Takambotaura kare kuti huwandu hwe tryptophan metabolite indole-3-propionic acid (IPA) muropa hunowanikwa mudumbu hwakaderera muvarwere vane fibrosis yechiropa. Muchidzidzo ichi, takaongorora transcriptome neDNA methylome muzviropa zvakafuta maererano nehuwandu hweIPA muropa, pamwe nebasa reIPA mukukonzera kushaikwa kwemasero ehepatic stellate (HSCs) mumuviri wemunhu.
Chidzidzo ichi chaisanganisira varwere 116 vakafuta vasina chirwere cheshuga cherudzi rwechipiri (T2DM) (makore 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m²) vakavhiyiwa bariatric paKuopio Bariatric Surgery Center (KOBS). Mazinga eIPA anotenderera akayerwa ne liquid chromatography-mass spectrometry (LC-MS), liver transcriptome analysis yakaitwa ne total RNA sequencing, uye DNA methylation analysis yakaitwa uchishandisa Infinium HumanMethylation450 BeadChip. Masero ehepatic stellate evanhu (LX-2) akashandiswa mukuyedza mu vitro.
Mazinga eIPA muropa aienderana nekuratidzwa kwemajini anobatanidzwa munzira dzeapoptotic, mitophagic, uye longevity muchiropa. Jini reAKT serine/threonine kinase 1 (AKT1) ndiro jini raive rakawanda uye rakanyanya kupindirana mu liver transcript uye DNA methylation profiles. Kurapwa kweIPA kwakakonzera apoptosis, kudzikira kwekufema kwemitochondrial, uye kwakachinja morphology yemasero uye mitochondrial dynamics nekuchinja kuratidzwa kwemajini anozivikanwa kuti anodzora fibrosis, apoptosis, uye kupona kwemasero eLX-2.
Kana zvabatanidzwa, data iri rinotsigira kuti IPA ine mhedzisiro yekurapa uye inogona kukonzera apoptosis uye kuchinja HSC phenotype kuenda kumamiriro asina basa, nokudaro zvichiwedzera mukana wekudzivirira liver fibrosis nekuvhiringidza HSC activation uye mitochondrial metabolism.
Kuwanda kwehuremu hwemafuta uye chirwere chemetabolic kwakabatana nekuwedzera kwehuwandu hwechirwere chechiropa chine mafuta (MASLD); chirwere ichi chinobata 25% kusvika 30% yevanhu vose [1]. Mugumisiro mukuru weMASLD i fibrosis yechiropa, maitiro anochinja-chinja anoratidzwa nekuunganidzwa kwe fibrous extracellular matrix (ECM) [2]. Masero makuru anobatanidzwa mu fibrosis yechiropa masero e hepatic stellate (HSCs), ayo anoratidza mana anozivikanwa phenotypes: quiescent, activated, inactivated, uye senescent [3, 4]. HSCs dzinogona kuvhurwa uye transdifferentiate kubva muchimiro che quiescent kuenda kumasero akaita se fibroblast-like ane simba rakawanda, nekuwedzera kuratidzwa kwe α-smooth muscle actin (α-SMA) uye type I collagen (Col-I) [5, 6]. Munguva yekudzoka kwe fibrosis yechiropa, HSCs dzinoshanda dzinobviswa kuburikidza ne apoptosis kana inactivation. Maitiro aya anosanganisira kudzikiswa kwemajini efibrogenic uye modulation yemajini eprosurvival (akadai seNF-κB uye PI3K/Akt signaling pathways) [7, 8], pamwe nekuchinja kwesimba remitochondrial uye mashandiro [9].
Mazinga emuropa e tryptophan metabolite indole-3-propionic acid (IPA) muropa, anogadzirwa mudumbu, akaonekwa kuti akaderera muzvirwere zvemuviri zvevanhu zvinosanganisira MASLD [10-13]. IPA inobatanidzwa nekudya fiber, inozivikanwa nemhedzisiro yayo ye antioxidant uye anti-inflammatory, uye inoderedza phenotype ye steatohepatitis (NASH) inokonzerwa nekudya mu vivo uye mu vitro [11-14]. Humwe humbowo hunobva muongororo yedu yapfuura, iyo yakaratidza kuti mazinga eIPA muropa aive akaderera muvarwere vane liver fibrosis pane muvarwere vakafuta vasina liver fibrosis muKuopio Bariatric Surgery Study (KOBS). Uyezve, takaratidza kuti kurapwa kweIPA kunogona kuderedza kutaura kwemajini ari zviratidzo zvechinyakare zvekunamatira kwemasero, kufamba kwemasero uye hematopoietic stem cell activation mu human hepatic stellate cell (LX-2) model uye inogona kuva metabolite inodzivirira hepatoprotective [15]. Zvisinei, hazvisati zvajeka kuti IPA inokonzera sei kudzoreredzwa kwe fibrosis muchiropa nekukurudzira HSC apoptosis uye mitochondrial bioenergetics.
Pano, tinoratidza kuti serum IPA inobatanidzwa nekuratidzwa kwemajini akapfuma muapoptosis, mitophagy, uye nzira dzehupenyu hurefu muchiropa chevanhu vane chirwere cheshuga chakakora asi chisiri cherudzi rwechipiri (KOBS). Uyezve, takaona kuti IPA inogona kukonzera kubviswa nekuderera kwemasero eropa akashanda (HSCs) kuburikidza nenzira yekusashanda. Izvi zvinoratidza basa idzva reIPA, zvichiita kuti ive chinangwa chekurapa chekusimudzira kudzoreredzwa kwefibrosis yechiropa.
Ongororo yakaitwa kare muboka reKOBS yakaratidza kuti varwere vane fibrosis yechiropa vaiva nemazinga eIPA akaderera anotenderera muropa zvichienzaniswa nevarwere vasina fibrosis yechiropa [15]. Kuti tibvise mhedzisiro inogona kuvhiringidzika yechirwere cheshuga cherudzi rwechipiri, takatora varwere 116 vakafuta vasina chirwere cheshuga cherudzi rwechipiri (avhareji yezera ± SD: 46.8 ± 9.3 years; BMI: 42.7 ± 5.0 kg/m2) (Tafura 1) kubva muchidzidzo cheKOBS chiri kuenderera mberi sevanhu vari muchidzidzo [16]. Vese vakatora chikamu vakapa mvumo yakanyorwa uye gwara rekudzidza rakatenderwa neEthics Committee yeNorth Savo County Hospital zvichienderana neDeclaration of Helsinki (54/2005, 104/2008 na27/2010).
Zviyero zve biopsy yechiropa zvakawanikwa panguva yekuvhiyiwa kwe bariatric uye zvakaongororwa ne histologicalist nenyanzvi dzezvirwere zvichienderana nezvinodiwa zvakatsanangurwa kare [17, 18]. Zvinodiwa zvekuongorora zvakapfupikiswa mu Supplementary Table S1 uye zvakatsanangurwa kare [19].
Sampuli dze serum yekutsanya dzakaongororwa ne untargeted liquid chromatography-mass spectrometry (LC-MS) yekuongorora metabolomics (n = 116). Sampuli dzakaongororwa uchishandisa UHPLC-qTOF-MS system (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) sezvakatsanangurwa kare19. Kuzivikanwa kwe isopropyl alcohol (IPA) kwakavakirwa panguva yekuchengetedza uye kuenzanisa MS/MS spectrum ne pure standards. IPA signal intensity (peak area) yakatariswa mune zvese zvakaongororwa [20].
Kuongororwa kweRNA yechiropa chose kwakaitwa uchishandisa Illumina HiSeq 2500 uye data rakagadziriswa sezvakatsanangurwa kare [19, 21, 22]. Takaita ongororo yekuratidza musiyano wezvinyorwa zvinokanganisa mashandiro emitochondrial/biogenesis tichishandisa majini e1957 akasarudzwa kubva kuMitoMiner 4.0 database [23]. Kuongororwa kwemethylation yeDNA yechiropa kwakaitwa uchishandisa Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) tichishandisa nzira imwecheteyo sezvakatsanangurwa kare [24, 25].
Masero esimbi echiropa emunhu (LX-2) akapihwa nemutsa naProf. Stefano Romeo uye akarimwa uye akachengetwa muDMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Kuti vasarudze dose yekushanda yeIPA, masero eLX-2 akarapwa nemazinga akasiyana eIPA (10 μM, 100 μM uye 1 mM; Sigma, 220027) muDMEM/F12 medium kwemaawa makumi maviri nemana. Uyezve, kuti vaongorore kugona kweIPA kumisa HSCs, masero eLX-2 akarapwa pamwe chete ne5 ng/ml TGF-β1 (R&D systems, 240-B-002/CF) uye 1 mM IPA mu serum-free medium kwemaawa makumi maviri nemana. Pakudzora mota zvinoenderana, 4 nM HCL ine 0.1% BSA yakashandiswa pakurapa TGF-β1 uye 0.05% DMSO yakashandiswa pakurapa IPA, uye ese ari maviri akashandiswa pamwe chete pakurapa kwakabatana.
Kubuda kwemasero eropa kwakaongororwa uchishandisa FITC Annexin V Apoptosis Detection Kit ine 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) zvichienderana nemirairo yemugadziri. Muchidimbu, LX-2 (1 × 105 cells/well) yakakurirwa husiku hwese mumaplates e12-well ndokuzorapwa nedosi dzakawanda dzeIPA kana IPA neTGF-β1. Zuva rakatevera, masero anoyangarara uye anonamatira akaunganidzwa, akashandiswa trypsin, akagezwa nePBS, akasungirirwazve muAnnexin V binding buffer, uye akachengetwa neFITC-Annexin V ne7-AAD kwemaminitsi gumi nemashanu.
Mitochondria mumasero mapenyu yakaiswa mavara ekuti ishande zvakanaka uchishandisa Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Pakuongorora MTR, masero eLX-2 akaiswa muhuremu hwakaenzana neIPA neTGF-β1. Mushure memaawa makumi maviri nemana, masero mapenyu akaongororwa, akagezwa nePBS, uye akazoiswa muhuremu hwe100 μM MTR mu serum-free medium pa37 °C kwemaminitsi makumi maviri sezvakatsanangurwa kare [26]. Pakuongorora morphology yemasero mapenyu, saizi yemasero uye kuomarara kwe cytoplasmic zvakaongororwa uchishandisa forward scatter (FSC) uye side scatter (SSC) parameters, zvichiteerana.
Ruzivo rwese (zviitiko 30,000) rwakatorwa uchishandisa NovoCyte Quanteon (Agilent) uye rwakaongororwa uchishandisa NovoExpress® 1.4.1 kana FlowJo V.10 software.
Mwero wekushandiswa kweokisijeni (OCR) uye mwero wekuwedzera acidification (ECAR) zvakayerwa panguva chaiyo uchishandisa Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) yakashongedzerwa neSeahorse XF Cell Mito Stress zvichienderana nemirairo yemugadziri. Muchidimbu, 2 × 104 LX-2 cells/well dzakadyarwa pamaplate eXF96 cell culture. Mushure mekuiswa mumasero husiku hwese, masero akarapwa neisopropanol (IPA) uye TGF-β1 (Supplementary Methods 1). Kuongororwa kwedata kwakaitwa uchishandisa software yeSeahorse XF Wave, inosanganisira Seahorse XF Cell Energy Phenotype Test Report Generator. Kubva pane izvi, Bioenergetic Health Index (BHI) yakaverengerwa [27].
RNA yose yakanyoreswa kuita cDNA. Kuti uwane nzira dzakatarwa, ona reference [15]. Mazana emuRNA emunhu 60S ribosomal acidic protein P0 (RPLP0) uye cyclophilin A1 (PPIA) akashandiswa seconstitutive gene controls. QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, The Netherlands) yakashandiswa neTaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) kana Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), uye relative gene expression fold yakaverengerwa uchishandisa comparative Ct value cycling parameters (ΔΔCt) uye ∆∆Ct method. Ruzivo rwema primers rwunopihwa muSupplementary Tables S2 neS3.
DNA yeNuclear (ncDNA) neDNA yemitochondrial (mtDNA) zvakatorwa uchishandisa DNeasy blood and tissue kit (Qiagen) sezvakatsanangurwa kare [28]. Huwandu hwemtDNA hwakaverengerwa nekuverenga chiyero chenzvimbo yega yega yemtDNA yakatarwa negeometric mean yenzvimbo nhatu dzeNuclear DNA (mtDNA/ncDNA), sezvakatsanangurwa muSupplementary Methods 2. Ruzivo rwema primers emtDNA nencDNA rwunopihwa muSupplementary Table S4.
Masero mapenyu akapendwa neMitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) kuti vaone network dzemitochondrial dziri pakati pemasero nedziri mukati memasero. Masero eLX-2 (1 × 104 masero/tsime) akakurirwa pamasiraidhi egirazi mumaplate ekurima ane pasi pegirazi anoenderana (Ibidi GmbH, Martinsried, Germany). Mushure memaawa makumi maviri nemana, masero mapenyu eLX-2 akapendwa ne100 μM MTR kwemaminitsi makumi maviri pa37 °C uye manuclei emasero akapendwa neDAPI (1 μg/ml, Sigma-Aldrich) sezvakatsanangurwa kare [29]. Network dzeMitochondrial dzakapendwa neZeiss Axio Observer inverted microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) dzakashongedzerwa neZeiss LSM 800 confocal module pa37 °C mumhepo ine humidity ine 5% CO2 uchishandisa 63×NA 1.3 objective. Takawana mifananidzo gumi yeZ-series yemhando yega yega yemuenzaniso. Imwe neimwe yeZ-series ine zvikamu makumi matatu, imwe neimwe ine ukobvu hwe9.86 μm. Pamuenzaniso wega wega, mifananidzo yenzvimbo gumi dzakasiyana dzemaonero yakawanikwa uchishandisa ZEN 2009 software (Carl Zeiss Microimaging GmbH, Jena, Germany), uye ongororo yemitochondrial morphology yakaitwa uchishandisa ImageJ software (v1.54d) [30, 31] zvichienderana nema parameter akatsanangurwa muSupplementary Methods 3.
Masero acho akasimbiswa ne2% glutaraldehyde mu 0.1 M phosphate buffer, zvichiteverwa nekugadziriswa ne 1% osmium tetroxide solution (Sigma Aldrich, MO, USA), zvishoma nezvishoma akabviswa mvura ne acetone (Merck, Darmstadt, Germany), uye pakupedzisira akaiswa mu epoxy resin. Zvikamu zvakatetepa zvakagadzirwa uye zvakapendwa ne 1% uranyl acetate (Merck, Darmstadt, Germany) uye 1% lead citrate (Merck, Darmstadt, Germany). Mifananidzo ye ultrastructural yakawanikwa uchishandisa JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) pa 80 kV inokurumidza.
Maumbirwo emasero eLX-2 akarapwa neIPA kwemaawa makumi maviri nemana akaongororwa nephase-contrast microscopy pa50x magnification uchishandisa Zeiss inverted light microscope (Zeiss Axio Vert.A1 naAxioCam MRm, Jena, Germany).
Ruzivo rwekiriniki rwakatsanangurwa seavhareji ± kutsauka kwakajairwa kana kuti median (interquartile range: IQR). Kuongororwa kwevariance nenzira imwe chete (continuous variables) kana χ² test (categorical variables) kwakashandiswa kuenzanisa mutsauko uripo pakati pemapoka matatu ekudzidza. False positive rate (FDR) yakashandiswa kugadzirisa bvunzo dzakawanda, uye majini ane FDR <0.05 akaonekwa seakakosha muhuwandu. Spearman correlation analysis yakashandiswa kubatanidza CpG DNA methylation neIPA signal intensity, ne nominal p values ​​​​(p <0.05) yakataurwa.
Kuongororwa kwenzira kwakaitwa pachishandiswa chishandiso chekuongorora majini (WebGestalt) chezvinyorwa 268 (zvisina kunyorwa p < 0.01), zvinyorwa 119 zvemitochondria (zvisina kunyorwa p < 0.05), uye 4350 CpGs kubva muzvinyorwa 3093 zvechiropa zvaive zvakabatana nemazinga eropa anotenderera muropa. Chishandiso cheVenny DB (vhezheni 2.1.0) chinowanikwa mahara chakashandiswa kuwana majini anopindirana, uye StringDB (vhezheni 11.5) chakashandiswa kufungidzira kudyidzana kwemapuroteni nemapuroteni.
Pakuyedza kweLX-2, sampuro dzakaedzwa kuti dzionekwe kuti dzakajairwa sei pachishandiswa bvunzo yeD'Agostino-Pearson. Data rakawanikwa kubva mumakopi matatu ebhayoloji uye rakaitwa ANOVA nenzira imwe chete neBonferroni post hoc test. P-value iri pasi pe0.05 yakaonekwa seyakakosha muhuwandu hwe ...
Kutanga, takaongorora hukama hwemazinga eIPA muropa nezvinyorwa zvechiropa, muviri wese, uye mitochondrial. Muhuwandu hwezvinyorwa zvese, jini rakasimba rakabatana nemazinga eIPA muropa raive MAPKAPK3 (FDR = 0.0077; mitogen-activated protein kinase-activated protein kinase 3); muhuwandu hwezvinyorwa zvine chekuita nemitochondria, jini rakasimba rakabatana raive AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Faira rekuwedzera 1 neFaira rekuwedzera 2).
Takazoongorora zvinyorwa zvepasi rose (n = 268; p < 0.01) uye zvinyorwa zvine chekuita ne mitochondria (n = 119; p < 0.05), pakupedzisira tichiona apoptosis senzira yakakosha zvikuru (p = 0.0089). Kune zvinyorwa zve mitochondrial zvine chekuita nemazinga e serum IPA, takatarisa pa apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), uye nzira dzeTNF signaling (FDR = 0.000006) (Mufananidzo 1A, Tafura 2, uye Supplementary Figures 1A-B).
Kuongororwa kwakabatana kwezvinyorwa zvepasi rose, zvine chekuita ne mitochondria, uye methylation yeDNA muchiropa chemunhu pamwe chete nemazinga e serum IPA. A inomiririra zvinyorwa zvepasi rose 268, zvinyorwa zve mitochondria 119 zvine chekuita ne mitochondria, uye zvinyorwa zve DNA methylated zvakaiswa pamepu kune 3092 CpG nzvimbo dzine chekuita nemazinga e serum IPA (p values ​​​​< 0.01 yezvinyorwa zvepasi rose uye DNA methylated, uye p values ​​​​< 0.05 yezvinyorwa zve mitochondrial). Zvinyorwa zvikuru zvinopindirana zvinoratidzwa pakati (AKT1 neYKT6). B Mepu yekudyidzana kwemajini gumi nematatu ane chikero chepamusoro chekudyidzana (0.900) nemamwe majini yakagadzirwa kubva kumajini makumi mashanu nematanhatu anopindirana (nzvimbo yemutsara mutema) ayo akabatanidzwa zvakanyanya nemazinga e serum IPA uchishandisa chishandiso che online StringDB. Green: Majini akaenzaniswa nechikamu chemasero cheGene Ontology (GO): mitochondria (GO:0005739). AKT1 ndiyo puroteni ine mamaki epamusoro (0.900) ekudyidzana nemamwe mapuroteni zvichibva padata (zvichibva pakutsvaga zvinyorwa, kuyedza, madhatabhesi, uye kutaurirana pamwe chete). Manodhi enetwork anomiririra mapuroteni, uye mipendero inomiririra kubatana pakati pemapuroteni.
Sezvo ma microbiota metabolites emudumbu achigona kudzora epigenetic composition kuburikidza neDNA methylation [32], takaongorora kana serum IPA levels dzaive dzakabatana ne liver DNA methylation. Takaona kuti nzvimbo mbiri huru dzemethylation dzakabatana ne serum IPA levels dzaive pedyo ne proline-serine-rich region 3 (C19orf55) uye heat shock protein family B (small) member 6 (HSPB6) (Additional file 3). DNA methylation ye4350 CpG (p < 0.01) yakabatana ne serum IPA levels uye yakapfuma munzira dzekutonga kwenguva refu (p = 0.006) (Mufananidzo 1A, Tafura 2, uye Supplementary Figure 1C).
Kuti tinzwisise mashandiro ehupenyu ari pasi pehukama huripo pakati pemazinga eIPA muropa, zvinyorwa zvepasi rose, zvinyorwa zvine chekuita ne mitochondria, uye methylation yeDNA muchiropa chemunhu, takaita ongororo yekubatanidza majini akaonekwa muongororo yapfuura yenzira (Mufananidzo 1A). Mhedzisiro yekuongorora kwekuwedzera nzira kwemajini 56 anopindirana (mukati memutsetse mutema muMufananidzo 1A) yakaratidza kuti nzira yeapoptosis (p = 0.00029) yakaratidza majini maviri akafanana neongororo nhatu: AKT1 neYKT6 (YKT6 v-SNARE homolog), sezvakaratidzwa muVenn diagram (Supplementary Picture 2 neMufananidzo 1A). Zvinofadza kuti takaona kuti AKT1 (cg19831386) neYKT6 (cg24161647) zvaive zvakabatana zvakanaka nemazinga eIPA muropa (Additional file 3). Kuti tione mukana wekudyidzana kwemapuroteni pakati pezvigadzirwa zvemajini, takasarudza majini gumi nematatu ane chibodzwa chepamusoro chenzvimbo (0.900) pakati pemajini 56 anopindirana sechikamu uye takagadzira mepu yekudyidzana. Zvichienderana nezinga rekuvimba (kuvimba kwemukati), jini reAKT1 rine mamakisi epamusoro (0.900) ndiro raiva pamusoro (Mufananidzo 1B).
Zvichibva pakuongorora nzira, takaona kuti apoptosis ndiyo nzira huru, saka takaongorora kana kurapwa kweIPA kwaizokanganisa apoptosis yeHSCs mu vitro. Takamboratidza kuti madosi akasiyana eIPA (10 μM, 100 μM, uye 1 mM) akanga asina chepfu kumasero eLX-2 [15]. Chidzidzo ichi chakaratidza kuti kurapwa kweIPA pa10 μM ne100 μM kwakawedzera huwandu hwemasero anoshanda uye anofa. Zvisinei, zvichienzaniswa neboka rekutonga, kushanda kwemasero kwakaderera pa1 mM IPA concentration, nepo chiyero che necrosis chemasero chisina kuchinja (Mufananidzo 2A, B). Tevere, kuti tiwane huwandu hwakakodzera hwekukonzera apoptosis mumasero eLX-2, takaedza 10 μM, 100 μM, uye 1 mM IPA kwemaawa makumi maviri nemana (Mufananidzo 2A-E neMufananidzo Wekuwedzera 3A-B). Zvinofadza kuti, IPA 10 μM ne100 μM zvakaderedza mwero weapoptosis (%), zvisinei, IPA 1 mM yakawedzera mwero weapoptosis uye apoptosis (%) zvichienzaniswa nekudzora uye nekudaro yakasarudzwa kuti iongororwezve (Mifananidzo 2A–D).
IPA inokonzera apoptosis yemasero eLX-2. Nzira yeAnnexin V ne7-AAD double staining yakashandiswa kuyedza apoptotic rate uye morphology yemasero kuburikidza ne flow cytometry. Masero eBA akaiswa mu incubated ne 10 μM, 100 μM uye 1 mM IPA kwemaawa makumi maviri nemana kana ne F–H TGF-β1 (5 ng/ml) uye 1 mM IPA mu serum-free medium kwemaawa makumi maviri nemana. A: masero mapenyu (Annexin V -/ 7AAD-); B: masero ane necrotic (Annexin V -/ 7AAD+); C, F: early (Annexin V +/ 7AAD-); D, G: late (Annexin V+/7AAD.+); E, H: percentage yemasero ese early nealate apoptotic mu apoptotic rate (%). Data rinoratidzwa seavhareji ± SD, n = 3 experiments dzakazvimirira. Kuenzanisa kwenhamba kwakaitwa uchishandisa nzira imwe chete yeANOVA neBonferroni post hoc test. *p < 0.05; ****p < 0.0001
Sezvatakamboratidza, 5 ng/ml TGF-β1 inogona kukonzera kushanda kweHSC nekuwedzera kuratidzwa kwemajini ekare ezviratidzo [15]. Masero eLX-2 akarapwa ne5 ng/ml TGF-β1 uye 1 mM IPA pamwe chete (Mufananidzo 2E–H). Kurapa kweTGF-β1 hakuna kuchinja mwero weapoptosis, zvisinei, kurapwa pamwe chete kweIPA kwakawedzera mwero weapoptosis uye apoptosis (%) zvichienzaniswa nekurapa kweTGF-β1 (Mufananidzo 2E–H). Mhedzisiro iyi inoratidza kuti 1 mM IPA inogona kukurudzira apoptosis mumasero eLX-2 pasina TGF-β1 induction.
Takaongorora zvakare mhedzisiro yeIPA pakufema kwemitochondrial mumasero eLX-2. Zvakabuda zvakaratidza kuti 1 mM IPA yakaderedza parameters ye oxygen consumption rate (OCR): kufema kusiri kwemitochondrial, kufema kwebasal uye kwakanyanya, kudonha kweproton uye kugadzirwa kweATP zvichienzaniswa neboka rekudzora (Mufananidzo 3A, B), nepo bioenergetic health index (BHI) isina kuchinja.
IPA inoderedza kufema kwemitochondrial mumasero eLX-2. Mutsetse wekufema kwemitochondrial (OCR) unoratidzwa semaparamita ekufema kwemitochondrial (kufema kusiri kwemitochondrial, kufema kwebasal, kufema kwakanyanya, kudonha kweproton, kugadzirwa kweATP, SRC neBHI). Masero A neB akaiswa mukati me10 μM, 100 μM uye 1 mM IPA kwemaawa makumi maviri nemana, zvichiteerana. Masero C neD akaiswa mukati meTGF-β1 (5 ng/ml) uye 1 mM IPA mu serum-free medium kwemaawa makumi maviri nemana, zvichiteerana. Kuyerwa kwese kwakagadziriswa kune huwandu hweDNA uchishandisa CyQuant kit. BHI: bioenergetic health index; SRC: respiratory reserve capacity; OCR: oxygen consumption rate. Data rinoratidzwa se mean ± standard deviation (SD), n = 5 independent experiments. Statistical comparisons dzakaitwa uchishandisa one-way ANOVA neBonferroni post hoc test. *p < 0.05; **p < 0.01; uye ***p < 0.001
Kuti tinzwisise zvizere nezvemhedzisiro yeIPA pa bioenergetic profile yeTGF-β1-activated LX-2 cells, takaongorora mitochondrial oxidative phosphorylation neOCR (Mufananidzo 3C, D). Zvakabuda zvakaratidza kuti kurapwa kweTGF-β1 kunogona kuderedza kufema kwakanyanya, respiratory reserve capacity (SRC) uye BHI zvichienzaniswa neboka rekutonga (Mufananidzo 3C, D). Pamusoro pezvo, kurapwa kwakabatana kwakaderedza basal respiratory, proton leak uye ATP production, asi SRC neBHI zvaive zvakakwirira zvakanyanya kupfuura zvakarapwa neTGF-β1 (Mufananidzo 3C, D).
Takaitawo "Cellular Energy Phenotype Test" yakapihwa neSeahorse software (Supplementary Fig. 4A–D). Sezvakaratidzwa muSupplementary Fig. 3B, OCR neECAR metabolic potentials dzakadzikira mushure mekurapwa kweTGF-β1, zvisinei, hapana musiyano wakaonekwa mumapoka ekurapa ekubatanidza neIPA zvichienzaniswa neboka rekutonga. Uyezve, ese ari maviri basal uye stress levels eOCR akadzikira mushure mekurapwa kwekubatanidza neIPA zvichienzaniswa neboka rekutonga (Supplementary Fig. 4C). Zvinofadza kuti, maitiro akafanana akaonekwa ne combination therapy, uko pasina shanduko mu basal uye stress levels yeECAR yakaonekwa zvichienzaniswa nekurapwa kweTGF-β1 (Supplementary Fig. 4C). MuHSCs, kudzikira kwe mitochondrial oxidative phosphorylation uye kugona kwe combination therapy kudzoreredza SCR neBHI mushure mekusangana neTGF-β1 therapy hazvina kuchinja metabolic potential (OCR neECAR). Kana zvabatanidzwa, mhedzisiro iyi inoratidza kuti IPA inogona kuderedza bioenergetics muHSCs, zvichiratidza kuti IPA inogona kukonzera kuderera kwesimba rinochinja HSC phenotype kuenda kuinactivation (Supplementary Picture 4D).
Mhedzisiro yeIPA pamitochondrial dynamics yakaongororwa uchishandisa quantification ye three-dimensional morphology uye network connections pamwe ne MTR staining (Mufananidzo 4 ne Supplementary Mufananidzo 5). Mufananidzo 4 unoratidza kuti, zvichienzaniswa neboka rekudzora, TGF-β1 treatment yakaderedza avhareji yenzvimbo yepamusoro, nhamba yebazi, kureba kwebazi rese, uye nhamba ye branch junction (Mufananidzo 4A na B) uye yakachinja huwandu hwe mitochondria kubva pa spherical kuenda pa intermediate morphology (Mufananidzo 4C). Kurapa kwe IPA chete ndiko kwakaderedza avhareji ye mitochondrial volume uye kwakachinja huwandu hwe mitochondria kubva pa spherical kuenda pa intermediate morphology zvichienzaniswa neboka rekudzora (Mufananidzo 4A). Kusiyana neizvi, sphericity, avhareji yerefu rebazi, uye mitochondrial activity yakaongororwa ne mitochondrial membrane potential-dependent MTR (Mufananidzo 4A na E) hazvina kuchinja uye ma parameter aya haana kusiyana pakati pemapoka. Kana zvabatanidzwa, mhedzisiro iyi inoratidza kuti kurapwa kwe TGF-β1 ne IPA kunoita sekunochinja chimiro che mitochondrial nehukuru pamwe nekuomarara kwe network mu LX-2 cells mhenyu.
IPA inochinja mashandiro emitochondrial uye kuwanda kweDNA yemitochondrial mumasero eLX-2. A. Mifananidzo inomiririra confocal yemasero mapenyu eLX-2 akaputirwa neTGF-β1 (5 ng/ml) uye 1 mM IPA kwemaawa makumi maviri nemana mu serum-free medium inoratidza mitochondrial networks dzakasvibiswa neMitotracker™ Red CMXRos uye nuclei dzakasvibiswa nebhuruu neDAPI. Data rese raive nemifananidzo inosvika gumi neshanu paboka rega rega. Takawana mifananidzo gumi yeZ-stack yemhando yega yega yesample. Sequence yega yega yeZ-axis yaive nezvidimbu makumi matatu, imwe neimwe ine ukobvu hwe9.86 μm. Scale bar: 10 μm. B. Zvinhu zvinomiririrwa (mitochondria chete) zvakaonekwa nekushandisa adaptive thresholding kumufananidzo. Kuongorora kwehuwandu uye kuenzanisa mitochondrial morphological network connections kwakaitwa kumasero ese ari muboka rega rega. C. Kuwanda kwe mitochondrial shape ratios. Values ​​​​pedyo ne0 zvinoratidza maumbirwo edenderedzwa, uye values ​​​​pedyo ne1 zvinoratidza maumbirwo efilamentous. D Zviri muMitochondrial DNA (mtDNA) zvakaongororwa sezvakatsanangurwa muZvinhu neNzira. E Mitotracker™ Red CMXRos analysis yakaitwa neflow cytometry (zviitiko 30,000) sezvakatsanangurwa muZvinhu neNzira. Data rinoratidzwa seavhareji ± SD, n = 3 miedzo yakazvimirira. Kuenzanisa kwestatistike kwakaitwa uchishandisa one-way ANOVA neBonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Takazoongorora huwandu hwemtDNA mumasero eLX-2 sechiratidzo chehuwandu hwemitochondrial. Kana tichienzanisa neboka rekutonga, huwandu hwemtDNA hwakawedzerwa muboka rakarapwa neTGF-β1 (Mufananidzo 4D). Kana tichienzanisa neboka rakarapwa neTGF-β1, huwandu hwemtDNA hwakadzikira muboka rekurapa kwakabatana (Mufananidzo 4D), zvichiratidza kuti IPA inogona kuderedza huwandu hwemtDNA uye pamwe huwandu hwemitochondrial pamwe nekufema kwemitochondrial (Mufananidzo 3C). Uyezve, IPA yakaita seyakaderedza huwandu hwemtDNA mukurapa kwakabatana asi haina kukanganisa mashandiro emitochondrial anokonzerwa neMTR (Mufananidzo 4A–C).
Takaongorora hukama hweIPA nemazinga e mRNA emajini ane chekuita ne fibrosis, apoptosis, survival, uye mitochondrial dynamics mumasero eLX-2 (Mufananidzo 5A–D). Zvichienzaniswa neboka rekutonga, boka rakarapwa neTGF-β1 rakaratidza kuwedzera kwekutaura kwemajini akadai se collagen type I α2 chain (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), tissue inhibitor ye metalloproteinase 1 (TIMP1), uye dynamin 1-like gene (DRP1), zvichiratidza kuwedzera kwe fibrosis uye activation. Uyezve, zvichienzaniswa neboka rekutonga, kurapwa kweTGF-β1 kwakaderedza mazinga e mRNA e nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, inhibitor ye B-cell α, enhancer ye nuclear factor κ gene light peptide (NFκB1A), uye inhibitor ye nuclear factor κB kinase subunit β (IKBKB) (Mufananidzo 5A–D). Zvichienzaniswa nekurapwa neTGF-β1, kurapwa kwakabatana neTGF-β1 neIPA kwakaderedza kuratidzwa kweCOL1A2 neMMP2, asi kwakawedzera mRNA levels dzePXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, uye IKBKB. Kurapa neIPA kwakaderedza zvakanyanya kuratidzwa kweMMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), uye mitochondrial fusion protein 2 (MFN2), nepo kuratidzwa kweCASP8, NFκB1A, NFκB1B, uye IKBKB kwakawedzerwa zvichienzaniswa neboka rekutonga. Zvisinei, hapana musiyano wakawanikwa mukuratidzwa kwecaspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), uye fission protein 1 (FIS1). Pamwe chete, mhinduro idzi dzinoratidza kuti kurapwa kweIPA kunogadzirisa kuratidzwa kwemajini ane chekuita ne fibrosis, apoptosis, kupona, uye mitochondrial dynamics. Ruzivo rwedu runoratidza kuti kurapwa kweIPA kunoderedza fibrosis mumasero eLX-2; panguva imwe chete, kunokurudzira kurarama nekushandura phenotype kuenda kune inactivation.
IPA inogadzirisa kuratidzwa kwemajini e fibroblast, apoptotic, viability, uye mitochondrial dynamics mumasero eLX-2. Histograms dzinoratidza mRNA expression maererano ne endogenous control (RPLP0 kana PPIA) mushure mekunge masero eLX-2 akurudzirwa neTGF-β1 neIPA mu serum-free medium kwemaawa makumi maviri nemana. A inoratidza fibroblasts, B inoratidza masero e apoptotic, C inoratidza masero ari mupenyu, uye D inoratidza mitochondrial dynamics gene expression. Data rinoratidzwa se mean ± standard deviation (SD), n = 3 independent experiments. Statistical comparisons dzakaitwa uchishandisa one-way ANOVA neBonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Zvadaro, shanduko muhukuru hwemasero (FSC-H) uye cytoplasmic complexity (SSC-H) dzakaongororwa ne flow cytometry (Mufananidzo 6A,B), uye shanduko mu morphology yemasero mushure mekurapwa kweIPA dzakaongororwa ne transmission electron microscopy (TEM) uye phase contrast microscopy (Mufananidzo 6A-B). Sezvaitarisirwa, masero ari muboka rakarapwa neTGF-β1 akawedzera muhukuru zvichienzaniswa neboka rekudzora (Mufananidzo 6A,B), zvichiratidza kuwedzera kwe rough endoplasmic reticulum (ER*) uye phagolysosomes (P), zvichiratidza kuvhurwa kwe hematopoietic stem cell (HSC) (Mufananidzo 6A). Zvisinei, zvichienzaniswa neboka rakarapwa neTGF-β1, hukuru hwemasero, cytoplasmic complexity (Mufananidzo 6A,B), uye ER* zvakadzikira muboka rekurapa reTGF-β1 neIPA (Mufananidzo 6A). Uyezve, kurapwa kweIPA kwakaderedza saizi yemasero, kuomarara kwe cytoplasmic (Mifananidzo 6A,B), P uye ER* (Mufananidzo Wekuwedzera 6A) zvichienzaniswa neboka rekudzora. Pamusoro pezvo, huwandu hwemasero e-apoptotic hwakawedzera mushure memaawa makumi maviri nemana ekurapwa kweIPA zvichienzaniswa neboka rekudzora (miseve chena, Mufananidzo Wekuwedzera 6B). Pamwe chete, mhedzisiro iyi inoratidza kuti 1 mM IPA inogona kukurudzira HSC apoptosis uye kudzosera shanduko mu morphological parameters dzemasero inokonzerwa neTGF-β1, nokudaro ichidzora saizi yemasero uye kuomarara, izvo zvinogona kunge zvakabatana neHSC inactivation.
IPA inochinja saizi yemasero uye kuomarara kwe cytoplasmic mumasero eLX-2. Mifananidzo inomiririra yekuongorora kwe flow cytometry. Ongororo iyi yakashandisa nzira yekutarisa magating yakanangana nemasero eLX-2: SSC-A/FSC-A kutsanangura huwandu hwemasero, FSC-H/FSC-A kuona ma doublet, uye SSC-H/FSC-H yekuongorora saizi yemasero uye kuomarara. Masero akaiswa mu incubation ne TGF-β1 (5 ng/ml) uye 1 mM IPA mu serum-free medium kwemaawa makumi maviri nemana. Masero eLX-2 akagoverwa mu lower left quadrant (SSC-H-/FSC-H-), upper left quadrant (SSC-H+/FSC-H-), lower right quadrant (SSC-H-/FSC-H+), uye upper right quadrant (SSC-H+/FSC-H+) yehukuru hwemasero uye cytoplasmic complexity analysis. B. Maumbirwo emasero akaongororwa ne flow cytometry uchishandisa FSC-H (forward scatter, cell size) uye SSC-H (side scatter, cytoplasmic complexity) (zviitiko 30,000). Data rinopihwa se mean ± SD, n = 3 experiments dzakazvimirira. Kuenzanisa kwestatistike kwakaitwa uchishandisa one-way ANOVA neBonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 uye ****p < 0.0001
Mametabolites emudumbu akadai seIPA ave nyaya inonyanya kufarirwa muongororo, zvichiratidza kuti zvinangwa zvitsva zvinogona kuwanikwa mugut microbiota. Saka zvinonakidza kuti IPA, metabolite yatakabatanidza ne fibrosis yechiropa muvanhu [15], yakaratidzwa kuva inogona kurwisa fibrosis mumhando dzemhuka [13, 14]. Pano, tinoratidza kekutanga hukama huripo pakati pe serum IPA ne global liver transcriptomics uye DNA methylation muvanhu vakafuta vasina chirwere cheshuga cherudzi rwechipiri (T2D), zvichiratidza apoptosis, mitophagy uye longevity, pamwe ne candidate gene AKT1 inodzora liver homeostasis. Chimwe chinhu chitsva chekudzidza kwedu ndechekuti takaratidza kudyidzana kwekurapwa kweIPA ne apoptosis, cell morphology, mitochondrial bioenergetics uye dynamics mumasero eLX-2, zvichiratidza simba shoma rinoshandura HSC phenotype kuenda ku inactivation, zvichiita kuti IPA ive mukana wekuvandudza liver fibrosis.
Takaona kuti apoptosis, mitophagy uye hupenyu hurefu ndiyo nzira dzakakosha dzecanonical dzakapfuma mumajini echiropa ane chekuita neropa rinotenderera IPA. Kukanganiswa kwehurongwa hwekudzora hunhu hwemitochondrial (MQC) kunogona kukonzera kusashanda zvakanaka kwemitochondrial, mitophagy uye apoptosis, nokudaro zvichikurudzira kuitika kweMASLD[33, 34]. Nokudaro, tinogona kufungidzira kuti IPA inogona kunge iine chekuita nekuchengetedza dynamics yemasero uye mitochondrial integrity kuburikidza neapoptosis, mitophagy uye hupenyu hurefu muchiropa. Data redu rakaratidza kuti majini maviri aiwanzoonekwa muongororo nhatu: YKT6 neAKT1. Zvakakosha kuziva kuti YKT6 ipuroteni yeSNARE inobatanidzwa mukuita kwekubatanidza membrane yemasero. Inoita basa mu autophagy uye mitophagy nekugadzira initiation complex ine STX17 neSNAP29 pa autophagosome, nokudaro ichikurudzira kubatanidzwa kwe autophagosomes ne lysosomes[35]. Uyezve, kurasikirwa nebasa reYKT6 kunokonzeresa kusashanda zvakanaka kwemasero [36], nepo kuwedzera kweYKT6 kuchibatanidzwa nekufambira mberi kwehepatocellular carcinoma (HCC), zvichiratidza kuwedzera kwekurarama kwemasero [37]. Kune rumwe rutivi, AKT1 ndiyo gene inonyanya kupindirana uye ine basa rakakosha muzvirwere zvechiropa, kusanganisira nzira yekutumira PI3K/AKT, kutenderera kwemasero, kufamba kwemasero, kuwanda, kunamatira kwemasero, basa remitochondrial, uye kuburitswa kwecollagen [38–40]. Nzira yekutumira PI3K/AKT inoshanda inogona kumutsa masero eropa (HSCs), ayo ari masero ane basa rekugadzira matrix yekunze (ECM), uye kusashanda kwayo kunogona kubatsira mukuitika nekufambira mberi kwefibrosis yechiropa [40]. Pamusoro pezvo, AKT ndeimwe yezvinhu zvakakosha zvekuchengetedza masero zvinodzivirira p53-dependent cell apoptosis, uye kuvhurwa kweAKT kunogona kusanganiswa nekudziviswa kweapoptosis yechiropa [41, 42]. Zvakawanikwa zvinoratidza kuti IPA inogona kunge iine chekuita nekubuda kwemasero eropa mumitochondria kuburikidza nekuchinja sarudzo ye hepatocytes pakati pekupinda mu apoptosis kana kupona. Mhedzisiro iyi inogona kudzorwa nemajini eAKT uye/kana YKT6, ayo akakosha pakugara zvakanaka kwechiropa.
Zvakabuda muongororo yedu zvakaratidza kuti 1 mM IPA yakakonzera apoptosis uye kudzikira kwekufema kwemitochondrial mumasero eLX-2 pasina kurapwa kweTGF-β1. Zvakakosha kuziva kuti apoptosis inzira huru yekugadzirisa fibrosis uye kuvhurwa kwemasero eropa (HSC), uye zvakare chiitiko chakakosha mukupindurwa kwemuviri kwefibrosis yechiropa [4, 43]. Uyezve, kudzorerwa kweBHI mumasero eLX-2 mushure mekurapwa kwakabatana kwakapa ruzivo rutsva nezvebasa rinogona kuitwa neIPA mukudzora bioenergetics yemitochondrial. Mumamiriro ekuzorora uye asina simba, masero eropa anowanzo shandisa mitochondrial oxidative phosphorylation kugadzira ATP uye ane basa shoma remetabolic. Kune rumwe rutivi, kuvhurwa kweHSC kunosimudzira kufema kwemitochondrial uye biosynthesis kutsiva zvinodiwa zvesimba zvekupinda mu glycolytic state [44]. Chokwadi chekuti IPA haina kukanganisa kugona kwemetabolic uye ECAR chinoratidza kuti nzira yeglycolytic haina kunyanya kukosheswa. Saizvozvowo, imwe ongororo yakaratidza kuti 1 mM IPA yakakwanisa kugadzirisa mashandiro emuchina wekufema wemitochondrial mu cardiomyocytes, human hepatocyte cell line (Huh7) uye human umbilical vein endothelial cells (HUVEC); Zvisinei, hapana mhedzisiro yeIPA yakawanikwa pa glycolysis mu cardiomyocytes, zvichiratidza kuti IPA inogona kukanganisa bioenergetics yemamwe marudzi emasero [45]. Nokudaro, tinofungidzira kuti 1 mM IPA inogona kushanda semuchina wemakemikari munyoro, sezvo ichigona kuderedza zvakanyanya fibrogenic gene expression, cell morphology uye mitochondrial bioenergetics pasina kuchinja huwandu hwe mtDNA [46]. Mitochondrial uncouplers inogona kudzivirira culture-induced fibrosis uye HSC activation [47] uye kuderedza mitochondrial ATP production inodzorwa kana kukonzerwa nemamwe mapuroteni akadai se uncoupling proteins (UCP) kana adenine nucleotide translocase (ANT). Zvichienderana nerudzi rwesero, chiitiko ichi chinogona kudzivirira masero kubva ku apoptosis uye/kana kukurudzira apoptosis [46]. Zvisinei, zvimwe zvidzidzo zvinodiwa kuti zvijekese basa reIPA semuchina wekubvisa mamitochondrial mukuita kuti masero eropa asashande.
Takazoongorora kana shanduko dziri mukufema kwemitochondrial dzichiratidzwa muchimiro chemitochondrial mumasero eLX-2 ari mupenyu. Zvinofadza kuti, kurapwa kweTGF-β1 kunochinja chiyero chemitochondrial kubva paspherical kuenda paintermediate, nekudzikira kwematavi emitochondrial uye kuwedzera kwekutaura kweDRP1, chinhu chikuru mukuputika kwemitochondrial [48]. Uyezve, kupatsanurwa kwemitochondrial kunobatanidzwa nekuoma kwenetwork yese, uye kuchinja kubva pakubatanidzwa kuenda pakuputika kwakakosha pakushanda kwemasero eropa (HSC), nepo kudziviswa kwekuputika kwemitochondrial kuchitungamira kuHSC apoptosis [49]. Saka, zvatakawana zvinoratidza kuti kurapwa kweTGF-β1 kunogona kukonzera kudzikira kwekubatana kwemitochondrial network nekudzikira kwematavi, izvo zvinonyanya kuitika mukuputika kwemitochondrial kwakabatana nemasero eropa activated (HSCs). Uyezve, data redu rakaratidza kuti IPA inogona kuchinja chiyero chemitochondria kubva paspherical kuenda pachimiro chepakati, nokudaro ichideredza kuratidzwa kweOPA1 neMFN2. Zvidzidzo zvakaratidza kuti kudzikiswa kweOPA1 kunogona kukonzera kudzikira kwesimba remitochondrial membrane uye kukonzera apoptosis yemasero [50]. MFN2 inozivikanwa nekubatsira mukubatanidza mitochondrial fusion uye apoptosis [51]. Zvakawanikwa zvinoratidza kuti kupinza kwemasero eLX-2 neTGF-β1 uye/kana IPA kunoita sekunge kunochinja chimiro nehukuru hwemitochondrial, pamwe nemamiriro ekushanda uye kuomarara kwenetwork.
Zvakawanikwa zvedu zvinoratidza kuti kurapa pamwe chete kweTGFβ-1 neIPA kunogona kuderedza mtDNA nemasero morphological parameters nekudzora mRNA expression ye fibrosis, apoptosis uye majini ane chekuita nehupenyu mumasero anodzivisa apoptosis. Zvechokwadi, IPA yakaderedza mRNA expression level yeAKT1 uye majini akakosha e fibrosis akadai seCOL1A2 neMMP2, asi yakawedzera expression level yeCASP8, iyo inobatanidzwa ne apoptosis. Zvakawanikwa zvedu zvakaratidza kuti mushure mekurapwa kweIPA, BAX expression yakaderera uye mRNA expression yeTIMP1 family subunits, BCL-2 neNF-κB yakawedzera, zvichiratidza kuti IPA inogona kukurudzira masaini ekupona mu hematopoietic stem cells (HSCs) anodzivisa apoptosis. Mamorekuru aya anogona kushanda semasaini ekutsigira kupona mu activated hematopoietic stem cells, izvo zvinogona kunge zvakabatana nekuwedzera mapuroteni ekurwisa apoptotic (akadai seBcl-2), kudzikira kwepro-apoptotic BAX, uye kubatana kwakaoma pakati peTIMP neNF-κB [5, 7]. IPA inoita mabasa ayo kuburikidza nePXR, uye takaona kuti kurapwa pamwe chete neTGF-β1 neIPA kwakawedzera huwandu hwePXR mRNA expression, zvichiratidza kudzikiswa kweHSC activation. Activated PXR signaling inozivikanwa kuti inodzivisa HSC activation mu vivo uye mu vitro [52, 53]. Zvatakawana zvinoratidza kuti IPA inogona kutora chikamu mukubviswa kweHSCs dzakashanda nekusimudzira apoptosis, kuderedza fibrosis uye mitochondrial metabolism, uye kuwedzera zviratidzo zvekurarama, izvo zvinova maitiro akajairika anoshandura phenotype yeHSC yakabatidzwa kuita isina kushanda. Imwe tsananguro inogona kuitika yemaitiro anogona uye basa reIPA mu apoptosis ndeyekuti inobvisa mitochondria isina kushanda zvakanyanya kuburikidza ne mitophagy (nzira yemukati) uye nzira yekutumira TNF (Tafura 1), iyo yakabatana zvakananga nenzira yekutumira NF-κB survival signaling (Mufananidzo Wekuwedzera 7). Zvinofadza kuti majini akapfuma ane chekuita neIPA anokwanisa kukurudzira zviratidzo zve-apoptotic uye zvinotsigira kupona munzira ye-apoptotic [54], zvichiratidza kuti IPA inogona kukurudzira nzira ye-apoptotic kana kupona nekubatana nemajini aya. Zvisinei, kuti IPA inokonzera sei apoptosis kana kupona panguva yekushanda kweHSC uye nzira dzayo dzekugadzira hazvisati zvajeka.
IPA imhando ye microbial metabolite inoumbwa kubva ku dietary tryptophan kuburikidza ne gut microbiota. Zvidzidzo zvakaratidza kuti ine anti-inflammatory, antioxidant, uye epigenetic regulatory properties mu intestine.[55] Zvidzidzo zvakaratidza kuti IPA inogona kugadzirisa mashandiro e intestinal barrier uye kuderedza oxidative stress, izvo zvinogona kubatsira kumhedzisiro yayo ye physiological.[56] Kutaura zvazviri, IPA inotakurwa kuenda kunhengo dzakananga kuburikidza nekutenderera kweropa, uye sezvo IPA ine chimiro chikuru che metabolite chakafanana ne tryptophan, serotonin, uye indole derivatives, IPA inoita metabolic actions zvichikonzera makwikwi e metabolic fates.[52] IPA inogona kukwikwidzana ne tryptophan-derived metabolites kuti isunge nzvimbo pama enzymes kana receptors, zvichikanganisa nzira dzakajairika dze metabolic. Izvi zvinoratidza kudiwa kwezvidzidzo zvakawanda pa pharmacokinetics yayo uye pharmacodynamics kuti inzwisise zviri nani hwindo rayo rekurapa.[57] Zvichiri kutarisirwa kuonekwa kana izvi zvichigonawo kuitika mu hematopoietic stem cells (HSCs).
Tinobvuma kuti chidzidzo chedu chine zvimwe zvipingamupinyi. Kuti tinyatsoongorora hukama hwakabatana neIPA, takabvisa varwere vane chirwere cheshuga cherudzi rwechipiri (T2DM). Tinobvuma kuti izvi zvinoderedza kushanda kwezvakawanikwa zvedu kuvarwere vane chirwere cheshuga cherudzi rwechipiri uye chirwere chechiropa chakakwira. Kunyangwe huwandu hweIPA mumuviri wemunhu huri pakati pe1–10 μM [11, 20], huwandu hwe1 mM IPA hwakasarudzwa zvichibva pane huwandu hwakanyanya husina chepfu [15] uye huwandu hwakanyanya hweapoptosis, pasina mutsauko muhuwandu hwemasero ane necrotic. Kunyangwe huwandu hwepamusoro hweIPA hwakashandiswa muchidzidzo ichi, parizvino hapana kubvumirana maererano nedosi inoshanda yeIPA [52]. Kunyangwe mhedzisiro yedu yakakosha, mhedzisiro yakakura yemetabolic yeIPA ichiri nzvimbo inoshanda yekutsvagisa. Uyezve, zvatakawana pakubatana pakati pemazinga eIPA muropa uye DNA methylation yezvinyorwa zvechiropa zvakawanikwa kwete kubva kumasero eropa (HSCs) chete asiwo kubva kumatishu echiropa. Takasarudza kushandisa masero eLX-2 evanhu zvichibva pane zvatakawana kare kubva mukuongorora kwetranscriptome kuti IPA ine chekuita nekushanda kwemasero eropa (HSC) [15], uye maHSC ndiwo masero makuru anobatanidzwa mukufambira mberi kwe fibrosis yechiropa. Chiropa chinoumbwa nemarudzi akawanda emasero, saka mamwe mamodheru emasero akadai se hepatocyte-HSC-immune cell co-culture system pamwe chete ne caspase activation uye DNA fragmentation pamwe chete nemaitiro ekuita kusanganisira protein level zvinofanirwa kufungwa kuti zvidzidze basa re IPA uye kudyidzana kwayo nemamwe marudzi emasero echiropa.