NMR-Based Metabolomic Analysis rau Cov teebmeem ntawm -Ketoglutarate Supplementation Ntawm C2C12 Myoblasts nyob rau hauv txawv zog xeev

Nyem qhov no kom tau txais Cistanche Anti-aging thiab qaug zog pab ntxiv

1. Taw qhia

Cov leeg pob txha yog lub cev loj tshaj plaws nyob rau hauv tib neeg lub cev thiab tswj kev ua neej ib txwm muaj.Kev cob qhia ua haujlwm ntevlos yog pathologyl cov txheej txheem ntawm cov kab mob ua rau cov leeg nqaij puaslos yogcov leeg nqaij tsis muaj zog txaus, nyob rau lub sijhawm no, kev rov ua kom cov leeg nqaij muaj zog thiab ua haujlwm tseem ceeb tshwj xeeb. Ntau yam khoom noj khoom haus tau ua haujlwm los txhawb cov leeg nqaij pob txha hypertrophy thiab txhim kho kev ua kis las [1]. Raws li kev sib tshuam ntawm cov organic carbon thiab nitrogen metabolism thiab ib txhij ib qho tseem ceeb nruab nrab ntawm TCA lub voj voog, -Ketoglutarate (AKG) tau pom cov teebmeem pleiotropic rau kev txhim kho cov leeg nqaij hauv kev sim tshuaj thiab tsiaj txhu [2–9]. Piv txwv li, AKG tuaj yeem txo cov plab hnyuv mucosal puas tsuaj [8] thiab o [9], ua rau txo qis.kev loj hlob ntawm mob qog nqaij hlav hauv plab[4] thiab daim siab fibrosis [5], txhawb kev loj hlob [6], thiab txo morbidity thiabncua kev laus[7]. Cov hauj lwm yav dhau los tau ua pov thawj tias AKG supplementation tuaj yeem txo qis cov leeg nqaij los ntawm kev tswj hwm ntawm parenteral hauv cov qauv kev raug mob ntawm cov neeg mob uas tau hloov tag nrho lub duav [2], thiab txhawb cov leeg nqaij hypertrophy thiab protein synthesis los ntawm Akt / mTOR signaling pathways [10,11]. Nyob rau hauv cov ntaub ntawv ntawm Duchenne cov leeg nqaij dystrophy, AKG supplementation tuaj yeem tiv thaiv cov leeg nqaij atrophy thiab kev ua haujlwm tsis zoo los ntawm PHD3 / ADRB2-txoj kev sib kho [12]. Peb cov hauj lwm yav dhau los kuj tau pom tias AKG supplementation tuaj yeem pab txhawb kev loj hlob ntawm C2C12 myoblasts, thiab txo qhov atrophy ntawm C2C12 myotubes kab lis kev cai hauv qhov nruab nrab tsis muaj piam thaj [13]. Muab hais tias cov piam thaj yog lub hauv paus tseem ceeb ntawm lub zog los tswj lub cev kev ua haujlwm ntawm cov leeg pob txha, cov teebmeem ntawm AKG supplementation rau kev txhim kho cov leeg nqaij yog nyob ntawm cov piam thaj hauv cov leeg pob txha. Qhov sib txawv ntawm AKG-induced los ntawm cov leeg pob txha ntawm lub zog sib txawv tsis meej.

AKG koom nrog hauv kev tsim cov amino acids, vitamins, thiaborganic acidsthiablub zog metabolismHauv lub cev, uas koom nrog kev hloov pauv ntawm AKG rau hauv glutamate los ntawm glutamate dehydrogenase, thiab tom qab amidation ntawm glutamate nrog ammonia los ntawm glutamine synthetase. AKG muab lub zog rau kev loj hlob ntawm tes los ntawm TCA lub voj voog thiab oxidative phosphorylation thiab txhawb nqa cov metabolism hauv cell thiab kev taw qhia los ntawm kev cuam tshuam nrog nws cov receptor OXGR (a G protein-coupled receptor) ntawm cell membrane [14,15]. Ntxiv mus, AKG tuaj yeem tswj hwm mitochondrial oxidative metabolism thiab txhawb kev tso cai ntawm lub xeev epigenetic los ntawm kev sib kho thaum ntxov embryonic cell xeev hloov thiab kab mob cell kev loj hlob [16]. Tsis tas li ntawd, kev tawm dag zog AKG tuaj yeem txhawb nqa nws cov receptor OXGR1 hauv cov qog adrenal los tswj cov thermogenesis thiab kev tawg ntawm triglycerides hauv cov ntaub so ntswg adipose thiab tsim cov txiaj ntsig zoo ntawm cov metabolism [17].

Txawm li cas los xij, ob peb txoj kev tshawb fawb tau ua los qhia txog AKG-vim kev hloov pauv ntawm cov leeg pob txha hauv lub zog sib txawv thiab cov txheej txheem metabolic hauv qab. Tsis ntev los no, kev tshuaj xyuas metabolomic tau ua haujlwm los ua kom pom tseeb cov txheej txheem molecular hauv qab cov txiaj ntsig zoo ntawm kev noj zaub mov ntxiv. Kev hloov pauv ntawm cov metabolites ua raws li cov khoom lag luam qis ntawm cov noob hloov pauv tuaj yeem cuam tshuam tag nrho cov kev hloov pauv hauv metabolic intuitively. Raws li cov txheej txheem tshwj xeeb tsim nyog rau kev txheeb xyuas qhov kev hloov pauv ntawm cov qib metabolite hauv biofluids, cov ntaub so ntswg, thiab cov hlwb, kev daws teeb meem siab, 1H nuclear magnetic resonance (NMR) spectroscopy tau siv dav hauv kev tshuaj ntsuam metabolomic. Qhov tseem ceeb, NMR-raws li metabolomic profileing muaj ntau yam zoo xws li kev tsim tawm siab, kev ntsuas ntau yam tsis muaj kev txiav txim siab, thiab kev npaj ua qauv yooj yim [18,19]. Yav dhau los, peb tau ua NMR-raws li kev tshuaj ntsuam xyuas metabolomic rau elucidating ob qho tib si cov teebmeem ntawm creatine supplementation ntawm C2C12 myoblasts [20], thiab cov teebmeem ntawm alanyl-glutamine supplementation ntawm myoblasts raug mob los ntawm lub zog deprivation [21].

2. Cov txiaj ntsig

2.1. Kev loj hlob thiab kev sib txawv ntawm C2C12 Myoblasts nrog AKG Supplementation

C2C12 myoblast cells cultured nyob rau hauv ib qho kev loj hlob nruab nrab nrog thiab tsis muaj AKG supplementation tau pab pawg raws li Nor-A thiab Nor, whereas cov nyob rau hauv qis-glucose loj hlob nruab nrab nrog los yog tsis muaj AKG supplementation tau pab pawg ua Low-A thiab Low. Consis tsev pheebsuab nrog rau yav dhau los txoj kev tshawb no [10], Nor-A hlwb nrog AKG supplementation ntawm ib tug concentration ntawm 2 hli nthuav tawm ib qho tseem ceeb ntawm cov cell viability txheeb ze rau Nor hlwb (Daim duab S1).

Yog li ntawd, AKG concentration ntawm 2 mm tau siv nyob rau hauv qhov kev sim rau kev soj ntsuam AKG-vim kev hloov pauv hauv kev loj hlob thiab kev sib txawv ntawm myoblasts. Piv nrog rau cov tsis muaj hlwb, Cov qes qes tau pom tias muaj kev cuam tshuam loj heev vim yog lub zog tsis muaj zog (Daim duab 1A). Txawm hais tias AKG supplementation tsis ua rau muaj qhov sib txawv morphology ntawm myoblasts nyob rau hauv ob lub zog lub xeev, nws tsis tsuas yog txhim kho tus nqi proliferation ntawm Low cells kab lis kev cai nyob rau hauv ib tug tsawg-qabzib nruab nrab tab sis kuj enhanced uas tsis yog cell cultured nyob rau hauv ib txwm nruab nrab raws li yav dhau los kev tshawb fawb [10,12]. Cov lej xov tooj ntawm ib cheeb tsam tau suav rau plaub pawg ntawm myoblasts (n=4 rau pawg): Tsis yog, 563.8 ± 10.4; Nor-A, 624.0 ± 10.8; Tsawg, 493.8 ± 14.5; Tsawg A, 547.0 ± 11.7 (Daim duab 1B). Nws yog ib qho tsim nyog sau cia tias Low-A cov hlwb tsis tau qhia cov lej sib txawv ntawm cov xov tooj ntawm tes los ntawm Nor hlwb, qhia tias AKG supplementation tuaj yeem rov qab cov xov tooj ntawm tes rau cov myoblasts hauv cov kab lis kev cai qis qis (Daim duab 1B).

Daim duab 1, Kev loj hlob thiab kev sib txawv muaj peev xwm ntawm C2C12 myoblasts nyob rau hauv cov xwm txheej ntawm ib txwm kab lis kev cai thiab qis. qabzib kab lis kev cai. (A) Myoblasts morphologies. (B) Cov xov tooj ntawm tes sib raug rau vaj huam sib luag A (n=4). (C) Cell viabilities txheeb ze tsis muaj hlwb soj ntsuam los ntawm MTS cell proliferation assay (n=5). (D) MyoD1 kab lus hauv myoblasts soj ntsuam los ntawm sab hnub poob blot. Cov tshuaj tiv thaiv GAPDH tau siv los ntsuas tus nqi ntawm cov protein hauv txhua txoj kab. (E) Kev txheeb xyuas txheeb cais sib raug rau cov vaj huam sib luag (D) (n=4). * p < 0 {{10}}5,** p < 0.01, *** p < 0.001, **** p < 0.0001.

Tsis tas li ntawd, qhov kev ntsuam xyuas MTS tau ua kom muaj nuj nqis los sib piv cov kev loj hlob ntawm plaub pawg hlwb (Daim duab 1C). Cov qe qes qis muaj qhov txo qis qis dua piv nrog cov tsis muaj hlwb, qhia tias cov piam thaj qis nruab nrab tsis txaus siab rau kev loj hlob ntawm cov hlwb. Qhov tseem ceeb, AKG supplementation txhim kho qhov kev loj hlob ntawm Nor-A thiab Low-A hlwb txheeb ze rau Nor thiab Low cells, feem. Nco ntsoov tias Low-A cov hlwb tau pom qhov muaj peev xwm loj hlob qis dua Nor hlwb, txhais tau hais tias AKG supplementation tsuas yog ib nrab rov qab los ntawm cov hlwb uas muaj kab mob hauv nruab nrab qis-qabzib.

.Qhov kev qhia ntawm myogenic sib txawv 1 (MyoD1) protein feem ntau yog siv los ua tus yam ntxwv ntawm lub peev xwm sib txawv ntawm cov hlwb. Peb, yog li ntawd, sib piv cov lus ntawm MyoD1 ntawm plaub pawg hlwb (Daim duab 1D). Cov hlwb qis tau pom tias muaj peev xwm txo qis qhov sib txawv ntawm qhov sib piv nrog Nor cell. Qhov tseem ceeb, AKG txhawb nqa kev tswj hwm kev sib txawv ntawm cov hlwb uas tau coj los ua ob qho tib si hauv nruab nrab thiab hauv nruab nrab cov piam thaj qis, raws li qhia los ntawm kwv yees li 30 feem pua ​​​​nce ntawm MyoD1expression hauv Nor-A thiab Low-A hlwb. Qhov tseem ceeb, Low-A cov hlwb tsis tau qhia qhov sib txawv ntawm MyoD1 qhia tawm los ntawm cov hlwb tsis, hais txog kev ua haujlwm siab ntawm AKG supplementation rau kev rov ua kom muaj peev xwm sib txawv ntawm cov hlwb hauv nruab nrab qis-qabzib.

Tsis tas li ntawd, peb tau txheeb xyuas cov peev xwm sib txawv ntawm myotube rau plaub pawg ntawm C2C12 myoblasts. Myotubes tau tsim los ntawm kev sib xyaw ntawm myoblasts kab lis kev cai hauv ib txwm thiab qis-qabzib sib txawv xov xwm nrog lossis tsis muaj AKG supplementationFigure S3). Morphologies ntawm C2C12 myotubes tau pom tias kab lis kev cai qis-qabzib cuam tshuam lub peev xwm ntawm myotube sib txawv ntawm cov hlwb, thiab AKG supplementation tuaj yeem txhawb kev sib txawv ntawm myotube ntawm myoblasts cultured ob qho tib si hauv nruab nrab nruab nrab thiab hauv nruab nrab ow-glucose.

2.2. NMR Spectra ntawm Aqueous Extracts ntawm C2C12 Myoblasts

Hom 850 MHz 1H NMR spectra tau sau tseg ntawm cov dej xa tawm los ntawm Nor, Nor-A, Low, thiab Low-A pawg ntawm C2C12 myoblasts (Daim duab 2A). Tag nrho ntawm 34 metabolites tau raug muab thiab sau tseg hauv Table S1. Cov haujlwm resonance ntawm cov metabolites tau lees paub los ntawm kev siv 2D 1H-13C HSOC thiab 1H-H TOCSY spectra (Figures S4 thiab S5). Kev soj ntsuam pom ntawm NMR spectra qhia tau hais tias culturing myoblasts nrog AKG supplementation ua rau cov khoom tseem ceeb ntawm intracellularAKG hauv Nor-A thiab Low-A hlwb (Daim duab 2B).

Daim duab 2, Nruab Nrab 850 MHz lH nuclear magnetic resonance (NMR) spectra sau tseg ntawm aqueous rho tawm los ntawm Nor, Nor-A, Low, thiab Low-A pawg ntawm C2C12 myoblasts. (A) Kev sib piv ntawm qhov nruab nrab NMR spectra ntawm plaub pawg. Cov nplai ntsug tau khaws cia tas li nyob rau hauv tag nrho cov lH NMR spectra. Lub cheeb tsam dej (4.7-5.2 ppm) raug tshem tawm (B) Cov cheeb tsam hauv zos amplified ntawm a-Ketoglutarate (AKG) peaks. Xiav / ntsuab / daj / liab kab: spectral cheeb tsam los ntawm Nor/Nor-A/Low/Low-A pawg. AKG, a-ketoglutarate; PC, O-phosphocholine; GPC, sn-glycero-3-phosphocholineUDP-glucose, Uridine diphosphate qabzib: CTP, sn-glycero-3-phosphocholine; NAD ntxiv, nicotinamide adenine dinucleotide AXP adenine mono /di /triphosphate.

2.3. Multivariate Data Analysis rau Kev Tshawb Fawb Cellular Metabolic Profiles

Peb txuas ntxiv ua cov ntaub ntawv sib txawv ntawm cov ntaub ntawv NMR spectral rau metabolic profiling ntawm plaub pawg ntawm C2C12 myoblasts. Peb thawj zaug tsim peb tus qauv PCA uas tsis muaj kev saib xyuas nrog thawj ob yam khoom (PC1, PC2) los saib xyuas cov kev sib koom ua ke thiab nthuav tawm qhov sib txawv ntawm cov metabolic ntawm cov pab pawg ntawm myoblasts. Cov qhab-nees ThePCA qhia tau hais tias cov metabolic profile ntawm cov cell kab lis kev cai nyob rau hauv ib tug tsawg-glucose nruab nrab yog txawv los ntawm cov kab lis kev cai nyob rau hauv ib tug ib txwm nruab nrab (Daim duab 3A), thiab AKCsupplementation ho hloov pauv cov qauv metabolic ntawm cov hlwb cultured ob qho tib si nyob rau hauv ib txwm nruab nrab thiab. hauv nruab nrab cov piam thaj qis (Daim duab 3B, C). Txawm li cas los xij, qhov sib txawv ntawm cov metabolic ntawm Nor-A thiab Nor pawg tau loj dua qhov nruab nrab ntawm pawg Low-A thiab Low pawg, txhais tau hais tias cov teebmeem ntawm AKG supplementation ntawm cov metabolic profile ntawm myoblasts zoo heev nyob ntawm lub zog ntawm lub hlwb.

Daim duab 3. Multivariate analyses rau 'HNMR spectra raug kaw nyob rau hauv aqueous rho tawm los ntawm C2C12 myoblasts ntawm Nor, Nor-A, Tsawg, thiab Low-A pawg. (AC) PCA cov qhab nia ntawm pawg qis thiab Tsis yog, pawg qis-A thiab qis pawg Nor-A thiab Nor; (DF) OPIS-DA cov qhab nia ntawm pawg Low thiab Nor (R2: 0.999, 02: 0.996), pawg Low-Aand Low (R2: 0.918: 02: 0.761), pawg Nor-A thiab Nor (R2: 0.927: 02: 0.838). Cov ellipses qhia txog 95 feem pua ​​​​kev ntseeg siab txwv.

Tsis tas li ntawd, peb tau tsim peb tus qauv saib xyuas OPLS-DA los qhia txog kev sib cais ntawm metabolic ntawm plaub pawg ntawm myoblasts (Daim duab 3D-F). Raws li qhov xav tau, OPLSDA qauv ua kom pom qhov sib txawv ntawm cov metabolic ntawm plaub pawg los ntawm kev khaws cov ntaub ntawv sib txawv orthogonal sib txawv thiab lim tawm cov ntaub ntawv tsis sib xws orthogonal sib txawv. Tsis tas li ntawd, peb tau ua qhov kev ntsuam xyuas random permutation (n=200) los ntsuas qhov kev ntseeg tau ntawm OPLS-DA qauv (Daim duab S6), uas qhia txog qhov siv tau ntawm cov qauv tsim OPLS-DA.

2.4. Kev txheeb xyuas qhov sib txawv thiab cov metabolite tseem ceeb

Txhawm rau kom muaj nuj nqis sib piv cov qib metabolite ntawm plaub pawg ntawm C2C12 myoblasts, peb suav cov qib txheeb ze ntawm cov metabolites txheeb xyuas raws li lawv cov txheeb ze sib xyaw (Table S2). Ua tau zoo AKG supplementation nce intracellularAKG qib hauv pawg Nor-A thiab qis-A tab sis tsis tau hloov pauv cov neeg hauv pawg Nor thiab qis. Peb tau ua tub ntxhais kawm qhov t-test los txheeb xyuas qhov sib txawv ntawm cov metabolites nrog cov qauv ntawm p < 0.05 (Daim duab 4). Kev sib piv ntawm Nor vs. Low tau txheeb xyuas 29 qhov sib txawv ntawm cov metabolites (Daim duab 4A), suav nrog 18 txhim kho metabolites (leucine, isoleucine, valine, acetate, glutamate, glutamine, methionine, aspartate, lysine, creatine, PC (O-phosphotyros). , phenylalanine, histidine, NAD ntxiv, formate, AXP), thiab 11 tsis kam Metabo. lites (glutathione, pyroglutamate, phosphocreatine, beta-alanine, GPC, qabzib, glycine, acetate, threonine, GTP, UDP-glucose). Kev sib piv ntawm Low-A vs. Low-identified differential metabolites (Daim duab 4B), suav nrog 7 cov metabolites ntxiv (ethanol, AKGbeta-alanine, PC, taurine, glycine, thiab GTP) thiab 3 txo cov metabolites (glutamine, lysine thiab myoinositol). Kev sib piv ntawm Nor-A vs. Tsis tau txheeb xyuas 18 qhov sib txawv ntawm cov metabolites (Daim duab 4C), suav nrog 6 cov txheej txheem metabolites (AKG, pyroglutamate, glutamine, lysine, qabzib, lactate), thiab 12 down-regulated metabolites (alanine, acetate, glutathione). methionine, phosphocreatine, PC, myoinositol, glycine, threonine, GTP, UDP-glucose, thiab AXP).

Daim duab 4. Cov txheeb ze sib txawv ntawm cov metabolites sib txawv tau txheeb xyuas los ntawm kev sib piv ua ke ntawm plaub pawg ntawm C2C12 myoblasts. (A) Tsawg vs. (B) Tsawg-A vs. Tsawg; (C) Nor-A vs. Nr. * p < 0 05, ** p < 0 01, *** p < 0.001, **** p < 0.0001.n { {13}} rau txhua pab pawg.

Tsis tas li ntawd, peb tau siv OPLS-DA qauv los txheeb xyuas cov metabolites tseem ceeb nrog cov qauv ntawm VIP> 1 (Daim duab 5). Tag nrho, 12, 10, thiab 10 cov metabolites tseem ceeb tau txheeb xyuas los ntawm OPLS-DA qauv ntawm Nor-A vs. Nor, Tsis yog vs. Tsawg, Tsawg-A vs. Tsawg.

Daim duab 5. VIP cov qhab nia ntawm cov metabolites tseem ceeb tau txheeb xyuas los ntawm kev sib piv ua ke ntawm plaub pawg ntawm C2C12myoblasts. (A) Tsawg vs. (B) Tsawg-A vs. Tsawg; (C) Nor-A vs. Nr. Liab / Xiav font qhia txog nce / txo qis ntawm cov metabolite.

Kev sib xyaw ua ke ntawm cov metabolites tseem ceeb thiab cov metabolites sib txawv tau muab cov yam ntxwv metabolites (Table 1). Qhov kev sib piv ntawm kev sib piv ntawm Low vs. Nor, Low-A vs. Low, thiab Nor-A vs. Tsis tau txheeb xyuas 10, 6, thiab 10 cov yam ntxwv metabolites, ntsig txog, qhia tias qhov cuam tshuam ntawm AKG supplementation ntawm myoblasts tau ze ze nrog lub zog. lub xeev ntawm cov hlwb.

2.5. Kev txheeb xyuas qhov tseem ceeb hloov pauv metabolic

Txoj Kev Peb tau ua cov txheej txheem metabolic txhawm rau txheeb xyuas cov kev hloov pauv tseem ceeb hauv cov txheej txheem metabolic (txoj hauv kev tseem ceeb) raws li qib ntawm cov metabolites txheeb xyuas los ntawm kev sib piv ua ke ntawm plaub pawg ntawm C1C12 myoblasts (Daim duab S7; Table 2 thiab Table S3). Kev tsom xam ntawm Nor vs. Low tau txheeb xyuas 11 txoj hauv kev tseem ceeb: (1) Alanine, aspartate, thiab glutamate metabolism; (2) Glycine, serine, thiab threonine metabolism; (3) Glutathione metabolism; (4) D-Glutamine thiab D-glutamate metabolism; (5) Starch thiab sucrose metabolism; (6) beta-Alanine metabolism; (7) Taurine thiab hypotaurine metabolism; (8) Phenylalanine metabolism; (9) Phenylalanine, tyrosine thiab tryptophan biosynthesis; (10) Nicotinate thiab nicotinamide metabolism; (11) Histidine metabolism. Txoj hauv kev tseem ceeb no tau cuam tshuam nrog lub zog metabolism, oxidative kev nyuaj siab, thiab TCA voj voog anaplerotic flux.

Kev tsom xam ntawm Nor-A vs. Tsis tsuas yog txheeb xyuas thawj tsib txoj hauv kev tseem ceeb 1–5 tsis suav nrog rau lwm txoj hauv kev 6–11. Qhov txawv, kev tsom xam ntawm Low-A vs. Low tsuas yog txheeb xyuas txog rau txoj hauv kev tseem ceeb: thawj plaub txoj hauv kev 1–4 qhia los ntawm kev sib piv ntawm Low vs. Nor, Nor-A vs. ob txoj kev 6–7 qhia los ntawm kev sib piv ntawm Low vs. Nor. Nco ntsoov tias AKG supplementation tsis tau hloov pauv txoj hauv kev 5 (Starch thiab sucrose metabolism) hauv cov hlwb uas muaj cov piam thaj hauv nruab nrab, tab sis cuam tshuam nrog ob txoj hauv kev (beta-Alanine metabolism thiab taurine thiab hypotaurine metabolism). Txhawm rau pom AKG-vim kev hloov pauv hauv cov yam ntxwv metabolites, peb tau npaj cov metabolites mus rau hauv daim ntawv qhia metabolic raws li Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Daim duab 6). KEGG tau siv dav los ua ib qho ntawm cov ntaub ntawv tseem ceeb los txhim kho cov kev sib txuas hauv metabolic thiab qhia txog cov txheej txheem metabolic tseem ceeb. Ob qho tib si hloov pauv cov yam ntxwv metabolites thiab hloov pauv txoj hauv kev metabolic tseem ceeb muab kev nkag siab tshiab rau hauv cov txheej txheem molecular hauv qab cov teebmeem ntawm AKG supplementation ntawm C2C12 myoblasts.