Tun hmain kan lo report tawh a, liver fibrosis vei zingah hian gut-derived tryptophan metabolite indole-3-propionic acid (IPA) serum level a hniam zawk tih kan lo sawi tawh a. He zirchiannaah hian serum IPA level nena inzawm, thau lutuk liver-a transcriptome leh DNA methylome te kan zirchiang a, chubakah in vitro-a hepatic stellate cells (HSCs) phenotypic inactivation thlentu IPA chanvo kan zirchiang bawk.
He zirchiannaah hian type 2 diabetes mellitus (T2DM) nei lo (kum 46.8 ± 9.3 kum; BMI: 42.7 ± 5.0 kg/m2) mi thau lutuk 116 te chu Kuopio Bariatric Surgery Center (KOBS)-ah bariatric surgery nei an ni. Circulating IPA level chu liquid chromatography-mass spectrometry (LC-MS) hmangin an teh a, liver transcriptome analysis chu total RNA sequencing hmangin an teh a, DNA methylation analysis chu Infinium HumanMethylation450 BeadChip hmangin an enfiah bawk. In vitro experiment atan hian mihring hepatic stellate cell (LX-2) hman a ni.
Serum IPA level hi liver-a apoptotic, mitophagic, leh longevity pathway-a inrawlh genes expression nen a inzawm a ni. Liver transcript leh DNA methylation profile-a interacting gene tam ber leh dominant ber chu AKT serine/threonine kinase 1 (AKT1) gene a ni. IPA hmanga enkawlna hian apoptosis a thlen a, mitochondrial thawkna a tihhniam a, LX-2 cells fibrosis, apoptosis leh dam khawchhuahna tidanglamtu nia hriat genes expression modulate-in cell morphology leh mitochondrial dynamics a tidanglam bawk.
Heng data zawng zawng hi khaikhawm ila, IPA hian therapeutic effects a nei thei tih a nemnghet a, apoptosis a thlen thei a, HSC phenotype chu inactive state lam hawiin a sawn thei a, chu chuan HSC activation leh mitochondrial metabolism tibuaitu liver fibrosis tihtawp theihna a tizau thei a ni.
Thau lutuk leh metabolic syndrome vei tamna hi metabolically associated fatty liver disease (MASLD) vei tamna nen a inzawm tih an sawi a; natna hian mipui zawng zawng 25% atanga 30% vel a nghawng a ni [1]. MASLD etiology rah chhuah ber chu liver fibrosis a ni a, hei hi dynamic process a ni a, fibrous extracellular matrix (ECM) a awm chhunzawm zel a ni Liver fibrosis-a inrawlh ber cells chu hepatic stellate cells (HSCs) te an ni a, phenotype hriat lar pali an nei a, chungte chu quiescent, activated, inactivated leh senescent te an ni [3, 4]. HSCs te hi activate leh transdifferentiate theih a ni a, quiescent form atanga proliferative fibroblast ang cells ah energy mamawhna sang tak neiin, α-smooth muscle actin (α-SMA) leh type I collagen (Col-I) expression a tipung thei a ni [5, Liver fibrosis reversal lai hian activated HSCs te chu apoptosis emaw inactivation emaw hmangin an paih chhuak thin. Heng process te hi fibrogenic genes downregulation leh prosurvival genes (NF-κB leh PI3K/Akt signaling pathways ang chi) modulation te a ni [7, 8], chubakah mitochondrial dynamics leh function inthlak danglamna te
Intestine-a siam tryptophan metabolite indole-3-propionic acid (IPA) serum level chu mihring metabolic natnaah MASLD telin a tlahniam tih hmuhchhuah a ni [10–13]. IPA hi dietary fiber intake nen a inzawm a, antioxidant leh anti-inflammatory effect neia hriat a ni a, diet-induced non-alcoholic steatohepatitis (NASH) phenotype in vivo leh in vitro-ah a ti tlahniam bawk [11– Evidence thenkhat chu kan zirchianna hmasa atanga kan hmuh a ni a, Kuopio Bariatric Surgery Study (KOBS)-a liver fibrosis nei damlo thau lutuk te aiin serum IPA level a hniam zawk tih a tarlang. Chubakah, IPA enkawlna hian mihring hepatic stellate cell (LX-2) model-a cell adhesion, cell migration leh hematopoietic stem cell activation-a classical marker ni thin genes expression a tihtlem theih thu leh hepatoprotective metabolite ni thei tur a nih thu kan hmuchhuak bawk Mahse, IPA hian HSC apoptosis leh mitochondrial bioenergetics activate-in engtin nge liver fibrosis regression a thlen tih erawh hriat chian a la ni lo.
Hetah hian serum IPA hi mi thau tak tak, mahse type 2 diabetes (KOBS) nei lote liver-a apoptosis, mitophagy, leh longevity pathways-a hausa genes expression nen a inzawm tih kan lantir a ni. Chubakah, IPA hian inactivation pathway hmangin activated hematopoietic stem cells (HSCs) clearance leh degradation a tichhuak thei tih kan hmu bawk. Heng results te hian IPA hian chanvo thar a nei tih a tilang a, hei hian liver fibrosis regression tihhmasawn nan therapeutic target awm thei a ni.
KOBS cohort-a zirchianna hmasaah chuan liver fibrosis nei damlote chu liver fibrosis nei lo damlote nena khaikhin chuan circulating IPA level a hniam zawk tih hmuhchhuah a ni Type 2 diabetes-in confounding effect a neih theihna tur chu paih chhuah tumin, KOBS study kalpui mek atang hian type 2 diabetes nei lo (mean age ± SD: 46.8 ± 9.3 years; BMI: 42.7 ± 5.0 kg/m2) (Table 1) mi thau lutuk 116 chu study population angin kan la a He huna tel zawng zawngte hian ziaka hriattirna (informed consent) an pe a, zirchianna protocol chu Declaration of Helsinki (54/2005, 104/2008 leh 27/2010) angin North Savo County Hospital-a Ethics Committee chuan a pawm a ni.
Bariatric surgery neih laiin liver biopsy specimen lak a ni a, a hmaa kan sawi tawh angin pathologist tawnhriat ngah tak takte chuan histologically assess a ni [17, 18]. Assessment criteria te hi Supplementary Table S1-ah hian tarlan a ni a, a hmain kan sawi tawh bawk [19].
Fasting serum sample te chu untargeted liquid chromatography-mass spectrometry (LC-MS) hmangin metabolomics analysis atan an zirchiang a (n = 116). Sample te chu a hmaa kan sawi tawh angin UHPLC-qTOF-MS system (1290 LC, 6540 qTOF-MS, Agilent Technologies, Waldbronn, Karlsruhe, Germany) hmangin an zirchiang a ni19. Isopropyl alcohol (IPA) hriatchhuahna chu retention time leh MS/MS spectrum chu pure standard nena khaikhin a ni. IPA signal intensity (peak area) chu analysis dang zawng zawngah ngaihtuah a ni [20].
Whole liver RNA sequencing chu Illumina HiSeq 2500 hmangin an ti a, a hmaa kan sawi tawh angin data chu preprocess a ni [19, 21, 22]. MitoMiner 4.0 database atanga gene 1957 thlan chhuah hmangin mitochondrial function/biogenesis nghawng thei transcripts targeted differential expression analysis kan nei a Liver DNA methylation analysis chu a hmaa kan sawi tawh ang bawkin Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) hmangin an ti a ni [24, 25].
Mihring hepatic stellate cells (LX-2) chu Prof. Stefano Romeo chuan khawngaihin a pe a, DMEM/F12 medium (Biowest, L0093-500, 1% Pen/Strep; Lonza, DE17-602E, 2% FBS; Gibco, 10270-106)-ah culture leh enkawl a ni. IPA working dose thlan nan LX-2 cells te chu IPA concentration hrang hrang (10 μM, 100 μM leh 1 mM; Sigma, 220027) hmangin DMEM/F12 medium ah 24 h chhung an enkawl a. Chubakah, IPA-in HSCs a inactivate theih dan zirchian nan LX-2 cells te chu 5 ng/ml TGF-β1 (R&D systems, 240-B-002/CF) leh 1 mM IPA nen serum-free medium-ah 24 h chhung co-treat an ni. Vehicle control inmil tan chuan TGF-β1 enkawlna atan 0.1% BSA awmna 4 nM HCL hman a ni a, 0.05% DMSO chu IPA enkawlna atan hman a ni a, an pahnih hian combination treatment atan hman dun a ni.
Apoptosis hi siamtu thupek angin FITC Annexin V Apoptosis Detection Kit 7-AAD (Biolegend, San Diego, CA, USA, Cat# 640922) hmanga teh a ni. A tawi zawngin, LX-2 (1 × 105 cells/well) chu 12-well plate-ah zan khat chhung culture a ni a, chutah chuan IPA emaw IPA leh TGF-β1 dose tam tak hmangin enkawl a ni. A tuk zingah chuan floating leh adherent cells te chu an la khawm a, trypsinized an ni a, PBS hmangin an silfai a, Annexin V binding buffer-ah resuspended an ni a, FITC-Annexin V leh 7-AAD nen 15 min chhung incubate a ni.
Cell nung chhunga mitochondria te chu MitotrackerTM Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) hmangin oxidative activity atan stain an ni. MTR assay atan chuan LX-2 cell te chu IPA leh TGF-β1 te nen density inangah an incubate a. 24 h hnuah cell nungte chu trypsinized an ni a, PBS hmangin an silfai a, chutah chuan a hmaa kan sawi tawh angin 100 μM MTR hmangin serum-free medium-ah 37 °C-ah min 20 chhung an incubate a ni Live cell morphology analysis atan chuan cell size leh cytoplasmic complexity te chu forward scatter (FSC) leh side scatter (SSC) parameter hmangin an zirchiang a.
Data zawng zawng (30,000 events) chu NovoCyte Quanteon (Agilent) hmanga lakkhawm a ni a, NovoExpress® 1.4.1 emaw FlowJo V.10 software hmanga zirchian a ni.
Oxygen consumption rate (OCR) leh extracellular acidification rate (ECAR) te chu Seahorse Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) hmanga siamtu thupek angin Seahorse XF Cell Mito Stress hmanga thuam hmangin real time-in an teh a ni. A tawi zawngin, 2 × 104 LX-2 cells/well chu XF96 cell culture plate-ah seeded a ni. Zan khat chhung an incubate hnuah cell te chu isopropanol (IPA) leh TGF-β1 hmangin an enkawl a (Supplementary Methods 1). Data thlirletna hi Seahorse XF Wave software hmanga tih a ni a, chutah chuan Seahorse XF Cell Energy Phenotype Test Report Generator pawh a tel a ni. Chuta tang chuan Bioenergetic Health Index (BHI) an chhut a ni [27].
Total RNA chu cDNA-ah transcribe a ni. Thiltih dan bikte chu reference [15] en rawh. Mihring 60S ribosomal acidic protein P0 (RPLP0) leh cyclophilin A1 (PPIA) mRNA level te chu constitutive gene control atan hman a ni. QuantStudio 6 pro Real-Time PCR System (Thermo Fisher, Landsmeer, The Netherlands) chu TaqManTM Fast Advanced Master Mix Kit (Applied Biosystems) emaw Sensifast SYBR Lo-ROX Kit (Bioline, BIO 94050) emaw nen hman a ni a, relative gene expression fold chu comparative Ct value cycling parameters (ΔΔCt) leh the ∆∆Ct hmanga tih dan. Primer chungchang chipchiar zawk chu Supplementary Table S2 leh S3-ah tarlan a ni.
Nuclear DNA (ncDNA) leh mitochondrial DNA (mtDNA) te chu a hmaa kan sawi tawh angin DNeasy blood and tissue kit (Qiagen) hmangin an la chhuak a ni [28]. mtDNA relative amount chu target mtDNA region tinte leh nuclear DNA region pathum (mtDNA/ncDNA) geometric mean ratio chhut a ni a, Supplementary Methods 2-ah chipchiar takin tarlan a ni a, mtDNA leh ncDNA tana primers kimchang chu Supplementary Table S4-ah tarlan a ni.
Cell nung te chu MitotrackerTM Red CMXRos (MTR) (Thermo Fisher Scientific, Carlsbad, CA) hmangin an stain a, chu chuan intercellular leh intracellular mitochondrial network te chu a hmu thei a ni. LX-2 cells (1 × 104 cells/well) chu glass slide-ah glass-bottomed culture plate inmil (Ibidi GmbH, Martinsried, Germany)-ah culture a ni. 24 h hnuah LX-2 cell nungte chu 100 μM MTR hmangin 37 °C-ah min 20 chhung incubate a ni a, cell nuclei chu a hmaa kan sawi tawh angin DAPI (1 μg/ml, Sigma-Aldrich) hmangin stain a ni Mitochondrial network te chu Zeiss Axio Observer inverted microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) hmangin Zeiss LSM 800 confocal module hmanga 37 °C-a humidified atmosphere 5% CO2 awmnaah 63×NA 1.3 objective hmangin hmuh theih a ni. Sample chi hrang hrangah Z-series image sawm kan la a. Z-series tin hian section 30 a nei a, a thuk zawng chu 9.86 μm a ni. Sample tin atan ZEN 2009 software (Carl Zeiss Microimaging GmbH, Jena, Germany) hmangin field of view hrang hrang sawm thlalak lak a ni a, Supplementary Methods 3-a parameter chipchiar taka tarlan angin ImageJ software (v1.54d) [30, 31] hmangin mitochondrial morphology analysis tih a ni.
Cell te chu 0.1 M phosphate buffer-ah 2% glutaraldehyde hmangin fixed a ni a, chumi hnuah 1% osmium tetroxide solution (Sigma Aldrich, MO, USA) hmangin fixation a ni a, acetone (Merck, Darmstadt, Germany) hmangin zawi zawiin dehydrated a ni a, a tawpah epoxy resin-ah embedded a ni. Ultrathin section siamin 1% uranyl acetate (Merck, Darmstadt, Germany) leh 1% lead citrate (Merck, Darmstadt, Germany) hmangin stain a ni. Ultrastructural images hi JEM 2100F EXII transmission electron microscope (JEOL Ltd, Tokyo, Japan) hmangin accelerating voltage 80 kV hmanga lak a ni.
IPA hmanga 24 h enkawl LX-2 cells morphology chu Zeiss inverted light microscope (Zeiss Axio Vert.A1 leh AxioCam MRm, Jena, Germany) hmangin 50x magnification-ah phase-contrast microscopy hmangin an zirchiang a.
Clinical data chu mean ± standard deviation emaw median (interquartile range: IQR) anga tarlan a ni. Study group pathumte inthlauhna tehkhin nan one-way analysis of variance (continuous variables) emaw χ2 test (categorical variables) emaw hman a ni. Multiple testing neihna atana siamthat nan false positive rate (FDR) hman a ni a, FDR < 0.05 nei gene te chu statistically significant anga ngaih a ni. Spearman correlation analysis hmangin CpG DNA methylation chu IPA signal intensity nen a inzawm tir a, nominal p value (p < 0.05) report a ni.
Pathway analysis hi web-based gene set analysis tool (WebGestalt) hmangin transcript 268 (nominal p < 0.01), mitochondria-associated transcript 119 (nominal p < 0.05), leh liver transcript 3093 zinga CpG 4350 te chu circulating serum IPA level nena inzawm a ni. Freely available Venny DB (version 2.1.0) tool hmangin overlapping genes an zawng a, StringDB (version 11.5) hmangin protein-protein interaction hmuh theih a ni bawk.
LX-2 experiment atan hian sample te chu D’Agostino-Pearson test hmangin normality test a ni. Data hi biological replicate pathum tal atanga lak niin Bonferroni post hoc test hmangin one-way ANOVA hmangin an enfiah a ni. p-value 0.05 aia tlem chu statistically significant anga ngaih a ni. Data chu mean ± SD anga tarlan a ni a, experiment zat chu figure tinah tarlan a ni. Analysis leh graph zawng zawng hi Windows tana GraphPad Prism 8 statistical software (GraphPad Software Inc., version 8.4.3, San Diego, USA) hmanga tih a ni.
A hmasa berin serum IPA level leh liver, whole-body, leh mitochondrial transcripts inzawmna kan zirchiang a. Total transcript profile-ah chuan serum IPA level nena inzawm gene chak ber chu MAPKAPK3 (FDR = 0.0077; mitogen-activated protein kinase-activated protein kinase 3) a ni a; mitochondria nena inzawm transcript profile-ah chuan associated gene chak ber chu AKT1 (FDR = 0.7621; AKT serine/threonine kinase 1) a ni (Additional file 1 leh Additional file 2).
Chumi hnuah global transcripts (n = 268; p < 0.01) leh mitochondria-associated transcripts (n = 119; p < 0.05) kan zirchiang a, a tawpah chuan apoptosis chu canonical pathway langsar ber (p = 0.0089) a nih thu kan hmuchhuak ta a ni. Serum IPA level nena inzawm mitochondrial transcripts atan chuan apoptosis (FDR = 0.00001), mitophagy (FDR = 0.00029), leh TNF signaling pathways (FDR = 0.000006) te kan ngaihtuah a (Figure 1A, Table 2, leh Supplementary Figures 1A-B).
Serum IPA level nena inzawm global, mitochondria-associated transcripts, leh mihring liver-a DNA methylation te overlapping analysis. A hian global transcript 268, mitochondria-associated transcript 119, leh DNA methylated transcripts a entir a, chu chu serum IPA level nena inzawm CpG site 3092-ah map a ni (global transcript leh DNA methylated tan p value < 0.01, mitochondrial transcript tan p value < 0.05). Overlapping transcript lian ber berte chu a laiah (AKT1 leh YKT6) tarlan a ni. B Gene dang nena interaction score sang ber (0.900) nei gene 13 te interaction map chu online tool StringDB hmangin serum IPA level nena inzawm tlat gene 56 (black line region) atanga siam a ni. Green: Gene Ontology (GO) cellular component: mitochondria (GO:0005739)-a gene map-a siam. AKT1 hi data (text mining, experiment, database, leh co-expression atanga chhut) atanga protein dang nena inzawmna neia score sang ber (0.900) nei protein a ni. Network node hian protein a entir a, edge hian protein inzawmna a entir bawk.
Gut microbiota metabolites hian DNA methylation hmangin epigenetic composition a tidanglam thei a ni [32], serum IPA level hi liver DNA methylation nen a inzawm em tih kan zirchiang a. Serum IPA level nena inzawm methylation site lian pahnih chu proline-serine-rich region 3 (C19orf55) leh heat shock protein family B (small) member 6 (HSPB6) bulah a awm tih kan hmuchhuak (Additional file 3). DNA methylation 4350 CpG (p < 0.01) chu serum IPA level nen a inzawm a, longevity regulatory pathways (p = 0.006)-ah a hausa hle (Figure 1A, Table 2, leh Supplementary Figure 1C).
Serum IPA level, global transcripts, mitochondria-associated transcripts, leh mihring liver-a DNA methylation inzawmna bulpui biological mechanism hriatthiam nan, a hmaa pathway analysis-a genes hmuhchhuah tawhte chu overlap analysis kan nei a (Figure 1A). Overlapping gene 56 (Figure 1A-a black line chhungah) pathway enrichment analysis result-ah chuan apoptosis pathway (p = 0.00029) hian analysis pathum-a common gene pahnih AKT1 leh YKT6 (YKT6 v-SNARE homolog) a tarlang a, Venn diagram-a kan hmuh angin (Supplementary Figure 2 leh Figure 1A). Ngaihven awm tak chu AKT1 (cg19831386) leh YKT6 (cg24161647) te hi serum IPA level nen a inzawm tha hle tih kan hmuchhuak (Additional file 3). Gene product hrang hrangte inkara protein inzawmna awm thei tur hriat theih nan, input atan overlapping gene 56 zingah common region score sang ber (0.900) nei gene 13 kan thlang a, interaction map kan siam a ni. Confidence level (marginal confidence) a zirin AKT1 gene score sang ber (0.900) chu a chung berah a awm a (Figure 1B).
Pathway analysis atanga kan hmuh dan chuan apoptosis chu pathway lian ber a nih avangin IPA hmanga enkawlna hian in vitro-a HSCs apoptosis a nghawng dawn em tih kan zirchiang a. Tun hmain IPA dose hrang hrang (10 μM, 100 μM, leh 1 mM) hi LX-2 cells tan nontoxic a ni tih kan lo hmuchhuak tawh a ni [15]. He zirchianna hian IPA 10 μM leh 100 μM-a enkawlna hian viable leh necrotic cells a tipung tih a tarlang. Mahse, control group nen khaikhin chuan 1 mM IPA concentration-ah cell viability a tlahniam a, cell necrosis rate erawh a danglam lo thung (Figure 2A, B). A dawtah chuan LX-2 cell-a apoptosis thlen thei tur concentration tha ber zawng turin 10 μM, 100 μM, leh 1 mM IPA chu 24 h chhung kan test a (Figure 2A-E leh Supplementary Figure 3A-B). Ngaihven awm tak chu IPA 10 μM leh 100 μM hian apoptosis rate (%) a tihhniam a, mahse, IPA 1 mM hian control nen khaikhin chuan late apoptosis leh apoptosis rate (%) a tisang a, chuvangin experiment dang neih lehna atan thlan a ni (Figures 2A–D).
IPA hian LX-2 cells te apoptosis a thlen thin. Annexin V leh 7-AAD double staining hmangin flow cytometry hmangin apoptotic rate leh cell morphology chu quantified a ni. BA cells chu 10 μM, 100 μM leh 1 mM IPA hmangin 24 h emaw, F–H TGF-β1 (5 ng/ml) leh 1 mM IPA nen serum-free medium-ah 24 h chhung emaw incubate a ni. A: cell nung (Annexin V -/ 7AAD-) te; B: cell thi tawh (Annexin V -/ 7AAD+) te; C, F: hmasa ber (Annexin V +/ 7AAD-); D, G: tlai lam (Annexin V+/7AAD.+); E, H: apoptotic rate-a early leh late apoptotic cell zawng zawng percentage (%). Data chu mean ± SD, n = 3 independent experiment anga tarlan a ni. Statistical comparisons chu Bonferroni post hoc test hmanga one-way ANOVA hmangin an teh a ni. *p < 0.05 a ni a; ****p < 0.0001 a ni
Tun hmaa kan lo tarlan tawh ang khan 5 ng/ml TGF-β1 hian classical marker genes expression tihpunna hmangin HSC activation a tichhuak thei a ni [15]. LX-2 cells te chu 5 ng/ml TGF-β1 leh 1 mM IPA inkawp hmangin an enkawl a (Fig. 2E–H). TGF-β1 enkawlna hian apoptosis rate a tidanglam lo a, mahse, IPA co-treatment hian TGF-β1 enkawlna nen khaikhin chuan late apoptosis leh apoptosis rate (%) a tisang zawk (Fig. 2E–H). Heng results te hian 1 mM IPA hian TGF-β1 induction atanga innghat lovin LX-2 cells ah apoptosis a tichak thei tih a tarlang.
LX-2 cell-a mitochondrial respiration-a IPA-in nghawng a neih dan kan zirchiang lehzual a. Results atanga a lan dan chuan 1 mM IPA hian oxygen consumption rate (OCR) parameters: non-mitochondrial respiration, basal leh maximal respiration, proton leak leh ATP production te chu control group nena khaikhin chuan a tihhniam thung (Figure 3A, B), bioenergetic health index (BHI) erawh a danglam lo thung.
IPA hian LX-2 cell-a mitochondrial respiration a ti tlem a ni. Mitochondrial respiration curve (OCR) hi mitochondrial respiration parameter (non-mitochondrial respiration, basal respiration, maximal respiration, proton leak, ATP generation, SRC leh BHI) angin tarlan a ni. Cell A leh B te chu 10 μM, 100 μM leh 1 mM IPA hmangin 24 h chhung an incubate a. Cell C leh D te chu TGF-β1 (5 ng/ml) leh 1 mM IPA te nen serum-free medium ah 24 h chhung an incubate a. Measurement zawng zawng chu CyQuant kit hmangin DNA content-ah normalized vek a ni. BHI: bioenergetic hriselna tehfung; SRC: thawkna lama reserve theihna; OCR: oxygen hman zat. Data chu mean ± standard deviation (SD) anga tarlan a ni a, n = independent experiment 5 a ni. Statistical comparisons chu one-way ANOVA leh Bonferroni post hoc test hmangin an teh a ni. *p < 0.05 a ni a; **p < 0.01 a ni a; leh ***p < 0.001 a ni
TGF-β1-activated LX-2 cells bioenergetic profile-a IPA-in nghawng a neih dan hriatthiamna zau zawk neih theih nan OCR hmangin mitochondrial oxidative phosphorylation kan zirchiang a (Fig. 3C,D). Results atanga a lan dan chuan TGF-β1 hmanga enkawlna hian control group nena khaikhin chuan maximum respiration, respiratory reserve capacity (SRC) leh BHI a tihhniam thei a ni (Fig. 3C,D). Tin, combination treatment hian basal respiration, proton leak leh ATP production a tihhniam a, mahse SRC leh BHI chu TGF-β1 hmanga enkawl aiin a sang zawk tih a chiang hle (Fig. 3C,D).
Seahorse software hmanga pek “Celular Energy Phenotype Test” pawh kan ti bawk (Supplementary Fig. 4A–D). Supplementary Fig. 3B-a kan hmuh angin TGF-β1 enkawlna hnuah OCR leh ECAR metabolic potential pahnih hi a tlahniam a, mahse, control group nena khaikhin chuan combination leh IPA hmanga enkawl group-ah hian danglamna a awm lo. Chubakah, control group nena khaikhin chuan combination leh IPA treatment hnuah OCR basal leh stress level pahnih a tlahniam a ni (Supplementary Fig. 4C). Ngaihven awm tak chu, combination therapy-ah pawh hetiang bawk hian a awm a, TGF-β1 enkawlna nena khaikhin chuan ECAR basal leh stress level-ah danglamna a awm lo (Supplementary Fig. 4C). HSCs-ah chuan mitochondrial oxidative phosphorylation tlahniam leh TGF-β1 treatment pek hnua combination treatment hmanga SCR leh BHI siam that theihna hian metabolic potential a tidanglam lo (OCR leh ECAR). Heng result zawng zawng hi khaikhawm ila, IPA hian HSCs-a bioenergetics a tihtlem thei tih a tilang a, hei hian IPA hian HSC phenotype chu inactivation lam hawia sawn tirtu energetic profile hniam zawk a thlen thei tih a tilang (Supplementary Figure 4D).
IPA hian mitochondrial dynamics a nghawng dan hi mitochondrial morphology leh network connection te three-dimensional quantification bakah MTR staining hmangin an zirchiang a (Figure 4 leh Supplementary Figure 5). Figure 4-ah hian control group nena khaikhin chuan TGF-β1 hmanga enkawlna hian mean surface area, branch number, total branch length, leh branch junction number a tihhniam bakah mitochondria proportion chu spherical atanga intermediate morphology-ah a thlak danglam tih a tarlang bawk (Figure 4C). IPA hmanga enkawlna chauh hian mean mitochondrial volume a tihhniam bakah control group nena khaikhin chuan mitochondria proportion chu spherical atanga intermediate morphology-ah a thlak danglam bawk (Figure 4A). Chumi danglamna chu mitochondrial membrane potential-dependent MTR (Figure 4A leh E) hmanga sphericity, mean branch length, leh mitochondrial activity te chu a danglam lo va, heng parameter te hi group hrang hrangah a danglam lo. Heng result zawng zawng hi khaikhawm ila, TGF-β1 leh IPA hmanga enkawlna hian mitochondrial shape leh size bakah LX-2 cell nung zingah network complexity a tidanglam niin a lang.
IPA hian LX-2 cell-a mitochondrial dynamics leh mitochondrial DNA tamna a tidanglam a ni. A. TGF-β1 (5 ng/ml) leh 1 mM IPA hmanga serum-free medium-a 24 h chhung incubated LX-2 cell nungte representative confocal images-ah chuan mitochondrial networks MitotrackerTM Red CMXRos hmanga stained leh nuclei DAPI hmanga blue stained te a lang. Data zawng zawngah hian group khatah image 15 tal a awm vek a ni. Sample chi hrang hrangah Z-stack image 10 kan la a. Z-axis sequence tinah hian slice 30 a awm a, a thuk zawng chu 9.86 μm a ni. Scale bar: 10 μm a ni. B. Representative objects (mitochondria chauh) image-a adaptive thresholding hmanga hriatchhuah. Group tina cell zawng zawng tan quantitative analysis leh mitochondrial morphological network connection tehkhin a ni. C. Mitochondrial shape ratio awm tam dan. Value 0 hnaih chuan spherical shape a tarlang a, 1 hnaih value chuan filamentous shape a tarlang bawk. D Mitochondrial DNA (mtDNA) awm zat chu Materials and Methods-a tarlan angin an chhut a. E MitotrackerTM Red CMXRos analysis hi Materials and Methods-a tarlan angin flow cytometry (thil 30,000) hmangin an ti a. Data chu mean ± SD, n = 3 independent experiment angin tarlan a ni. Statistical comparisons chu one-way ANOVA leh Bonferroni post hoc test hmangin an teh a ni. *p < 0.05 a ni a; **p < 0.01 a ni a; ***p < 0.001 a ni a; ****p < 0.0001 a ni
Chumi hnuah LX-2 cell-a mtDNA awm zat chu mitochondrial number chhinchhiahna atan kan zirchiang a. Control group nen khaikhin chuan TGF-β1 hmanga enkawl group-ah mtDNA content a sang zawk a ni (Figure 4D). TGF-β1 hmanga enkawl group nen khaikhin chuan combination treatment group-ah mtDNA content a tlahniam a (Figure 4D), hei hian IPA hian mtDNA content leh mitochondrial number bakah mitochondrial respiration a tihhniam thei tih a tilang (Figure 3C). Chubakah, IPA hian combination treatment-ah mtDNA awm zat a tihtlem niin a lang a, mahse MTR-mediated mitochondrial activity erawh a nghawng lo (Figures 4A–C).
LX-2 cells-a fibrosis, apoptosis, survival, leh mitochondrial dynamics nena inzawm genes mRNA level nena IPA inzawmna kan zirchiang a (Figure 5A–D). Control group nena khaikhin chuan TGF-β1 hmanga enkawl group-ah hian collagen type I α2 chain (COL1A2), α-smooth muscle actin (αSMA), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP1), leh dynamin 1-like gene (DRP1) te ang gene expression a sang zawk tih hmuhchhuah a ni a, hei hian a pung tih a tilang fibrosis leh activation te a awm bawk. Chubakah, control group nena khaikhin chuan TGF-β1 enkawlna hian nuclear pregnane X receptor (PXR), caspase 8 (CASP8), MAPKAPK3, B-cell α inhibitor, nuclear factor κ gene light peptide (NFκB1A) enhancer, leh nuclear factor κB kinase subunit β (IKBKB) inhibitor mRNA level a tihhniam bawk (Figure 5A–D). TGF-β1 enkawlna nena khaikhin chuan TGF-β1 leh IPA hmanga enkawlna hian COL1A2 leh MMP2 expression a ti tlem a, mahse PXR, TIMP1, B-cell lymphoma-2 (BCL-2), CASP8, NFκB1A, NFκB1-β, leh IKBKB mRNA level a tisang thung. IPA hmanga enkawlna hian MMP2, Bcl-2-associated protein X (BAX), AKT1, optic atrophy protein 1 (OPA1), leh mitochondrial fusion protein 2 (MFN2) expression nasa takin a tihhniam a, chutih laiin control group nena khaikhin chuan CASP8, NFκB1A, NFκB1B, leh IKBKB te expression a tipung thung. Mahse, caspase-3 (CASP3), apoptotic peptidase activating factor 1 (APAF1), mitochondrial fusion protein 1 (MFN1), leh fission protein 1 (FIS1) te expression-ah danglamna a awm lo. Heng result zawng zawng hi a pum puia thlir chuan IPA enkawlna hian fibrosis, apoptosis, survival, leh mitochondrial dynamics nena inzawm genes expression a modulate tih a tilang. Kan data atanga a lan dan chuan IPA enkawlna hian LX-2 cell-a fibrosis a tihziaawm a; chutih rual chuan phenotype chu inactivation lam hawiin a sawn a, dam khawchhuahna a tichak bawk.
IPA hian LX-2 cell-a fibroblast, apoptotic, viability, leh mitochondrial dynamics genes expression a modulate a ni. Histogram-ah hian LX-2 cells te chu serum-free medium-ah 24 h chhung TGF-β1 leh IPA hmanga induce a nih hnuah endogenous control (RPLP0 emaw PPIA) nena khaikhin chuan mRNA expression a lang. A hian fibroblasts a tarlang a, B hian apoptotic cells a tarlang a, C hian cell damchhuak a tarlang a, D hian mitochondrial dynamics gene expression a tarlang bawk. Data chu mean ± standard deviation (SD) anga tarlan a ni a, n = independent experiment 3 a ni. Statistical comparisons chu one-way ANOVA leh Bonferroni post hoc test hmangin an teh a ni. *p < 0.05 a ni a; **p < 0.01 a ni a; ***p < 0.001 a ni a; ****p < 0.0001 a ni
Tichuan, cell size (FSC-H) leh cytoplasmic complexity (SSC-H) inthlak danglamna chu flow cytometry hmangin an zirchiang a (Figure 6A,B), IPA treatment hnua cell morphology inthlak danglamna chu transmission electron microscopy (TEM) leh phase contrast microscopy hmangin an zirchiang bawk (Supplementary Figure 6A-B). Beisei ang ngeiin TGF-β1 hmanga enkawl group-a cell-te chu control group nena khaikhin chuan an lian zawk a (Figure 6A,B), hei hian rough endoplasmic reticulum (ER*) leh phagolysosomes (P) te classic expansion a lantir a, hei hian hematopoietic stem cell (HSC) activation a tilang a ni (Supplementary Figure 6A). Mahse, TGF-β1 hmanga enkawl group nen khaikhin chuan TGF-β1 leh IPA combination treatment group-ah hian cell size, cytoplasmic complexity (Fig. 6A,B), leh ER* content a tlahniam a (Supplementary Fig. 6A). Chubakah, IPA hmanga enkawlna hian control group nena khaikhin chuan cell size, cytoplasmic complexity (Figs. 6A,B), P leh ER* content (Supplementary Fig. 6A) a tihhniam bawk. Chu bakah, IPA enkawlna 24 h hnuah control group nena khaikhin chuan apoptotic cells awm zat a pung bawk (white arrows, Supplementary Fig. 6B). Heng result zawng zawng hi a pum puia thlir chuan 1 mM IPA hian HSC apoptosis a tichak thei a, TGF-β1-in a thlen cell morphological parameters inthlak danglamna chu a tidanglam thei a, chu chuan cell size leh complexity a tidanglam thei a, hei hi HSC inactivation nen a inzawm thei a ni.
IPA hian LX-2 cell-a cell size leh cytoplasmic complexity a tidanglam thin. Flow cytometry hmanga zirchianna aiawhtu thlalak. Analysis-ah hian LX-2 cells tana bik gating strategy hman a ni a, chungte chu cell population sawifiahna atan SSC-A/FSC-A, doublets hriatchhuahna atan FSC-H/FSC-A, leh cell size leh complexity analysis atan SSC-H/FSC-H te hman a ni. Cell te chu TGF-β1 (5 ng/ml) leh 1 mM IPA nen serum-free medium-ah 24 h chhung an incubate a. LX-2 cells te chu cell size leh cytoplasmic complexity analysis atan lower left quadrant (SSC-H-/FSC-H-), upper left quadrant (SSC-H+/FSC-H-), lower right quadrant (SSC-H-/FSC-H+), leh upper right quadrant (SSC-H+/FSC-H+) ah te sem a ni. B. Cell morphology chu flow cytometry hmangin FSC-H (forward scatter, cell size) leh SSC-H (side scatter, cytoplasmic complexity) (30,000 events) hmangin an zirchiang a. Data chu mean ± SD, n = 3 independent experiment angin tarlan a ni. Statistical comparisons chu one-way ANOVA leh Bonferroni post hoc test hmangin an teh a ni. *p < 0.05 a ni a; **p < 0.01 a ni a; ***p < 0.001 leh ****p < 0.0001 a ni
Gut metabolites IPA ang chi hi zirchiannaah chuan thupui lar tak a ni tawh a, hei hian gut microbiota-ah hian target thar hmuhchhuah a nih theih thu a sawi. Chuvangin mihringah liver fibrosis nena kan inzawm metabolite IPA hi rannung model-ah anti-fibrotic compound a nih theih thu hmuhchhuah a ni tih hi ngaihnawm tak a ni [13, Hetah hian a vawi khat nan type 2 diabetes (T2D) nei lo mi thau tak takte serum IPA leh global liver transcriptomics leh DNA methylation inzawmna kan entir a, apoptosis, mitophagy leh dam rei theihna te, chubakah liver homeostasis tidanglamtu candidate gene awm thei AKT1 te pawh a tarlang bawk. Kan zirchianna thil thar dang chu IPA enkawlna leh LX-2 cell-a apoptosis, cell morphology, mitochondrial bioenergetics leh dynamics inzawmna kan lantir a, hei hian HSC phenotype chu inactivation lam hawia sawn tirtu energy spectrum hniam zawk a nih thu a tarlang a, hei hian IPA hi liver fibrosis tihchangtlunna atana candidate awm thei a ni.
Kan hmuh dan chuan apoptosis, mitophagy leh dam rei te hi circulating serum IPA nena inzawm liver genes enriched canonical pathway pawimawh ber an ni. Mitochondrial quality control (MQC) system tihbuai hian mitochondrial dysfunction, mitophagy leh apoptosis a thlen thei a, chu chuan MASLD lo awm theihna a tipung thei a ni[33, Chuvangin, IPA hi apoptosis, mitophagy leh liver-a dam rei tak hmanga cell dynamics leh mitochondrial integrity vawn thatnaah a inrawlh thei tih kan ngaihtuah thei a ni. Kan data atanga a lan dan chuan assay pathum zingah hian gene pahnih a awm tlangpui a, chungte chu YKT6 leh AKT1 te an ni. Hriat tur chu YKT6 hi SNARE protein a ni a, cell membrane fusion kalpui danah a inrawlh a ni. Autophagosome-a STX17 leh SNAP29 te nen initiation complex siamin autophagy leh mitophagy-ah chanvo a nei a, chu chuan autophagosomes leh lysosomes te fusion a tichak a ni Chubakah, YKT6 hnathawh hloh hian mitophagy a tichhe a ni A lehlamah chuan AKT1 hi interacting gene pawimawh ber a ni a, liver natnaah pawh hmun pawimawh tak a chang a, chung zingah chuan PI3K/AKT signaling pathway, cell cycle, cell migration, proliferation, focal adhesion, mitochondrial function, leh collagen secretion te pawh a tel Activated PI3K/AKT signaling pathway hian hematopoietic stem cells (HSCs) a tichak thei a, chungte chu extracellular matrix (ECM) siamna atana mawhphurtu cell an ni a, a dysregulation hian liver fibrosis lo awm leh kal zelnaah a pui thei bawk Chu bakah, AKT hi p53-dependent cell apoptosis titawptu cell dam khawchhuahna atana pawimawh ber pakhat a ni a, AKT activation hi liver cell apoptosis titawptu nen a inzawm thei bawk[41, Results hmuh chhuah atang hian IPA hian hepatocytes te apoptosis luh emaw damchhuah emaw inkara thutlukna siamna a nghawng avangin liver mitochondria-associated apoptosis ah a inrawlh thei tih a tarlang. Heng effects te hi AKT leh/ emaw YKT6 candidate genes te hian an control thei a, hei hi liver homeostasis atan a pawimawh hle.
Kan result atanga kan hmuh dan chuan 1 mM IPA hian TGF-β1 treatment atanga innghat lo LX-2 cells ah apoptosis a thlen a, mitochondrial respiration a tihhniam bawk. Hriat tur pawimawh tak chu apoptosis hi fibrosis resolution leh hematopoietic stem cell (HSC) activation kawng lian tak a ni a, liver fibrosis reversible physiological response-a thil thleng pawimawh tak a ni bawk [4, Chubakah, combination treatment hnua LX-2 cells-a BHI lo awm lehna hian mitochondrial bioenergetics regulation-a IPA-in a chanvo a neih theih dan tur hriatna thar a pe a ni. Chawlh leh thawk lo dinhmunah chuan hematopoietic cells te hian mitochondrial oxidative phosphorylation hmangin ATP an siam chhuak tlangpui a, metabolic activity pawh an nei tlem hle. A lehlamah chuan HSC activation hian mitochondrial respiration leh biosynthesis a tichak a, chu chuan glycolytic state-a luh theihna tura energy mamawh chu a phuhruk thei a ni [44]. IPA hian metabolic potential leh ECAR a nghawng lo tih hian glycolytic pathway hi ngaih pawimawh a ni lo tih a tilang. Chutiang bawkin zirchianna dang pakhatah chuan 1 mM IPA hian cardiomyocytes, human hepatocyte cell line (Huh7) leh human umbilical vein endothelial cells (HUVEC)-a mitochondrial respiratory chain activity chu a tidanglam thei tih hmuhchhuah a ni a Mahse, IPA hian cardiomyocyte-a glycolysis-ah nghawng a nei lo tih hmuhchhuah a ni a, hei hian IPA hian cell chi dang bioenergetics a nghawng thei tih a tilang Chuvangin, 1 mM IPA hian mtDNA awm zat tidanglam lovin fibrogenic gene expression, cell morphology leh mitochondrial bioenergetics te nasa takin a tihtlem theih avangin mild chemical uncoupler angin hna a thawk thei niin kan ngai Mitochondrial uncouplers hian culture-induced fibrosis leh HSC activation te a titawp thei a ni Cell chi hrang hrang a zirin he thil thleng hian cell te chu apoptosis lakah a veng thei a, apoptosis a tichak thei bawk [46]. Mahse, hematopoietic stem cell inactivation-a mitochondrial uncoupler anga IPA chanvo hriat chian nan zirchianna dang neih belh a ngai a ni.
Chumi hnuah chuan mitochondrial respiration inthlak danglamna hi LX-2 cell nung mitochondrial morphology-ah a lang em tih kan zirchiang a. Ngaihven awm tak chu TGF-β1 enkawlna hian mitochondrial proportion chu spherical atanga intermediate-ah a thlak danglam a, mitochondrial branching a tlahniam a, mitochondrial fission-a thil pawimawh tak DRP1 expression a tipung bawk Chubakah, mitochondrial fragmentation hi network complexity pum pui nen a inzawm a, fusion atanga fission a inthlak hi hematopoietic stem cell (HSC) activation atan a pawimawh hle a, mitochondrial fission inhibition erawh chuan HSC apoptosis a thlen thung Chutiang chuan kan results chuan TGF-β1 enkawlna hian branching tlahniam nen mitochondrial network complexity tihhniam a thlen thei tih a tarlang a, hei hi activated hematopoietic stem cells (HSCs) nena inzawm mitochondrial fission-ah a tam zawk a ni. Chubakah, kan data atanga a lan dan chuan IPA hian mitochondria proportion chu spherical atanga intermediate shape ah a thlak thei a, chu chuan OPA1 leh MFN2 expression a ti tlem thei a ni. Zirna hrang hrangah OPA1 downregulation hian mitochondrial membrane potential a tihhniam thei a, cell apoptosis a tichhuak thei tih hmuhchhuah a ni MFN2 hian mitochondrial fusion leh apoptosis a mediate tih hriat a ni[51]. Results hmuh chhuah dan chuan TGF-β1 leh/ emaw IPA hmanga LX-2 cells induction hian mitochondrial shape leh size a modulate niin a lang a, chubakah activation state leh network complexity pawh a modulate niin a lang.
Kan results chuan TGFβ-1 leh IPA te combination treatment hian apoptosis-evading cells-a fibrosis, apoptosis leh survival-related genes mRNA expression regulate-in mtDNA leh cell morphological parameters a tihhniam thei tih a tarlang. Dik tak chuan IPA hian AKT1 leh fibrosis gene pawimawh COL1A2 leh MMP2 te mRNA expression level a tihhniam a, mahse CASP8 expression level a tisang a, hei hi apoptosis nen a inzawm a ni. Kan result-ah chuan IPA hmanga enkawl hnuah BAX expression a tlahniam a, TIMP1 family subunit, BCL-2 leh NF-κB mRNA expression a pung a, hei hian IPA hian apoptosis pumpelh thei hematopoietic stem cells (HSCs)-a survival signal a tichak thei tih a tilang. Heng molecule te hian activated hematopoietic stem cells-ah pro-survival signal angin hna an thawk thei a, hei hi anti-apoptotic proteins (Bcl-2 ang chi) expression tihpunna, pro-apoptotic BAX expression tlahniam, leh TIMP leh NF-κB inkara complex interplay te nen a inzawm thei a ni [5, IPA hian PXR hmangin a nghawng a nei a, TGF-β1 leh IPA hmanga enkawlna hian PXR mRNA expression level a tisang tih kan hmu a, hei hian HSC activation tihtawp a nih thu a tarlang. Activated PXR signaling hian in vivo leh in vitro-ah HSC activation a titawp tih hriat a ni [52, 53]. Kan result hian IPA hian activated HSCs clearance-ah a tel thei tih a tarlang a, apoptosis a tichak a, fibrosis leh mitochondrial metabolism a tihtlem bakah survival signals a tichak a, hei hi activated HSC phenotype chu inactivated-a chantirtu process pangngai a ni. IPA hian apoptosis-a a awm theih mechanism leh a chanvo sawifiahna dang chu mitochondria hnathawk thei lo chu mitophagy (intrinsic pathway) leh extrinsic TNF signaling pathway (Table 1) hmangin a scavenge ber a, hei hi NF-κB survival signaling pathway nen direct-a inzawm a ni (Supplementary Figure 7). Ngaihven awm tak chu IPA nena inzawm enriched genes te hian apoptotic pathway ah pro-apoptotic leh pro-survival signals an siam thei a ni [54], hei hian IPA hian heng genes te nena inzawmna neiin apoptotic pathway emaw survival emaw a tichhuak thei tih a tilang a ni. Mahse, HSC activation laiin IPA hian engtin nge apoptosis emaw damchhuahna emaw a thlen tih leh a mechanistic pathways te hi a la chiang lo.
IPA hi microbial metabolite a ni a, dietary tryptophan atanga gut microbiota hmanga siam a ni. Zirna hrang hrangah chuan intestinal environment-ah anti-inflammatory, antioxidant, leh epigenetic regulatory property a nei tih hmuhchhuah a ni.[55] Zirna hrang hrangah IPA hian intestinal barrier function a tidanglam thei a, oxidative stress a tihziaawm thei a, hei hian a local physiological effects a thlen thei a ni. Dik tak chuan IPA hi circulation kaltlangin target organs-ah a kal a, IPA hian tryptophan, serotonin leh indole derivatives te nen hian major metabolite structure inang chiah chiah a neih avangin IPA hian metabolic action a nei a, chu chuan competitive metabolic fates a thlen a ni. IPA hian tryptophan atanga chhuak metabolites te nen enzyme emaw receptor emaw a binding sites atan an inel thei a, hei hian metabolic pathway pangngai a tibuai thei a ni. Hei hian a therapeutic window hriatthiamna tha zawk neih theih nan a pharmacokinetics leh pharmacodynamics chungchanga zirchianna neih belh a tulzia a tilang chiang hle.[57] Hei hi hematopoietic stem cells (HSCs)-ah pawh a thleng thei em tih chu hriat a la ni lo.
Kan zirlai hian tihkhawtlai engemaw zat a nei tih kan pawm a ni. IPA nena inzawm inzawmnate chiang taka enfiah turin type 2 diabetes mellitus (T2DM) vei te chu kan paih chhuak a ni. Hei hian kan thil hmuhchhuahte hi type 2 diabetes mellitus leh advanced liver disease vei damlote tana hman theihna zau tak a tikhawtlai tih kan pawm a ni. Mihring serum-a IPA physiological concentration chu 1–10 μM ni mahse [11, 20], non-toxic concentration sang ber [15] leh apoptosis rate sang ber atanga thlirin 1 mM IPA concentration chu thlan a ni a, necrotic cell population percentage-ah danglamna a awm lo. He zirchiannaah hian IPA supraphysiological levels hman ni mah se, tunah hian IPA hman tangkai dan tur chungchangah hian inremna a awm lo [52]. Kan result hi a langsar hle nachungin, IPA metabolic fate zau zawk hi zirchianna active area a la ni reng a ni. Chubakah, serum IPA level leh liver transcripts DNA methylation inzawmna chungchanga kan thil hmuhchhuahte hi hematopoietic stem cells (HSCs) atang chauh ni lovin liver tissue atang pawhin kan hmu chhuak bawk. IPA hi hematopoietic stem cell (HSC) activation nen a inzawm tih transcriptome analysis atanga kan hmuhchhuah tawh atanga mihring LX-2 cells hman kan thlang a ni Liver hi cell chi hrang hrang atanga siam a nih avangin cell model dang hepatocyte-HSC-immune cell co-culture system te chu caspase activation leh DNA fragmentation nena inzawm bakah protein level telin a hnathawh dan te ngaihtuah a ngai a, IPA chanvo leh liver cell chi dang nena a inzawmna zir chian a ngai a ni.