A baya mun bayar da rahoton cewa matakan sinadarin tryptophan metabolite indole-3-propionic acid (IPA) da aka samo daga cikin hanji sun yi ƙasa a cikin marasa lafiya da ke fama da cutar hanta. A cikin wannan binciken, mun binciki sinadarin methylome na transcriptome da DNA a cikin hanta masu kiba dangane da matakan IPA na jini, da kuma rawar da IPA ke takawa wajen haifar da rashin aiki na ƙwayoyin hepatic stellate (HSCs) a cikin jini.
Binciken ya haɗa da marasa lafiya 116 masu kiba waɗanda ba su da ciwon suga na nau'in 2 (T2DM) (shekaru 46.8 ± 9.3; BMI: 42.7 ± 5.0 kg/m²) waɗanda aka yi musu tiyatar bariatric a Cibiyar Tiyatar Kuopio Bariatric (KOBS). An auna matakan IPA da ke yawo ta hanyar amfani da liquid chromatography-mass spectrometry (LC-MS), an yi nazarin hanta ta hanyar jimillar jerin RNA, kuma an yi nazarin methylation na DNA ta amfani da Infinium HumanMethylation450 BeadChip. An yi amfani da ƙwayoyin stellate na hanta na ɗan adam (LX-2) don gwaje-gwajen in vitro.
Matakan IPA na jini suna da alaƙa da bayyanar kwayoyin halitta da ke da hannu a cikin hanyoyin apoptotic, mitophagic, da tsawon rai a cikin hanta. Kwayar halittar AKT serine/threonine kinase 1 (AKT1) ita ce kwayar halittar da ta fi yawa kuma mafi rinjaye da ke hulɗa a cikin bayanan hanta da bayanan DNA methylation. Maganin IPA ya haifar da apoptosis, raguwar numfashi na mitochondrial, da kuma canza yanayin ƙwayoyin halitta da yanayin mitochondrial ta hanyar daidaita bayyanar kwayoyin halitta da aka sani don daidaita fibrosis, apoptosis, da kuma tsira daga ƙwayoyin LX-2.
Idan aka haɗa waɗannan bayanai, waɗannan bayanai suna goyan bayan cewa IPA tana da tasirin warkewa mai yuwuwa kuma tana iya haifar da apoptosis da kuma canza yanayin HSC zuwa yanayin rashin aiki, ta haka ne ke faɗaɗa yuwuwar hana fibrosis na hanta ta hanyar tsoma baki ga kunna HSC da metabolism na mitochondrial.
Yaɗuwar kiba da ciwon metabolism yana da alaƙa da ƙaruwar kamuwa da cutar hanta mai kitse (MASLD); cutar tana shafar kashi 25% zuwa 30% na yawan jama'a [1]. Babban sakamakon cutar MASLD shine fibrosis na hanta, wani tsari mai ƙarfi wanda ke nuna ci gaba da tarin ƙwayoyin fibrous extracellular matrix (ECM) [2]. Manyan ƙwayoyin da ke cikin fibrosis na hanta sune ƙwayoyin hepatic stellate (HSCs), waɗanda ke nuna nau'ikan halittu guda huɗu da aka sani: quiescent, activated, inactivated, da senescent [3, 4]. Ana iya kunna HSCs kuma a canza su daga siffar quiescent zuwa ƙwayoyin fibroblast masu yaduwa tare da buƙatar kuzari mai yawa, tare da ƙaruwar bayyanar α-smooth muscle actin (α-SMA) da nau'in I collagen (Col-I) [5, 6]. A lokacin juyawar fibrosis na hanta, ana kawar da HSCs da aka kunna ta hanyar apoptosis ko rashin kunnawa. Waɗannan hanyoyin sun haɗa da rage yawan ƙwayoyin halittar fibrogenic da kuma daidaita kwayoyin halittar prosurvival (kamar hanyoyin siginar NF-κB da PI3K/Akt) [7, 8], da kuma canje-canje a cikin yanayin aiki da aiki na mitochondrial [9].
An gano cewa matakan sinadarin tryptophan metabolite indole-3-propionic acid (IPA) a cikin jini, wanda ake samarwa a cikin hanji, sun ragu a cikin cututtukan metabolism na ɗan adam, ciki har da MASLD [10-13]. IPA yana da alaƙa da shan fiber na abinci, an san shi da tasirin antioxidant da anti-inflammatory, kuma yana rage yanayin steatohepatitis (NASH) wanda abinci ke haifarwa a cikin jiki da kuma a cikin vitro [11-14]. Wasu shaidu sun fito ne daga bincikenmu na baya, wanda ya nuna cewa matakan IPA na jini sun yi ƙasa a cikin marasa lafiya da ke fama da fibrosis na hanta fiye da marasa lafiya masu kiba ba tare da fibrosis na hanta ba a cikin Nazarin Tiyatar Kuopio Bariatric (KOBS). Bugu da ƙari, mun nuna cewa maganin IPA zai iya rage bayyanar kwayoyin halitta waɗanda ke nuna alamun mannewar tantanin halitta, ƙaura ta tantanin halitta da kunna ƙwayoyin halitta na hepatic stem a cikin samfurin tantanin halitta na hanta (LX-2) kuma yana iya zama metabolite mai kare hepato [15]. Duk da haka, har yanzu ba a san yadda IPA ke haifar da koma baya ga fibrosis na hanta ta hanyar kunna apoptosis na HSC da bioenergetics na mitochondrial ba.
A nan, mun nuna cewa IPA na jini yana da alaƙa da bayyanar kwayoyin halitta da aka wadatar a cikin hanyoyin apoptosis, mitophagy, da tsawon rai a cikin hanta na mutanen da ke da ciwon sukari na 2 (KOBS) masu kiba amma waɗanda ba su da nau'in 2. Bugu da ƙari, mun gano cewa IPA na iya haifar da sharewa da lalata ƙwayoyin halittar hematopoietic da aka kunna (HSCs) ta hanyar hanyar hana aiki. Waɗannan sakamakon sun bayyana sabon rawar da IPA ke takawa, wanda hakan ya sa ya zama babban abin da za a iya cimmawa don haɓaka koma-baya a cikin fibrosis na hanta.
Wani bincike da aka yi a baya a ƙungiyar KOBS ya nuna cewa marasa lafiya da ke fama da cutar fibrosis ta hanta suna da ƙarancin matakan IPA da ke yawo idan aka kwatanta da marasa lafiya da ba su da cutar fibrosis ta hanta [15]. Domin kawar da tasirin da ke tattare da ciwon suga na nau'in 2, mun ɗauki marasa lafiya 116 masu kiba waɗanda ba su da ciwon suga na nau'in 2 (matsakaicin shekaru ± SD: 46.8 ± 9.3 shekaru; BMI: 42.7 ± 5.0 kg/m2) (Tebur 1) daga binciken KOBS da ake ci gaba da yi a matsayin yawan mutanen da aka yi binciken [16]. Duk mahalarta sun ba da izini a rubuce kuma Kwamitin Ɗabi'a na Asibitin Gundumar North Savo ya amince da yarjejeniyar binciken bisa ga Sanarwar Helsinki (54/2005, 104/2008 da 27/2010).
An samo samfuran biopsy na hanta a lokacin tiyatar bariatric kuma ƙwararrun likitoci sun tantance su ta hanyar histological bisa ga sharuɗɗan da aka bayyana a baya [17, 18]. An taƙaita sharuɗɗan kimantawa a cikin Ƙarin Tebur S1 kuma an bayyana su a baya [19].
An yi nazarin samfuran jinin azumi ta hanyar amfani da na'urar auna kitse mai yawa (LC-MS) don nazarin metabolomics (n = 116). An yi nazarin samfuran ta amfani da tsarin UHPLC-qTOF-MS (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Jamus) kamar yadda aka bayyana a baya19. Gano isopropyl barasa (IPA) ya dogara ne akan lokacin riƙewa da kwatanta bakan MS/MS tare da tsarkakkun ƙa'idodi. An yi la'akari da ƙarfin siginar IPA (yankin kololuwa) a cikin duk ƙarin bincike [20].
An yi amfani da Illumina HiSeq 2500 wajen yin jerin RNA na hanta gaba ɗaya kuma an riga an sarrafa bayanai kamar yadda aka bayyana a baya [19, 21, 22]. Mun yi nazarin bambance-bambancen maganganu na rubuce-rubucen da suka shafi aikin mitochondrial/biogenesis ta amfani da kwayoyin halitta 1957 da aka zaɓa daga bayanan MitoMiner 4.0 [23]. An yi nazarin methylation na DNA na hanta ta amfani da Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, Amurka) ta amfani da hanya ɗaya kamar yadda aka bayyana a baya [24, 25].
Farfesa Stefano Romeo ya samar da ƙwayoyin halittar hanta na ɗan adam (LX-2) cikin alheri kuma an noma su kuma an kula da su a cikin matsakaiciyar DMEM/F12 (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106). Don zaɓar yawan aikin IPA, an yi wa ƙwayoyin LX-2 magani da yawan IPA daban-daban (10 μM, 100 μM da 1 mM; Sigma, 220027) a cikin matsakaicin DMEM/F12 na tsawon awanni 24. Bugu da ƙari, don bincika ikon IPA na hana HSCs, an yi wa ƙwayoyin LX-2 magani tare da 5 ng/ml TGF-β1 (tsarin R&D, 240-B-002/CF) da 1 mM IPA a cikin matsakaiciyar da ba ta da jini na tsawon awanni 24. Ga masu kula da abin hawa da suka dace, an yi amfani da 4 nM HCL mai ɗauke da 0.1% BSA don maganin TGF-β1 kuma an yi amfani da 0.05% DMSO don maganin IPA, kuma an yi amfani da duka biyun tare don maganin haɗin gwiwa.
An tantance Apoptosis ta amfani da FITC Annexin V Apoptosis Detection Kit tare da 7-AAD (Biolegend, San Diego, CA, Amurka, Cat# 640922) bisa ga umarnin masana'anta. A takaice, an shuka LX-2 (ƙwayoyin 1 × 105 a kowace rijiya) cikin dare a cikin faranti 12 sannan aka yi wa magani da allurai da yawa na IPA ko IPA da TGF-β1. Washegari, an tattara ƙwayoyin da ke iyo da manne, an yi musu trypsin, an wanke su da PBS, an sake daure su a cikin ma'ajiyar Annexin V, sannan aka saka su da FITC-Annexin V da 7-AAD na tsawon minti 15.
An yi wa Mitochondria a cikin ƙwayoyin halitta masu rai fenti don aikin oxidative ta amfani da Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA). Don gwaje-gwajen MTR, an saka ƙwayoyin LX-2 a daidai adadin IPA da TGF-β1. Bayan awanni 24, an yi wa ƙwayoyin halitta masu rai trypsin, an wanke su da PBS, sannan aka saka su da 100 μM MTR a cikin matsakaici mara serum a 37 °C na tsawon mintuna 20 kamar yadda aka bayyana a baya [26]. Don nazarin yanayin ƙwayoyin halitta masu rai, an yi nazarin girman ƙwayoyin halitta da sarkakiyar cytoplasm ta amfani da sigogin watsawa gaba (FSC) da watsawa gefe (SSC), bi da bi.
An samo dukkan bayanai (abubuwan da suka faru 30,000) ta amfani da NovoCyte Quanteon (Agilent) kuma an yi nazari a kansu ta amfani da manhajar NovoExpress® 1.4.1 ko FlowJo V.10.
An auna yawan amfani da iskar oxygen (OCR) da kuma yawan acidification na ƙwayoyin halitta (ECAR) a ainihin lokacin ta amfani da na'urar nazarin iskar oxygen ta Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) wacce aka sanya mata Seahorse XF Cell Mito Stress bisa ga umarnin masana'anta. A takaice, an shuka ƙwayoyin 2 × 104 LX-2 a cikin faranti na al'adar ƙwayoyin XF96. Bayan an dasa su cikin dare ɗaya, an yi wa ƙwayoyin magani da isopropanol (IPA) da TGF-β1 (Hanyoyin Ƙarin 1). An gudanar da nazarin bayanai ta amfani da manhajar Seahorse XF Wave, wadda ta haɗa da janareta na rahoton gwajin kuzarin ƙwayoyin halitta na Seahorse XF. Daga wannan, an ƙididdige Manhajar Lafiya ta Bioenergetic (BHI) [27].
An rubuta jimlar RNA zuwa cDNA. Don takamaiman hanyoyi, duba nassoshi [15]. An yi amfani da matakan furotin ribosomal acidic na ɗan adam 60S P0 (RPLP0) da cyclophilin A1 (PPIA) mRNA azaman sarrafa kwayar halitta. An yi amfani da Tsarin PCR na QuantStudio 6 pro Real-Time (Thermo Fisher, Landsmeer, Netherlands) tare da Kit ɗin TaqMan™ Fast Advanced Master Mix (Applied Biosystems) ko Kit ɗin Sensifast SYBR Lo-ROX (Bioline, BIO 94050), kuma an ƙididdige ninki na bayyanar kwayar halitta ta amfani da sigogin zagayowar ƙimar Ct (ΔΔCt) da hanyar ∆∆Ct. An bayar da cikakkun bayanai game da firam ɗin a cikin Teburin Ƙarin S2 da S3.
An cire DNA na nukiliya (ncDNA) da kuma mitochondrial DNA (mtDNA) ta amfani da kayan aikin jini da nama na DNeasy (Qiagen) kamar yadda aka bayyana a baya [28]. An ƙididdige adadin mtDNA ta hanyar ƙididdige rabon kowane yanki na mtDNA da aka yi niyya zuwa matsakaicin geometric na yankuna uku na DNA na nukiliya (mtDNA/ncDNA), kamar yadda aka yi bayani dalla-dalla a cikin Hanyoyin Ƙarin 2. An bayar da cikakkun bayanai game da primers na mtDNA da ncDNA a cikin Teburin Ƙarin S4.
An yi wa ƙwayoyin halitta masu rai fenti da Mitotracker™ Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) don ganin hanyoyin sadarwa na mitochondrial tsakanin ƙwayoyin halitta da na cikin ƙwayoyin halitta. An yi wa ƙwayoyin LX-2 (ƙwayoyin 1 × 104/rijiya) fenti a kan gilashin a cikin faranti na al'ada da ke ƙasa da gilashi (Ibidi GmbH, Martinsried, Jamus). Bayan awanni 24, an saka ƙwayoyin LX-2 masu rai da 100 μM MTR na tsawon mintuna 20 a zafin jiki na 37 °C kuma an yi wa ƙwayoyin halitta fenti da DAPI (1 μg/ml, Sigma-Aldrich) kamar yadda aka bayyana a baya [29]. An nuna hanyoyin sadarwa na Mitochondrial ta amfani da na'urar hangen nesa ta Zeiss Axio Observer (Carl Zeiss Microimaging GmbH, Jena, Jamus) wacce aka sanye da na'urar Zeiss LSM 800 confocal module a zafin jiki na 37 °C a cikin yanayi mai laushi tare da 5% CO2 ta amfani da maƙasudin 63 × NA 1.3. Mun sami hotuna goma na jerin Z don kowane nau'in samfurin. Kowace jerin Z ta ƙunshi sassa 30, kowannensu yana da kauri na 9.86 μm. Ga kowane samfurin, an samo hotunan fannoni daban-daban guda goma na gani ta amfani da software na ZEN 2009 (Carl Zeiss Microimaging GmbH, Jena, Jamus), kuma an gudanar da nazarin yanayin mitochondrial ta amfani da software na ImageJ (v1.54d) [30, 31] bisa ga sigogin da aka bayyana a cikin Ƙarin Hanyoyi 3.
An gyara ƙwayoyin halittar da kashi 2% na glutaraldehyde a cikin 0.1 M phosphate buffer, sannan aka haɗa shi da maganin osmium tetroxide na 1% (Sigma Aldrich, MO, Amurka), a hankali aka cire shi da acetone (Merck, Darmstadt, Jamus), sannan a ƙarshe aka saka shi a cikin epoxy resin. An shirya sassan da suka yi siriri sosai kuma aka yi musu fenti da kashi 1% uranyl acetate (Merck, Darmstadt, Jamus) da kuma citrate na gubar 1% (Merck, Darmstadt, Jamus). An samo hotunan ultrastructural ta amfani da na'urar hangen nesa ta JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) a ƙarfin lantarki mai sauri na 80 kV.
An yi nazarin yanayin ƙwayoyin LX-2 da aka yi wa magani da IPA na tsawon awanni 24 ta hanyar amfani da na'urar hangen nesa mai bambanci a mataki-mataki a girman sau 50 ta amfani da na'urar hangen nesa mai haske ta Zeiss (Zeiss Axio Vert.A1 da AxioCam MRm, Jena, Jamus).
An bayyana bayanan asibiti a matsayin matsakaicin ± karkacewar daidaito ko matsakaici (tsakanin tsakanin ƙungiyoyi: IQR). An yi amfani da nazarin bambanci na hanya ɗaya (masu canzawa masu ci gaba) ko gwajin χ² (masu canji na rukuni) don kwatanta bambance-bambance tsakanin ƙungiyoyin binciken guda uku. An yi amfani da ƙimar tabbataccen ƙarya (FDR) don gyara don gwaji da yawa, kuma an ɗauki kwayoyin halitta tare da FDR < 0.05 a matsayin mahimmanci a kididdiga. An yi amfani da nazarin hulɗar Spearman don daidaita methylation na DNA na CpG tare da ƙarfin siginar IPA, tare da rahoton ƙimar p mara suna (p < 0.05).
An gudanar da nazarin hanyoyin ta amfani da kayan aikin nazarin kwayoyin halitta na yanar gizo (WebGestalt) don rubutattun bayanai 268 (p < 0.01 na lamba), rubuce-rubuce 119 da ke da alaƙa da mitochondria (p < 0.05 na lamba), da kuma 4350 CpGs daga cikin rubuce-rubucen hanta 3093 waɗanda ke da alaƙa da matakan IPA na jini. An yi amfani da kayan aikin Venny DB (sigar 2.1.0) da ake samu kyauta don nemo kwayoyin halitta masu haɗuwa, kuma an yi amfani da StringDB (sigar 11.5) don ganin hulɗar furotin da furotin.
Don gwajin LX-2, an gwada samfurori don tabbatar da daidaito ta amfani da gwajin D'Agostino-Pearson. An samo bayanai daga aƙalla kwafi uku na halittu kuma an yi musu ANOVA ta hanya ɗaya tare da gwajin Bonferroni bayan hoc. An ɗauki ƙimar p ƙasa da 0.05 a matsayin mahimmanci a kididdiga. An gabatar da bayanai a matsayin matsakaicin ± SD, kuma an nuna adadin gwaje-gwajen a cikin kowane adadi. An yi duk nazarin da zane-zane ta amfani da software na ƙididdiga na GraphPad Prism 8 na Windows (GraphPad Software Inc., sigar 8.4.3, San Diego, Amurka).
Da farko, mun binciki alaƙar matakan IPA na jini da hanta, dukkan jiki, da kuma bayanan mitochondrial. A cikin jimlar bayanan bayanai, mafi ƙarfin kwayar halitta da ke da alaƙa da matakan IPA na jini shine MAPKPK3 (FDR = 0.0077; furotin kinase-activated mitogen-activated protein kinase-activated protein kinase 3); a cikin bayanin bayanai da ke da alaƙa da mitochondria, mafi ƙarfin kwayar halitta da ke da alaƙa ita ce AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) (Ƙarin fayil 1 da Ƙarin fayil 2).
Sai muka yi nazarin bayanan duniya (n = 268; p < 0.01) da bayanan da suka shafi mitochondria (n = 119; p < 0.05), a ƙarshe muka gano apoptosis a matsayin hanya mafi mahimmanci ta canonical (p = 0.0089). Ga bayanan mitochondrial da ke da alaƙa da matakan IPA na jini, mun mayar da hankali kan apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), da hanyoyin siginar TNF (FDR = 0.000006) (Hoto na 1A, Tebur na 2, da Hotunan Ƙarin 1A-B).
Binciken da aka yi game da bayanan da suka shafi mitochondria, da kuma methylation na DNA a cikin hanta ta ɗan adam dangane da matakan IPA na jini. A yana wakiltar bayanan da suka shafi mitochondria guda 119, da bayanan da suka shafi mitochondria guda 119, da kuma bayanan da suka shafi DNA guda 119 waɗanda aka tsara su zuwa wuraren CpG guda 3092 da ke da alaƙa da matakan IPA na jini (ƙimar p < 0.01 don bayanan da suka shafi duniya da DNA methylated, da kuma dabi'un p < 0.05 don bayanan mitochondrial). An nuna manyan bayanan da suka shafi mitochondria a tsakiya (AKT1 da YKT6). B An gina taswirar hulɗar kwayoyin halitta guda 13 tare da mafi girman maki na hulɗa (0.900) tare da wasu kwayoyin halitta daga kwayoyin halitta guda 56 da suka haɗu (yankin layin baƙi) waɗanda ke da alaƙa da matakan IPA na jini ta amfani da kayan aikin kan layi StringDB. Kore: Kwayoyin halitta da aka tsara zuwa ga sashin ƙwayoyin halitta na Gene Ontology (GO): mitochondria (GO: 0005739). AKT1 shine furotin mai maki mafi girma (0.900) don hulɗa da wasu sunadarai bisa ga bayanai (bisa ga haƙar rubutu, gwaje-gwaje, bayanai, da kuma haɗin gwiwa). Ƙungiyoyin sadarwa suna wakiltar sunadarai, kuma gefuna suna wakiltar haɗi tsakanin sunadarai.
Tunda metabolites na ƙwayoyin cuta na hanji na iya daidaita tsarin halittar epigenetic ta hanyar methylation na DNA [32], mun bincika ko matakan IPA na jini suna da alaƙa da methylation na DNA na hanta. Mun gano cewa manyan wuraren methylation guda biyu da ke da alaƙa da matakan IPA na jini suna kusa da yankin proline-serine mai arzikin serine 3 (C19orf55) da kuma dangin furotin mai girgiza zafi B (ƙaramin) memba 6 (HSPB6) (Ƙarin fayil na 3). methylation na DNA na 4350 CpG (p < 0.01) yana da alaƙa da matakan IPA na jini kuma an wadatar da shi a cikin hanyoyin daidaita tsawon rai (p = 0.006) (Hoto na 1A, Tebur na 2, da Ƙarin Hoto na 1C).
Domin fahimtar hanyoyin nazarin halittu da ke ƙarƙashin alaƙar da ke tsakanin matakan IPA na jini, bayanan da aka tattara a duniya, bayanan da suka shafi mitochondria, da kuma methylation na DNA a cikin hanta ɗan adam, mun yi nazarin kwayoyin halittar da aka gano a cikin binciken hanyoyin da suka gabata (Hoto na 1A). Sakamakon nazarin wadatar hanyoyin kwayoyin halittar da suka haɗu guda 56 (a cikin layin baƙi a Hoto na 1A) ya nuna cewa hanyar apoptosis (p = 0.00029) ta nuna kwayoyin halitta guda biyu da suka zama ruwan dare a cikin binciken uku: AKT1 da YKT6 (YKT6 v-SNARE homolog), kamar yadda aka nuna a cikin zane na Venn (Hoto na Ƙarin 2 da Hoto na 1A). Abin sha'awa, mun gano cewa AKT1 (cg19831386) da YKT6 (cg24161647) suna da alaƙa mai kyau da matakan IPA na jini (Ƙarin fayil na 3). Don gano yiwuwar hulɗar furotin tsakanin samfuran kwayoyin halitta, mun zaɓi kwayoyin halitta 13 tare da mafi girman maki na yanki (0.900) tsakanin kwayoyin halitta 56 da suka haɗu a matsayin shigarwa kuma muka gina taswirar hulɗa. Dangane da matakin amincewa (kwarin gwiwa mai nisa), kwayar halittar AKT1 mai maki mafi girma (0.900) ita ce a saman (Hoto na 1B).
Dangane da nazarin hanyoyin, mun gano cewa apoptosis ita ce babbar hanyar, don haka mun bincika ko maganin IPA zai shafi apoptosis na HSCs a cikin vitro. A baya mun nuna cewa allurai daban-daban na IPA (10 μM, 100 μM, da 1 mM) ba su da guba ga ƙwayoyin LX-2 [15]. Wannan binciken ya nuna cewa maganin IPA a 10 μM da 100 μM ya ƙara yawan ƙwayoyin da ke rayuwa da kuma necrotic. Duk da haka, idan aka kwatanta da ƙungiyar kulawa, ƙarfin ƙwayoyin halitta ya ragu a yawan IPA na 1 mM, yayin da ƙimar necrosis na ƙwayoyin halitta bai canza ba (Hoto na 2A, B). Na gaba, don nemo mafi kyawun yawan da zai haifar da apoptosis a cikin ƙwayoyin LX-2, mun gwada 10 μM, 100 μM, da 1 mM IPA na tsawon awanni 24 (Hoto na 2A-E da Ƙarin Hoto na 3A-B). Abin sha'awa, IPA 10 μM da 100 μM sun rage ƙimar apoptosis (%), duk da haka, IPA 1 mM ta ƙara yawan apoptosis da apoptosis na ƙarshen lokaci (%) idan aka kwatanta da sarrafawa kuma saboda haka aka zaɓi shi don ƙarin gwaje-gwaje (Figures 2A–D).
IPA yana haifar da apoptosis na ƙwayoyin LX-2. An yi amfani da hanyar fenti mai launi biyu ta Annexin V da 7-AAD don auna ƙimar apoptotic da yanayin ƙwayoyin ta hanyar cytometry mai gudana. An saka ƙwayoyin BA da 10 μM, 100 μM da 1 mM IPA na tsawon awanni 24 ko kuma tare da F–H TGF-β1 (5 ng/ml) da 1 mM IPA a cikin matsakaici mara serum na tsawon awanni 24. A: ƙwayoyin halitta masu rai (Annexin V -/ 7AAD-); B: ƙwayoyin necrotic (Annexin V -/ 7AAD+); C, F: farkon (Annexin V +/ 7AAD-); D, G: marigayi (Annexin V+/7AAD.+); E, H: kashi na jimillar ƙwayoyin apoptotic na farko da na ƙarshe a cikin ƙimar apoptotic (%). Ana bayyana bayanai a matsayin matsakaicin ± SD, n = gwaje-gwaje 3 masu zaman kansu. An yi kwatancen ƙididdiga ta amfani da ANOVA ta hanya ɗaya tare da gwajin Bonferroni bayan hoc. *p < 0.05; ****p < 0.0001
Kamar yadda muka nuna a baya, TGF-β1 5 ng/ml na iya haifar da kunna HSC ta hanyar ƙara bayyanar kwayoyin halittar alamun gargajiya [15]. An yi wa ƙwayoyin LX-2 magani da 5 ng/ml TGF-β1 da 1 mM IPA a hade (Hoto na 2E–H). Maganin TGF-β1 bai canza ƙimar apoptosis ba, duk da haka, maganin haɗin gwiwa na IPA ya ƙara yawan apoptosis da apoptosis a ƙarshen (%) idan aka kwatanta da maganin TGF-β1 (Hoto na 2E–H). Waɗannan sakamakon sun nuna cewa 1 mM IPA na iya haɓaka apoptosis a cikin ƙwayoyin LX-2 ba tare da la'akari da shigar TGF-β1 ba.
Mun ƙara bincika tasirin IPA akan numfashin mitochondrial a cikin ƙwayoyin LX-2. Sakamakon ya nuna cewa 1 mM IPA ya rage ma'aunin yawan amfani da iskar oxygen (OCR): numfashin da ba na mitochondrial ba, numfashin basal da mafi girma, fitar da proton da samar da ATP idan aka kwatanta da ƙungiyar kulawa (Hoto na 3A, B), yayin da ma'aunin lafiyar bioenergetic (BHI) bai canza ba.
IPA yana rage numfashin mitochondrial a cikin ƙwayoyin LX-2. An gabatar da lanƙwasa na numfashin mitochondrial (OCR) a matsayin ma'aunin numfashin mitochondrial (numfashi mara mitochondrial, numfashin basal, numfashi mafi girma, zubewar proton, samar da ATP, SRC da BHI). An haɗa ƙwayoyin A da B da 10 μM, 100 μM da 1 mM IPA na tsawon awanni 24, bi da bi. An haɗa ƙwayoyin C da D da TGF-β1 (5 ng/ml) da 1 mM IPA a cikin matsakaici mara serum na tsawon awanni 24, bi da bi. An daidaita duk ma'auni zuwa abubuwan da ke cikin DNA ta amfani da kayan CyQuant. BHI: ma'aunin lafiya mai kuzari; SRC: ƙarfin ajiyar numfashi; OCR: ƙimar amfani da iskar oxygen. An gabatar da bayanai a matsayin matsakaicin ± karkacewar misali (SD), n = gwaje-gwaje 5 masu zaman kansu. An yi kwatancen ƙididdiga ta amfani da gwajin ANOVA da Bonferroni na hanya ɗaya. *p < 0.05; **p < 0.01; kuma ***p < 0.001
Domin samun cikakkiyar fahimtar tasirin IPA akan yanayin bioenergetic na ƙwayoyin LX-2 masu aiki da TGF-β1, mun yi nazarin mitochondrial oxidative phosphorylation ta hanyar OCR (Hoto na 3C,D). Sakamakon ya nuna cewa maganin TGF-β1 zai iya rage matsakaicin numfashi, ƙarfin ajiyar numfashi (SRC) da BHI idan aka kwatanta da ƙungiyar kulawa (Hoto na 3C,D). Bugu da ƙari, maganin haɗin gwiwa ya rage numfashin basal, zubar da proton da samar da ATP, amma SRC da BHI sun fi waɗanda aka yi wa magani da TGF-β1 girma sosai (Hoto na 3C,D).
Mun kuma yi "Gwajin Tsarin Halittar Makamashi na Cellular" wanda software na Seahorse ya bayar (Ƙarin Hoto na 4A–D). Kamar yadda aka nuna a cikin Ƙarin Hoto na 3B, an rage ƙarfin OCR da ECAR na metabolism bayan maganin TGF-β1, duk da haka, babu wani bambanci da aka gani a cikin haɗin gwiwa da ƙungiyoyin maganin IPA idan aka kwatanta da ƙungiyar kulawa. Bugu da ƙari, matakan basal da damuwa na OCR sun ragu bayan haɗin gwiwa da maganin IPA idan aka kwatanta da ƙungiyar kulawa (Ƙarin Hoto na 4C). Abin sha'awa, an lura da irin wannan tsari tare da maganin haɗin gwiwa, inda ba a lura da wani canji a matakan basal da damuwa na ECAR ba idan aka kwatanta da maganin TGF-β1 (Ƙarin Hoto na 4C). A cikin HSCs, raguwar phosphorylation na oxidative mitochondrial da ikon maganin haɗin gwiwa don dawo da SCR da BHI bayan fallasa ga maganin TGF-β1 bai canza ƙarfin metabolism ba (OCR da ECAR). Idan aka haɗa waɗannan sakamakon, waɗannan sakamakon sun nuna cewa IPA na iya rage bioenergetics a cikin HSCs, yana nuna cewa IPA na iya haifar da ƙarancin bayanin kuzari wanda ke canza yanayin HSC zuwa rashin aiki (Karin Hoto na 4D).
An binciki tasirin IPA akan yanayin mitochondrial ta amfani da ƙididdige girman siffofi uku na siffar mitochondrial da haɗin hanyar sadarwa da kuma tabon MTR (Hoto na 4 da Hoto na Ƙari na 5). Hoto na 4 ya nuna cewa, idan aka kwatanta da ƙungiyar kulawa, maganin TGF-β1 ya rage matsakaicin yankin saman, adadin reshe, jimlar tsawon reshe, da lambar mahaɗin reshe (Hoto na 4A da B) kuma ya canza rabon mitochondria daga siffar sphere zuwa matsakaici (Hoto na 4C). Maganin IPA kawai ya rage matsakaicin girman mitochondrial kuma ya canza rabon mitochondria daga siffar sphere zuwa matsakaici idan aka kwatanta da ƙungiyar kulawa (Hoto na 4A). Sabanin haka, sphericity, matsakaicin tsawon reshe, da aikin mitochondrial da aka kimanta ta hanyar MTR mai yuwuwar dogara da membrane na mitochondrial (Hoto na 4A da E) sun kasance ba su canza ba kuma waɗannan sigogi ba su bambanta tsakanin ƙungiyoyi ba. Idan aka haɗa su, waɗannan sakamakon sun nuna cewa maganin TGF-β1 da IPA sun bayyana suna daidaita siffar mitochondrial da girmansa da kuma sarkakiyar hanyar sadarwa a cikin ƙwayoyin LX-2 masu rai.
IPA yana canza yanayin mitochondrial da yawan DNA na mitochondrial a cikin ƙwayoyin LX-2. A. Hotunan confocal masu wakiltar ƙwayoyin LX-2 masu rai waɗanda aka haɗa da TGF-β1 (5 ng/ml) da 1 mM IPA na tsawon awanni 24 a cikin matsakaici mara serum wanda ke nuna cibiyoyin sadarwar mitochondrial da aka fentin da Mitotracker™ Red CMXRos da shuɗi mai nuclei tare da DAPI. Duk bayanan sun ƙunshi aƙalla hotuna 15 a kowace rukuni. Mun sami hotuna 10 na Z-stack don kowane nau'in samfurin. Kowane jerin Z-axis ya ƙunshi yanka 30, kowannensu yana da kauri na 9.86 μm. Ma'aunin sikelin: 10 μm. B. Abubuwan wakilci (mitochondria kawai) waɗanda aka gano ta hanyar amfani da ma'aunin daidaitawa ga hoton. An gudanar da bincike mai ƙima da kwatanta haɗin hanyar sadarwa ta mitochondrial morphological ga duk ƙwayoyin halitta a cikin kowane rukuni. C. Yawan rabon siffar mitochondrial. Ƙimar kusa da 0 tana nuna siffofi masu siffar ƙwallo, kuma ƙimar kusa da 1 tana nuna siffofi masu siffar filamentous. An tantance abubuwan da ke cikin DNA na Mitochondrial (mtDNA) kamar yadda aka bayyana a cikin Kayayyaki da Hanyoyi. An gudanar da nazarin E Mitotracker™ Red CMXRos ta hanyar amfani da cytometry na kwarara (abubuwa 30,000) kamar yadda aka bayyana a cikin Kayayyaki da Hanyoyi. An gabatar da bayanai a matsayin matsakaicin ± SD, n = gwaje-gwaje 3 masu zaman kansu. An gudanar da kwatancen ƙididdiga ta amfani da gwajin ANOVA na hanya ɗaya da Bonferroni bayan hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Sai muka yi nazarin abubuwan da ke cikin mtDNA a cikin ƙwayoyin LX-2 a matsayin alamar lambar mitochondrial. Idan aka kwatanta da ƙungiyar kulawa, abubuwan da ke cikin mtDNA sun ƙaru a cikin ƙungiyar da aka yi wa TGF-β1 magani (Hoto na 4D). Idan aka kwatanta da ƙungiyar da aka yi wa TGF-β1 magani, abubuwan da ke cikin mtDNA sun ragu a cikin ƙungiyar maganin haɗin gwiwa (Hoto na 4D), wanda ke nuna cewa IPA na iya rage abubuwan da ke cikin mtDNA da kuma wataƙila adadin mitochondria da kuma numfashin mitochondrial (Hoto na 3C). Bugu da ƙari, IPA ya yi kama da ya rage abubuwan da ke cikin mtDNA a cikin maganin haɗin gwiwa amma bai shafi ayyukan mitochondrial da MTR ke jagoranta ba (Hoto na 4A–C).
Mun binciki alaƙar IPA da matakan mRNA na kwayoyin halitta da ke da alaƙa da fibrosis, apoptosis, rayuwa, da kuma yanayin mitochondrial a cikin ƙwayoyin LX-2 (Hoto na 5A–D). Idan aka kwatanta da ƙungiyar kulawa, ƙungiyar da aka yi wa magani da TGF-β1 ta nuna ƙaruwar bayyanar kwayoyin halitta kamar su collagen type I α2 chain (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), hana nama na metalloproteinase 1 (TIMP1), da kuma dynamin 1-like gene (DRP1), wanda ke nuna ƙaruwar fibrosis da kunnawa. Bugu da ƙari, idan aka kwatanta da ƙungiyar kulawa, maganin TGF-β1 ya rage matakan mRNA na mai karɓar X na ciki na nukiliya (PXR), caspase 8 (CASP8), MAPKPK3, mai hana B-cell α, mai haɓaka peptide na haske na kwayar halitta na nukiliya (NFκB1A), da kuma mai hana β na kinase subunit na nukiliya (IKBKB) (Hoto na 5A–D). Idan aka kwatanta da maganin TGF-β1, haɗin gwiwa da TGF-β1 da IPA ya rage bayyanar COL1A2 da MMP2, amma ya ƙara matakan mRNA na PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, da IKBKB. Maganin IPA ya rage bayyanar MMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), da mitochondrial fusion protein 2 (MFN2) sosai, yayin da aka ƙara bayyanar CASP8, NFκB1A, NFκB1B, da IKBKB idan aka kwatanta da ƙungiyar kulawa. Duk da haka, babu wani bambanci da aka samu a cikin bayyanar caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), da fission protein 1 (FIS1). A hade, waɗannan sakamakon sun nuna cewa maganin IPA yana daidaita bayyanar kwayoyin halitta da ke da alaƙa da fibrosis, apoptosis, survival, da mitochondrial dynamics. Bayananmu sun nuna cewa maganin IPA yana rage fibrosis a cikin ƙwayoyin LX-2; a lokaci guda, yana ƙarfafa rayuwa ta hanyar canza yanayin zuwa rashin aiki.
IPA tana daidaita bayyanar kwayoyin halittar fibroblast, apoptotic, viability, da mitochondrial dynamics a cikin ƙwayoyin LX-2. Histograms suna nuna bayyanar mRNA dangane da sarrafa endogenous (RPLP0 ko PPIA) bayan an haifar da ƙwayoyin LX-2 tare da TGF-β1 da IPA a cikin matsakaici mara serum na tsawon awanni 24. A yana nuna fibroblasts, B yana nuna ƙwayoyin apoptotic, C yana nuna ƙwayoyin da suka tsira, kuma D yana nuna bayyanar kwayar halittar mitochondrial dynamics. An gabatar da bayanai azaman matsakaicin ± karkacewar daidaito (SD), n = gwaje-gwaje 3 masu zaman kansu. An gudanar da kwatancen ƙididdiga ta amfani da gwajin ANOVA na hanya ɗaya da Bonferroni bayan hoc. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Bayan haka, an tantance canje-canje a girman tantanin halitta (FSC-H) da sarkakiyar cytoplasmic (SSC-H) ta hanyar amfani da cytometry mai gudana (Hoto na 6A,B), kuma an tantance canje-canje a cikin yanayin tantanin halitta bayan maganin IPA ta hanyar amfani da na'urar daukar hoto ta electron transmission electron (TEM) da kuma na'urar daukar hoto mai bambanci (Hoto na Karin Bayani na 6A-B). Kamar yadda aka zata, ƙwayoyin halitta a cikin rukunin da aka yi wa magani da TGF-β1 sun ƙaru a girma idan aka kwatanta da rukunin sarrafawa (Hoto na 6A,B), suna nuna faɗaɗawar gargajiya ta reticulum mai ƙarfi na endoplasmic reticulum (ER*) da phagolysosomes (P), suna nuna kunna ƙwayar halittar hematopoietic (HSC) (Hoto na Karin Bayani na 6A). Duk da haka, idan aka kwatanta da rukunin da aka yi wa magani da TGF-β1, girman tantanin halitta, sarkakiyar cytoplasmic (Hoto na 6A,B), da abun ciki na ER* sun ragu a cikin rukunin maganin haɗin gwiwa na TGF-β1 da IPA (Hoto na Karin Bayani na 6A). Bugu da ƙari, maganin IPA ya rage girman tantanin halitta, sarkakiyar cytoplasm (Hotuna 6A,B), abun ciki na P da ER* (Hoto na Ƙarin 6A) idan aka kwatanta da ƙungiyar sarrafawa. Bugu da ƙari, abun ciki na ƙwayoyin apoptotic ya ƙaru bayan awanni 24 na maganin IPA idan aka kwatanta da ƙungiyar sarrafawa (kibiyoyi fari, Hoto na Ƙarin 6B). Gabaɗaya, waɗannan sakamakon sun nuna cewa 1 mM IPA na iya ƙarfafa apoptosis na HSC da kuma juya canje-canje a cikin sigogin siffa ta tantanin halitta da TGF-β1 ya haifar, ta haka yana daidaita girman tantanin halitta da sarkakiyar tantanin halitta, wanda ƙila yana da alaƙa da rashin kunna HSC.
IPA tana canza girman tantanin halitta da sarkakiyar cytoplasm a cikin ƙwayoyin LX-2. Hotunan wakilci na nazarin cytometry na kwarara. Binciken ya yi amfani da dabarun gating na musamman ga ƙwayoyin LX-2: SSC-A/FSC-A don ayyana yawan tantanin halitta, FSC-H/FSC-A don gano doublets, da SSC-H/FSC-H don nazarin girman tantanin halitta da sarkakiyar tantanin halitta. An haɗa ƙwayoyin halitta da TGF-β1 (5 ng/ml) da 1 mM IPA a cikin matsakaici mara serum na tsawon awanni 24. An rarraba ƙwayoyin LX-2 zuwa ƙananan quarant na hagu (SSC-H-/FSC-H-), quarant na sama na hagu (SSC-H+/FSC-H-), quarant na ƙasa na dama (SSC-H-/FSC-H+), da quarant na sama na dama (SSC-H+/FSC-H+) don nazarin girman tantanin halitta da sarkakiyar cytoplasmic. B. An yi nazarin yanayin ƙwayoyin halitta ta hanyar amfani da tsarin kwararar ƙwayoyin halitta ta amfani da FSC-H (watsawa gaba, girman ƙwayoyin halitta) da SSC-H (watsawa ta gefe, sarkakiyar cytoplasmic) (abubuwan da suka faru 30,000). An gabatar da bayanai a matsayin matsakaicin ± SD, n = gwaje-gwaje 3 masu zaman kansu. An yi kwatancen ƙididdiga ta amfani da gwajin ANOVA na hanya ɗaya da Bonferroni bayan hoc. *p < 0.05; **p < 0.01; ***p < 0.001 da ****p < 0.0001
Abubuwan da ke haifar da ƙwayoyin cuta na hanji kamar IPA sun zama abin da ake tattaunawa a kai, wanda ke nuna cewa ana iya gano sabbin abubuwan da ake so a cikin ƙwayoyin cuta na hanji. Saboda haka, abin sha'awa ne cewa IPA, wani metabolite da muka haɗa shi da fibrosis na hanta a cikin mutane [15], an nuna cewa yana iya zama wani abu mai hana fibrosis a cikin samfuran dabbobi [13, 14]. A nan, mun nuna a karon farko alaƙa tsakanin IPA na jini da transcriptomics na hanta na duniya da methylation na DNA a cikin mutanen da ke da kiba ba tare da ciwon sukari na nau'in 2 ba (T2D), yana nuna apoptosis, mitophagy da tsawon rai, da kuma yiwuwar kwayar halittar AKT1 da ke daidaita homeostasis na hanta. Wani sabon abu na bincikenmu shine cewa mun nuna hulɗar maganin IPA tare da apoptosis, yanayin ƙwayoyin halitta, mitochondrial bioenergetics da dynamics a cikin ƙwayoyin LX-2, yana nuna ƙarancin kuzari wanda ke canza yanayin HSC zuwa rashin aiki, yana mai da IPA zaɓi mai yuwuwa don inganta fibrosis na hanta.
Mun gano cewa apoptosis, mitophagy da tsawon rai sune mafi mahimmancin hanyoyin canonical da aka wadatar a cikin kwayoyin halittar hanta da ke da alaƙa da zagayawa a cikin jini IPA. Rushewar tsarin kula da ingancin mitochondrial (MQC) na iya haifar da rashin aiki na mitochondrial, mitophagy da apoptosis, ta haka yana haɓaka faruwar MASLD[33, 34]. Saboda haka, za mu iya hasashen cewa IPA na iya shiga cikin kiyaye yanayin ƙwayoyin halitta da amincin mitochondrial ta hanyar apoptosis, mitophagy da tsawon rai a cikin hanta. Bayananmu sun nuna cewa kwayoyin halitta guda biyu sun zama ruwan dare a cikin gwaje-gwaje uku: YKT6 da AKT1. Ya kamata a lura cewa YKT6 furotin ne na SNARE da ke da hannu a cikin tsarin haɗakar membrane na tantanin halitta. Yana taka rawa a cikin autophagy da mitophagy ta hanyar ƙirƙirar hadaddun farawa tare da STX17 da SNAP29 akan autophagosome, ta haka yana haɓaka haɗakar autophagosomes da lysosomes [35]. Bugu da ƙari, asarar aikin YKT6 yana haifar da rashin aiki na mitophagy [36], yayin da ƙaruwar YKT6 yana da alaƙa da ci gaban cutar kansar hepatocellular (HCC), wanda ke nuna ƙaruwar rayuwar ƙwayoyin halitta [37]. A gefe guda kuma, AKT1 shine mafi mahimmancin kwayar halitta mai hulɗa kuma yana taka muhimmiyar rawa a cikin cututtukan hanta, gami da hanyar siginar PI3K/AKT, zagayowar ƙwayoyin halitta, ƙaura ta ƙwayoyin halitta, yaduwa, manne mai da hankali, aikin mitochondrial, da fitar da collagen [38-40]. Hanyar siginar PI3K/AKT mai kunnawa na iya kunna ƙwayoyin tushe na hematopoietic (HSCs), waɗanda sune ƙwayoyin da ke da alhakin samar da matrix na extracellular (ECM), kuma rashin daidaituwarsa na iya taimakawa wajen faruwa da ci gaban fibrosis na hanta [40]. Bugu da ƙari, AKT yana ɗaya daga cikin mahimman abubuwan da ke haifar da rayuwa ta ƙwayoyin halitta waɗanda ke hana apoptosis na ƙwayoyin halitta masu dogaro da p53, kuma kunna AKT na iya dangantawa da hana apoptosis na ƙwayoyin hanta [41, 42]. Sakamakon da aka samu ya nuna cewa IPA na iya shiga cikin apoptosis da ke da alaƙa da mitochondria ta hanyar shafar shawarar hepatocytes tsakanin shiga apoptosis ko rayuwa. Waɗannan tasirin na iya kasancewa ƙarƙashin tsarin kwayoyin halittar AKT da/ko YKT6, waɗanda ke da mahimmanci ga homeostasis na hanta.
Sakamakonmu ya nuna cewa 1 mM IPA ya haifar da apoptosis da raguwar numfashin mitochondrial a cikin ƙwayoyin LX-2 ba tare da la'akari da maganin TGF-β1 ba. Abin lura ne cewa apoptosis babbar hanya ce ta warware fibrosis da kunna ƙwayoyin halittar hematopoietic (HSC), kuma muhimmin abu ne a cikin amsawar ilimin halittar hanta mai canzawa [4, 43]. Bugu da ƙari, dawo da BHI a cikin ƙwayoyin LX-2 bayan maganin haɗin gwiwa ya samar da sabbin fahimta game da yuwuwar rawar da IPA ke takawa a cikin daidaita bioenergetics na mitochondrial. A ƙarƙashin yanayin hutawa da rashin aiki, ƙwayoyin hematopoietic yawanci suna amfani da mitochondrial oxidative phosphorylation don samar da ATP kuma suna da ƙarancin aikin metabolism. A gefe guda kuma, kunna HSC yana haɓaka numfashin mitochondrial da biosynthesis don rama buƙatun kuzari na shiga yanayin glycolytic [44]. Gaskiyar cewa IPA bai shafi ƙarfin metabolism da ECAR yana nuna cewa hanyar glycolytic ba ta da fifiko sosai. Hakazalika, wani bincike ya nuna cewa 1 mM IPA ya sami damar daidaita ayyukan sarkar numfashi na mitochondrial a cikin cardiomyocytes, layin ƙwayoyin hepatocyte na ɗan adam (Huh7) da ƙwayoyin endothelial na jijiyoyin jini na ɗan adam (HUVEC); Duk da haka, ba a sami wani tasirin IPA akan glycolysis a cikin cardiomyocytes ba, yana nuna cewa IPA na iya shafar bioenergetics na wasu nau'ikan ƙwayoyin halitta [45]. Saboda haka, muna hasashen cewa 1 mM IPA na iya aiki azaman mai cire sinadarai mai sauƙi, tunda yana iya rage yawan bayyanar kwayoyin fibrogenic, yanayin ƙwayoyin halitta da bioenergetics na mitochondrial ba tare da canza adadin mtDNA ba [46]. Masu cire Mitochondrial na iya hana fibrosis da aka haifar da al'ada da kunna HSC [47] da rage samar da ATP na mitochondrial wanda wasu sunadarai kamar su uncoupling proteins (UCP) ko adenine nucleotide translocase (ANT) ke sarrafawa ko haifarwa. Dangane da nau'in ƙwayoyin halitta, wannan lamari na iya kare ƙwayoyin halitta daga apoptosis da/ko haɓaka apoptosis [46]. Duk da haka, ana buƙatar ƙarin bincike don fayyace rawar da IPA ke takawa a matsayin mai haɗa mitochondrial a cikin rashin aiki na ƙwayoyin halittar jini.
Sai muka binciki ko canje-canje a cikin numfashin mitochondrial suna bayyana a cikin yanayin mitochondrial a cikin ƙwayoyin LX-2 masu rai. Abin sha'awa, maganin TGF-β1 yana canza rabon mitochondrial daga mai siffar ƙwallo zuwa matsakaici, tare da raguwar reshe na mitochondrial da ƙaruwar bayyanar DRP1, babban abin da ke haifar da fission na mitochondrial [48]. Bugu da ƙari, rarrabuwar mitochondrial yana da alaƙa da rikitarwar hanyar sadarwa gabaɗaya, kuma sauyawa daga haɗuwa zuwa fission yana da mahimmanci don kunna ƙwayoyin tushe na hematopoietic (HSC), yayin da hana fission na mitochondrial yana haifar da apoptosis na HSC [49]. Don haka, sakamakonmu ya nuna cewa maganin TGF-β1 na iya haifar da raguwar rikitarwar hanyar sadarwa ta mitochondrial tare da raguwar reshe, wanda ya fi yawa a cikin fission na mitochondrial da ke da alaƙa da ƙwayoyin tushe na hematopoietic da aka kunna (HSCs). Bugu da ƙari, bayananmu sun nuna cewa IPA na iya canza rabon mitochondria daga siffar ƙwallo zuwa matsakaici, ta haka rage bayyanar OPA1 da MFN2. Nazarin ya nuna cewa raguwar OPA1 na iya haifar da raguwar yuwuwar membrane na mitochondrial da kuma haifar da apoptosis na ƙwayoyin halitta [50]. An san MFN2 yana daidaita haɗin mitochondrial da apoptosis [51]. Sakamakon da aka samu ya nuna cewa shigar da ƙwayoyin LX-2 ta hanyar TGF-β1 da/ko IPA yana daidaita siffar da girman mitochondrial, da kuma yanayin kunnawa da sarkakiyar hanyar sadarwa.
Sakamakon bincikenmu ya nuna cewa haɗin gwiwar maganin TGFβ-1 da IPA na iya rage siginar mtDNA da siginar halittar tantanin halitta ta hanyar daidaita bayyanar mRNA na fibrosis, apoptosis da kwayoyin halittar da ke da alaƙa da rayuwa a cikin ƙwayoyin da ke guje wa apoptosis. Hakika, IPA ta rage matakin bayyanar mRNA na AKT1 da mahimman kwayoyin halittar fibrosis kamar COL1A2 da MMP2, amma ta ƙara matakin bayyanar CASP8, wanda ke da alaƙa da apoptosis. Sakamakon bincikenmu ya nuna cewa bayan maganin IPA, bayyanar BAX ta ragu kuma bayyanar mRNA na ƙananan rukunin iyali na TIMP1, BCL-2 da NF-κB ta ƙaru, yana nuna cewa IPA na iya ƙarfafa siginar tsira a cikin ƙwayoyin halittar hematopoietic (HSCs) waɗanda ke guje wa apoptosis. Waɗannan ƙwayoyin na iya aiki azaman siginar tsira a cikin ƙwayoyin halittar hematopoietic da aka kunna, wanda ƙila yana da alaƙa da ƙaruwar bayyanar sunadaran anti-apoptosis (kamar Bcl-2), raguwar bayyanar pro-apoptosis BAX, da kuma haɗakar da ke tsakanin TIMP da NF-κB [5, 7]. IPA tana yin tasirinta ta hanyar PXR, kuma mun gano cewa haɗin gwiwa tare da TGF-β1 da IPA ya ƙara matakan bayyanar PXR mRNA, wanda ke nuna danne kunna HSC. An san cewa siginar PXR da aka kunna tana hana kunna HSC duka a cikin vivo da kuma a cikin vitro [52, 53]. Sakamakonmu ya nuna cewa IPA na iya shiga cikin share HSCs da aka kunna ta hanyar haɓaka apoptosis, rage fibrosis da metabolism na mitochondrial, da haɓaka siginar rayuwa, waɗanda sune hanyoyin da suka saba da ke canza yanayin HSC da aka kunna zuwa wanda ba a kunna ba. Wani bayani mai yuwuwa game da yuwuwar hanyar da rawar IPA ke takawa a cikin apoptosis shine cewa yana tona mitochondria mara aiki galibi ta hanyar mitophagy (hanyar ciki) da hanyar siginar TNF ta waje (Tebur 1), wanda ke da alaƙa kai tsaye da hanyar siginar tsira ta NF-κB (Hoto na Ƙari na 7). Abin sha'awa, kwayoyin halittar da suka wadatar da IPA suna iya haifar da siginar pro-apoptotic da pro-raivival a cikin hanyar apoptotic [54], wanda ke nuna cewa IPA na iya haifar da hanyar apoptotic ko rayuwa ta hanyar hulɗa da waɗannan kwayoyin halitta. Duk da haka, yadda IPA ke haifar da apoptosis ko rayuwa yayin kunna HSC da hanyoyinta na injiniya har yanzu ba a fayyace ba.
IPA wani nau'in metabolite ne na ƙwayoyin cuta da aka samar daga tryptophan na abinci ta hanyar ƙwayoyin cuta na hanji. Nazarin ya nuna cewa yana da kaddarorin hana kumburi, antioxidant, da kuma tsarin epigenetic a cikin yanayin hanji.[55] Nazarin ya nuna cewa IPA na iya daidaita aikin shingen hanji da rage damuwa ta oxidative, wanda zai iya ba da gudummawa ga tasirin ilimin halittarsa ​​na gida.[56] A zahiri, ana jigilar IPA zuwa ga gabobin da aka nufa ta hanyar zagayawar jini, kuma tunda IPA tana da irin wannan babban tsarin metabolite tare da tryptophan, serotonin, da abubuwan da aka samo daga indole, IPA tana yin ayyukan metabolism wanda ke haifar da ƙaddarar metabolism mai gasa.[52] IPA na iya yin gasa da metabolites da aka samo daga tryptophan don wuraren ɗaurewa akan enzymes ko masu karɓa, wanda hakan na iya kawo cikas ga hanyoyin metabolism na yau da kullun. Wannan yana nuna buƙatar ƙarin bincike kan pharmacokinetics da pharmacodynamics don fahimtar taga ta magani mafi kyau.[57] Har yanzu ba a ga ko wannan na iya faruwa a cikin ƙwayoyin tushe na hematopoietic (HSCs).
Mun yarda cewa bincikenmu yana da wasu iyakoki. Domin mu bincika alaƙar da ke da alaƙa da IPA, mun ware marasa lafiya da ke da ciwon suga na nau'in 2 (T2DM). Mun yarda cewa wannan yana iyakance fa'idar bincikenmu ga marasa lafiya da ke da ciwon suga na nau'in 2 da kuma ci gaba da cutar hanta. Duk da cewa yawan sinadarin IPA a cikin jinin ɗan adam shine 1-10 μM [11, 20], an zaɓi yawan sinadarin IPA na 1 mM bisa ga mafi girman yawan da ba shi da guba [15] da kuma mafi girman adadin apoptosis, ba tare da wani bambanci a cikin adadin yawan ƙwayoyin necrotic ba. Duk da cewa an yi amfani da matakan IPA na supraphysiological a cikin wannan binciken, a halin yanzu babu wata yarjejeniya game da ingantaccen adadin IPA [52]. Kodayake sakamakonmu yana da mahimmanci, babban ƙaddarar metabolism na IPA ya kasance wani yanki mai aiki na bincike. Bugu da ƙari, bincikenmu kan alaƙar da ke tsakanin matakan IPA na jini da methylation na DNA na bayanan hanta an samo su ba kawai daga ƙwayoyin tushe na hematopoietic (HSCs) ba har ma daga kyallen hanta. Mun zaɓi amfani da ƙwayoyin LX-2 na ɗan adam bisa ga binciken da muka yi a baya daga nazarin transcriptome cewa IPA tana da alaƙa da kunna ƙwayoyin halitta na hematopoietic (HSC) [15], kuma HSCs su ne manyan ƙwayoyin halitta da ke da hannu a ci gaban fibrosis na hanta. Hanta ta ƙunshi nau'ikan ƙwayoyin halitta da yawa, don haka ya kamata a yi la'akari da wasu samfuran ƙwayoyin halitta kamar tsarin haɗin gwiwar ƙwayoyin halitta na hepatocyte-HSC tare da kunna caspase da rarrabuwar DNA da kuma tsarin aiki gami da matakin furotin don nazarin rawar da IPA ke takawa da hulɗarta da sauran nau'ikan ƙwayoyin hanta.