Anyị kọrọ na mbụ na ọkwa serum nke tryptophan metabolite indole-3-propionic acid (IPA) nke sitere na eriri afọ dị ala karịa na ndị ọrịa nwere fibrosis imeju. N'ọmụmụ ihe a, anyị nyochachara transcriptome na DNA methylome na imeju buru ibu n'ihe gbasara ọkwa IPA serum, yana ọrụ IPA na-arụ n'ịkpalite phenotypic inactivation nke hepatic stellate cells (HSCs) na vitro.
Nnyocha ahụ gụnyere ndị ọrịa buru ibu 116 na-enweghị ụdị ọrịa shuga mellitus nke abụọ (T2DM) (afọ 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m²) bụ ndị a wachara ahụ bariatric na Kuopio Bariatric Surgery Center (KOBS). E jiri mmiri mmiri chromatography-mass spectrometry (LC-MS) tụọ ọkwa IPA na-agagharị agagharị, e mere nyocha imeju site na ngụkọta RNA sequencing, e mekwara nyocha methylation DNA site na iji Infinium HumanMethylation450 BeadChip. E jiri mkpụrụ ndụ stellate hepatic mmadụ (LX-2) mee nnwale in vitro.
Ọkwa IPA dị n'ime ọbara jikọtara ya na ngosipụta nke mkpụrụ ndụ ihe nketa metụtara ụzọ apoptotic, mitophagic, na ogologo ndụ n'ime imeju. Mkpụrụ ndụ ihe nketa AKT serine/threonine kinase 1 (AKT1) bụ mkpụrụ ndụ ihe nketa kachasị baa ụba ma na-emekọrịta ihe na nhazi imeju na profaịlụ methylation DNA. Ọgwụgwọ IPA kpatara apoptosis, mbelata iku ume mitochondrial, na mgbanwe morphology sel na mitochondrial dynamics site na ịgbanwe ngosipụta nke mkpụrụ ndụ ihe nketa a maara na-achịkwa fibrosis, apoptosis, na ndụ nke mkpụrụ ndụ LX-2.
N'ịchịkọta data ndị a, data ndị a na-akwado na IPA nwere ike inwe mmetụta ọgwụgwọ ma nwee ike ịkpalite apoptosis ma gbanwee ụdị HSC gaa na ọnọdụ na-adịghị arụ ọrụ, si otú a na-agbasa ohere nke igbochi fibrosis imeju site na igbochi ọrụ HSC na metabolism mitochondrial.
A na-ejikọta oke ibu na ọrịa metabolic na mmụba nke ọrịa imeju abụba metụtara metabolic (MASLD); ọrịa ahụ na-emetụta 25% ruo 30% nke ndị mmadụ n'ozuzu [1]. Isi ihe na-esi na MASLD apụta bụ imeju fibrosis, usoro na-agbanwe agbanwe nke a na-eji mkpokọta fibrous extracellular matrix (ECM) na-aga n'ihu [2]. Mkpụrụ ndụ ndị bụ isi metụtara imeju fibrosis bụ mkpụrụ ndụ hepatic stellate (HSCs), nke na-egosipụta ụdị ihe anọ a maara: quiescent, activated, inactivated, na senescent [3, 4]. Enwere ike ịgbalite HSCs ma gbanwee ha site na ụdị quiescent gaa na mkpụrụ ndụ dị ka fibroblast proliferative nwere oke ike, yana mmụba nke α-smooth muscle actin (α-SMA) na ụdị I collagen (Col-I) [5, 6]. N'oge mgbanwe fibrosis imeju, a na-ewepụ HSCs emelitere site na apoptosis ma ọ bụ inactivation. Usoro ndị a gụnyere mbelata nke mkpụrụ ndụ ihe nketa fibrogenic na mgbanwe nke mkpụrụ ndụ ihe nketa prosurvival (dịka NF-κB na PI3K/Akt signaling pathways) [7, 8], yana mgbanwe na mitochondrial dynamics na function [9].
A chọpụtala na ọkwa serum nke tryptophan metabolite indole-3-propionic acid (IPA), nke a na-emepụta n'ime eriri afọ, ebelatala na ọrịa metabolic mmadụ gụnyere MASLD [10-13]. IPA na-ejikọta ya na oriri eriri nri, a maara ya maka mmetụta antioxidant na mgbochi mkpali ya, ma na-ebelata ụdị steatohepatitis (NASH) nke na-abụghị mmanya na-akpata na vivo na in vitro [11-14]. Ụfọdụ ihe akaebe sitere na ọmụmụ ihe anyị gara aga, nke gosipụtara na ọkwa IPA serum dị ala na ndị ọrịa nwere fibrosis imeju karịa na ndị ọrịa buru ibu na-enweghị fibrosis imeju na Kuopio Bariatric Surgery Study (KOBS). Ọzọkwa, anyị gosiri na ọgwụgwọ IPA nwere ike ibelata ngosipụta nke mkpụrụ ndụ ihe nketa ndị bụ akara ochie nke njikọta sel, mbugharị sel na mkpali mkpụrụ ndụ hepatic stem cell na ụdị hepatic stem cell (LX-2) ma bụrụ metabolite nwere ike igbochi hepatoprotective [15]. Agbanyeghị, ọ ka edoghị anya otu IPA si ebute regression fibrosis imeju site na ịgbalite apoptosis HSC na mitochondrial bioenergetics.
N'ebe a, anyị na-egosi na IPA serum na-ejikọta ya na ngosipụta nke mkpụrụ ndụ ihe nketa bara ụba na apoptosis, mitophagy, na ụzọ ogologo ndụ n'ime imeju nke ndị nwere oke ibu mana ndị na-abụghị ụdị shuga nke abụọ (KOBS). Ọzọkwa, anyị chọpụtara na IPA nwere ike ịkpalite mwepụ na mmebi nke mkpụrụ ndụ hematopoietic stem (HSCs) na-arụ ọrụ site na ụzọ inactivation. Nsonaazụ ndị a na-ekpughe ọrụ ọhụrụ maka IPA, na-eme ka ọ bụrụ ihe mgbaru ọsọ ọgwụgwọ iji kwalite nlọghachi nke fibrosis imeju.
Nnyocha e mere n'oge gara aga n'òtù KOBS gosiri na ndị ọrịa nwere fibrosis imeju nwere ọkwa IPA dị ala ma e jiri ya tụnyere ndị na-enweghị fibrosis imeju [15]. Iji wepụ mmetụta mgbagwoju anya nke ọrịa shuga ụdị nke abụọ, anyị kpọbatara ndị ọrịa buru ibu 116 na-enweghị ọrịa shuga ụdị nke abụọ (afọ nkezi ± SD: 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m2) (Tebụl 1) site na ọmụmụ KOBS na-aga n'ihu dị ka ndị mmadụ na-amụ ihe [16]. Ndị niile sonyere nyere nkwenye ederede ma Kọmitii Omume nke Ụlọ Ọgwụ North Savo County kwadoro usoro ọmụmụ ahụ dịka Nkwupụta Helsinki (54/2005, 104/2008 na 27/2010) si kwuo.
E nwetara ihe nlele biopsy imeju n'oge ịwa ahụ bariatric ma ndị ọkachamara n'ihe gbasara ọrịa nyochara ya site na histological dịka ụkpụrụ akọwara na mbụ si dị [17, 18]. A chịkọtara ihe ndị a na-enyocha na Tebụl Mgbakwunye S1 ma akọpụtala ha na mbụ [19].
E jiri usoro chromatography-mass spectrometry (LC-MS) nke a na-anaghị atụle anya mee nyocha ọbara maka ihe nlele ọbara maka nyocha metabolomics (n = 116). E jiri usoro UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) nyochaa ihe nlele ọbara dịka akọwara na mbụ19. Nchọpụta nke mmanya isopropyl (IPA) dabere na oge nchekwa na ntụnyere nke ụdị MS/MS na ọkọlọtọ dị ọcha. E tụlere ike mgbaàmà IPA (mpaghara kachasị elu) na nyocha ndị ọzọ niile [20].
E jiri Illumina HiSeq 2500 mee usoro RNA imeju dum, e wee hazie data ahụ dịka akọwara na mbụ [19, 21, 22]. Anyị mere nyocha ngosipụta dị iche iche nke akwụkwọ ndị metụtara ọrụ/biogenesis mitochondrial site na iji mkpụrụ ndụ ihe nketa 1957 ahọpụtara site na nchekwa data MitoMiner 4.0 [23]. E jiri otu usoro ahụ akọwara na mbụ mee nyocha methylation DNA imeju site na iji Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) mee nyocha methylation DNA imeju.
Prọfesọ Stefano Romeo ji obiọma nye mkpụrụ ndụ hepatic stellate mmadụ (LX-2) ma kọọ ma chekwaa ya na DMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Iji họrọ oke ọrụ nke IPA, a gwọrọ mkpụrụ ndụ LX-2 site na iji ọtụtụ IPA dị iche iche (10 μM, 100 μM na 1 mM; Sigma, 220027) na DMEM/F12 medium ruo awa 24. Ọzọkwa, iji nyochaa ikike IPA nwere igbochi HSCs, ejiri 5 ng/ml TGF-β1 (usoro R&D, 240-B-002/CF) na 1 mM IPA gwọọ mkpụrụ ndụ LX-2 n'ime ihe na-enweghị serum ruo awa 24. Maka njikwa ụgbọala ndị kwekọrọ, ejiri 4 nM HCL nwere 0.1% BSA mee ọgwụgwọ TGF-β1 ebe ejiri 0.05% DMSO mee ọgwụgwọ IPA, ejiri ha abụọ mee ihe ọnụ maka ọgwụgwọ njikọta.
E jiri FITC Annexin V Apoptosis Detection Kit mee nyocha Apoptosis site na iji 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) dịka ntuziaka onye nrụpụta si dị. N'ụzọ dị mkpirikpi, a kụrụ LX-2 (1 × 105 mkpụrụ ndụ kwa olulu mmiri) n'abalị n'ime efere olulu mmiri 12 wee gwọọ ya na ọtụtụ doses nke IPA ma ọ bụ IPA na TGF-β1. N'echi ya, a chịkọtara mkpụrụ ndụ ndị na-ese n'elu mmiri na ndị na-arapara n'ahụ, trypsin, saa ya na PBS, tinyeghachi ha na Annexin V binding buffer, ma tinye FITC-Annexin V na 7-AAD n'ime ya maka nkeji iri na ise.
E ji Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) mee ka mkpụrụ ndụ dị ndụ tee Mitochondria n'ime mkpụrụ ndụ maka ọrụ oxidative. Maka nnwale MTR, e tinyere mkpụrụ ndụ LX-2 n'otu njupụta ha na IPA na TGF-β1. Mgbe awa 24 gachara, e tinyere mkpụrụ ndụ trypsin, saa ha na PBS, wee tinye ha na 100 μM MTR n'ime ihe na-enweghị serum na okpomọkụ 37 °C ruo nkeji 20 dịka akọwara na mbụ [26]. Maka nyocha morphology nke mkpụrụ ndụ dị ndụ, e jiri paramita mgbasa ozi n'ihu (FSC) na mgbasa ozi n'akụkụ (SSC) nyochaa nha mkpụrụ ndụ na mgbagwoju anya cytoplasmic, n'otu n'otu.
E jiri NovoCyte Quanteon (Agilent) nweta data niile (ihe omume 30,000) ma jiri ngwanrọ NovoExpress® 1.4.1 ma ọ bụ FlowJo V.10 nyochaa ha.
A tụrụ ọnụego oriri oxygen (OCR) na ọnụego asịd extracellular (ECAR) n'oge a na-ahụ anya site na iji Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) nke nwere Seahorse XF Cell Mito Stress dịka ntuziaka onye nrụpụta si dị. N'ụzọ dị mkpirikpi, a kụrụ mkpụrụ ndụ 2 × 104 LX-2/olulu mmiri n'elu efere ọdịbendị mkpụrụ ndụ XF96. Mgbe a kụchara mkpụrụ ndụ n'abalị, a na-eji isopropanol (IPA) na TGF-β1 (Usoro Mgbakwunye 1) agwọ mkpụrụ ndụ. E mere nyocha data site na iji ngwanrọ Seahorse XF Wave, nke gụnyere Seahorse XF Cell Energy Phenotype Test Report Generator. Site na nke a, a gbakọrọ Bioenergetic Health Index (BHI) [27].
E dekọrọ RNA zuru oke n'ime cDNA. Maka usoro ụfọdụ, lee ntụaka [15]. E jiri ọkwa mRNA nke 60S ribosomal acidic P0 (RPLP0) na cyclophilin A1 (PPIA) mee ihe dị ka njikwa mkpụrụ ndụ ihe nketa. Ejiri QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, Netherlands) mee ihe na TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) ma ọ bụ Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050), a gbakọọkwara mkpụkọ ngosipụta mkpụrụ ndụ ihe nketa site na iji paramita okirikiri Ct (ΔΔCt) na usoro ∆∆Ct. E nyere nkọwa nke praịmarị na Tebụl Mgbakwunye S2 na S3.
E jiri ngwa DNeasy blood and tissue kit (Qiagen) wepụta DNA nuklia (ncDNA) na mitochondrial DNA (mtDNA) dịka akọwara na mbụ [28]. A gbakọrọ ọnụọgụ mtDNA site na ịgbakọ oke nke mpaghara mtDNA ọ bụla a na-elekwasị anya na nkezi geometric nke mpaghara DNA nuklia atọ (mtDNA/ncDNA), dịka akọwara na Usoro Mgbakwunye 2. Nkọwa nke primers maka mtDNA na ncDNA dị na Tebụl Mgbakwunye S4.
E ji Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) mee ka mkpụrụ ndụ dị ndụ tee agba iji hụ anya na netwọk mitochondrial dị n'etiti sel na nke dị n'ime sel. E ji iko kpụọ mkpụrụ ndụ LX-2 (1 × 104 mkpụrụ ndụ/olulu) n'elu ihe ngosi iko n'ime efere omenala dị n'okpuru iko (Ibidi GmbH, Martinsried, Germany). Mgbe awa 24 gachara, e ji 100 μM MTR tinye mkpụrụ ndụ LX-2 dị ndụ ruo nkeji 20 na 37 °C ma tinye DAPI (1 μg/ml, Sigma-Aldrich) n'ime mkpụrụ ndụ dịka akọwara na mbụ [29]. E jiri igwe onyonyo Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, Germany) nke nwere modulu confocal Zeiss LSM 800 na 37 °C n'ime ikuku mmiri mmiri nwere 5% CO2 site na iji ihe mgbaru ọsọ 63 × NA 1.3. Anyị nwetara onyonyo usoro Z iri maka ụdị ihe nlele ọ bụla. Usoro Z ọ bụla nwere ngalaba iri atọ, nke ọ bụla nwere ọkpụrụkpụ nke 9.86 μm. Maka ihe nlele ọ bụla, ejiri ngwanrọ ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany) nweta onyonyo nke mpaghara iri dị iche iche nke echiche, e jirikwa ngwanrọ ImageJ (v1.54d) mee nyocha morphology mitochondrial [30, 31] dịka paramita ndị a kọwara na Usoro Mgbakwunye 3 si dị.
E ji glutaraldehyde 2% dozie mkpụrụ ndụ ndị ahụ na 0.1 M phosphate buffer, wee tinye ya na 1% osmium tetroxide solution (Sigma Aldrich, MO, USA), jiri nwayọ nwayọ gbazee mmiri na acetone (Merck, Darmstadt, Germany), ma mechaa tinye ya na epoxy resin. E ji 1% uranyl acetate (Merck, Darmstadt, Germany) na 1% lead citrate (Merck, Darmstadt, Germany) kwadebe ma tee ya agba. E ji igwe microscope elektrọn nnyefe JEM 2100F EXII (JEOL Ltd, Tokyo, Japan) nweta onyonyo Ultrastructural na voltaji na-eme ka ọ dị ngwa ngwa nke 80 kV.
A nyochachara ọdịdị nke mkpụrụ ndụ LX-2 ejiri IPA gwọọ ruo awa 24 site na iji igwe onyonyo ọdịiche nke usoro na mmụba ugboro 50 site na iji igwe onyonyo ìhè Zeiss inverted (Zeiss Axio Vert.A1 na AxioCam MRm, Jena, Germany).
E gosipụtara data ahụike dị ka nkezi ± nkewa ọkọlọtọ ma ọ bụ etiti (oke dị n'etiti: IQR). E jiri otu ụzọ nyocha nke mgbanwe (ọnọdụ na-aga n'ihu) ma ọ bụ ule χ² (ọnọdụ dị iche iche) tụnyere ọdịiche dị n'etiti otu ọmụmụ ihe atọ ahụ. E jiri ọnụego ụgha dị mma (FDR) dozie maka ọtụtụ nnwale, a na-ewerekwa mkpụrụ ndụ ihe nketa nwere FDR < 0.05 dị ka ihe dị mkpa na ọnụọgụgụ. E jiri nyocha mmekọrịta Spearman jikọta methylation DNA CpG na ike mgbaàmà IPA, yana ụkpụrụ p aha (p < 0.05) kọrọ.
E jiri ngwa nyocha mkpụrụ ndụ ihe nketa dị na weebụ (WebGestalt) mee nyocha ụzọ maka akwụkwọ edemede 268 (aha p < 0.01), akwụkwọ edemede 119 metụtara mitochondria (aha p < 0.05), na 4350 CpGs n'ime akwụkwọ edemede imeju 3093 nke jikọtara ya na ọkwa IPA serum na-agagharị. E jiri ngwa Venny DB (aha 2.1.0) dị n'efu chọta mkpụrụ ndụ ihe nketa ndị na-ekpuchi ibe ha, e jikwa StringDB (aha 11.5) mee ihe iji hụ mmekọrịta protein na protein.
Maka nnwale LX-2, a nwalere ihe nlele maka ihe ndị a na-eme n'otu oge site na iji ule D'Agostino-Pearson. E nwetara data site na opekata mpe ihe atọ e ji mee ihe na bayọlọji ma nye ya ANOVA otu ụzọ site na iji ule Bonferroni mgbe hoc gasịrị. A na-ewere uru p nke na-erughị 0.05 dị ka ihe dị mkpa na ọnụọgụgụ. E gosipụtara data dị ka nkezi ± SD, a na-egosikwa ọnụọgụgụ nnwale na eserese ọ bụla. E mere nyocha na eserese niile site na iji ngwanrọ statistical GraphPad Prism 8 maka Windows (GraphPad Software Inc., ụdị 8.4.3, San Diego, USA).
Nke mbụ, anyị nyochara njikọ dị n'etiti ọkwa IPA serum na imeju, ahụ dum, na akwụkwọ ndekọ mitochondrial. Na mkpokọta profaịlụ akwụkwọ ndekọ, mkpụrụ ndụ ihe nketa kachasị ike jikọtara ya na ọkwa IPA serum bụ MAPKPK3 (FDR = 0.0077; protein kinase-activated mitogen-activated protein kinase-activated protein kinase 3); na profaịlụ akwụkwọ ndekọ mitochondria metụtara, mkpụrụ ndụ ihe nketa kachasị sie ike jikọtara ya bụ AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Faịlụ mgbakwunye 1 na faịlụ mgbakwunye 2).
Anyị nyochachara akwụkwọ ndị e dere n'ụwa niile (n = 268; p < 0.01) na akwụkwọ ndị e dere n'ime mitochondria (n = 119; p < 0.05), wee chọpụta apoptosis dị ka ụzọ kacha mkpa (p = 0.0089). Maka akwụkwọ ndị e dere n'ime mitochondria metụtara ọkwa IPA serum, anyị lebara anya na apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), na ụzọ TNF signaling (FDR = 0.000006) (Foto 1A, Tebụl 2, na Foto Ndị Ọzọ 1A-B).
Nnyocha zuru ụwa ọnụ, nke metụtara mitochondria, na methylation DNA n'ime imeju mmadụ na njikọ ya na ọkwa IPA serum. A na-anọchite anya akwụkwọ 268 zuru ụwa ọnụ, akwụkwọ 119 metụtara mitochondria, na akwụkwọ methylated DNA nke e sere na saịtị CpG 3092 jikọtara ya na ọkwa IPA serum (uru p < 0.01 maka akwụkwọ global na DNA methylated, na uru p < 0.05 maka akwụkwọ mitochondrial). E gosipụtara akwụkwọ global ndị a na-ekpuchi n'etiti (AKT1 na YKT6). B E wuru maapụ mmekọrịta nke mkpụrụ ndụ ihe nketa 13 nwere akara mmekọrịta kachasị elu (0.900) na mkpụrụ ndụ ihe nketa ndị ọzọ site na mkpụrụ ndụ ihe nketa 56 na-ekpuchi (mpaghara ahịrị ojii) nke jikọtara nke ukwuu na ọkwa IPA serum site na iji ngwa ịntanetị StringDB. Akwụkwọ ndụ akwụkwọ ndụ: Mkpụrụ ndụ ihe nketa e sere na akụkụ sel Gene Ontology (GO): mitochondria (GO: 0005739). AKT1 bụ protein nwere akara kachasị elu (0.900) maka mmekọrịta ya na protein ndị ọzọ dabere na data (dabere na nchọta ederede, nnwale, nchekwa data, na ngosipụta ọnụ). Nọdụ netwọk na-anọchite anya protein, na nsọtụ na-anọchite anya njikọ dị n'etiti protein.
Ebe ọ bụ na metabolites microbiota nke eriri afọ nwere ike ịchịkwa ihe mejupụtara epigenetic site na DNA methylation [32], anyị nyochachara ma ọkwa IPA nke serum nwere njikọ na methylation DNA imeju. Anyị chọpụtara na ebe abụọ dị mkpa nke methylation jikọtara ya na ọkwa IPA serum dị nso na mpaghara 3 nke proline-serine-rich (C19orf55) na ezinụlọ protein heat shock ezinụlọ B (obere) onye otu 6 (HSPB6) (Faịlụ ọzọ 3). Methylation DNA nke 4350 CpG (p < 0.01) nwere njikọ na ọkwa IPA serum ma emelitere ya na ụzọ nchịkwa ogologo oge (p = 0.006) (Foto 1A, Tebụl 2, na Foto Mgbakwunye 1C).
Iji ghọta usoro ihe ndị dị ndụ nke na-akpata njikọ dị n'etiti ọkwa IPA serum, akwụkwọ ndị e dere n'ụwa niile, akwụkwọ ndị e dere n'ime mitochondria, na methylation DNA n'ime imeju mmadụ, anyị mere nyocha nke mkpụrụ ndụ ihe nketa ndị a chọpụtara na nyocha ụzọ gara aga (Foto 1A). Nsonaazụ nke nyocha mmụba ụzọ nke mkpụrụ ndụ ihe nketa 56 na-ekpuchi (n'ime ahịrị ojii dị na Foto 1A) gosiri na ụzọ apoptosis (p = 0.00029) gosipụtara mkpụrụ ndụ ihe nketa abụọ a na-ahụkarị na nyocha atọ ahụ: AKT1 na YKT6 (YKT6 v-SNARE homolog), dịka egosiri na eserese Venn (Foto mgbakwunye 2 na Foto 1A). N'ụzọ na-akpali mmasị, anyị chọpụtara na AKT1 (cg19831386) na YKT6 (cg24161647) nwere njikọ dị mma na ọkwa IPA serum (Foto mgbakwunye 3). Iji chọpụta mmekọrịta protein nwere ike ịdị n'etiti ngwaahịa mkpụrụ ndụ ihe nketa, anyị họọrọ mkpụrụ ndụ ihe nketa 13 nwere akara mpaghara kachasị elu (0.900) n'etiti mkpụrụ ndụ ihe nketa 56 na-ekpuchi dị ka ntinye ma wuo maapụ mmekọrịta. Dịka ọkwa ntụkwasị obi si dị (obi ike dị n'akụkụ), mkpụrụ ndụ ihe nketa AKT1 nwere akara kachasị elu (0.900) nọ n'elu (Foto 1B).
Dabere na nyocha ụzọ, anyị chọpụtara na apoptosis bụ ụzọ kachasị mkpa, yabụ anyị nyochachara ma ọgwụgwọ IPA ga-emetụta apoptosis nke HSCs na vitro. Anyị gosiri na mbụ na doses dị iche iche nke IPA (10 μM, 100 μM, na 1 mM) anaghị egbu mkpụrụ ndụ LX-2 [15]. Ọmụmụ ihe a gosiri na ọgwụgwọ IPA na 10 μM na 100 μM mụbara ọnụọgụ nke mkpụrụ ndụ dị ndụ na necrotic. Agbanyeghị, ma e jiri ya tụnyere otu njikwa, ike mkpụrụ ndụ belatara na ntinye IPA 1 mM, ebe ọnụego necrosis sel ka na-agbanweghị agbanwe (Foto 2A, B). Na-esote, iji chọta ntinye kachasị mma iji kpalite apoptosis na mkpụrụ ndụ LX-2, anyị nwalere 10 μM, 100 μM, na 1 mM IPA maka awa 24 (Foto 2A-E na Foto Mgbakwunye 3A-B). N'ụzọ na-akpali mmasị, IPA 10 μM na 100 μM belatara ọnụego apoptosis (%), agbanyeghị, IPA 1 mM mụbara ọnụego apoptosis late na apoptosis (%) ma e jiri ya tụnyere njikwa, ya mere a họọrọ ya maka nnwale ndị ọzọ (Foto 2A–D).
IPA na-akpali apoptosis nke mkpụrụ ndụ LX-2. E jiri usoro Annexin V na 7-AAD double staining mee ihe iji tụọ ọnụego apoptotic na ọdịdị mkpụrụ ndụ site na cytometry flow. E tinyere mkpụrụ ndụ BA na 10 μM, 100 μM na 1 mM IPA n'ime ihe na-enweghị serum ruo awa 24 ma ọ bụ site na F–H TGF-β1 (5 ng/ml) na 1 mM IPA n'ime ihe na-enweghị serum ruo awa 24. A: mkpụrụ ndụ dị ndụ (Annexin V -/ 7AAD-); B: mkpụrụ ndụ necrotic (Annexin V -/ 7AAD+); C, F: mmalite (Annexin V +/ 7AAD-); D, G: late (Annexin V+/7AAD.+); E, H: pasent nke mkpokọta mkpụrụ ndụ apoptotic mbụ na late na ọnụego apoptotic (%). A na-egosipụta data dị ka nkezi ± SD, n = nnwale onwe onye 3. E jiri otu ụzọ ANOVA mee ntụnyere ọnụọgụgụ na ule Bonferroni mgbe hoc gasịrị. *p < 0.05; ****p < 0.0001
Dịka anyị gosiri na mbụ, TGF-β1 5 ng/ml nwere ike ịkpalite ọrụ HSC site n'ịbawanye ngosipụta nke mkpụrụ ndụ ihe nrịbama klasịk [15]. E jiri TGF-β1 5 ng/ml na 1 mM IPA gwọọ mkpụrụ ndụ LX-2 (Foto 2E–H). Ọgwụgwọ TGF-β1 agbanweghị ọnụego apoptosis, agbanyeghị, ọgwụgwọ IPA mụbara oke apoptosis late apoptosis na apoptosis (%) ma e jiri ya tụnyere ọgwụgwọ TGF-β1 (Foto 2E–H). Nsonaazụ ndị a na-egosi na 1 mM IPA nwere ike ịkwalite apoptosis na mkpụrụ ndụ LX-2 n'adabereghị na TGF-β1 induction.
Anyị nyochakwara mmetụta IPA nwere n'iku ume mitochondrial na mkpụrụ ndụ LX-2. Nsonaazụ gosiri na 1 mM IPA belatara oke oriri oxygen (OCR) paramita: iku ume na-abụghị mitochondrial, iku ume basal na maximal, ntapu proton na mmepụta ATP ma e jiri ya tụnyere otu njikwa (Foto 3A, B), ebe ndeksi ahụike bioenergetic (BHI) agbanweghị.
IPA na-ebelata iku ume mitochondrial na mkpụrụ ndụ LX-2. A na-egosi usoro iku ume mitochondrial (OCR) dị ka paramita iku ume mitochondrial (iku ume mitochondrial na-abụghị mitochondrial, iku ume basal, iku ume kachasị elu, ntapu proton, mmepụta ATP, SRC na BHI). E tinyere 10 μM, 100 μM na 1 mM IPA n'ime mkpụrụ ndụ A na B n'ime awa 24, n'otu n'otu. E tinyere TGF-β1 (5 ng/ml) na 1 mM IPA n'ime mkpụrụ ndụ C na D n'ime usoro enweghị serum ruo awa 24, n'otu n'otu. E mere ka nha niile dabara na ọdịnaya DNA site na iji kit CyQuant. BHI: bioenergetic health index; SRC: ikike nchekwa iku ume; OCR: ọnụego oriri oxygen. E gosipụtara data dị ka nkezi ± ọkọlọtọ diversion (SD), n = nnwale ndị nọọrọ onwe ha 5. E mere ntụnyere ọnụ ọgụgụ site na iji otu ụzọ ANOVA na Bonferroni post hoc test. *p < 0.05; **p < 0.01; na ***p < 0.001
Iji nweta nghọta zuru oke gbasara mmetụta IPA na profaịlụ bioenergetic nke mkpụrụ ndụ LX-2 TGF-β1-activated, anyị nyochara mitochondrial oxidative phosphorylation site na OCR (Fig. 3C,D). Nsonaazụ gosiri na ọgwụgwọ TGF-β1 nwere ike ibelata oke iku ume, ikike nchekwa iku ume (SRC) na BHI ma e jiri ya tụnyere otu njikwa (Fig. 3C,D). Na mgbakwunye, ọgwụgwọ njikọta ahụ belatara iku ume basal, ntapu proton na mmepụta ATP, mana SRC na BHI dị elu karịa nke ejiri TGF-β1 gwọọ (Fig. 3C,D).
Anyị mekwara "Ule Mkpụrụ ndụ Ike Cellular" nke ngwanrọ Seahorse nyere (Foto mgbakwunye 4A–D). Dịka egosiri na Foto mgbakwunye 3B, ma ikike OCR na ECAR metabolic potentials belatara mgbe ọgwụgwọ TGF-β1 gasịrị, agbanyeghị, ọ dịghị ihe dị iche na njikọta na otu ọgwụgwọ IPA ma e jiri ya tụnyere otu njikwa. Ọzọkwa, ma ọkwa basal na nrụgide nke OCR belatara mgbe njikọta na ọgwụgwọ IPA ma e jiri ya tụnyere otu njikwa (Foto mgbakwunye 4C). N'ụzọ na-adọrọ mmasị, a hụrụ usoro yiri nke ahụ na ọgwụgwọ njikọta, ebe a hụghị mgbanwe na ọkwa basal na nrụgide nke ECAR ma e jiri ya tụnyere ọgwụgwọ TGF-β1 (Foto mgbakwunye 4C). Na HSCs, mbelata na mitochondrial oxidative phosphorylation na ikike nke ọgwụgwọ njikọta iji weghachi SCR na BHI mgbe ekpughere ya na ọgwụgwọ TGF-β1 agbanweghị ikike metabolic (OCR na ECAR). N'ịchịkọta ha, nsonaazụ ndị a na-egosi na IPA nwere ike ibelata bioenergetics na HSCs, na-atụ aro na IPA nwere ike ịkpalite obere profaịlụ ike nke na-agbanwe ụdị HSC gaa na enweghị ọrụ (Foto mgbakwunye 4D).
E jiri ọnụọgụ atọ nke ụdị mitochondrial morphology na njikọ netwọk yana MTR staining nyochaa mmetụta nke IPA na dynamics mitochondrial (Foto 4 na Foto Mgbakwunye 5). Foto 4 na-egosi na, ma e jiri ya tụnyere otu njikwa, ọgwụgwọ TGF-β1 belatara mpaghara elu nkezi, ọnụọgụ alaka, ogologo alaka zuru oke, na nọmba njikọ alaka (Foto 4A na B) ma gbanwee oke nke mitochondria site na ụdị gburugburu ruo na ụdị etiti (Foto 4C). Naanị ọgwụgwọ IPA belatara olu mitochondrial nkezi ma gbanwee oke nke mitochondria site na ụdị gburugburu ruo na ụdị etiti ma e jiri ya tụnyere otu njikwa (Foto 4A). N'ụzọ dị iche, sphericity, ogologo alaka nkezi, na ọrụ mitochondrial nke a tụlere site na MTR nke mitochondrial potential-dependent MTR (Foto 4A na E) ka dị iche, paramita ndị a adịghịkwa iche n'etiti otu. A na-ejikọta ha ọnụ, nsonaazụ ndị a na-egosi na ọgwụgwọ TGF-β1 na IPA yiri ka ọ na-agbanwe ọdịdị na nha mitochondrial yana mgbagwoju anya netwọk na mkpụrụ ndụ LX-2 dị ndụ.
IPA na-agbanwe usoro mitochondrial na oke DNA mitochondrial na mkpụrụ ndụ LX-2. A. Foto confocal ndị a na-anọchite anya nke mkpụrụ ndụ LX-2 dị ndụ nke ejiri TGF-β1 (5 ng/ml) na 1 mM IPA mee ihe ruo awa 24 n'ime ihe na-enweghị serum na-egosi netwọk mitochondrial nke ejiri Mitotracker™ Red CMXRos na nuclei acha anụnụ anụnụ mee ihe na DAPI. Data niile nwere opekata mpe onyonyo 15 kwa otu. Anyị nwetara onyonyo Z-stack 10 maka ụdị nlele ọ bụla. Usoro Z-axis ọ bụla nwere mpekere 30, nke ọ bụla nwere ọkpụrụkpụ nke 9.86 μm. Ogwe nha: 10 μm. B. Ihe nnọchi anya (mitochondria naanị) achọpụtara site na itinye oke mgbanwe na onyonyo ahụ. E mere nyocha ọnụọgụgụ na ntụnyere nke njikọ netwọkụ morphological mitochondrial maka mkpụrụ ndụ niile dị na otu ọ bụla. C. Ugboro nke oke ọdịdị mitochondrial. Uru dị nso na 0 na-egosi ọdịdị gburugburu, ụkpụrụ dị nso na 1 na-egosi ọdịdị filamentous. A chọpụtara ọdịnaya nke DNA Mitochondrial (mtDNA) D dịka akọwara na ihe ndị dị na ya na usoro na ụzọ. E mere nyocha E Mitotracker™ Red CMXRos site na iji usoro cytometry (ihe omume 30,000) dịka akọwara na ihe ndị dị na usoro na ụzọ. E gosipụtara data dị ka nkezi ± SD, n = nnwale ndị nọọrọ onwe ha atọ. E mere ntụnyere ọnụọgụgụ site na iji otu ụzọ ANOVA na Bonferroni mgbe ule hoc gasịrị. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Anyị nyochachara ọdịnaya mtDNA dị na mkpụrụ ndụ LX-2 dị ka ihe ngosi nke ọnụọgụ mitochondrial. Ma e jiri ya tụnyere otu njikwa, ọnụọgụ mtDNA mụbara na otu ejiri TGF-β1 gwọọ (Foto 4D). Ma e jiri ya tụnyere otu ejiri TGF-β1 gwọọ, ọnụọgụ mtDNA belatara na otu ọgwụgwọ njikọta (Foto 4D), na-egosi na IPA nwere ike ibelata ọdịnaya mtDNA na ikekwe ọnụọgụ mitochondrial yana iku ume mitochondrial (Foto 3C). Ọzọkwa, IPA yiri ka ọ na-ebelata ọdịnaya mtDNA na ọgwụgwọ njikọta mana ọ metụtaghị ọrụ mitochondrial nke MTR na-akwalite (Foto 4A-C).
Anyị nyochachara njikọ IPA na ọkwa mRNA nke mkpụrụ ndụ ihe nketa jikọtara ya na fibrosis, apoptosis, ndụ, na mitochondrial dynamics na mkpụrụ ndụ LX-2 (Foto 5A–D). Ma e jiri ya tụnyere otu njikwa, otu a gwọrọ TGF-β1 gosipụtara mmụba nke ngosipụta nke mkpụrụ ndụ ihe nketa dịka collagen type I α2 yinye (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), ihe mgbochi anụ ahụ nke metalloproteinase 1 (TIMP1), na dynamin 1-like gene (DRP1), na-egosi mmụba nke fibrosis na mkpali. Ọzọkwa, ma e jiri ya tụnyere otu njikwa, ọgwụgwọ TGF-β1 belatara ọkwa mRNA nke ihe nnabata X pregnane nuklia (PXR), caspase 8 (CASP8), MAPKPK3, ihe na-egbochi B-cell α, ihe na-eme ka nuclear factor κ gene light peptide (NFκB1A), na ihe na-egbochi nuclear factor κB kinase subunit β (IKBKB) (Foto 5A–D). Ma e jiri ya tụnyere ọgwụgwọ TGF-β1, ọgwụgwọ njikọta na TGF-β1 na IPA belatara ngosipụta nke COL1A2 na MMP2, mana mụbara ọkwa mRNA nke PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, na IKBKB. Ọgwụgwọ IPA belatara ngosipụta nke MMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), na mitochondrial fusion protein 2 (MFN2), ebe ngosipụta nke CASP8, NFκB1A, NFκB1B, na IKBKB mụbara ma e jiri ya tụnyere otu njikwa. Agbanyeghị, achọpụtaghị ọdịiche ọ bụla na ngosipụta nke caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), na fission protein 1 (FIS1). N'ozuzu, nsonaazụ ndị a na-egosi na ọgwụgwọ IPA na-agbanwe ngosipụta nke mkpụrụ ndụ ihe nketa metụtara fibrosis, apoptosis, survival, na mitochondrial dynamics. Data anyị na-egosi na ọgwụgwọ IPA na-ebelata fibrosis na mkpụrụ ndụ LX-2; n'otu oge ahụ, ọ na-akpali ndụ site na ịgbanwe ụdị ahụ gaa na enweghị ọrụ.
IPA na-agbanwe ngosipụta nke mkpụrụ ndụ ihe nketa fibroblast, apoptotic, viability, na mitochondrial dynamics na mkpụrụ ndụ LX-2. Histograms na-egosipụta ngosipụta mRNA ma e jiri ya tụnyere njikwa endogenous (RPLP0 ma ọ bụ PPIA) mgbe ejiri TGF-β1 na IPA kpalite mkpụrụ ndụ LX-2 n'ime ihe na-enweghị serum ruo awa 24. A na-egosi fibroblast, B na-egosi mkpụrụ ndụ apoptotic, C na-egosi mkpụrụ ndụ ndị dị ndụ, na D na-egosi ngosipụta mkpụrụ ndụ ihe nketa mitochondrial dynamics. A na-egosi data dị ka nkezi ± ọkọlọtọ divideration (SD), n = nnwale onwe onye 3. E mere ntụnyere ọnụ ọgụgụ site na iji otu ụzọ ANOVA na Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Mgbe ahụ, e jiri usoro cytometry flow nyochaa mgbanwe dị na nha mkpụrụ ndụ (FSC-H) na mgbagwoju anya cytoplasmic (SSC-H) (Foto 6A,B), a tụlekwara mgbanwe dị na ọdịdị mkpụrụ ndụ mgbe ọgwụgwọ IPA gasịrị site na iji transmission electron microscopy (TEM) na phase contrast microscopy (Foto Mgbakwunye 6A-B). Dịka a tụrụ anya ya, mkpụrụ ndụ dị na otu a gwọrọ TGF-β1 mụbara na nha ma e jiri ya tụnyere otu njikwa (Foto 6A,B), na-egosi mgbasawanye ochie nke rough endoplasmic reticulum (ER*) na phagolysosomes (P), na-egosi ọrụ hematopoietic stem cell (HSC) (Foto Mgbakwunye 6A). Agbanyeghị, ma e jiri ya tụnyere otu a gwọrọ TGF-β1, nha mkpụrụ ndụ, mgbagwoju anya cytoplasmic (Foto 6A,B), na ọdịnaya ER* belatara na otu ọgwụgwọ njikọta TGF-β1 na IPA (Foto Mgbakwunye 6A). Ọzọkwa, ọgwụgwọ IPA belatara nha mkpụrụ ndụ, mgbagwoju anya cytoplasmic (Figs. 6A,B), ọdịnaya P na ER* (Fig. 6A mgbakwunye) ma e jiri ya tụnyere otu njikwa. Na mgbakwunye, ọdịnaya nke mkpụrụ ndụ apoptotic mụbara mgbe awa 24 nke ọgwụgwọ IPA gasịrị ma e jiri ya tụnyere otu njikwa (akụ ọcha, Fig. 6B). N'ozuzu, nsonaazụ ndị a na-egosi na 1 mM IPA nwere ike ịkpalite apoptosis HSC ma gbanwee mgbanwe na paramita morphological cell nke TGF-β1 kpatara, si otú a na-achịkwa nha na mgbagwoju anya nke mkpụrụ ndụ, nke nwere ike ijikọ ya na mmechi HSC.
IPA na-agbanwe nha mkpụrụ ndụ na mgbagwoju anya cytoplasmic na mkpụrụ ndụ LX-2. Foto ndị nnọchiteanya nke nyocha cytometry mmiri. Nyocha ahụ jiri usoro nhazi nke akọwapụtara maka mkpụrụ ndụ LX-2: SSC-A/FSC-A iji kọwaa ọnụọgụgụ mkpụrụ ndụ, FSC-H/FSC-A iji chọpụta doublets, na SSC-H/FSC-H maka nha mkpụrụ ndụ na nyocha mgbagwoju anya. E tinyere TGF-β1 (5 ng/ml) na 1 mM IPA n'ime sel n'enweghị serum ruo awa 24. E kesara mkpụrụ ndụ LX-2 n'ime quadrant aka ekpe dị ala (SSC-H-/FSC-H-), quadrant aka ekpe dị elu (SSC-H+/FSC-H-), quadrant aka nri dị ala (SSC-H-/FSC-H+), na quadrant aka nri elu (SSC-H+/FSC-H+) maka nha mkpụrụ ndụ na nyocha mgbagwoju anya cytoplasmic. B. E jiri FSC-H (ịgbasa n'ihu, nha mkpụrụ ndụ) na SSC-H (ịgbasa n'akụkụ, mgbagwoju anya cytoplasmic) (ihe omume 30,000) nyochaa ọdịdị mkpụrụ ndụ. E gosipụtara data dị ka nkezi ± SD, n = nnwale atọ nọọrọ onwe ha. E jiri otu ụzọ ANOVA na Bonferroni mee ntụnyere ọnụọgụgụ. *p < 0.05; **p < 0.01; ***p < 0.001 na ****p < 0.0001
Metabolites afọ dịka IPA aghọọla isiokwu a na-eme nnyocha nke ọma, na-egosi na enwere ike ịchọta ihe ọhụrụ na microbiota eriri afọ. Ya mere, ọ na-atọ ụtọ na egosila na IPA, metabolite anyị jikọtara ya na fibrosis imeju n'ime mmadụ [15], bụ ihe nwere ike igbochi fibrotic n'ụdị anụmanụ [13, 14]. N'ebe a, anyị na-egosi maka oge mbụ njikọ dị n'etiti IPA serum na transcriptomics imeju zuru ụwa ọnụ na DNA methylation na ndị buru ibu na-enweghị ụdị ọrịa shuga nke abụọ (T2D), na-egosipụta apoptosis, mitophagy na ogologo ndụ, yana mkpụrụ ndụ ihe nketa AKT1 nwere ike ịchịkwa homeostasis imeju. Ihe ọhụrụ ọzọ nke ọmụmụ anyị bụ na anyị gosipụtara mmekọrịta nke ọgwụgwọ IPA na apoptosis, morphology sel, mitochondrial bioenergetics na dynamics na mkpụrụ ndụ LX-2, na-egosi obere ike spectrum nke na-agbanwe phenotype HSC gaa na inactivation, na-eme IPA onye nwere ike imeziwanye fibrosis imeju.
Anyị chọpụtara na apoptosis, mitophagy na ogologo ndụ bụ ụzọ kacha mkpa dị mkpa nke mkpụrụ ndụ ihe nketa imeju nke jikọtara ya na IPA serum na-agagharị. Mmebi nke sistemụ njikwa mma mitochondrial (MQC) nwere ike ibute nsogbu mitochondrial, mitophagy na apoptosis, si otú a na-akwalite mmepe nke MASLD[33, 34]. Ya mere, anyị nwere ike iche na IPA nwere ike itinye aka na ịnọgide na-enwe usoro mkpụrụ ndụ na iguzosi ike n'ezi ihe mitochondrial site na apoptosis, mitophagy na ogologo ndụ n'ime imeju. Data anyị gosiri na mkpụrụ ndụ ihe nketa abụọ bụ ihe a na-ahụkarị n'ime nnwale atọ ahụ: YKT6 na AKT1. Ọ dị mkpa ịmara na YKT6 bụ protein SNARE nke na-etinye aka na usoro nke njikọta akpụkpọ ahụ sel. Ọ na-arụ ọrụ na autophagy na mitophagy site na ịmepụta mgbagwoju anya mmalite na STX17 na SNAP29 na autophagosome, si otú a na-akwalite njikọta nke autophagosomes na lysosomes[35]. Ọzọkwa, mfu nke ọrụ YKT6 na-ebute nsogbu nke mitophagy [36], ebe mmụba nke YKT6 na-ejikọta ya na ọganihu nke ọrịa kansa hepatocellular (HCC), na-egosi mmụba nke ndụ sel [37]. N'aka nke ọzọ, AKT1 bụ mkpụrụ ndụ ihe nketa kachasị mkpa na-akpakọrịta ma na-arụ ọrụ dị mkpa na ọrịa imeju, gụnyere ụzọ mgbaàmà PI3K/AKT, okirikiri sel, mbugharị sel, mmụba, njikọta focal, ọrụ mitochondrial, na mwepụ collagen [38-40]. Ụzọ mgbaàmà PI3K/AKT arụ ọrụ nwere ike ime ka mkpụrụ ndụ hematopoietic stem (HSCs) rụọ ọrụ, nke bụ mkpụrụ ndụ na-ahụ maka mmepụta nke extracellular matrix (ECM), na dysregulation ya nwere ike itinye aka na mmepe na ọganihu nke fibrosis imeju [40]. Na mgbakwunye, AKT bụ otu n'ime ihe ndị bụ isi na-azọpụta sel nke na-egbochi apoptosis sel p53, yana mmụba AKT nwere ike ijikọ ya na mgbochi nke apoptosis sel imeju [41, 42]. Nsonaazụ enwetara na-egosi na IPA nwere ike itinye aka na apoptosis nke metụtara mitochondria imeju site na imetụta mkpebi nke hepatocytes n'etiti ịbanye na apoptosis ma ọ bụ ndụ. Enwere ike ịchịkwa mmetụta ndị a site na mkpụrụ ndụ ihe nketa AKT na/ma ọ bụ YKT6, nke dị oke mkpa maka homeostasis imeju.
Nsonaazụ anyị gosiri na 1 mM IPA butere apoptosis ma belata iku ume mitochondrial n'ime mkpụrụ ndụ LX-2 na-adabereghị na ọgwụgwọ TGF-β1. Ọ dị mkpa ịmara na apoptosis bụ ụzọ dị mkpa maka mkpebi fibrosis na mkpali mkpụrụ ndụ hematopoietic (HSC), ọ bụkwa ihe dị mkpa na nzaghachi physiological nke fibrosis imeju [4, 43]. Ọzọkwa, mweghachi nke BHI na mkpụrụ ndụ LX-2 mgbe ọgwụgwọ jikọtara ọnụ nyere nghọta ọhụrụ banyere ọrụ IPA nwere ike ịrụ na nhazi nke bioenergetics mitochondrial. N'okpuru ọnọdụ izu ike na enweghị ọrụ, mkpụrụ ndụ hematopoietic na-ejikarị mitochondrial oxidative phosphorylation emepụta ATP ma nwee obere ọrụ metabolic. N'aka nke ọzọ, mmegharị HSC na-eme ka iku ume mitochondrial na biosynthesis dịkwuo mma iji kwụọ ụgwọ ike nke ịbanye na steeti glycolytic [44]. Eziokwu ahụ bụ na IPA emetụtaghị ikike metabolic na ECAR na-egosi na ụzọ glycolytic adịghị mkpa. N'otu aka ahụ, nnyocha ọzọ gosiri na 1 mM IPA nwere ike ịgbanwe ọrụ mitochondrial respiratory yinye na cardiomyocytes, mkpụrụ ndụ hepatocyte mmadụ (Huh7) na mkpụrụ ndụ endothelial vein umbilical (HUVEC); Agbanyeghị, ahụghị mmetụta IPA na glycolysis na cardiomyocytes, na-atụ aro na IPA nwere ike imetụta bioenergetics nke ụdị mkpụrụ ndụ ndị ọzọ [45]. Ya mere, anyị na-eche na 1 mM IPA nwere ike ịrụ ọrụ dị ka ihe na-adịghị mma na kemịkalụ, ebe ọ nwere ike ibelata ngosipụta mkpụrụ ndụ fibrogenic nke ukwuu, ọdịdị mkpụrụ ndụ na bioenergetics mitochondrial na-enweghị mgbanwe nke mtDNA [46]. Ndị na-anaghị ejikọta Mitochondrial nwere ike igbochi fibrosis nke ọdịbendị kpatara na mkpali HSC [47] ma belata mmepụta ATP mitochondrial nke ụfọdụ protein dị ka uncoupling protein (UCP) ma ọ bụ adenine nucleotide translocase (ANT) na-achịkwa ma ọ bụ na-akpali. Dabere na ụdị mkpụrụ ndụ, ihe a nwere ike ichebe mkpụrụ ndụ site na apoptosis na/ma ọ bụ kwalite apoptosis [46]. Agbanyeghị, a chọrọ ọmụmụ ihe ndị ọzọ iji kọwaa ọrụ IPA dị ka ihe na-egbochi njikọ mitochondrial na mmechi nke mkpụrụ ndụ hematopoietic.
Anyị mechara nyochaa ma mgbanwe ndị dị na iku ume mitochondrial na-apụta ìhè na ọdịdị mitochondrial na mkpụrụ ndụ LX-2 dị ndụ. N'ụzọ na-adọrọ mmasị, ọgwụgwọ TGF-β1 na-agbanwe oke mitochondrial site na gburugburu ruo etiti, yana mbelata alaka mitochondrial na mmụba ngosipụta nke DRP1, ihe dị mkpa na mitochondrial fission [48]. Ọzọkwa, nkewa mitochondrial na-ejikọta ya na mgbagwoju anya netwọk zuru oke, mgbanwe site na njikọ gaa na fission dị oke mkpa maka mkpali sel hematopoietic (HSC), ebe mgbochi nke fission mitochondrial na-eduga na apoptosis HSC [49]. Ya mere, nsonaazụ anyị na-egosi na ọgwụgwọ TGF-β1 nwere ike ibute mbelata na mgbagwoju anya netwọk mitochondrial yana mbelata alaka, nke a na-ahụkarị na fission mitochondrial jikọtara ya na mkpụrụ ndụ hematopoietic activated (HSCs). Ọzọkwa, data anyị gosiri na IPA nwere ike ịgbanwe oke nke mitochondria site na ọdịdị gburugburu ruo etiti, si otú a belata ngosipụta nke OPA1 na MFN2. Ọmụmụ ihe egosila na mbelata nke OPA1 nwere ike ibute mbelata na ikike membrane mitochondrial ma kpalite apoptosis sel [50]. A maara MFN2 dị ka ihe na-akwalite njikọ mitochondrial na apoptosis[51]. Nsonaazụ enwetara na-egosi na TGF-β1 na/ma ọ bụ IPA na-akpali mkpụrụ ndụ LX-2 dịka ọ na-agbanwe ọdịdị na nha mitochondrial, yana ọnọdụ mkpali na mgbagwoju anya netwọk.
Nsonaazụ anyị gosiri na ọgwụgwọ TGFβ-1 na IPA nwere ike ibelata mtDNA na paramita morphological nke mkpụrụ ndụ site na ịchịkwa ngosipụta mRNA nke fibrosis, apoptosis na mkpụrụ ndụ ihe nketa metụtara ndụ na mkpụrụ ndụ ndị na-ezere apoptosis. N'ezie, IPA belatara ọkwa ngosipụta mRNA nke AKT1 na mkpụrụ ndụ ihe nketa fibrosis dị mkpa dịka COL1A2 na MMP2, mana mụbara ọkwa ngosipụta nke CASP8, nke jikọtara ya na apoptosis. Nsonaazụ anyị gosiri na mgbe ọgwụgwọ IPA gasịrị, ngosipụta BAX belatara ma ngosipụta mRNA nke mkpụrụ ndụ ezinụlọ TIMP1, BCL-2 na NF-κB mụbara, na-egosi na IPA nwere ike ịkpalite akara ndụ na mkpụrụ ndụ ihe nketa hematopoietic (HSCs) nke na-ezere apoptosis. Mkpụrụ ndụ ndị a nwere ike ịrụ ọrụ dị ka akara pro-lanarị na mkpụrụ ndụ ihe nketa hematopoietic arụ ọrụ, nke nwere ike ijikọ ya na mmụba ngosipụta nke protein anti-apoptotic (dị ka Bcl-2), mbelata ngosipụta nke pro-apoptotic BAX, na mmekọrịta dị mgbagwoju anya n'etiti TIMP na NF-κB [5, 7]. IPA na-arụ ọrụ site na PXR, anyị chọpụtakwara na ọgwụgwọ njikọta na TGF-β1 na IPA mụbara ọkwa ngosipụta PXR mRNA, na-egosi mgbochi nke mkpali HSC. A maara na mgbaàmà PXR arụ ọrụ na-egbochi mmegharị HSC ma na vivo na in vitro [52, 53]. Nsonaazụ anyị na-egosi na IPA nwere ike isonye na mwepụ nke HSCs arụ ọrụ site na ịkwalite apoptosis, ibelata fibrosis na metabolism mitochondrial, na ime ka akara ndụ dịkwuo mma, nke bụ usoro nkịtị nke na-agbanwe ụdị HSC arụ ọrụ ka ọ bụrụ nke na-anaghị arụ ọrụ. Nkọwa ọzọ enwere ike maka usoro na ọrụ IPA nwere ike ịrụ na apoptosis bụ na ọ na-ewepụ mitochondria na-adịghị arụ ọrụ nke ọma site na mitophagy (ụzọ dị n'ime) na ụzọ mgbaàmà TNF mpụga (Tebụl 1), nke ejikọtara kpọmkwem na ụzọ mgbaàmà ndụ NF-κB (Foto mgbakwunye 7). N'ụzọ na-adọrọ mmasị, mkpụrụ ndụ ihe nketa ndị metụtara IPA nwere ike ịkpalite ihe mgbaàmà pro-apoptotic na pro-laventory n'ụzọ apoptotic [54], na-atụ aro na IPA nwere ike ịkpalite ụzọ apoptotic ma ọ bụ ndụ site na mmekọrịta na mkpụrụ ndụ ihe nketa ndị a. Agbanyeghị, otu IPA si akpalite apoptosis ma ọ bụ ndụ n'oge ọrụ HSC na ụzọ usoro ya ka edoghị anya.
IPA bụ ihe na-eme ka nje bacteria dị na ya pụta site na nri tryptophan site na microbiota eriri afọ. Ọmụmụ ihe egosila na o nwere ihe mgbochi mkpali, antioxidant, na epigenetic na gburugburu eriri afọ.[55] Ọmụmụ ihe egosila na IPA nwere ike ịgbanwe ọrụ mgbochi eriri afọ ma belata nrụgide oxidative, nke nwere ike itinye aka na mmetụta anụ ahụ mpaghara ya.[56] N'ezie, a na-ebuga IPA gaa n'akụkụ ahụ ndị a na-elekwasị anya site na mgbasa ozi, ebe ọ bụ na IPA nwere nnukwu usoro metabolite yiri nke ahụ na tryptophan, serotonin, na indole derivatives, IPA na-eme ihe omume metabolic nke na-ebute akara aka metabolic asọmpi.[52] IPA nwere ike ịsọ mpi na metabolites sitere na tryptophan maka ebe ejikọtara na enzymes ma ọ bụ ndị nnata, nke nwere ike imebi ụzọ metabolic nkịtị. Nke a na-egosi mkpa ọ dị maka ọmụmụ ihe ndị ọzọ na pharmacokinetics na pharmacodynamics ya iji ghọta windo ọgwụgwọ ya nke ọma.[57] A ka ga-ahụ ma nke a nwekwara ike ime na mkpụrụ ndụ hematopoietic stem (HSCs).
Anyị kwetara na ọmụmụ ihe anyị nwere ụfọdụ oke. Iji nyochaa njikọ metụtara IPA kpọmkwem, anyị wepụrụ ndị ọrịa nwere ụdị ọrịa shuga nke abụọ (T2DM). Anyị kwetara na nke a na-egbochi ojiji sara mbara nke nchọpụta anyị nye ndị ọrịa nwere ụdị ọrịa shuga nke abụọ na ọrịa imeju dị elu. Ọ bụ ezie na ntinye ahụ ike nke IPA na serum mmadụ bụ 1-10 μM [11, 20], a họọrọ ntinye ahụ nke 1 mM IPA dabere na ntinye kachasị elu na-adịghị egbu egbu [15] na ọnụego kachasị elu nke apoptosis, na-enweghị ọdịiche na pasent nke ọnụọgụgụ mkpụrụ ndụ necrotic. Ọ bụ ezie na ejiri ọkwa supraphysiological nke IPA mee ihe na ọmụmụ ihe a, ugbu a enweghị nkwekọrịta gbasara dose dị irè nke IPA [52]. Ọ bụ ezie na nsonaazụ anyị dị mkpa, oke metabolic sara mbara nke IPA ka bụ mpaghara nyocha na-arụ ọrụ. Ọzọkwa, enwetara nchọpụta anyị na njikọ dị n'etiti ọkwa IPA serum na DNA methylation nke transcripts imeju ọ bụghị naanị site na mkpụrụ ndụ hematopoietic stem (HSCs) kamakwa site na anụ ahụ imeju. Anyị họọrọ iji mkpụrụ ndụ LX-2 mmadụ dabere na nchọpụta anyị gara aga site na nyocha transcriptome na IPA na-ejikọta ya na mkpali nke mkpụrụ ndụ hematopoietic (HSC) [15], HSC bụkwa mkpụrụ ndụ bụ isi na-etinye aka na mmepe nke fibrosis imeju. Imeju nwere ọtụtụ ụdị mkpụrụ ndụ, yabụ a ga-atụle ụdị mkpụrụ ndụ ndị ọzọ dị ka sistemụ hepatocyte-HSC-immune cell co-culture yana mkpali caspase na nkewa DNA yana usoro ọrụ gụnyere ọkwa protein iji mụọ ọrụ nke IPA na mmekọrịta ya na ụdị mkpụrụ ndụ imeju ndị ọzọ.