me ka hoʻohana ʻana i nā palena hoʻohālikelike Ct value cycling (ΔΔCt) a me ke ʻano ∆∆Ct. Hāʻawi ʻia nā kikoʻī o nā primers ma nā Papa Hoʻohui S2 a me S3.
Ua unuhi ʻia ka DNA Nuklea (ncDNA) a me ka DNA mitochondrial (mtDNA) me ka hoʻohana ʻana i ka pahu koko a me nā ʻiʻo DNeasy (Qiagen) e like me ka mea i wehewehe mua ʻia [28]. Ua helu ʻia ka nui o ka mtDNA ma ka helu ʻana i ka lakio o kēlā me kēia ʻāpana mtDNA i manaʻo ʻia i ka awelika geometric o nā ʻāpana DNA nuklea ʻekolu (mtDNA/ncDNA), e like me ka mea i hōʻike kikoʻī ʻia ma nā ʻAno Hana Hoʻohui 2. Hāʻawi ʻia nā kikoʻī o nā primers no mtDNA a me ncDNA ma ka Papa Hoʻohui S4.
Ua hoʻoluʻu ʻia nā pūnaewele ola me Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) e ʻike i nā pūnaewele mitochondrial intercellular a me intracellular. Ua hoʻoulu ʻia nā pūnaewele LX-2 (1 × 104 mau pūnaewele/luawai) ma nā paheʻe aniani i loko o nā papa moʻomeheu aniani-lalo like (Ibidi GmbH, Martinsried, Kelemānia). Ma hope o 24 hola, ua hoʻoulu ʻia nā pūnaewele LX-2 ola me 100 μM MTR no 20 min ma 37 °C a ua hoʻoluʻu ʻia nā nuclei cell me DAPI (1 μg/ml, Sigma-Aldrich) e like me ka mea i wehewehe mua ʻia [29]. Ua ʻike ʻia nā pūnaewele Mitochondrial me ka hoʻohana ʻana i kahi microscope inverted Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, Kelemānia) i lako me kahi module confocal Zeiss LSM 800 ma 37 °C i loko o kahi lewa humidified me 5% CO2 me ka hoʻohana ʻana i kahi pahuhopu 63 × NA 1.3. Ua loaʻa iā mākou he ʻumi kiʻi Z-series no kēlā me kēia ʻano laʻana. Loaʻa i kēlā me kēia Z-series he 30 mau ʻāpana, me ka mānoanoa o 9.86 μm kēlā me kēia. No kēlā me kēia laʻana, ua loaʻa nā kiʻi o nā ʻano ʻike like ʻole he ʻumi me ka hoʻohana ʻana i ka polokalamu ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Kelemānia), a ua hana ʻia ka loiloi morphology mitochondrial me ka hoʻohana ʻana i ka polokalamu ImageJ (v1.54d) [30, 31] e like me nā palena i hōʻike kikoʻī ʻia ma nā ʻAno Hoʻohui 3.
Ua hoʻopaʻa ʻia nā pūnaewele me 2% glutaraldehyde i loko o ka 0.1 M phosphate buffer, a ukali ʻia e ka hoʻopaʻa ʻana me 1% osmium tetroxide solution (Sigma Aldrich, MO, USA), hoʻomaloʻo mālie ʻia me ka acetone (Merck, Darmstadt, Kelemānia), a ma hope ua hoʻokomo ʻia i loko o ka epoxy resin. Ua hoʻomākaukau ʻia nā ʻāpana Ultrathin a ua hoʻoluʻu ʻia me 1% uranyl acetate (Merck, Darmstadt, Kelemānia) a me 1% lead citrate (Merck, Darmstadt, Kelemānia). Ua loaʻa nā kiʻi Ultrastructural me ka hoʻohana ʻana i kahi microscope electron transmission JEM 2100F EXII (JEOL Ltd, Tokyo, Iapana) ma kahi volta wikiwiki o 80 kV.
Ua kālailai ʻia ke ʻano o nā pūnaewele LX-2 i mālama ʻia me IPA no 24 h e ka microscopy phase-contrast ma 50x magnification me ka hoʻohana ʻana i kahi microscope māmā Zeiss inverted (Zeiss Axio Vert.A1 a me AxioCam MRm, Jena, Kelemānia).
Ua hōʻike ʻia nā ʻikepili lapaʻau ma ke ʻano he awelika ± maʻamau deviation a i ʻole median (interquartile range: IQR). Ua hoʻohana ʻia ka loiloi hoʻokahi ala o ka variance (nā loli hoʻomau) a i ʻole ka hoʻāʻo χ² (nā loli mahele) e hoʻohālikelike i nā ʻokoʻa ma waena o nā hui noiʻi ʻekolu. Ua hoʻohana ʻia ka helu maikaʻi wahaheʻe (FDR) e hoʻoponopono no nā hoʻāʻo he nui, a ua manaʻo ʻia he koʻikoʻi nā genes me FDR < 0.05. Ua hoʻohana ʻia ka loiloi correlation Spearman e hoʻopili i ka methylation CpG DNA me ka ikaika hōʻailona IPA, me nā waiwai p nominal (p < 0.05) i hōʻike ʻia.
Ua hana ʻia ke kālailai ala me ka hoʻohana ʻana i kahi mea hana loiloi hoʻonohonoho gene ma ka pūnaewele (WebGestalt) no 268 mau palapala (nominal p < 0.01), 119 mau palapala e pili ana i ka mitochondria (nominal p < 0.05), a me 4350 CpGs mai loko mai o 3093 mau palapala akepaʻa e pili ana me nā pae IPA serum e kaapuni ana. Ua hoʻohana ʻia ka mea hana Venny DB (mana 2.1.0) i loaʻa manuahi e ʻimi i nā genes e pili ana, a ua hoʻohana ʻia ʻo StringDB (mana 11.5) e nānā i nā pilina protein-protein.
No ka hoʻokolohua LX-2, ua hoʻāʻo ʻia nā laʻana no ka maʻamau me ka hoʻohana ʻana i ka hoʻāʻo D'Agostino-Pearson. Ua loaʻa nā ʻikepili mai ka liʻiliʻi he ʻekolu mau replicates biological a ua hoʻokau ʻia i hoʻokahi ala ANOVA me ka hoʻāʻo post hoc Bonferroni. Ua manaʻo ʻia he koʻikoʻi ka p-value ma lalo o 0.05. Hōʻike ʻia nā ʻikepili ma ke ʻano he mean ± SD, a ua hōʻike ʻia ka helu o nā hoʻokolohua ma kēlā me kēia kiʻi. Ua hana ʻia nā loiloi a me nā kiʻi āpau me ka hoʻohana ʻana i ka polokalamu helu GraphPad Prism 8 no Windows (GraphPad Software Inc., mana 8.4.3, San Diego, USA).
ʻO ka mea mua, ua noiʻi mākou i ka pilina o nā pae serum IPA me ke akepaʻa, ke kino holoʻokoʻa, a me nā transcripts mitochondrial. Ma ka ʻikepili transcript holoʻokoʻa, ʻo ka gene ikaika loa e pili ana me nā pae serum IPA ʻo MAPKAPK3 (FDR = 0.0077; mitogen-activated protein kinase-activated protein kinase 3); ma ka ʻikepili transcript e pili ana i ka mitochondria, ʻo ka gene ikaika loa e pili ana ʻo AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Faila Hou 1 a me ka faila Hou 2).
A laila ua kālailai mākou i nā palapala honua (n = 268; p < 0.01) a me nā palapala e pili ana i ka mitochondria (n = 119; p < 0.05), a ʻike hope loa i ka apoptosis ʻo ia ke ala canonical koʻikoʻi loa (p = 0.0089). No nā palapala mitochondrial e pili ana me nā pae IPA serum, ua kālele mākou i ka apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), a me nā ala hōʻailona TNF (FDR = 0.000006) (Kiʻi 1A, Papa 2, a me nā Kiʻi Hoʻohui 1A-B).
ʻO ka loiloi pili ʻana o nā palapala honua, nā mitochondria e pili ana, a me ka methylation DNA i loko o ke ake kanaka e pili ana me nā pae serum IPA. Hōʻike ʻo A i 268 mau palapala honua, 119 mau palapala e pili ana i ka mitochondria, a me nā palapala DNA methylated i hoʻopaʻa ʻia i 3092 mau wahi CpG e pili ana me nā pae serum IPA (nā waiwai p < 0.01 no nā palapala honua a me ka DNA methylated, a me nā waiwai p < 0.05 no nā palapala mitochondrial). Hōʻike ʻia nā palapala nui e pili ana ma waena (AKT1 a me YKT6). B Ua kūkulu ʻia ka palapala pilina o nā genes 13 me ka helu pilina kiʻekiʻe loa (0.900) me nā genes ʻē aʻe mai nā genes e pili ana he 56 (ʻāpana laina ʻeleʻele) i pili nui me nā pae serum IPA me ka hoʻohana ʻana i ka mea hana pūnaewele StringDB. ʻŌmaʻomaʻo: Nā Genes i hoʻopaʻa ʻia i ka ʻāpana cellular Gene Ontology (GO): mitochondria (GO:0005739). ʻO AKT1 ka protein me ka helu kiʻekiʻe loa (0.900) no nā pilina me nā protein ʻē aʻe e pili ana i ka ʻikepili (e pili ana i ka ʻeli kikokikona, nā hoʻokolohua, nā waihona ʻikepili, a me ka co-expression). Hōʻike nā kikowaena pūnaewele i nā protein, a hōʻike nā lihi i nā pilina ma waena o nā protein.
ʻOiai hiki i nā metabolites microbiota gut ke hoʻoponopono i ka haku mele epigenetic ma o ka methylation DNA [32], ua noiʻi mākou inā pili nā pae serum IPA me ka methylation DNA ate. Ua ʻike mākou ʻo nā wahi methylation nui ʻelua e pili ana me nā pae serum IPA kokoke i ka proline-serine-rich region 3 (C19orf55) a me ka ʻohana protein shock heat B (liʻiliʻi) lālā 6 (HSPB6) (Faila hou 3). Ua pili ka methylation DNA o 4350 CpG (p < 0.01) me nā pae serum IPA a ua hoʻonui ʻia i nā ala hoʻoponopono lōʻihi (p = 0.006) (Kiʻi 1A, Papa 2, a me ke Kiʻi Hoʻohui 1C).
No ka hoʻomaopopo ʻana i nā ʻano hana olaola e pili ana i nā pilina ma waena o nā pae IPA serum, nā transcripts honua, nā transcripts e pili ana i ka mitochondria, a me ka methylation DNA i loko o ke ake kanaka, ua hana mākou i kahi loiloi overlap o nā genes i ʻike ʻia ma ka loiloi ala mua (Kiʻi 1A). Ua hōʻike nā hopena o ka loiloi hoʻonui ala o nā genes overlapping 56 (i loko o ka laina ʻeleʻele ma ke Kiʻi 1A) ua hōʻike ke ala apoptosis (p = 0.00029) i ʻelua mau genes i maʻamau i nā loiloi ʻekolu: AKT1 a me YKT6 (YKT6 v-SNARE homolog), e like me ka mea i hōʻike ʻia ma ka kiʻikuhi Venn (Kiʻi Hoʻohui 2 a me ke Kiʻi 1A). ʻO ka mea hoihoi, ua ʻike mākou ua pili pono ʻo AKT1 (cg19831386) a me YKT6 (cg24161647) me nā pae IPA serum (Faila Hoʻohui 3). No ka ʻike ʻana i nā pilina protein hiki ke hana ma waena o nā huahana gene, ua koho mākou i 13 mau genes me ka helu ʻāpana maʻamau kiʻekiʻe loa (0.900) ma waena o 56 mau genes overlapping ma ke ʻano he input a kūkulu i kahi palapala ʻāina pilina. Wahi a ka pae hilinaʻi (hilinaʻi marginal), ʻo ka gene AKT1 me ka helu kiʻekiʻe loa (0.900) aia ma ka piko (Kiʻi 1B).
Ma muli o ke kālailai ala, ua ʻike mākou ʻo ka apoptosis ke ala nui, no laila ua noiʻi mākou inā e hoʻopilikia ka mālama ʻana o IPA i ka apoptosis o HSCs in vitro. Ua hōʻike mua mākou he ʻona ʻole nā ​​​​​​lāʻau like ʻole o IPA (10 μM, 100 μM, a me 1 mM) i nā cell LX-2 [15]. Ua hōʻike kēia haʻawina ua hoʻonui ka mālama ʻana o IPA ma 10 μM a me 100 μM i ka nui o nā cell ola a me nā necrotic. Eia naʻe, i hoʻohālikelike ʻia me ka hui kaohi, ua emi ke ola o nā cell ma ka 1 mM IPA concentration, ʻoiai ʻaʻole i loli ka nui o ka necrosis cell (Kiʻi 2A, B). ʻO ka mea aʻe, no ka ʻike ʻana i ka concentration kūpono e hoʻoulu ai i ka apoptosis i nā cell LX-2, ua hoʻāʻo mākou i 10 μM, 100 μM, a me 1 mM IPA no 24 h (Kiʻi 2A-E a me ke Kiʻi Hoʻohui 3A-B). ʻO ka mea hoihoi, ua hoʻemi ʻo IPA 10 μM a me 100 μM i ka helu apoptosis (%), akā naʻe, ua hoʻonui ʻo IPA 1 mM i ka apoptosis hope a me ka helu apoptosis (%) i hoʻohālikelike ʻia me ka kaohi a no laila ua koho ʻia no nā hoʻokolohua hou aʻe (Nā Kiʻi 2A–D).
Hoʻoulu ʻo IPA i ka apoptosis o nā pūnaewele LX-2. Ua hoʻohana ʻia ke ʻano hoʻoluʻu pālua ʻo Annexin V a me 7-AAD e helu i ka nui o ka apoptotic a me ke ʻano o ke kelepona ma o ka cytometry kahe. Ua hoʻoulu ʻia nā pūnaewele BA me 10 μM, 100 μM a me 1 mM IPA no 24 hola a i ʻole me F–H TGF-β1 (5 ng/ml) a me 1 mM IPA i loko o ka medium serum-free no 24 hola. A: nā pūnaewele ola (Annexin V -/ 7AAD-); B: nā pūnaewele necrotic (Annexin V -/ 7AAD+); C, F: mua (Annexin V +/ 7AAD-); D, G: lohi (Annexin V+/7AAD.+); E, H: pakeneka o ka huina o nā pūnaewele apoptotic mua a me ka hope i ka nui o ka apoptotic (%). Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± SD, n = 3 mau hoʻokolohua kūʻokoʻa. Ua hana ʻia nā hoʻohālikelike helu me ka hoʻohana ʻana i hoʻokahi ala ANOVA me ka hoʻāʻo post hoc Bonferroni. *p < 0.05; ****p < 0.0001
E like me kā mākou i hōʻike ai ma mua, hiki i ka 5 ng/ml TGF-β1 ke hoʻoulu i ka hoʻoulu ʻana o ka HSC ma ka hoʻonui ʻana i ka hōʻike ʻana o nā genes marker maʻamau [15]. Ua mālama ʻia nā pūnaewele LX-2 me 5 ng/ml TGF-β1 a me 1 mM IPA i ka hui pū ʻana (Kiʻi 2E–H). ʻAʻole i hoʻololi ka mālama ʻana o TGF-β1 i ka helu apoptosis, akā naʻe, ua hoʻonui ka mālama ʻana o IPA i ka apoptosis hope a me ka helu apoptosis (%) i hoʻohālikelike ʻia me ka mālama ʻana o TGF-β1 (Kiʻi 2E–H). Hōʻike kēia mau hopena hiki i ka 1 mM IPA ke hoʻolaha i ka apoptosis i loko o nā pūnaewele LX-2 me ke kūʻokoʻa o ka hoʻoulu ʻana o TGF-β1.
Ua noiʻi hou mākou i ka hopena o IPA ma ka hanu mitochondrial i loko o nā pūnaewele LX-2. Ua hōʻike nā hopena ua hoʻemi ka 1 mM IPA i nā palena o ka nui o ka hoʻohana ʻana i ka oxygen (OCR): ka hanu ʻole-mitochondrial, ka hanu basal a me ka maximal, ka leak proton a me ka hana ʻana o ATP i hoʻohālikelike ʻia me ka hui kaohi (Kiʻi 3A, B), ʻoiai ʻaʻole i loli ka helu olakino bioenergetic (BHI).
Hoʻemi ka IPA i ka hanu mitochondrial i loko o nā pūnaewele LX-2. Hōʻike ʻia ke kiʻikuhi hanu mitochondrial (OCR) ma ke ʻano he mau palena hanu mitochondrial (hanu ʻole mitochondrial, hanu basal, hanu nui loa, leak proton, hanauna ATP, SRC a me BHI). Ua hoʻoulu ʻia nā pūnaewele A a me B me 10 μM, 100 μM a me 1 mM IPA no 24 hola, kēlā me kēia. Ua hoʻoulu ʻia nā pūnaewele C a me D me TGF-β1 (5 ng/ml) a me 1 mM IPA i loko o ka medium serum-free no 24 hola, kēlā me kēia. Ua hoʻohālikelike ʻia nā ana āpau i ka ʻike DNA me ka hoʻohana ʻana i ka pahu CyQuant. BHI: helu olakino bioenergetic; SRC: hiki ke mālama hanu; OCR: helu hoʻohana oxygen. Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± standard deviation (SD), n = 5 mau hoʻokolohua kūʻokoʻa. Ua hana ʻia nā hoʻohālikelike helu me ka hoʻohana ʻana i ka hoʻāʻo hoʻokahi ala ANOVA a me Bonferroni post hoc. *p < 0.05; **p < 0.01; a me ***p < 0.001
No ka loaʻa ʻana o kahi ʻike piha o ka hopena o IPA ma ka ʻaoʻao bioenergetic o nā pūnaewele LX-2 i hoʻāla ʻia e TGF-β1, ua kālailai mākou i ka phosphorylation oxidative mitochondrial e OCR (Kiʻi 3C, D). Ua hōʻike nā hopena e hiki i ka mālama ʻana iā TGF-β1 ke hōʻemi i ka hanu kiʻekiʻe loa, ka hiki ke mālama i ka hanu (SRC) a me BHI i hoʻohālikelike ʻia me ka hui kaohi (Kiʻi 3C, D). Eia kekahi, ua hōʻemi ka mālama hui pū ʻana i ka hanu basal, ka leak proton a me ka hana ʻana o ATP, akā ʻoi aku ka kiʻekiʻe o SRC a me BHI ma mua o nā mea i mālama ʻia me TGF-β1 (Kiʻi 3C, D).
Ua hana pū mākou i ka "Cellular Energy Phenotype Test" i hāʻawi ʻia e ka polokalamu Seahorse (Kiʻi Hoʻohui 4A–D). E like me ka mea i hōʻike ʻia ma ke Kiʻi Hoʻohui 3B, ua emi nā hiki ke hoʻololi i ka metabolism o OCR a me ECAR ma hope o ka mālama ʻana iā TGF-β1, akā naʻe, ʻaʻohe ʻokoʻa i ʻike ʻia ma nā hui mālama hui a me IPA i hoʻohālikelike ʻia me ka hui kaohi. Eia kekahi, ua emi nā pae basal a me ke kaumaha o OCR ma hope o ka hui pū ʻana a me ka mālama ʻana iā IPA i hoʻohālikelike ʻia me ka hui kaohi (Kiʻi Hoʻohui 4C). ʻO ka mea hoihoi, ua ʻike ʻia kahi ʻano like me ka hui pū ʻana, kahi i ʻike ʻole ʻia ai kahi loli i nā pae basal a me ke kaumaha o ECAR i hoʻohālikelike ʻia me ka mālama ʻana iā TGF-β1 (Kiʻi Hoʻohui 4C). I loko o nā HSC, ʻo ka emi ʻana o ka mitochondrial oxidative phosphorylation a me ka hiki o ka hui pū ʻana ke hoʻihoʻi iā SCR a me BHI ma hope o ka hōʻike ʻana i ka mālama ʻana iā TGF-β1 ʻaʻole i hoʻololi i ka hiki ke hoʻololi i ka metabolic potential (OCR a me ECAR). I ka hui pū ʻia ʻana, hōʻike kēia mau hopena e hiki i ka IPA ke hōʻemi i ka bioenergetics ma HSCs, e hōʻike ana e hiki i ka IPA ke hoʻoulu i kahi ʻano ikehu haʻahaʻa e hoʻololi i ka phenotype HSC i ka inactivation (Kiʻi Hoʻohui 4D).
Ua noiʻi ʻia ka hopena o IPA ma ka dynamics mitochondrial me ka hoʻohana ʻana i ka helu ʻekolu-dimensional o ke ʻano mitochondrial a me nā pilina pūnaewele a me ka hoʻoluʻu MTR (Kiʻi 4 a me ke Kiʻi Hoʻohui 5). Hōʻike ka Kiʻi 4, i ka hoʻohālikelike ʻia me ka hui kaohi, ua hoʻemi ka mālama ʻana o TGF-β1 i ka awelika o ka ʻili, ka helu lālā, ka lōʻihi lālā holoʻokoʻa, a me ka helu hui lālā (Kiʻi 4A a me B) a ua hoʻololi i ka hapa o mitochondria mai ka spherical a i ka morphology waena (Kiʻi 4C). ʻO ka mālama ʻana o IPA wale nō i hoʻemi i ka awelika o ka nui mitochondrial a ua hoʻololi i ka hapa o mitochondria mai ka spherical a i ka morphology waena i hoʻohālikelike ʻia me ka hui kaohi (Kiʻi 4A). I ​​ka hoʻohālikelike ʻana, ua noho ka sphericity, ka awelika o ka lālā, a me ka hana mitochondrial i loiloi ʻia e ka mitochondrial membrane potential-dependent MTR (Kiʻi 4A a me E) i hoʻololi ʻole ʻia a ʻaʻole ʻokoʻa kēia mau palena ma waena o nā hui. I ka hui pū ʻia ʻana, hōʻike kēia mau hopena e ʻike ʻia ana ka mālama ʻana o TGF-β1 a me IPA e hoʻololi i ke ʻano a me ka nui o ka mitochondrial a me ka paʻakikī o ka pūnaewele i nā cell LX-2 ola.
Hoʻololi ka IPA i ka dynamics mitochondrial a me ka nui o ka DNA mitochondrial i loko o nā pūnaewele LX-2. A. Nā kiʻi confocal hōʻike o nā pūnaewele LX-2 ola i hoʻoulu ʻia me TGF-β1 (5 ng/ml) a me 1 mM IPA no 24 h i loko o ka serum-free medium e hōʻike ana i nā pūnaewele mitochondrial i hoʻoluʻu ʻia me Mitotracker™ Red CMXRos a me nā nuclei i hoʻoluʻu ʻia i ka uliuli me DAPI. Loaʻa i nā ʻikepili āpau ma ka liʻiliʻi he 15 mau kiʻi no kēlā me kēia hui. Ua loaʻa iā mākou he 10 mau kiʻi Z-stack no kēlā me kēia ʻano laʻana. Loaʻa i kēlā me kēia moʻo Z-axis he 30 mau ʻāpana, kēlā me kēia me ka mānoanoa o 9.86 μm. Pā unahi: 10 μm. B. Nā mea hōʻike (mitochondria wale nō) i ʻike ʻia ma ka hoʻopili ʻana i ka adaptive thresholding i ke kiʻi. Ua hana ʻia ka loiloi quantitative a me ka hoʻohālikelike ʻana o nā pilina pūnaewele morphological mitochondrial no nā pūnaewele āpau i kēlā me kēia hui. C. Alapine (pinepine) o nā lakio ʻano mitochondrial. ʻO nā waiwai kokoke i ka 0 e hōʻike ana i nā ʻano spherical, a ʻo nā waiwai kokoke i ka 1 e hōʻike ana i nā ʻano filamentous. D Ua hoʻoholo ʻia ka ʻike Mitochondrial DNA (mtDNA) e like me ka mea i wehewehe ʻia ma nā Mea Hana a me nā Hana. Ua hana ʻia ka loiloi E Mitotracker™ Red CMXRos e ka flow cytometry (30,000 mau hanana) e like me ka mea i wehewehe ʻia ma nā Materials and Methods. Hōʻike ʻia nā ʻikepili ma ke ʻano he mean ± SD, n = 3 mau hoʻokolohua kūʻokoʻa. Ua hana ʻia nā hoʻohālikelike helu me ka hoʻohana ʻana i ka hoʻāʻo hoʻokahi ala ANOVA a me Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
A laila ua kālailai mākou i ka ʻike mtDNA i loko o nā pūnaewele LX-2 ma ke ʻano he hōʻailona o ka helu mitochondrial. Ke hoʻohālikelike ʻia me ka hui kaohi, ua hoʻonui ʻia ka ʻike mtDNA i ka hui i mālama ʻia me TGF-β1 (Kiʻi 4D). Ke hoʻohālikelike ʻia me ka hui i mālama ʻia me TGF-β1, ua emi ka ʻike mtDNA i ka hui mālama hui pū ʻia (Kiʻi 4D), e hōʻike ana e hiki i ka IPA ke hōʻemi i ka ʻike mtDNA a me ka helu mitochondrial a me ka hanu mitochondrial (Kiʻi 3C). Eia kekahi, me he mea lā ua hōʻemi ka IPA i ka ʻike mtDNA i ka mālama hui pū ʻana akā ʻaʻole i hoʻopilikia i ka hana mitochondrial i hoʻopili ʻia e MTR (Kiʻi 4A–C).
Ua noiʻi mākou i ka pilina o IPA me nā pae mRNA o nā genes e pili ana me ka fibrosis, apoptosis, ke ola ʻana, a me nā dynamics mitochondrial i loko o nā cell LX-2 (Kiʻi 5A–D). Ke hoʻohālikelike ʻia me ka hui kaohi, ua hōʻike ka hui i mālama ʻia me TGF-β1 i ka hoʻonui ʻia ʻana o ka hōʻike ʻana o nā genes e like me ke kaulahao collagen type I α2 (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP1), a me ka dynamin 1-like gene (DRP1), e hōʻike ana i ka hoʻonui ʻia ʻana o ka fibrosis a me ka hoʻāla ʻana. Eia kekahi, ke hoʻohālikelike ʻia me ka hui kaohi, ua hoʻemi ka mālama ʻana me TGF-β1 i nā pae mRNA o ka nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, inhibitor of B-cell α, enhancer of nuclear factor κ gene light peptide (NFκB1A), a me ka inhibitor of nuclear factor κB kinase subunit β (IKBKB) (Kiʻi 5A–D). I ka hoʻohālikelike ʻia me ka lāʻau TGF-β1, ua hoʻemi ka lāʻau hui pū ʻana me TGF-β1 a me IPA i ka hōʻike ʻana o COL1A2 a me MMP2, akā ua hoʻonui i nā pae mRNA o PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, a me IKBKB. Ua hoʻemi nui ka lāʻau IPA i ka hōʻike ʻana o MMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), a me ka mitochondrial fusion protein 2 (MFN2), ʻoiai ua hoʻonui ʻia ka hōʻike ʻana o CASP8, NFκB1A, NFκB1B, a me IKBKB i ka hoʻohālikelike ʻia me ka hui kaohi. Eia naʻe, ʻaʻohe ʻokoʻa i loaʻa i ka hōʻike ʻana o caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), a me ka fission protein 1 (FIS1). Ma ke ʻano hui pū ʻia, hōʻike kēia mau hopena e hoʻololi ka mālama ʻana o IPA i ka hōʻike ʻana o nā genes e pili ana me ka fibrosis, apoptosis, ke ola ʻana, a me nā dynamics mitochondrial. Hōʻike kā mākou ʻikepili e hōʻemi ana ka mālama ʻana o IPA i ka fibrosis i loko o nā pūnaewele LX-2; i ka manawa like, hoʻoulu ia i ke ola ʻana ma ka hoʻololi ʻana i ka phenotype i ka inactivation.
Hoʻololi ʻo IPA i ka hōʻike ʻana o nā genes fibroblast, apoptotic, viability, a me mitochondrial dynamics i loko o nā cell LX-2. Hōʻike nā Histograms i ka hōʻike mRNA e pili ana i ka mana endogenous (RPLP0 a i ʻole PPIA) ma hope o ka hoʻoulu ʻia ʻana o nā cell LX-2 me TGF-β1 a me IPA i loko o ka serum-free medium no 24 h. Hōʻike ʻo A i nā fibroblasts, hōʻike ʻo B i nā cell apoptotic, hōʻike ʻo C i nā cell ola, a hōʻike ʻo D i ka hōʻike ʻana o ka gene mitochondrial dynamics. Hōʻike ʻia nā ʻikepili ma ke ʻano he mean ± standard deviation (SD), n = 3 mau hoʻokolohua kūʻokoʻa. Ua hana ʻia nā hoʻohālikelike helu me ka hoʻohana ʻana i ka hoʻāʻo hoʻokahi ala ANOVA a me Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
A laila, ua loiloi ʻia nā loli i ka nui o ke kelepona (FSC-H) a me ka paʻakikī cytoplasmic (SSC-H) e ka cytometry kahe (Kiʻi 6A,B), a ua loiloi ʻia nā loli i ke ʻano o ke kelepona ma hope o ka mālama ʻana o IPA e ka microscopy electron transmission (TEM) a me ka microscopy ʻokoʻa pae (Kiʻi Hoʻohui 6A-B). E like me ka mea i manaʻo ʻia, ua piʻi ka nui o nā kelepona i loko o ka hui i mālama ʻia me TGF-β1 i hoʻohālikelike ʻia me ka hui kaohi (Kiʻi 6A,B), e hōʻike ana i ka hoʻonui maʻamau o ka reticulum endoplasmic rough (ER*) a me nā phagolysosomes (P), e hōʻike ana i ka hoʻāla ʻana o ke kelepona kumu hematopoietic (HSC) (Kiʻi Hoʻohui 6A). Eia naʻe, i hoʻohālikelike ʻia me ka hui i mālama ʻia me TGF-β1, ua emi ka nui o ke kelepona, ka paʻakikī o ka cytoplasmic (Kiʻi 6A,B), a me ka ʻike o ER* i loko o ka hui mālama hui pū ʻana o TGF-β1 a me IPA (Kiʻi Hoʻohui 6A). Eia kekahi, ua hoʻemi ka mālama ʻana me IPA i ka nui o ke kelepona, ka paʻakikī o ka cytoplasmic (Kiʻi 6A,B), P a me ka ʻike o ER* (Kiʻi Hoʻohui 6A) i hoʻohālikelike ʻia me ka hui kaohi. Eia kekahi, ua piʻi ka nui o nā cell apoptotic ma hope o 24 mau hola o ka mālama ʻana iā IPA i hoʻohālikelike ʻia me ka hui kaohi (nā pua keʻokeʻo, Supplementary Fig. 6B). Ma ke ʻano hui, hōʻike kēia mau hopena e hiki i ka 1 mM IPA ke hoʻoulu i ka apoptosis HSC a hoʻohuli i nā loli i nā ʻano morphological cell i hoʻokumu ʻia e TGF-β1, no laila e hoʻoponopono ai i ka nui o ka cell a me ka paʻakikī, hiki ke pili me ka inactivation HSC.
Hoʻololi ka IPA i ka nui o ke kelepona a me ka paʻakikī o ka cytoplasm i loko o nā pūnaewele LX-2. Nā kiʻi hōʻike o ka nānā ʻana i ka cytometry kahe. Ua hoʻohana ka nānā ʻana i kahi hoʻolālā gating kūikawā no nā pūnaewele LX-2: SSC-A/FSC-A e wehewehe i ka heluna o ke kelepona, FSC-H/FSC-A e ʻike i nā pālua, a me SSC-H/FSC-H no ka nānā ʻana i ka nui o ke kelepona a me ka paʻakikī. Ua hoʻoulu ʻia nā pūnaewele me TGF-β1 (5 ng/ml) a me 1 mM IPA i loko o ka medium serum-free no 24 h. Ua hoʻolaha ʻia nā pūnaewele LX-2 i ka quadrant hema haʻahaʻa (SSC-H-/FSC-H-), ka quadrant hema kiʻekiʻe (SSC-H+/FSC-H-), ka quadrant ʻākau haʻahaʻa (SSC-H-/FSC-H+), a me ka quadrant ʻākau kiʻekiʻe (SSC-H+/FSC-H+) no ka nui o ke kelepona a me ka nānā ʻana i ka paʻakikī o ka cytoplasm. B. Ua kālailai ʻia ke ʻano o ke kelepona e ka cytometry kahe me ka hoʻohana ʻana iā FSC-H (hoʻopuehu mua, ka nui o ke kelepona) a me SSC-H (hoʻopuehu ʻaoʻao, paʻakikī o ka cytoplasm) (30,000 mau hanana). Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± SD, n = 3 mau hoʻokolohua kūʻokoʻa. Ua hana ʻia nā hoʻohālikelike helu me ka hoʻohana ʻana i ka hoʻāʻo hoʻokahi ala ANOVA a me Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001 a me ****p < 0.0001
Ua lilo nā metabolites ʻōpū e like me IPA i kumuhana wela o ka noiʻi, e hōʻike ana e ʻike ʻia paha nā pahuhopu hou i loko o ka microbiota ʻōpū. No laila he mea hoihoi ia ua hōʻike ʻia ʻo IPA, kahi metabolite a mākou i hoʻopili ai i ka fibrosis akepaʻa i loko o ke kanaka [15], he hui anti-fibrotic hiki ke loaʻa i nā hiʻohiʻona holoholona [13, 14]. Maanei, hōʻike mākou no ka manawa mua i kahi pilina ma waena o ka serum IPA a me ka transcriptomics akepaʻa honua a me ka methylation DNA i nā poʻe momona me ka ʻole o ka maʻi diabetes type 2 (T2D), e hōʻike ana i ka apoptosis, mitophagy a me ka lōʻihi o ke ola, a me kahi gene moho AKT1 e hoʻoponopono ana i ka homeostasis akepaʻa. ʻO kekahi mea hou o kā mākou noiʻi ʻana, ua hōʻike mākou i ka pilina o ka mālama ʻana o IPA me ka apoptosis, morphology cell, mitochondrial bioenergetics a me nā dynamics i loko o nā cell LX-2, e hōʻike ana i kahi spectrum ikehu haʻahaʻa e hoʻololi i ka phenotype HSC i ka inactivation, e hana ana iā IPA i moho kūpono no ka hoʻomaikaʻi ʻana i ka fibrosis akepaʻa.
Ua ʻike mākou ʻo ka apoptosis, mitophagy a me ke ola lōʻihi nā ala canonical koʻikoʻi i hoʻonui ʻia i nā genes ate e pili ana me ka serum IPA e kahe ana. ʻO ka hoʻopilikia ʻana o ka ʻōnaehana mitochondrial quality control (MQC) hiki ke alakaʻi i ka mitochondrial dysfunction, mitophagy a me apoptosis, no laila e hoʻolaha ana i ka hanana o MASLD [33, 34]. No laila, hiki iā mākou ke kuhi e pili ana paha ʻo IPA i ka mālama ʻana i nā dynamics cell a me ka pono mitochondrial ma o apoptosis, mitophagy a me ke ola lōʻihi i loko o ke ake. Ua hōʻike kā mākou ʻikepili he ʻelua mau genes i maʻamau ma waena o nā assays ʻekolu: YKT6 a me AKT1. He mea kūpono ke hoʻomaopopo ʻana he protein SNARE ʻo YKT6 i komo i ke kaʻina hana o ka fusion membrane cell. He kuleana kona i ka autophagy a me ka mitophagy ma ka hoʻokumu ʻana i kahi hui hoʻomaka me STX17 a me SNAP29 ma ka autophagosome, no laila e hoʻolaha ana i ka fusion o nā autophagosomes a me nā lysosomes [35]. Eia kekahi, ʻo ka nalowale o ka hana YKT6 e hopena i ka mitophagy impaired [36], ʻoiai ʻo ka upregulation o YKT6 e pili ana me ka holomua o ka hepatocellular carcinoma (HCC), e hōʻike ana i ka hoʻonui ʻia o ke ola cell [37]. Ma ka ʻaoʻao ʻē aʻe, ʻo AKT1 ka gene launa pū koʻikoʻi loa a he kuleana koʻikoʻi kāna i nā maʻi akepaʻa, me ke ala hōʻailona PI3K/AKT, ke kaʻina hana o ke kelepona, ka neʻe ʻana o ke kelepona, ka hoʻonui ʻana, ka hoʻopili ʻana o ka poʻe, ka hana mitochondrial, a me ka hoʻokaʻawale ʻana o ka collagen [38–40]. Hiki i ke ala hōʻailona PI3K/AKT i hoʻāla ʻia ke hoʻāla i nā cell stem hematopoietic (HSCs), ʻo ia nā cell e kuleana no ka hana ʻana o ka matrix extracellular (ECM), a ʻo kona dysregulation e kōkua paha i ka hanana a me ka holomua o ka fibrosis akepaʻa [40]. Eia kekahi, ʻo AKT kekahi o nā mea ola nui o ke kelepona e kāohi ana i ka apoptosis cell p53-dependent, a hiki ke pili ka hoʻāla ʻana o AKT me ka pale ʻana i ka apoptosis cell akepaʻa [41, 42]. Hōʻike nā hopena i loaʻa e pili ana paha ʻo IPA i ka apoptosis e pili ana i ka mitochondria akepaʻa ma ka hoʻopili ʻana i ka hoʻoholo ʻana o nā hepatocytes ma waena o ke komo ʻana i ka apoptosis a i ʻole ke ola ʻana. Hiki ke hoʻoponopono ʻia kēia mau hopena e nā genes moho AKT a/a i ʻole YKT6, he mea koʻikoʻi no ka homeostasis akepaʻa.
Ua hōʻike ʻia kā mākou mau hopena ua hoʻoulu ka 1 mM IPA i ka apoptosis a ua hoʻemi i ka hanu mitochondrial i loko o nā pūnaewele LX-2 me ke kūʻokoʻa o ka mālama ʻana iā TGF-β1. He mea kūpono ke hoʻomaopopo ʻia ʻo ka apoptosis kahi ala nui no ka hoʻonā ʻana o ka fibrosis a me ka hoʻāla ʻana o ke kelepona kumu hematopoietic (HSC), a he hanana koʻikoʻi hoʻi ia i ka pane physiological reversible o ka fibrosis ake [4, 43]. Eia kekahi, ʻo ka hoʻihoʻi ʻana o BHI i loko o nā pūnaewele LX-2 ma hope o ka hui pū ʻana o ka mālama ʻana ua hāʻawi i nā ʻike hou i ke kuleana hiki o IPA i ka hoʻoponopono ʻana i nā bioenergetics mitochondrial. Ma lalo o nā kūlana hoʻomaha a me ka hana ʻole, hoʻohana maʻamau nā pūnaewele hematopoietic i ka phosphorylation oxidative mitochondrial e hana i ka ATP a loaʻa ka hana metabolic haʻahaʻa. Ma ka ʻaoʻao ʻē aʻe, hoʻonui ka hoʻāla ʻana o HSC i ka hanu mitochondrial a me ka biosynthesis e uku no nā koi ikehu o ke komo ʻana i ke kūlana glycolytic [44]. ʻO ka ʻoiaʻiʻo ʻaʻole i hoʻopilikia ka IPA i ka hiki ke metabolic a me ECAR e hōʻike ana ʻaʻole i hoʻonohonoho mua ʻia ke ala glycolytic. Pēlā nō, ua hōʻike kekahi noiʻi ʻē aʻe ua hiki iā 1 mM IPA ke hoʻololi i ka hana kaulahao hanu mitochondrial i loko o nā cardiomyocytes, ka laina cell hepatocyte kanaka (Huh7) a me nā cell endothelial vein umbilical kanaka (HUVEC); Eia naʻe, ʻaʻohe hopena o IPA i loaʻa ma ka glycolysis i loko o nā cardiomyocytes, e hōʻike ana e hiki i ka IPA ke hoʻopilikia i ka bioenergetics o nā ʻano cell ʻē aʻe [45]. No laila, ke kuhi nei mākou e hana paha ʻo 1 mM IPA ma ke ʻano he uncoupler kemika akahai, ʻoiai hiki iā ia ke hōʻemi nui i ka hōʻike ʻana o ka gene fibrogenic, ke ʻano o ke kelepona a me nā bioenergetics mitochondrial me ka hoʻololi ʻole i ka nui o mtDNA [46]. Hiki i nā uncouplers Mitochondrial ke kāohi i ka fibrosis i hoʻokomo ʻia e ka moʻomeheu a me ka hoʻāla ʻana o HSC [47] a hōʻemi i ka hana ATP mitochondrial i hoʻoponopono ʻia a hoʻoulu ʻia paha e kekahi mau protein e like me nā protein uncoupling (UCP) a i ʻole adenine nucleotide translocase (ANT). Ma muli o ke ʻano o ke kelepona, hiki i kēia hanana ke pale i nā cell mai ka apoptosis a/a i ʻole e hoʻolaha i ka apoptosis [46]. Eia nō naʻe, pono nā haʻawina hou aʻe e wehewehe i ke kuleana o IPA ma ke ʻano he uncoupler mitochondrial i ka inactivation o ke kelepona kumu hematopoietic.
A laila ua noiʻi mākou inā paha e hōʻike ʻia nā loli i ka hanu mitochondrial i ke ʻano mitochondrial i loko o nā cell LX-2 ola. ʻO ka mea hoihoi, hoʻololi ka mālama ʻana o TGF-β1 i ka hapa mitochondrial mai ka spherical a i ka waena, me ka emi ʻana o ka lālā mitochondrial a me ka hoʻonui ʻana i ka hōʻike ʻana o DRP1, kahi kumu nui i ka fission mitochondrial [48]. Eia kekahi, pili ka fragmentation mitochondrial me ka paʻakikī o ka pūnaewele holoʻokoʻa, a ʻo ka hoʻololi ʻana mai ka fusion a i ka fission he mea koʻikoʻi no ka hoʻoulu ʻana o ke cell stem hematopoietic (HSC), ʻoiai ʻo ka pale ʻana i ka fission mitochondrial e alakaʻi i ka apoptosis HSC [49]. No laila, hōʻike kā mākou mau hopena e hiki i ka mālama ʻana o TGF-β1 ke hoʻoulu i ka emi ʻana o ka paʻakikī o ka pūnaewele mitochondrial me ka emi ʻana o ka lālā, ʻoi aku ka maʻamau i ka fission mitochondrial e pili ana me nā cell stem hematopoietic i hoʻāla ʻia (HSCs). Eia kekahi, ua hōʻike kā mākou ʻikepili hiki i ka IPA ke hoʻololi i ka hapa o ka mitochondria mai ka spherical a i ke ʻano waena, no laila e hōʻemi ana i ka hōʻike ʻana o OPA1 a me MFN2. Ua hōʻike nā haʻawina e hiki i ka downregulation o OPA1 ke hoʻoulu i ka emi ʻana o ka hiki ke hoʻopili ʻia ka mitochondrial membrane a hoʻoulu i ka apoptosis cell [50]. Ua ʻike ʻia ʻo MFN2 e hoʻoponopono i ka fusion mitochondrial a me ka apoptosis [51]. Hōʻike nā hopena i loaʻa e like me ka hoʻololi ʻana o ka hoʻokomo ʻana o nā pūnaewele LX-2 e TGF-β1 a/a i ʻole IPA i ke ʻano a me ka nui o ka mitochondrial, a me ke kūlana hoʻāla a me ka paʻakikī o ka pūnaewele.
Hōʻike kā mākou mau hopena e hiki i ka hui pū ʻana o ka mālama ʻana o TGFβ-1 a me IPA ke hōʻemi i ka mtDNA a me nā ʻano morphological o ke kelepona ma o ka hoʻoponopono ʻana i ka hōʻike mRNA o ka fibrosis, apoptosis a me nā genes e pili ana i ke ola ʻana i nā cell e pale ana i ka apoptosis. ʻOiaʻiʻo, ua hoʻemi ʻo IPA i ka pae hōʻike mRNA o AKT1 a me nā genes fibrosis koʻikoʻi e like me COL1A2 a me MMP2, akā ua hoʻonui i ka pae hōʻike o CASP8, ka mea e pili ana me ka apoptosis. Ua hōʻike kā mākou mau hopena ma hope o ka mālama ʻana o IPA, ua emi ka hōʻike ʻana o BAX a ua hoʻonui ʻia ka hōʻike mRNA o nā subunits ʻohana TIMP1, BCL-2 a me NF-κB, e hōʻike ana e hiki i ka IPA ke hoʻoulu i nā hōʻailona ola i nā cell kumu hematopoietic (HSCs) e pale ana i ka apoptosis. Hiki i kēia mau mole ke hana ma ke ʻano he mau hōʻailona pro-survival i nā cell kumu hematopoietic i hoʻāla ʻia, hiki ke pili me ka hoʻonui ʻia o ka hōʻike ʻana o nā protein anti-apoptotic (e like me Bcl-2), ka emi ʻana o ka hōʻike ʻana o ka pro-apoptotic BAX, a me kahi pilina paʻakikī ma waena o TIMP a me NF-κB [5, 7]. Hoʻopuka ka IPA i kona hopena ma o PXR, a ua ʻike mākou ua hoʻonui ka hui pū ʻana me TGF-β1 a me IPA i nā pae hōʻike mRNA PXR, e hōʻike ana i ka hoʻopau ʻana i ka hoʻoulu ʻana o HSC. Ua ʻike ʻia ka hōʻailona PXR i hoʻāla ʻia e kāohi i ka hoʻoulu ʻana o HSC ma vivo a ma vitro [52, 53]. Hōʻike kā mākou mau hopena e komo paha ʻo IPA i ka hoʻomaʻemaʻe ʻana o nā HSC i hoʻāla ʻia ma ka hoʻolaha ʻana i ka apoptosis, ka hōʻemi ʻana i ka fibrosis a me ka metabolism mitochondrial, a me ka hoʻonui ʻana i nā hōʻailona ola, ʻo ia nā kaʻina hana maʻamau e hoʻololi i ka phenotype HSC i hoʻāla ʻia i kahi mea i hoʻopau ʻia. ʻO kekahi wehewehe kūpono no ka mīkini hiki a me ke kuleana o IPA i ka apoptosis ʻo ia ka scavenges mitochondria dysfunctional ma o ka mitophagy (ala kūloko) a me ke ala hōʻailona TNF extrinsic (Papa 1), kahi i pili pono i ke ala hōʻailona ola NF-κB (Kiʻi Hoʻohui 7). ʻO ka mea hoihoi, hiki i nā genes i hoʻonui ʻia e pili ana i ka IPA ke hoʻoulu i nā hōʻailona pro-apoptotic a me pro-survival i ke ala apoptotic [54], e hōʻike ana e hiki i ka IPA ke hoʻoulu i ke ala apoptotic a i ʻole ke ola ma ka launa pū ʻana me kēia mau genes. Eia nō naʻe, pehea e hoʻoulu ai ʻo IPA i ka apoptosis a i ʻole ke ola ʻana i ka wā o ka hoʻoulu ʻana o HSC a me kona mau ala mechanistic e noho maopopo ʻole ana.
He metabolite microbial ka IPA i hoʻokumu ʻia mai ka tryptophan meaʻai ma o ka microbiota ʻōpū. Ua hōʻike ʻia nā haʻawina he mau waiwai anti-inflammatory, antioxidant, a me epigenetic regulatory i loko o ke kaiapuni ʻōpū.[55] Ua hōʻike ʻia nā haʻawina hiki i ka IPA ke hoʻololi i ka hana pale ʻōpū a hōʻemi i ke kaumaha oxidative, kahi e hiki ai ke kōkua i kona hopena physiological kūloko.[56] ʻOiaʻiʻo, lawe ʻia ka IPA i nā ʻōpū i manaʻo ʻia ma o ke kahe ʻana o ke koko, a no ka mea ua like ka IPA me kahi ʻano metabolite nui me ka tryptophan, serotonin, a me nā derivatives indole, hoʻokō ʻo IPA i nā hana metabolic e hopena i nā hopena metabolic hoʻokūkū.[52] Hiki i ka IPA ke hoʻokūkū me nā metabolites i loaʻa mai ka tryptophan no nā wahi hoʻopaʻa ma nā enzymes a i ʻole nā ​​​​mea loaʻa, e hoʻopilikia paha i nā ala metabolic maʻamau. Hōʻike kēia i ka pono no nā haʻawina hou aʻe e pili ana i kāna pharmacokinetics a me pharmacodynamics e hoʻomaopopo maikaʻi i kona puka makani therapeutic.[57] Ke kali nei e ʻike ʻia inā hiki ke hana pū kēia i nā cell stem hematopoietic (HSCs).
Ke ʻae nei mākou he mau palena ko kā mākou noiʻi. No ka nānā pono ʻana i nā pilina e pili ana i ka IPA, ua kāpae mākou i nā poʻe maʻi me ka maʻi diabetes mellitus type 2 (T2DM). Ke ʻae nei mākou e kaupalena ana kēia i ka hoʻohana ākea o kā mākou mau ʻike i nā poʻe maʻi me ka maʻi diabetes mellitus type 2 a me ka maʻi akepaʻa holomua. ʻOiai ʻo ka nui o ka IPA i loko o ke koko kanaka he 1-10 μM [11, 20], ua koho ʻia kahi nui o 1 mM IPA ma muli o ka nui kiʻekiʻe loa ʻaʻole ʻona [15] a me ka nui kiʻekiʻe o ka apoptosis, me ka ʻole o ka ʻokoʻa i ka pakeneka o ka heluna cell necrotic. ʻOiai ua hoʻohana ʻia nā pae supraphysiological o IPA i kēia noiʻi, ʻaʻohe manaʻo like i kēia manawa e pili ana i ka mahele maikaʻi o IPA [52]. ʻOiai he koʻikoʻi kā mākou mau hopena, ʻo ka hopena metabolic ākea o IPA e mau ana kahi wahi noiʻi ikaika. Eia kekahi, ʻo kā mākou mau ʻike ma ka pilina ma waena o nā pae serum IPA a me ka methylation DNA o nā transcripts ate i loaʻa ʻole mai nā cell stem hematopoietic (HSCs) akā mai nā ʻiʻo ate. Ua koho mākou e hoʻohana i nā pūnaewele LX-2 kanaka ma muli o kā mākou mau ʻike mua mai ka loiloi transcriptome e pili ana ʻo IPA me ka hoʻāla ʻana o ke kelepona kumu hematopoietic (HSC) [15], a ʻo HSC nā pūnaewele nui i komo i ka holomua o ka fibrosis ake. Ua haku ʻia ke akepaʻa me nā ʻano kelepona he nui, no laila pono e noʻonoʻo ʻia nā hiʻohiʻona kelepona ʻē aʻe e like me ka ʻōnaehana co-culture cell hepatocyte-HSC-immune i hui pū ʻia me ka hoʻāla ʻana o caspase a me ka fragmentation DNA a me ke ʻano o ka hana me ka pae protein e aʻo ai i ke kuleana o IPA a me kāna pilina me nā ʻano kelepona akepaʻa ʻē aʻe.