I kōrerotia e mātou i mua ko ngā taumata toto o te metabolite tryptophan i ahu mai i te puku indole-3-propionic acid (IPA) he iti ake i roto i ngā tūroro e pāngia ana e te fibrosis ate. I roto i tēnei rangahau, i tirotirohia e mātou te transcriptome me te DNA methylome i roto i ngā ate momona e pā ana ki ngā taumata IPA toto, me te mahi a te IPA ki te whakaoho i te whakakorenga phenotypic o ngā pūtau stellate ate (HSC) i roto i te vitro.
I whakaurua ki te rangahau he 116 ngā tūroro momona kāore he mate huka momo 2 (T2DM) (tau 46.8 ± 9.3 tau; BMI: 42.7 ± 5.0 kg/m²) i pokangahia ki te pokanga bariatric i te Kuopio Bariatric Surgery Center (KOBS). I inehia ngā taumata IPA e rere ana mā te chromatography wai-spectrometry papatipu (LC-MS), i mahia te tātaritanga transcriptome ate mā te raupapatanga RNA katoa, ā, i mahia te tātaritanga methylation DNA mā te whakamahi i te Infinium HumanMethylation450 BeadChip. I whakamahia ngā pūtau stellate ate tangata (LX-2) mō ngā whakamātautau in vitro.
I hono ngā taumata IPA toto ki te whakaaturanga o ngā ira e whai wāhi ana ki ngā ara apoptotic, mitophagic, me te roa o te ora i roto i te ate. Ko te ira AKT serine/threonine kinase 1 (AKT1) te ira taunekeneke tino nui me te tino kaha i roto i te tuhinga ate me ngā kōtaha methylation DNA. Nā te maimoatanga IPA i whakaoho te apoptosis, i whakaiti i te manawa mitochondrial, ā, i whakarerekē i te āhua o te pūtau me te hihiri mitochondrial mā te whakarerekē i te whakaaturanga o ngā ira e mōhiotia ana hei whakahaere i te fibrosis, te apoptosis, me te oranga o ngā pūtau LX-2.
Ina huihuia ēnei raraunga, e tautoko ana te IPA i ngā pānga rongoā pea, ā, ka taea e ia te whakaoho i te apoptosis me te neke i te momo HSC ki te āhua kore mahi, ā, ka whānui ake te āheinga ki te aukati i te fibrosis ate mā te whakararuraru i te whakahohenga HSC me te pākia o te mitochondrial.
Kua honoa te horapa o te momona me te mate pūmua ki te pikinga ake o te mate ate momona e pā ana ki te pūmua (MASLD); ka pāngia e te mate te 25% ki te 30% o te taupori whānui [1]. Ko te hua matua o te MASLD ko te fibrosis ate, he tukanga hihiri e tohuhia ana e te kohikohi tonu o te matrix extracellular fibrous (ECM) [2]. Ko ngā pūtau matua e uru ana ki te fibrosis ate ko ngā pūtau stellate ate (HSC), e whakaatu ana i ngā momo āhua e whā e mōhiotia ana: te ata noho, te whakahohe, te kore whakahohe, me te koroheke [3, 4]. Ka taea te whakahohe i ngā HSC me te whakawhiti i te rerekētanga mai i te āhua ata noho ki ngā pūtau rite ki te fibroblast e tipu haere ana me te nui o te hiahia pūngao, me te pikinga ake o te whakaaturanga o te α-smooth muscle actin (α-SMA) me te momo I collagen (Col-I) [5, 6]. I te wā o te whakahurihanga o te fibrosis ate, ka whakakorea ngā HSC whakahohe mā te apoptosis, te kore whakahohe rānei. Kei roto i ēnei tukanga te whakahekenga i ngā ira fibrogenic me te whakarerekētanga o ngā ira prosurvival (pērā i ngā ara tohu NF-κB me PI3K/Akt) [7, 8], tae atu ki ngā huringa o ngā mahi me ngā mahi mitochondrial [9].
Kua kitea kua heke ngā taumata toto o te matū tryptophan indole-3-propionic acid (IPA), i hangaia i roto i te whekau, i roto i ngā mate pākia tangata tae atu ki te MASLD [10–13]. E hono ana te IPA ki te kai muka kai, e mōhiotia ana mō ōna pānga antioxidant me te anti-inflammatory, ā, ka whakaiti i te momo mate steatohepatitis kore-waipiro (NASH) i puta mai i te kai i roto i te vivo me te in vitro [11–14]. Ko ētahi taunakitanga i ahu mai i tā mātou rangahau o mua, i whakaatu he iti ake ngā taumata IPA toto i roto i ngā tūroro he fibrosis ate i ngā tūroro momona kāore he fibrosis ate i roto i te Kuopio Bariatric Surgery Study (KOBS). I tua atu, i whakaaturia e mātou ka taea e te maimoatanga IPA te whakaiti i te whakaaturanga o ngā ira he tohu tawhito o te piri o te pūtau, te hekenga o te pūtau me te whakahohenga o te pūtau take hematopoietic i roto i te tauira pūtau stellate ate tangata (LX-2) ā, he matū tiaki hepato pea [15]. Heoi, kāore anō kia mārama me pēhea te whakaoho a te IPA i te hokinga mai o te fibrosis ate mā te whakahohe i te apoptosis HSC me te bioenergetics mitochondrial.
I konei, ka whakaatuhia e mātou he hononga te IPA toto ki te whakaaturanga o ngā ira e whai kiko ana ki te apoptosis, te mitophagy, me ngā ara roa o te ora i roto i te ate o te hunga momona engari ehara i te mate huka momo 2 (KOBS). I tua atu, i kitea e mātou ka taea e te IPA te whakaoho i te whakakorenga me te whakahekenga o ngā pūtau take hematopoietic (HSC) kua whakahohehia mā te ara whakakorenga. E whakaatu ana ēnei hua i tētahi mahi hou mō te IPA, ka waiho hei whāinga rongoā pea hei whakatairanga i te hokinga mai o te fibrosis ate.
I whakaatuhia i roto i tētahi rangahau o mua i roto i te rōpū KOBS he iti ake ngā taumata IPA tohanga toto o ngā tūroro he mate pukupuku ate ki te whakataurite ki ngā tūroro kāore he mate pukupuku ate [15]. Hei whakakore i te pānga whakararuraru pea o te mate huka momo 2, i tohua e mātou he 116 ngā tūroro momona kāore he mate huka momo 2 (tau toharite ± SD: 46.8 ± 9.3 tau; BMI: 42.7 ± 5.0 kg/m2) (Ripanga 1) mai i te rangahau KOBS e haere tonu ana hei taupori rangahau [16]. I tukuna e ngā kaiuru katoa he whakaaetanga ā-tuhi, ā, i whakaaetia te kawa rangahau e te Komiti Matatika o te Hōhipera o North Savo County i runga anō i te Whakapuakitanga o Helsinki (54/2005, 104/2008 me 27/2010).
I tangohia ngā tauira koiora ate i te wā o te pokanga bariatric, ā, i arotakehia ngā tauira histological e ngā tohunga pathology mātau i runga anō i ngā paearu kua whakaahuatia i mua [17, 18]. Kua whakarāpopototia ngā paearu aromatawai ki te Ripanga Tāpiri S1, ā, kua whakaahuatia i mua [19].
I tātarihia ngā tauira toto nohopuku mā te whakamahi i te whakamātautau chromatography-papatipu wai kore-taunga (LC-MS) mō te tātari metabolomics (n = 116). I tātarihia ngā tauira mā te whakamahi i te pūnaha UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Tiamana) e ai ki te whakaahuatanga o mua19. Ko te tautuhi i te waipiro isopropyl (IPA) i ahu mai i te wā pupuri me te whakataurite i te whānuitanga MS/MS me ngā paerewa parakore. I whakaarohia te kaha o te tohu IPA (te rohe tihi) i roto i ngā tātaritanga katoa [20].
I whakahaerehia te raupapatanga RNA ate katoa mā te whakamahi i te Illumina HiSeq 2500, ā, i tukatukahia ngā raraunga i mua i te whakaahuatanga o mua [19, 21, 22]. I whakahaerehia e mātou he tātaritanga rerekētanga arotahi o ngā tuhinga e pā ana ki te mahi/koiogenesis mitochondrial mā te whakamahi i ngā ira 1957 i tīpakohia mai i te pātengi raraunga MitoMiner 4.0 [23]. I whakahaerehia te tātaritanga methylation DNA ate mā te whakamahi i te Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) mā te whakamahi i te tikanga kotahi i whakaahuatia o mua [24, 25].
Nā Ahorangi Stefano Romeo i tuku mai ngā pūtau whetu ate tangata (LX-2), ā, i whakatipuria, i puritia hoki i roto i te reo DMEM/F12 (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Hei whiriwhiri i te horopeta mahi o te IPA, i rongoatia ngā pūtau LX-2 ki ngā kukū rerekē o te IPA (10 μM, 100 μM me te 1 mM; Sigma, 220027) i roto i te reo DMEM/F12 mō ngā haora 24. Hei tāpiri, hei tirotiro i te kaha o te IPA ki te whakakore i ngā HSC, i rongoatia ngā pūtau LX-2 ki te 5 ng/ml TGF-β1 (ngā pūnaha R&D, 240-B-002/CF) me te 1 mM IPA i roto i te reo kore-huka mō ngā haora 24. Mō ngā mana whakahaere waka e pā ana, i whakamahia te 4 nM HCL kei roto ko te 0.1% BSA mō te maimoatanga TGF-β1 me te 0.05% DMSO mō te maimoatanga IPA, ā, i whakamahia ngātahitia ngā mea e rua mō te maimoatanga whakakotahi.
I aromatawaihia te apoptosis mā te whakamahi i te FITC Annexin V Apoptosis Detection Kit me te 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) e ai ki ngā tohutohu a te kaihanga. I roto i te poto, i whakatipuria a LX-2 (1 × 105 pūtau/pū) i te pō i roto i ngā pereti 12-pū, kātahi ka rongoatia ki ngā horopeta maha o te IPA, te IPA rānei me te TGF-β1. I te rā i muri mai, i kohia ngā pūtau e mānu ana me ngā pūtau piri, i whakamātauria, i horoia ki te PBS, i whakatārewatia anō ki roto i te arai here Annexin V, ā, i whakatōkia ki te FITC-Annexin V me te 7-AAD mō te 15 meneti.
I pania ngā mitochondria i roto i ngā pūtau ora mō te mahi waikura mā te whakamahi i te Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Mō ngā whakamātautau MTR, i whaowhia ngā pūtau LX-2 ki ngā kiato ōrite ki te IPA me te TGF-β1. I muri i te 24 hāora, i trypsinized ngā pūtau ora, i horoia ki te PBS, kātahi ka whaowhia ki te 100 μM MTR i roto i te reo kore-serum i te 37 °C mō te 20 meneti e ai ki te whakaahuatanga o mua [26]. Mō te tātari āhua pūtau ora, i tātarihia te rahi o te pūtau me te uaua o te cytoplasm mā te whakamahi i ngā tawhā marara whakamua (FSC) me te marara taha (SSC).
I kohia ngā raraunga katoa (30,000 ngā takahanga) mā te whakamahi i te NovoCyte Quanteon (Agilent) ā, i tātarihia mā te whakamahi i te pūmanawa NovoExpress® 1.4.1, FlowJo V.10 rānei.
I inehia te tere o te kai hāora (OCR) me te tere waikawa o waho o te pūtau (ECAR) i te wā tūturu mā te whakamahi i tētahi Kaitātari Rererangi o waho o te pūtau Seahorse (Agilent Technologies, Santa Clara, CA) kua rite ki tētahi Kaitātari Rererangi XF Pūtau Seahorse e ai ki ngā tohutohu a te kaihanga. I roto i te poto, i whakatōngia kia 2 × 104 ngā pūtau LX-2/pūtau ki runga i ngā pereti ahurea pūtau XF96. I muri i te whakatōnga i te pō, i rongoatia ngā pūtau ki te isopropanol (IPA) me te TGF-β1 (Ngā Tikanga Tāpiri 1). I whakahaerehia te tātari raraunga mā te whakamahi i te pūmanawa Seahorse XF Wave, kei roto ko te Kaihanga Pūrongo Whakamātautau Phenotype Pūtau Seahorse XF. Mai i tēnei, i tatauhia he Taupū Hauora Bioenergetic (BHI) [27].
I tāruatia te RNA katoa ki roto i te cDNA. Mō ngā tikanga motuhake, tirohia te tohutoro [15]. I whakamahia ngā taumata mRNA o te Human 60S ribosomal acidic protein P0 (RPLP0) me te cyclophilin A1 (PPIA) hei mana whakahaere ira tuturu. I whakamahia te QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, Netherlands) me te TaqMan™ Fast Advanced Master Mix Kit (Applied Biosystems) me te Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050) rānei, ā, i tatauhia te whakapūtanga ira whanaunga mā te whakamahi i ngā tawhā huringa uara Ct whakatairite (ΔΔCt) me te tikanga ∆∆Ct. Kei roto i ngā Ripanga Tāpiri S2 me S3 ngā taipitopito o ngā paraimere.
I tangohia te DNA karihi (ncDNA) me te DNA mitochondrial (mtDNA) mā te whakamahi i te kete toto me te kiko DNeasy (Qiagen) e ai ki te whakaahuatanga o mua [28]. I tatauhia te nui o te mtDNA mā te tatau i te ōwehenga o ia rohe mtDNA ūnga ki te toharite āhuahanga o ngā rohe DNA karihi e toru (mtDNA/ncDNA), e ai ki ngā Tikanga Tāpiri 2. Kei te Ripanga Tāpiri S4 ngā taipitopito o ngā paraimere mō te mtDNA me te ncDNA.
I pania ngā pūtau ora ki te Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) hei tiro i ngā whatunga mitochondrial i waenga i ngā pūtau me ngā pūtau o roto. I whakatōngia ngā pūtau LX-2 (1 × 104 pūtau/pūnga) ki runga i ngā kiriata karāhe i roto i ngā pereti ahurea raro-karāhe e rite ana (Ibidi GmbH, Martinsried, Tiamana). I muri i te 24 hāora, i whakatōngia ngā pūtau LX-2 ora ki te 100 μM MTR mō te 20 meneti i te 37 °C, ā, i pania ngā karihi pūtau ki te DAPI (1 μg/ml, Sigma-Aldrich) e ai ki te whakaahuatanga o mua [29]. I tirongia ngā whatunga mitochondrial mā te whakamahi i te karuārai Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, Tiamana) kua rite ki te kōwae confocal Zeiss LSM 800 i te 37 °C i roto i te hau makuku me te 5% CO2 mā te whakamahi i te whāinga 63×NA 1.3. I whiwhi mātou i ngā whakaahua raupapa-Z tekau mō ia momo tauira. E 30 ngā wāhanga o ia raupapa-Z, ā, ko te matotoru o ia wāhanga he 9.86 μm. Mō ia tauira, i tangohia ngā whakaahua o ngā mara tirohanga tekau mā te whakamahi i te pūmanawa ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Tiamana), ā, i mahia te tātaritanga āhua mitochondrial mā te whakamahi i te pūmanawa ImageJ (v1.54d) [30, 31] e ai ki ngā tawhā kua whakamāramahia i roto i ngā Tikanga Tāpiri 3.
I whakapūmauhia ngā pūtau ki te 2% glutaraldehyde i roto i te 0.1 M phosphate buffer, ā, i muri mai ka whakapūmauhia ki te 1% osmium tetroxide solution (Sigma Aldrich, MO, USA), ka whakamaroketia mārire ki te acetone (Merck, Darmstadt, Tiamana), ā, i te mutunga ka whakauruhia ki roto i te epoxy resin. I whakaritea ngā wāhanga tino angiangi, ā, i pania ki te 1% uranyl acetate (Merck, Darmstadt, Tiamana) me te 1% lead citrate (Merck, Darmstadt, Tiamana). I whiwhihia ngā whakaahua ultrastructure mā te whakamahi i te JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Hapani) i te ngaohiko whakatere o te 80 kV.
I tātarihia te āhua o ngā pūtau LX-2 i rongoatia ki te IPA mō te 24 hāora mā te karuārai-whakarerekētanga ā-wāhanga i te whakanuinga 50x mā te whakamahi i te karuārai mārama whakahurihuri Zeiss (Zeiss Axio Vert.A1 me AxioCam MRm, Jena, Tiamana).
I whakaatuhia ngā raraunga haumanu hei toharite ± paerewa rerekētanga, hei tau waenga rānei (awhe interquartile: IQR). I whakamahia te tātaritanga rerekētanga kotahi-ara (ngā taurangi tonu) te whakamātautau χ² rānei (ngā taurangi kāwai) hei whakatairite i ngā rerekētanga i waenga i ngā rōpū rangahau e toru. I whakamahia te reiti pai teka (FDR) hei whakatika i ngā whakamātautau maha, ā, i kiia he hiranga tatauranga ngā ira me te FDR < 0.05. I whakamahia te tātaritanga hononga Spearman hei hono i te methylation DNA CpG me te kaha tohu IPA, me ngā uara p ingoa (p < 0.05) i pūrongohia.
I whakahaerehia te tātari ara mā te whakamahi i tētahi taputapu tātari huinga ira tukutuku (WebGestalt) mō ngā tuhinga 268 (nama p < 0.01), 119 ngā tuhinga e pā ana ki te mitochondria (nama p < 0.05), me te 4350 CpG i roto i te 3093 ngā tuhinga ate e pā ana ki ngā taumata IPA toto e rere ana. I whakamahia te taputapu Venny DB (putanga 2.1.0) e wātea ana mō te kore utu hei kimi i ngā ira e taupatupatu ana, ā, i whakamahia a StringDB (putanga 11.5) hei tiro i ngā taunekeneke pūmua-pūmua.
Mō te whakamātautau LX-2, i whakamātauria ngā tauira mō te āhua noa mā te whakamahi i te whakamātautau D'Agostino-Pearson. I tangohia ngā raraunga mai i te iti rawa e toru ngā tāruarua koiora, ā, i tukuna ki te ANOVA kotahi-ara me te whakamātautau Bonferroni post hoc. I kiia he uara-p iti iho i te 0.05 he mea nui ki te tatauranga. Ka whakaatuhia ngā raraunga hei toharite ± SD, ā, ko te maha o ngā whakamātautau kei roto i ia ahua. I mahia ngā tātaritanga me ngā kauwhata katoa mā te whakamahi i te pūmanawa tatauranga GraphPad Prism 8 mō Windows (GraphPad Software Inc., putanga 8.4.3, San Diego, USA).
Tuatahi, i tirotirohia e mātou te hononga o ngā taumata IPA toto ki ngā tuhinga ate, tinana katoa, me ngā tuhinga mitochondrial. I roto i te kōtaha tuhinga katoa, ko te ira kaha rawa atu e pā ana ki ngā taumata IPA toto ko MAPKAPK3 (FDR = 0.0077; mitogen-activated protein kinase-activated protein kinase 3); i roto i te kōtaha tuhinga e pā ana ki te mitochondria, ko te ira kaha rawa atu e pā ana ko AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Kōnae Tāpiri 1 me te Kōnae Tāpiri 2).
Kātahi ka tātarihia e mātou ngā tuhinga ā-ao (n = 268; p < 0.01) me ngā tuhinga e pā ana ki te mitochondria (n = 119; p < 0.05), ā, i te mutunga ka kitea te apoptosis hei ara matua tino nui (p = 0.0089). Mō ngā tuhinga mitochondrial e pā ana ki ngā taumata IPA toto, i arotahi mātou ki te apoptosis (FDR = 0.00001), te mitophagy (FDR = 0.00029), me ngā ara tohu TNF (FDR = 0.000006) (Pikitia 1A, Ripanga 2, me ngā Pikitia Tāpiri 1A-B).
Te tātaritanga taupatupatu o ngā tuhinga ā-ao, e pā ana ki te mitochondria, me te methylation DNA i roto i te ate tangata e pā ana ki ngā taumata IPA toto. E tohu ana a A i te 268 ngā tuhinga ā-ao, 119 ngā tuhinga e pā ana ki te mitochondria, me ngā tuhinga methylated DNA kua maheretia ki ngā pae CpG 3092 e pā ana ki ngā taumata IPA toto (ngā uara p < 0.01 mō ngā tuhinga ā-ao me te DNA methylated, me ngā uara p < 0.05 mō ngā tuhinga mitochondrial). Kei waenganui ngā tuhinga taupatupatu matua (AKT1 me YKT6). B Ko te mahere taunekeneke o ngā ira 13 me te kaute taunekeneke teitei rawa (0.900) me ētahi atu ira i hangaia mai i ngā ira taupatupatu 56 (rohe rārangi pango) i tino pā ana ki ngā taumata IPA toto mā te whakamahi i te taputapu ipurangi StringDB. Kakariki: Ngā ira kua maheretia ki te wāhanga pūtau Gene Ontology (GO): mitochondria (GO:0005739). Ko AKT1 te pūmua me te kaute teitei rawa (0.900) mō ngā taunekeneke me ētahi atu pūmua i runga i ngā raraunga (i runga i te keri tuhinga, ngā whakamātautau, ngā pātengi raraunga, me te whakaaturanga tahi). Ko ngā pona whatunga e tohu ana i ngā pūmua, ā, ko ngā tapa e tohu ana i ngā hononga i waenga i ngā pūmua.
I te mea ka taea e ngā matūriki o te puku te whakahaere i te hanganga epigenetic mā te methylation DNA [32], i tirotirohia e mātou mēnā he hononga ngā taumata IPA toto ki te methylation DNA ate. I kitea e mātou ko ngā pae methylation matua e rua e pā ana ki ngā taumata IPA toto e tata ana ki te rohe 3 whai rawa i te proline-serine (C19orf55) me te mema whānau B (iti) pūmua ru wera 6 (HSPB6) (Kōnae Tāpiri 3). Ko te methylation DNA o te 4350 CpG (p < 0.01) i honoa ki ngā taumata IPA toto, ā, i whakanuia i roto i ngā ara whakahaere roa (p = 0.006) (Pikitia 1A, Ripanga 2, me te Pikitia Tāpiri 1C).
Hei mārama ki ngā tikanga koiora e tūāpapa ana i ngā hononga i waenga i ngā taumata IPA toto, ngā tuhinga ā-ao, ngā tuhinga e pā ana ki te mitochondria, me te methylation DNA i roto i te ate tangata, i whakahaerehia e mātou he tātaritanga taupatupatu o ngā ira i tautuhia i roto i te tātaritanga ara o mua (Pikitia 1A). Ko ngā hua o te tātaritanga whakarei ara o ngā ira taupatupatu e 56 (i roto i te rārangi pango i te Pikitia 1A) i whakaatu ko te ara apoptosis (p = 0.00029) i whakaatu i ngā ira e rua e rite ana ki ngā tātaritanga e toru: AKT1 me YKT6 (YKT6 v-SNARE homolog), e whakaaturia ana i roto i te tūtohi Venn (Pikitia Tāpiri 2 me te Pikitia 1A). He mea whakamīharo, i kitea e mātou he hononga pai a AKT1 (cg19831386) me YKT6 (cg24161647) ki ngā taumata IPA toto (Kōnae Tāpiri 3). Hei tautuhi i ngā taunekeneke pūmua pea i waenga i ngā hua ira, i tīpakohia e mātou he 13 ngā ira me te kaute rohe noa teitei rawa (0.900) i roto i te 56 ngā ira taupatupatu hei whakauru, ā, i hangaia he mahere taunekeneke. E ai ki te taumata whakapono (whakapono taha), ko te ira AKT1 me te kaute teitei rawa (0.900) i te tihi (Pikitia 1B).
I runga i te tātaritanga ara, i kitea e mātou ko te apoptosis te ara matua, nō reira i tirotirohia e mātou mēnā ka pā te maimoatanga IPA ki te apoptosis o ngā HSC i roto i te vitro. I whakaaturia e mātou i mua ko ngā horopeta rerekē o te IPA (10 μM, 100 μM, me te 1 mM) kāore he paitini ki ngā pūtau LX-2 [15]. I whakaatuhia e tēnei rangahau ko te maimoatanga IPA i te 10 μM me te 100 μM i whakanui ake i te maha o ngā pūtau ora me ngā pūtau necrotic. Heoi, ki te whakatauritea ki te rōpū whakahaere, i heke te ora o ngā pūtau i te 1 mM kukū IPA, i te mea kāore i rerekē te tere necrosis pūtau (Pikitia 2A, B). Muri iho, ki te kimi i te kukū tino pai hei whakaoho i te apoptosis i roto i ngā pūtau LX-2, i whakamatauria e mātou te 10 μM, 100 μM, me te 1 mM IPA mō te 24 hāora (Pikitia 2A-E me te Pikitia Tāpiri 3A-B). He mea whakamīharo, i whakaitihia e te IPA 10 μM me te 100 μM te tere apoptosis (%), heoi, i whakapiki ake te IPA 1 mM i te tere apoptosis me te apoptosis (%) i te whakataurite ki te mana whakahaere, nō reira i whiriwhiria ai mō ētahi atu whakamātautau (Pikitia 2A–D).
Ka whakaoho te IPA i te apoptosis o ngā pūtau LX-2. I whakamahia te tikanga tāima takirua Annexin V me te 7-AAD hei ine i te tere apoptotic me te āhua o te pūtau mā te cytometry rere. I whaowhia ngā pūtau BA ki te 10 μM, 100 μM me te 1 mM IPA mō te 24 hāora, ki te F–H TGF-β1 rānei (5 ng/ml) me te 1 mM IPA i roto i te reo kore-serum mō te 24 hāora. A: ngā pūtau ora (Annexin V -/ 7AAD-); B: ngā pūtau necrotic (Annexin V -/ 7AAD+); C, F: wawe (Annexin V +/ 7AAD-); D, G: tōmuri (Annexin V+/7AAD.+); E, H: ōrau o ngā pūtau apoptotic wawe me te tōmuri katoa i roto i te tere apoptotic (%). Ka whakaatuhia ngā raraunga hei toharite ± SD, n = 3 ngā whakamātautau motuhake. I mahia ngā whakatairite tatauranga mā te whakamahi i te ANOVA kotahi-ara me te whakamātautau Bonferroni post hoc. *p < 0.05; ****p < 0.0001
E ai ki tā mātou i whakaatu i mua, ka taea e te 5 ng/ml TGF-β1 te whakaoho i te whakahohenga HSC mā te whakanui ake i te whakaaturanga o ngā ira tohu tawhito [15]. I rongoatia ngā pūtau LX-2 ki te 5 ng/ml TGF-β1 me te 1 mM IPA i te whakakotahitanga (Pikitia 2E–H). Kāore te maimoatanga TGF-β1 i whakarerekē i te tere apoptosis, heoi, i whakanuia e te maimoatanga-tahi IPA te apoptosis mutunga me te tere apoptosis (%) ki te whakataurite ki te maimoatanga TGF-β1 (Pikitia 2E–H). E whakaatu ana ēnei hua ka taea e te 1 mM IPA te whakatairanga i te apoptosis i roto i ngā pūtau LX-2 ahakoa te whakaoho TGF-β1.
I tirotirohia anō e mātou te pānga o te IPA ki te manawa mitochondrial i roto i ngā pūtau LX-2. I whakaatuhia e ngā hua i whakaitihia e te 1 mM IPA ngā tawhā o te tere o te whakamahinga hāora (OCR): te manawa kore-mitochondrial, te manawa taketake me te manawa nui, te turuturu proton me te whakaputa ATP ki te whakataurite ki te rōpū whakahaere (Pikitia 3A, B), engari kāore i rerekē te taupū hauora koiora-pūngao (BHI).
Ka whakaitihia e te IPA te manawa mitochondrial i roto i ngā pūtau LX-2. Ka whakaatuhia te pihi manawa mitochondrial (OCR) hei tawhā manawa mitochondrial (manawa kore-mitochondrial, manawa taketake, manawa nui rawa, turuturu proton, whakaputa ATP, SRC me te BHI). I whaowhia ngā pūtau A me B ki te 10 μM, 100 μM me te 1 mM IPA mō te 24 hāora, ia. I whaowhia ngā pūtau C me D ki te TGF-β1 (5 ng/ml) me te 1 mM IPA i roto i te reo kore-serum mō te 24 hāora, ia. I whakatauritea ngā inenga katoa ki te ihirangi DNA mā te whakamahi i te kete CyQuant. BHI: taupū hauora koiora; SRC: kaha rahui manawa; OCR: tere kai hāora. Ka whakaatuhia ngā raraunga hei toharite ± paerewa rerekētanga (SD), n = 5 ngā whakamātautau motuhake. I mahia ngā whakatairite tatauranga mā te whakamahi i te ANOVA kotahi-ara me te whakamātautau Bonferroni post hoc. *p < 0.05; **p < 0.01; me ***p < 0.001
Hei whiwhi i tētahi māramatanga whānui ake mō te pānga o te IPA ki te kōtaha koiora o ngā pūtau LX-2 kua whakahohehia e TGF-β1, i tātarihia e mātou te phosphorylation oxidative mitochondrial mā te OCR (Pikitia 3C,D). I whakaatuhia e ngā hua ka taea e te maimoatanga TGF-β1 te whakaiti i te manawa mōrahi, te kaha rahui manawa (SRC) me te BHI ki te whakataurite ki te rōpū whakahaere (Pikitia 3C,D). Hei tāpiri, i whakaitihia e te maimoatanga whakakotahi te manawa taketake, te turuturu proton me te whakaputa ATP, engari he nui ake te SRC me te BHI i ērā i rongoatia ki te TGF-β1 (Pikitia 3C,D).
I whakahaerehia hoki e mātou te "Whakamātautau Āhuatanga Pūngao Pūtau" i whakaratohia e te pūmanawa Seahorse (Pikitia Tāpiri 4A–D). E ai ki te Pikitia Tāpiri 3B, i heke ngā pūmanawa pākia OCR me ECAR i muri i te maimoatanga TGF-β1, heoi, kāore he rerekētanga i kitea i roto i ngā rōpū maimoatanga whakakotahi me te IPA ki te rōpū whakahaere. I tua atu, i heke ngā taumata turanga me te ahotea o OCR i muri i te maimoatanga whakakotahi me te IPA ki te rōpū whakahaere (Pikitia Tāpiri 4C). He mea whakamīharo, i kitea he tauira rite ki te maimoatanga whakakotahi, i reira kāore i kitea he huringa i roto i ngā taumata turanga me te ahotea o ECAR ki te maimoatanga TGF-β1 (Pikitia Tāpiri 4C). I roto i ngā HSC, ko te whakahekenga o te phosphorylation oxidative mitochondrial me te kaha o te maimoatanga whakakotahi ki te whakaora i te SCR me te BHI i muri i te pānga ki te maimoatanga TGF-β1 kāore i whakarerekē i te pūmanawa pākia (OCR me ECAR). Ina huihuia, e tohu ana ēnei hua ka whakaiti pea te IPA i ngā bioenergetics i roto i ngā HSC, e kī ana ka puta pea i te IPA he kōtaha pūngao iti ake ka neke te āhuatanga HSC ki te whakakorenga (Pikitia Tāpiri 4D).
I tirotirohia te pānga o te IPA ki ngā whanaketanga mitochondrial mā te whakamahi i te ine-toru-ahu o te āhua mitochondrial me ngā hononga whatunga me te tae MTR (Pikitia 4 me te Pikitia Tāpiri 5). E whakaatu ana te Pikitia 4, ki te whakatauritea ki te rōpū whakahaere, i whakaitihia e te maimoatanga TGF-β1 te horahanga mata toharite, te maha o ngā peka, te roa katoa o ngā peka, me te maha o ngā hononga peka (Pikitia 4A me B) ā, i whakarerekētia te ōwehenga o ngā mitochondria mai i te porowhita ki te āhua waenga (Pikitia 4C). Ko te maimoatanga IPA anake i whakaiti i te rahinga mitochondrial toharite, ā, i whakarerekētia te ōwehenga o ngā mitochondria mai i te porowhita ki te āhua waenga ki te whakatauritea ki te rōpū whakahaere (Pikitia 4A). He rerekē, ko te porowhita, te roa toharite o ngā peka, me te mahi mitochondrial i aromatawaitia e te MTR e whakawhirinaki ana ki te kaha o te membrane mitochondrial (Pikitia 4A me E) i noho tonu kāore i rerekē, ā, kāore ēnei tawhā i rerekē i waenga i ngā rōpū. Ina huihuia, e kī ana ēnei hua ko te maimoatanga TGF-β1 me te IPA te āhua nei ka whakarerekē i te āhua me te rahi o ngā mitochondrial me te uauatanga o te whatunga i roto i ngā pūtau LX-2 ora.
Ka whakarerekē te IPA i ngā mahi mitochondrial me te nui o te DNA mitochondrial i roto i ngā pūtau LX-2. A. Ngā whakaahua confocal māngai o ngā pūtau LX-2 ora i whakatipuria ki te TGF-β1 (5 ng/ml) me te 1 mM IPA mō te 24 hāora i roto i te reo kore-serum e whakaatu ana i ngā whatunga mitochondrial i pania ki te Mitotracker™ Whero CMXRos me ngā karihi i pania ki te kikorangi ki te DAPI. I roto i ngā raraunga katoa he 15 ngā whakaahua mō ia rōpū. I whiwhi mātou i te 10 ngā whakaahua Z-stack mō ia momo tauira. I roto i ia raupapa tuaka-Z he 30 ngā poro, ia he matotoru o te 9.86 μm. Pae tauine: 10 μm. B. Ngā mea māngai (mitochondria anake) i tautuhia mā te whakamahi i te paepae urutau ki te whakaahua. I mahia te tātari tauanga me te whakataurite i ngā hononga whatunga morphological mitochondrial mō ngā pūtau katoa i roto i ia rōpū. C. Te auau o ngā ōwehenga āhua mitochondrial. Ko ngā uara e tata ana ki te 0 e tohu ana i ngā āhua porowhita, ā, ko ngā uara e tata ana ki te 1 e tohu ana i ngā āhua miro. D I whakatauhia te ihirangi DNA Mitochondrial (mtDNA) e ai ki te whakaahuatanga i roto i ngā Rauemi me ngā Tikanga. I whakahaerehia te tātaritanga E Mitotracker™ Red CMXRos mā te whakamahi i te flow cytometry (30,000 ngā huihuinga) e ai ki te whakaahuatanga i roto i ngā Rauemi me ngā Tikanga. Ka whakaatuhia ngā raraunga hei toharite ± SD, n = 3 ngā whakamātautau motuhake. I whakahaerehia ngā whakatairite tatauranga mā te whakamahi i te ANOVA kotahi-ara me te whakamātautau Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Kātahi ka tātarihia e mātou te ihirangi mtDNA i roto i ngā pūtau LX-2 hei tohu mō te maha o ngā mitochondrial. Ki te whakatauritea ki te rōpū whakahaere, i piki ake te ihirangi mtDNA i roto i te rōpū i rongoatia ki te TGF-β1 (Pikitia 4D). Ki te whakatauritea ki te rōpū i rongoatia ki te TGF-β1, i heke te ihirangi mtDNA i roto i te rōpū maimoatanga whakakotahi (Pikitia 4D), e kī ana ka whakaiti pea te IPA i te ihirangi mtDNA me te maha pea o ngā mitochondrial me te manawa mitochondrial (Pikitia 3C). I tua atu, te āhua nei i whakaitihia e te IPA te ihirangi mtDNA i roto i te maimoatanga whakakotahi engari kāore i pā ki te mahi mitochondrial i whakahaerehia e te MTR (Pikitia 4A–C).
I tirotirohia e mātou te hononga o te IPA ki ngā taumata mRNA o ngā ira e pā ana ki te fibrosis, te apoptosis, te oranga, me ngā whanaketanga mitochondrial i roto i ngā pūtau LX-2 (Pikitia 5A–D). Ki te whakatauritea ki te rōpū whakahaere, i whakaatu te rōpū i rongoatia ki te TGF-β1 i te pikinga ake o te whakaaturanga o ngā ira pēnei i te mekameka α2 momo collagen I (COL1A2), te α-smooth muscle actin (αSMA), te matrix metalloproteinase 2 (MMP2), te aukati kiko o te metalloproteinase 1 (TIMP1), me te ira dynamin 1-rite (DRP1), e tohu ana i te pikinga ake o te fibrosis me te whakahohenga. I tua atu, ki te whakatauritea ki te rōpū whakahaere, i whakaitihia e te maimoatanga TGF-β1 ngā taumata mRNA o te nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, te aukati o te B-pūtau α, te whakarei ake i te nuclear factor κ gene light peptide (NFκB1A), me te aukati o te nuclear factor κB kinase subunit β (IKBKB) (Pikitia 5A–D). Ki te whakatauritea ki te maimoatanga TGF-β1, i whakaitihia e te maimoatanga whakakotahi me te TGF-β1 me te IPA te whakaaturanga o COL1A2 me te MMP2, engari i whakapiki ake i ngā taumata mRNA o PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, me IKBKB. I tino whakaitihia e te maimoatanga IPA te whakaaturanga o MMP2, te pūmua X e pā ana ki te Bcl-2 (BAX), AKT1, te pūmua optic atrophy 1 (OPA1), me te pūmua whakakotahitanga mitochondrial 2 (MFN2), engari i piki ake te whakaaturanga o CASP8, NFκB1A, NFκB1B, me IKBKB ki te whakatauritea ki te rōpū whakahaere. Heoi, kāore i kitea he rerekētanga i roto i te whakaaturanga o caspase-3 (CASP3), te apoptotic peptidase activating factor 1 (APAF1), te pūmua whakakotahitanga mitochondrial 1 (MFN1), me te pūmua fission 1 (FIS1). I te katoa, e whakaatu ana ēnei hua ka whakatikatika te maimoatanga IPA i te whakaaturanga o ngā ira e pā ana ki te fibrosis, te apoptosis, te oranga tonutanga, me ngā whanaketanga mitochondrial. E whakaatu ana ā mātou raraunga ka whakaitihia e te maimoatanga IPA te fibrosis i roto i ngā pūtau LX-2; i te wa ano, ka whakaoho i te oranga tonutanga mā te neke i te phenotype ki te whakakorenga.
Ka whakarerekē te IPA i te whakaaturanga o ngā ira fibroblast, apoptotic, viability, me te mitochondrial dynamics i roto i ngā pūtau LX-2. Ka whakaatuhia e ngā histograms te whakaaturanga mRNA e pā ana ki te mana whakahaere taketake (RPLP0, PPIA rānei) i muri i te whakaohokia o ngā pūtau LX-2 ki te TGF-β1 me te IPA i roto i te reo kore-serum mō te 24 hāora. Ko A e tohu ana i ngā fibroblast, ko B e tohu ana i ngā pūtau apoptotic, ko C e tohu ana i ngā pūtau ora, ā, ko D e tohu ana i te whakaaturanga ira mitochondrial dynamics. Ka whakaatuhia ngā raraunga hei toharite ± paerewa rerekētanga (SD), n = 3 ngā whakamātautau motuhake. I whakahaerehia ngā whakataurite tatauranga mā te whakamahi i te ANOVA kotahi-ara me te whakamātautau Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Kātahi ka aromatawaihia ngā panonitanga o te rahi o te pūtau (FSC-H) me te uauatanga o te cytoplasm (SSC-H) mā te cytometry rere (Pikitia 6A,B), ā, ko ngā panonitanga o te āhua o te pūtau i muri i te maimoatanga IPA i aromatawaihia mā te karu hiko tuku (TEM) me te karu rerekētanga ā-wāhanga (Pikitia Tāpiri 6A-B). E ai ki te whakaaro, i piki ake te rahi o ngā pūtau i roto i te rōpū i rongoatia ki te TGF-β1 ki te rōpū whakahaere (Pikitia 6A,B), e whakaatu ana i te whānui matarohia o te reticulum endoplasmic taratara (ER*) me ngā phagolysosomes (P), e tohu ana i te whakahohenga o te pūtau take hematopoietic (HSC) (Pikitia Tāpiri 6A). Heoi, ki te whakatauritea ki te rōpū i rongoatia ki te TGF-β1, i heke te rahi o te pūtau, te uauatanga o te cytoplasm (Pikitia 6A,B), me te ihirangi ER* i roto i te rōpū maimoatanga whakakotahitanga TGF-β1 me te IPA (Pikitia Tāpiri 6A). I ​​tua atu, i whakaitihia e te maimoatanga IPA te rahi o te pūtau, te uauatanga o te cytoplasm (Pikitia 6A,B), te ihirangi P me te ER* (Pikitia Tāpiri 6A) ki te rōpū whakahaere. Hei tāpiritanga, i piki ake te nui o ngā pūtau apoptotic i muri i te 24 hāora o te maimoatanga IPA ki te whakataurite ki te rōpū whakahaere (ngā pere mā, Tāpiritanga Pikitia 6B). I te katoa, e kī ana ēnei hua ka taea e te 1 mM IPA te whakaoho i te apoptosis HSC me te whakakore i ngā huringa o ngā tawhā āhua pūtau i puta mai i te TGF-β1, ā, mā reira ka whakahaere i te rahi me te uaua o te pūtau, e pā ana pea ki te whakakorenga o te HSC.
Ka whakarerekē te IPA i te rahi o te pūtau me te uauatanga o te cytoplasm i roto i ngā pūtau LX-2. Ngā whakaahua māngai o te tātaritanga cytometry rere. I whakamahia e te tātaritanga he rautaki kuaha motuhake mō ngā pūtau LX-2: SSC-A/FSC-A hei tautuhi i te taupori pūtau, FSC-H/FSC-A hei tautuhi i ngā takirua, me te SSC-H/FSC-H mō te tātaritanga rahi me te uauatanga o te pūtau. I whakatōngia ngā pūtau ki te TGF-β1 (5 ng/ml) me te 1 mM IPA i roto i te reo kore-serum mō te 24 hāora. I tohatohahia ngā pūtau LX-2 ki te hauwhā maui o raro (SSC-H-/FSC-H-), te hauwhā maui o runga (SSC-H+/FSC-H-), te hauwhā matau o raro (SSC-H-/FSC-H+), me te hauwhā matau o runga (SSC-H+/FSC-H+) mō te tātaritanga rahi o te pūtau me te uauatanga o te cytoplasm. B. I tātarihia te āhua o te pūtau mā te cytometry rere mā te whakamahi i te FSC-H (marara whakamua, rahi pūtau) me te SSC-H (marara taha, uauatanga o te cytoplasm) (30,000 ngā huihuinga). Ka whakaaturia ngā raraunga hei toharite ± SD, n = 3 ngā whakamātautau motuhake. I whakahaerehia ngā whakatairite tatauranga mā te whakamahi i te ANOVA kotahi-ara me te whakamātautau Bonferroni post hoc. *p < 0.05; **p < 0.01; ***p < 0.001 me ****p < 0.0001
Kua noho ngā matūkai o te puku pērā i te IPA hei kaupapa rangahau wera, e kī ana tera pea ka kitea he whāinga hou i roto i te microbiota puku. Nō reira, he mea whakamere te whakaatu ko te IPA, he matūkai kua honoa e mātou ki te fibrosis ate i roto i te tangata [15], kua whakaatuhia he matū ārai-fibrotic pea i roto i ngā tauira kararehe [13, 14]. I konei, ka whakaatuhia e mātou mō te wā tuatahi he hononga i waenga i te IPA toto me te transcriptomics ate ā-ao me te methylation DNA i roto i ngā tāngata momona kāore he mate huka momo 2 (T2D), e whakaatu ana i te apoptosis, te mitophagy me te roa o te ora, me te ira kaitono pea AKT1 e whakahaere ana i te homeostasis ate. Ko tētahi atu mea hou o tā mātou rangahau ko tā mātou whakaatu i te taunekeneke o te maimoatanga IPA me te apoptosis, te āhua o te pūtau, te bioenergetics mitochondrial me ngā hihiri i roto i ngā pūtau LX-2, e tohu ana i te whānuitanga pūngao iti ake e neke ana i te phenotype HSC ki te whakakorenga, e hanga ana i te IPA hei kaitono pea mō te whakapai ake i te fibrosis ate.
I kitea e mātou ko te apoptosis, te mitophagy me te roa o te ora ngā ara matua nui rawa atu i roto i ngā ira ate e pā ana ki te toto totoro IPA. Ko te whakararuraru i te pūnaha mana kounga mitochondrial (MQC) ka arahi pea ki te ngoikore o te mitochondrial, te mitophagy me te apoptosis, ā, ka whakatairanga i te putanga mai o te MASLD [33, 34]. Nō reira, ka taea e mātou te whakapae he wāhi pea a IPA ki te pupuri i te hihiri pūtau me te pono o te mitochondrial mā te apoptosis, te mitophagy me te roa o te ora i roto i te ate. I whakaatuhia e ā mātou raraunga e rua ngā ira e kitea ana puta noa i ngā whakamātautau e toru: YKT6 me AKT1. He mea tika kia mōhio ko te YKT6 he pūmua SNARE e uru ana ki te tukanga whakakotahitanga o te membrane pūtau. He wāhi nui tāna ki te autophagy me te mitophagy mā te hanga i tētahi matatini tīmatanga me STX17 me SNAP29 i runga i te autophagosome, ā, ka whakatairanga i te whakakotahitanga o ngā autophagosome me ngā lysosomes [35]. I tua atu, ko te ngaronga o te mahi YKT6 ka hua ake te ngoikore o te mitophagy [36], ko te pikinga ake o te YKT6 e pā ana ki te haere whakamua o te hepatocellular carcinoma (HCC), e whakaatu ana i te pikinga o te oranga o te pūtau [37]. I tētahi atu taha, ko te AKT1 te ira taunekeneke tino nui, ā, he mahi nui tāna i roto i ngā mate ate, tae atu ki te ara tohu PI3K/AKT, te huringa pūtau, te hekenga pūtau, te tipu, te piri arotahi, te mahi mitochondrial, me te whakaputanga collagen[38–40]. Ka taea e te ara tohu PI3K/AKT whakahohe te whakahohe i ngā pūtau take hematopoietic (HSC), ko ngā pūtau te kawenga mō te hanga o te matrix extracellular (ECM), ā, ko tōna koretake pea ka whai wāhi ki te putanga me te haere whakamua o te fibrosis ate[40]. Hei tāpiri, ko te AKT tētahi o ngā mea matua e ora ai ngā pūtau e aukati ana i te apoptosis pūtau e whakawhirinaki ana ki te p53, ā, ko te whakahohenga AKT pea e hono ana ki te aukati i te apoptosis pūtau ate[41, 42]. E kī ana ngā hua i puta ko te IPA pea kei roto i te apoptosis e pā ana ki te mitochondria ate mā te pā ki te whakatau o ngā hepatocytes i waenga i te urunga ki te apoptosis, te ora rānei. Ka taea te whakahaere i ēnei pānga e ngā ira kaitono AKT me/ranei YKT6, he mea nui mō te homeostasis ate.
I whakaatuhia e ā mātou hua i puta ai te apoptosis i te 1 mM IPA, ā, i heke iho te manawa mitochondrial i roto i ngā pūtau LX-2, ahakoa te maimoatanga TGF-β1. He mea nui kia mōhiotia ko te apoptosis te ara matua mō te whakatau i te fibrosis me te whakahohenga pūtau take hematopoietic (HSC), ā, he kaupapa matua anō hoki i roto i te urupare ā-tinana whakahurihuri o te fibrosis ate [4, 43]. I tua atu, ko te whakahokinga mai o te BHI i roto i ngā pūtau LX-2 i muri i te maimoatanga whakakotahi i whakarato i ngā māramatanga hou ki te tūranga pea o te IPA i roto i te whakahaere i ngā bioenergetics mitochondrial. I raro i ngā āhuatanga okiokinga me te kore mahi, ka whakamahia e ngā pūtau hematopoietic te phosphorylation oxidative mitochondrial hei whakaputa ATP, ā, he iti te mahi metabolic. I tētahi atu taha, ka whakarei ake te whakahohenga HSC i te manawa mitochondrial me te biosynthesis hei utu mō ngā hiahia pūngao o te tomo ki te āhua glycolytic [44]. Ko te meka kāore te IPA i pā ki te kaha metabolic me te ECAR e kī ana he iti ake te kaupapa matua o te ara glycolytic. Waihoki, i whakaatuhia e tētahi atu rangahau he kaha te 1 mM IPA ki te whakarerekē i te mahi mekameka manawa mitochondrial i roto i ngā cardiomyocytes, te raina pūtau hepatocyte tangata (Huh7) me ngā pūtau endothelial uaua umbilical tangata (HUVEC); Heoi, kāore i kitea he pānga o te IPA ki te glycolysis i roto i ngā cardiomyocytes, e kī ana ka pā pea te IPA ki te bioenergetics o ētahi atu momo pūtau [45]. Nō reira, e whakapae ana mātou ka mahi pea te 1 mM IPA hei wetewete matū ngawari, nā te mea ka taea e ia te whakaiti nui i te whakaaturanga ira fibrogenic, te āhua o te pūtau me te bioenergetics mitochondrial me te kore e whakarerekē i te nui o te mtDNA [46]. Ka taea e ngā wetewete mitochondrial te aukati i te fibrosis me te whakahohenga HSC i puta mai i te ahurea [47] me te whakaiti i te hanga ATP mitochondrial e whakahaerehia ana, e whakaohohia ana rānei e ētahi pūmua pēnei i te wetewete pūmua (UCP) te adenine nucleotide translocase (ANT) rānei. I runga i te momo pūtau, ka taea e tēnei āhuatanga te tiaki i ngā pūtau mai i te apoptosis me/te whakatairanga rānei i te apoptosis [46]. Heoi, me whai atu rangahau hei whakamārama i te tūranga o te IPA hei wetewete mitochondrial i roto i te whakakorenga pūtau take hematopoietic.
I muri mai ka tirotirohia e mātou mēnā kei te whakaatahia ngā huringa o te manawa mitochondrial i roto i te āhua mitochondrial i roto i ngā pūtau LX-2 ora. He mea whakamīharo, ka whakarerekē te maimoatanga TGF-β1 i te ōwehenga mitochondrial mai i te porowhita ki te waenga, me te hekenga o te peka mitochondrial me te pikinga o te whakaaturanga o DRP1, he mea matua i roto i te wehenga mitochondrial [48]. I tua atu, ko te wehewehenga mitochondrial e pā ana ki te uauatanga whānui o te whatunga, ā, he mea nui te whakawhiti mai i te hanumi ki te wehenga mō te whakahohenga o te pūtau take hematopoietic (HSC), ko te aukati i te wehenga mitochondrial ka arahi ki te apoptosis HSC [49]. Nō reira, e whakaatu ana ā mātou hua ka taea e te maimoatanga TGF-β1 te whakaheke i te uauatanga o te whatunga mitochondrial me te hekenga o te peka, he mea noa ake i roto i te wehenga mitochondrial e pā ana ki ngā pūtau take hematopoietic (HSC) kua whakahohehia. I tua atu, i whakaatuhia e ā mātou raraunga ka taea e te IPA te whakarerekē i te ōwehenga o te mitochondria mai i te porowhita ki te āhua waenga, ā, ka whakaitihia te whakaaturanga o OPA1 me MFN2. Kua whakaatuhia e ngā rangahau ko te whakahekenga o te OPA1 ka taea te whakaheke i te kaha o te membrane mitochondrial me te whakaoho i te apoptosis pūtau [50]. E mōhiotia ana ko MFN2 te kaiwhakahaere i te hanumi mitochondrial me te apoptosis [51]. E whakaatu ana ngā hua i puta ko te whakaoho o ngā pūtau LX-2 e TGF-β1 me/ranei IPA te āhua nei ka whakarerekē i te āhua me te rahi o ngā mitochondrial, tae atu ki te āhua whakahohenga me te uauatanga o te whatunga.
E whakaatu ana ā mātou hua ka taea e te maimoatanga whakakotahi o te TGFβ-1 me te IPA te whakaiti i ngā tawhā āhua o te mtDNA me te pūtau mā te whakahaere i te whakaaturanga mRNA o te fibrosis, te apoptosis me ngā ira e pā ana ki te oranga i roto i ngā pūtau e karo ana i te apoptosis. Ina hoki, i whakaitihia e te IPA te taumata whakaaturanga mRNA o te AKT1 me ngā ira fibrosis nui pēnei i te COL1A2 me te MMP2, engari i whakapiki ake i te taumata whakaaturanga o te CASP8, e pā ana ki te apoptosis. I whakaatu ā mātou hua i muri i te maimoatanga IPA, i heke te whakaaturanga BAX me te pikinga o te whakaaturanga mRNA o ngā waeine whānau TIMP1, BCL-2 me te NF-κB, e kī ana ka taea e te IPA te whakaoho i ngā tohu oranga i roto i ngā pūtau take hematopoietic (HSC) e karo ana i te apoptosis. Ka mahi pea ēnei ngota hei tohu pro-ora i roto i ngā pūtau take hematopoietic kua whakahohehia, e pā ana pea ki te pikinga o te whakaaturanga o ngā pūmua anti-apoptotic (pēnei i te Bcl-2), te hekenga o te whakaaturanga o te pro-apoptotic BAX, me te taunekeneke uaua i waenga i te TIMP me te NF-κB [5, 7]. Ka puta ngā pānga o te IPA mā te PXR, ā, i kitea e mātou ko te maimoatanga whakakotahi me te TGF-β1 me te IPA i whakapiki ake i ngā taumata whakaaturanga mRNA PXR, e tohu ana i te pehitanga o te whakahohenga HSC. E mōhiotia ana ka aukati te tohu PXR whakahohe i te whakahohenga HSC i roto i te tinana ora me te tinana vitro [52, 53]. E whakaatu ana ā mātou hua ka whai wāhi pea te IPA ki te whakakore i ngā HSC whakahohe mā te whakatairanga i te apoptosis, te whakaiti i te fibrosis me te pākia o te mitochondrial, me te whakarei ake i ngā tohu oranga, he tukanga noa e huri ai te momo HSC whakahohe ki tētahi mea kāore i whakahohea. Ko tētahi atu whakamārama pea mō te tikanga me te tūranga pea o te IPA i roto i te apoptosis ko tana kohikohi i ngā mitochondria kore mahi i te nuinga o te wā mā te mitophagy (ara ā-roto) me te ara tohu TNF o waho (Ripanga 1), e hono tika ana ki te ara tohu oranga NF-κB (Pikitia Tāpiri 7). He mea whakamīharo, ka taea e ngā ira whai rawa e pā ana ki te IPA te whakaoho i ngā tohu pro-apoptotic me te pro-ora i roto i te ara apoptotic [54], e kī ana ka whakaoho pea te IPA i te ara apoptotic, te oranga rānei mā te taunekeneke ki ēnei ira. Heoi, kāore anō kia mārama me pēhea te whakaoho a te IPA i te apoptosis, i te oranga rānei i te wā e whakahohehia ana te HSC me ōna ara tikanga.
He matūriki koiora te IPA i hangaia mai i te tryptophan kai mā roto i te microbiota puku. Kua whakaatuhia e ngā rangahau he āhuatanga ārai-mumura, ārai-waikura, me te whakahaere epigenetic i roto i te taiao puku.[55] Kua whakaatuhia e ngā rangahau ka taea e te IPA te whakarerekē i te mahi ārai puku me te whakaiti i te ahotea waikura, ka whai wāhi pea ki ōna pānga ā-rohe.[56] Inaa, ka haria te IPA ki ngā whekau ūnga mā roto i te tohanga toto, ā, nā te mea he rite te hanganga matūriki nui o te IPA ki te tryptophan, te serotonin, me ngā indole derivatives, ka mahi te IPA i ngā mahi matūriki ka hua ake he whakataetae pānga matūriki.[52] Ka whakataetae pea te IPA ki ngā matūriki i ahu mai i te tryptophan mō ngā pae here ki ngā whākōkī, ki ngā kaiwhakawhiwhi rānei, ka whakararuraru pea i ngā ara matūriki noa. E whakaatu ana tēnei i te hiahia mō ētahi atu rangahau mō ōna pharmacokinetics me te pharmacodynamics kia pai ake ai te mārama ki tōna matapihi rongoā.[57] Me kite tonu mēnā ka taea hoki tēnei te puta i roto i ngā pūtau take hematopoietic (HSC).
E whakaae ana mātou he iti noa ngā herenga o tā mātou rangahau. Hei tirotiro i ngā hononga e pā ana ki te IPA, i tangohia e mātou ngā tūroro he mate huka momo 2 (T2DM). E whakaae ana mātou he iti noa te whānuitanga o ā mātou kitenga ki ngā tūroro he mate huka momo 2 me te mate ate kua whanake. Ahakoa ko te kukū ā-tinana o te IPA i roto i te toto tangata he 1–10 μM [11, 20], i whiriwhiria he kukū 1 mM IPA i runga i te kukū teitei rawa atu kāore i te paitini [15] me te tere teitei rawa atu o te apoptosis, kāore he rerekētanga i roto i te ōrau o te taupori pūtau necrotic. Ahakoa i whakamahia ngā taumata supraphysiological o te IPA i roto i tēnei rangahau, kāore anō kia whakaaetia te horopeta whai hua o te IPA i tēnei wā [52]. Ahakoa he mea nui ā mātou hua, ko te whānuitanga o te mate pākia o te IPA he wāhi rangahau tonu. I tua atu, ko ā mātou kitenga mō te hononga i waenga i ngā taumata IPA toto me te methylation DNA o ngā tuhinga ate i whiwhihia ehara i te mea mai i ngā pūtau take hematopoietic (HSC) anake engari mai i ngā kiko ate hoki. I whiriwhiria e mātou te whakamahi i ngā pūtau LX-2 tangata i runga i ā mātou kitenga o mua mai i te tātaritanga transcriptome e kī ana ko te IPA e hono ana ki te whakahohenga o ngā pūtau take hematopoietic (HSC) [15], ā, ko ngā HSC ngā pūtau matua e uru ana ki te whanaketanga o te fibrosis ate. He maha ngā momo pūtau o te ate, nō reira me whai whakaaro ki ētahi atu tauira pūtau pēnei i te pūnaha whakatipu pūtau ārai mate hepatocyte-HSC me te whakahohenga caspase me te wehewehenga DNA me te tikanga mahi tae atu ki te taumata pūmua hei ako i te mahi a te IPA me tana taunekeneke ki ētahi atu momo pūtau ate.