Spermidine Alleviates Intrauterine Hypoxia-Induced Offspring Newborn Myocardial Mitochondrial Damage nyob rau hauv nas Los ntawm Inhibiting Oxidative Stress Thiab Regulating Mitochondrial Quality Control Part 1

Abstract

Keeb kwm: Intrauterine hypoxia (IUH) ua rau muaj kev pheej hmoo ntawm cov kab mob plawv hauv cov me nyuam. Raws li ib hom reactive oxygen (ROS) scavenger, polyamine spermidine (SPD) yog qhov tseem ceeb rau embryonic thiab fetal ciaj sia taus thiab loj hlob. Txawm li cas los xij, kev tshawb fawb ntxiv txog kev tiv thaiv SPD thiab cov txheej txheem rau IUH-vim lub plawv puas hauv cov xeeb ntxwv yog xav tau.

Glycoside ntawm cistanche tuaj yeem ua rau muaj kev ua haujlwm ntawm SOD hauv plawv thiab daim siab cov ntaub so ntswg, thiab txo cov ntsiab lus ntawm lipofuscin thiab MDA hauv txhua cov ntaub so ntswg, tshem tawm ntau yam reactive oxygen radicals (OH-, H₂O₂, thiab lwm yam) thiab tiv thaiv DNA puas. los ntawm OH-radicals. Cistanche phenylethanoid glycosides muaj peev xwm tshem tawm cov dawb radicals, muaj peev xwm txo tau ntau dua li cov vitamin C, txhim kho cov haujlwm ntawm SOD hauv cov phev ncua, txo cov ntsiab lus ntawm MDA, thiab muaj qee yam kev tiv thaiv ntawm cov phev membrane muaj nuj nqi. Cistanche polysaccharides tuaj yeem txhim kho kev ua haujlwm ntawm SOD thiab GSH-Px hauv erythrocytes thiab ntsws cov ntaub so ntswg ntawm cov nas senescent sim los ntawm D-galactose, nrog rau txo cov ntsiab lus ntawm MDA thiab collagen hauv lub ntsws thiab ntshav, thiab nce cov ntsiab lus ntawm elastin, muaj Cov nyhuv scavenging zoo ntawm DPPH, ncua lub sijhawm ntawm hypoxia hauv cov nas senescent, txhim kho cov haujlwm ntawm SOD hauv cov ntshav, thiab ncua lub physiological degeneration ntawm lub ntsws hauv kev sim cov nas nrog cellular morphological degeneration, kev sim tau pom tias Cistanche muaj peev xwm antioxidant zoo. thiab muaj peev xwm los ua ib qho tshuaj tiv thaiv thiab kho cov kab mob ntawm daim tawv nqaij laus. Nyob rau tib lub sijhawm, echinacoside hauv Cistanche muaj lub peev xwm tseem ceeb txhawm rau txhawm rau DPPH dawb radicals thiab muaj peev xwm tshem tawm cov pa oxygen reactive thiab tiv thaiv dawb radical-induced collagen degradation, thiab kuj muaj kev kho zoo ntawm thymine dawb radical anion puas.

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Lub hom phiaj: Txoj kev tshawb no tsom los tshawb xyuas cov kev tiv thaiv kev tiv thaiv ntawm prenatal SPD kev kho mob ntawm IUH-vim lub plawv puas nyob rau hauv cov me nyuam yug nas nas thiab nws cov hauv paus ntsiab lus ntawm mitochondrial mechanism.

Cov txheej txheem: Tus qauv nas ntawm IUH tau tsim los ntawm kev raug rau 10 feem pua ​​O2 xya hnub ua ntej lub sijhawm. Lub caij no, rau xya hnub, cov nas cev xeeb tub tau muab SPD (5 mg.kg-1.d-1; ip). Cov nas tsuag ib hnub tau muab fij los ntsuam xyuas ntau yam, suav nrog kev loj hlob, kev puas tsuaj hauv plawv, cardiomyocytes proliferation, myocardial oxidative stress, cell apoptosis, thiab mitochondrial function, thiab muaj mitochondrial zoo tswj (MQC), nrog rau mitophagy, mitochondrial biogenesis, thiab mitochondrial fusion / fission. Hauv kev sim hauv vitro, thawj cardiomyocytes tau raug hypoxia nrog lossis tsis muaj SPD rau 24 teev.

Cov txiaj ntsig: IUH poob lub cev hnyav, lub plawv hnyav, lub plawv Ki67 qhia, kev ua ntawm SOD, thiab CAT thiab adenosine 5'- triphosphate (ATP) ntau ntau thiab nce BAX/BCL2 qhia thiab TUNEL-zoo nuclei tooj. Tsis tas li ntawd, IUH kuj ua rau cov qauv mitochondrial abnormality, dysfunction, thiab txo mitophagy (tsawg tus naj npawb ntawm mitophagosomes), declined mitochondrial biogenesis (tso qhia ntawm SIRT-1, PGC-1, NRF-2, TFAM), thiab ua rau fission / fusion imbalance (nce feem pua ​​​​ntawm mitochondrial fragments, nce DRP1 qhia, thiab txo MFN2 qhia) hauv myocardium. Kuj ceeb tias, kev kho SPD normalizes cov kev hloov pauv hauv IUH-vim tsis tau. Tsis tas li ntawd, SPD kuj tiv thaiv hypoxia-induced ROS tsub zuj zuj, mitochondrial membrane muaj peev xwm lwj, thiab mitophagy txo qis hauv cardiomyocytes.

Xaus: Maternal SPD kev kho mob ua rau IUH-vim lub plawv puas hauv cov menyuam yug tshiab nas los ntawm kev txhim kho myocardial mitochondrial muaj nuj nqi los ntawm kev tiv thaiv oxidation thiab anti-apoptosis, thiab tswj MQC.

1. Keeb kwm

Epidemiological thiab tsiaj kev tshawb fawb qhia tias ib qho adv se intrauterine ib puag ncig cuam tshuam nrog kev pheej hmoo ntawm cov kab mob plawv hauv cov neeg laus (1). Prenatal hypoxia, qhov feem ntau tsis zoo nyob rau hauv lub tsev menyuam ib puag ncig, tuaj yeem ua rau tus menyuam hauv plab tsis tuaj yeem paub txog nws txoj kev loj hlob ntawm caj ces, tshwm sim raws li kev loj hlob qeeb thiab kev ua haujlwm ntawm lub cev raug mob hauv lub caij nplooj ntoo hlav (2). Tus me nyuam hauv plab hypoxia feem ntau tshwm sim los ntawm kev xeeb tub nrog cov placental vascular tsis kam xws li pre-clampsia, inter-pregnancy at high altitude, or maternal respiratory disease. Qhov tseem ceeb tshaj, ib feem peb ntawm lawv raug cuam tshuam los ntawm obstructive pw tsaug zog apnea hypoventilation syndrome (OSAHS) thaum cev xeeb tub lig, thiab nws kuj tseem cuam tshuam nrog kev loj hlob ntawm gestational hypertension syndrome. Qhov tseem ceeb tshaj plaws kev hloov pauv pathophysiological SAHS yog qhov tsis sib xws ntawm hypoxia (3, 4). Hauv ntau tus qauv tsiaj, intrauterine hypoxic (IUH) ua rau lub plawv txo qis, diastolic thiab systolic dysfunction, hypertrophic kev loj hlob, txo cardiomyocyte proliferation, thiab qeeb cardiomyocyte maturation nyob rau hauv lub fetal thiab neonatal lub plawv, yog li ua rau muaj kev pheej hmoo ntawm lub plawv mob nyob rau hauv cov neeg laus ( 5). Cov txiaj ntsig ntawm kev ua haujlwm ntawm kev mob hypoxia tuaj yeem piav qhia txog cov txheej txheem ntawm cov theem tseem ceeb ntawm kev loj hlob ntawm lub plawv, uas ua rau lub plawv oxidative kev nyuaj siab txhim kho thiab cell apoptosis nce hauv cov xeeb ntxwv (6-9). Lub con pt ntawm kev loj hlob programming ua rau kev nkag siab vim tias peb lub cev muaj zog ntau dua thiab cov yas thaum lub neej thaum ntxov. Tus mob intrauterine txiav txim siab thiab pab cov ntsiab lus ntawm lub cev thiab cov metabolism hauv peb lub neej. Yog li, qhov kev hloov pauv ntawm tus menyuam hauv plab rau IUH ua rau muaj kev loj hlob ntawm cov neeg laus cov kab mob plawv (10). Mitochondria yog cov organelles nyuaj ua si lub luag haujlwm tseem ceeb hauv cellular zog-generating thiab ib tug myriad ntawm cellar signaling txheej xwm. Cov txheej txheem thiab kev ua haujlwm zoo ntawm mitochondria yog qhov tseem ceeb rau kev ciaj sia ntawm cardiomyocytes, thiab thaum muaj kev cuam tshuam, kev cuam tshuam ntawm mit-hondrial homeostasis ua rau kev loj hlob ntawm cov kab mob carac. Tsis ntev los no, ntau qhov kev tshawb fawb tau tsom mus rau lub luag haujlwm ntawm kev ua haujlwm tsis zoo ntawm lub cev xeeb tub hauv plab hypoxia-inducing mitochondrial dysfunction hauv kev kho cov menyuam lub plawv puas. Nws tau raug tshaj tawm tias qhov kev ua haujlwm mitochondrialiratory tau ua tsis taus, thiab kev qhia ntawm ntau txoj cai mitochondrial mol hloov lub plawv perinatal raug rau IUH (11). Ntxiv mus, IUH ua rau txo qis mitochondrial complex enz e kev ua haujlwm thiab hlais lub plawv tsis ua haujlwm hauv cov xeeb ntxwv tom qab myocardial ischemia (12). Raws li, txoj kev tshawb no tsom mus nrhiav cov molecular txuas amo prenatal hypoxia, oxidative stress, mitochondrial dys action, thiab mob plawv hauv cov xeeb ntxwv tshiab (13).

Txoj kev loj hlob thiab kev loj hlob ntawm no high-volume mitochondrial system nyob rau hauv lub myocardium feem ntau tshwm sim nyob rau hauv lub perinatal thiab postnatal txoj kev loj hlob theem, feem ntau yog vim lub plawv metabolic hloov los ntawm kev siv qabzib o fatty acids rau lub adenosine 5'-triphosphate (ATP) tiam tom qab yug los. (14). Cov ntaub ntawv pov thawj pom tau hais tias mitochondrial zoo tswj (MQC) cov txheej txheem yog qhov tseem ceeb rau kev loj hlob ntawm cardiomyocytes (15). Mitochondrial kev tswj kom zoo, feem ntau suav nrog mitochondrial biogenesis, mitophagy ua ke nrog kev ua kom muaj zog, kev tswj xyuas kev sib txuas lus zoo, orchestrates kom muaj nuj nqis thiab zoo ntawm mitochondria thiab txhim kho mitochondrial muaj nuj nqi thiab cardiomyocyte ciaj sia nyob rau hauv kev ntxhov siab (16). Peroxisome proliferators-activate receptors (PPAR) coactivator 1 (PGC-1) ​​ua lub luag haujlwm tseem ceeb hauv kev tsav tsheb mitochondrial biogenesis thiab ua haujlwm hauv lub plawv (17). Peroxisome proliferators-activate receptors coactivator 1 / (-/-) nas lub siab nthuav tawm cov npe kos npe f qhov kev loj hlob tsis zoo thiab qhov txawv txav loj hauv kev ua haujlwm mitochondrial thiab ntom (18). Tsis tas li ntawd, tshem tawm mitophagy-mediator Parkin tiv thaiv morphological thiab functional mitochondria maturation nyob rau hauv lub cev xeeb tub theem (19). Mitochondria yog cov organelles uas muaj zog heev, ua rau muaj kev cuam tshuam tas li thiab cov xwm txheej fusion cuam tshuam nrog lawv txoj haujlwm. Thaum lub sij hawm embryonic lig, lub tsheb ac-specific genetic ablation ntawm mitochondrial fusion pro nyob rau hauv MFN1 thiab MFN2 nyob rau hauv cov nas pom loj mitochondrial dysfunction tom qab yug me nyuam thiab tsim cardiomyopathy (20). Strikingly, ib txoj kev tshawb fawb tsis ntev los no tau qhia tias mob hyp ia cuam tshuam mitochondrial dynamics nyob rau hauv lub fetal gui a npua forebrain (21). Raws li, MQC tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev txhim kho myocardium raug mob hauv IUH cov xeeb ntxwv. Txawm li cas los xij, me ntsis paub txog qhov cuam tshuam ntawm prenatal hypoxia ntawm neonatal cardiac MQC system thiab cov txiaj ntsig ntawm MQC ua haujlwm tsis zoo ntawm lub plawv neonatal. Txoj kev tshawb no tau coj lub luag haujlwm no los tshawb txog mitochondrial-related mechanism ntawm IUH-induced myocardial puas nyob rau hauv cov me nyuam tshiab thiab ntxiv tshawb nrhiav cov tswv yim tiv thaiv kom txo tau mob plawv ntawm IUH.

Polyamines (PAs), suav nrog putrescine (PU), spermidine (SPD), thiab spermine (SP), muaj nyob hauv yuav luag txhua yam kab mob nyob thiab yog qhov tseem ceeb rau embryonic thiab fetal surval, kev loj hlob, thiab kev loj hlob (22-24) . Cov kev tshawb fawb pom tias yaj raug IUH tuaj yeem txhim kho embryonic dysplasia los ntawm kev ntxiv exogenous polyamines (25, 26). Tsis tas li ntawd, lub cev loj hlob ntawm cov ntaub ntawv qhia txog lub luag haujlwm ntawm polyamines hauv kev tshem tawm cov dawb radicals thiab tiv thaiv DNA, proteins, thiab lipids los ntawm kev puas tsuaj ntawm oxidative kev nyuaj siab. Nws kuj tau qhia tias polyamine supplementation nce lub neej ncua ntawm cov qauv kab mob los ntawm antioxidant, anti-inflammatory, thiab mitophagy-inducing pro-sim (27-29). Cov kev tshawb fawb tsis ntev los no tau sau tseg tias sup mentation nrog SPD hauv kev noj haus yog cardioprotective thiab prolongs lifespan nyob rau hauv ob leeg nas thiab tib neeg los ntawm stimulating mitophagy thiab mitochondrial respiration thiab txhim kho lub plawv (30, 31). Qhov tseem ceeb tshaj, peb yav dhau los tau tshaj tawm tias kev kis tus poj niam hypoxic thaum lub sij hawm lig ntawm kev loj hlob ntawm fetal ua rau cov npe dased ana thiab nce catabolism ntawm polyamines nyob rau hauv cov ntaub so ntswg carac ntawm cov nas yug tshiab. SPD tiv thaiv lub plawv raug mob nyob rau hauv ven-hnub cov me nyuam nas raug rau IUH los ntawm inhibiting mitochondrial mentation (32).

2. Lub hom phiaj

Hauv txoj kev tshawb no, IUH yog qhov xav tias yuav ua rau cov qauv mitochondrial thiab kev ua haujlwm tsis txaus los ntawm kev ua kom oxidative kev nyuaj siab thiab rhuav tshem MQC cov txheej txheem hauv cov menyuam yug tshiab lub siab, thiab kev kho SPD niam txiv hauv utero yog xav tias yuav txo tau qhov mob myocardial los ntawm kev txo qis kev txhim kho ntawm mitochondria. Txoj kev tshawb no yuav pab txhawb kev txhim kho kev tiv thaiv lossis kho cov tswv yim rau IUH cov xeeb ntxwv los tiv thaiv cov neeg laus cov kab mob plawv.

3. Cov txheej txheem

3.1. Tsiaj

Cov txiv neej thiab poj niam Wistar nas (3 lub hlis) tau yuav los ntawm Lub Tsev Haujlwm Saib Xyuas Kev Tshawb Fawb Tsiaj ntawm Harbin Medical University. Tag nrho cov txheej txheem tau pom zoo los ntawm Pawg Saib Xyuas Kev Ncaj Ncees ntawm Harbin Medical University (Tuam Tshoj), thiab txhua qhov kev sim tau ua los ntawm National Institutes of Health cov lus qhia. Cov nas uas muaj tus txiv neej-rau-poj niam piv ntawm 2: 1 tau muab tso rau hauv lub tawb rau mating. Qhov chaw mos smears tau ua rau hnub tom qab txhawm rau kuaj pom cov phev hauv qhov chaw mos lossis qhov chaw mos smears, uas tau lees paub tias yog hnub xoom-hnub ntawm cev xeeb tub. Cov nas cev xeeb tub tau khaws cia rau hauv ib chav uas tswj cov av noo (60 feem pua) thiab tswj qhov kub thiab txias (21 degree), thiab lub voj voog lub teeb-tso yog 12: 12 teev.

3.2. Intrauterine Hypoxia Model

Txij hnub tim 15 mus txog rau hnub 21 ntawm cev xeeb tub, cov nas nyob hauv pawg hypoxia (n=10) tau muab tso rau hauv qhov chaw kaw plexiglass, txhaj nrog huab cua thiab nitrogen, thiab saib xyuas los ntawm lub ntsuas pa oxygen (Pro OX12{{13). } }; BioSpherix, New York, USA), thiab nqus tau cov pa oxygen ntawm 10 feem pua ​​​​rau plaub teev hauv ib hnub. Cov ntshav kuaj ntshav tau raug coj los ntawm txoj cai femoral artery, cov ntshav roj thiab pH qhov tseem ceeb tau ntsuas los tswj cov hlab ntsha oxygen ib feem ntawm 50 - 55 mmHg, cov ntshav oxygen saturation tau khaws cia ntawm 80 - 85 feem pua, thiab cov txheej txheem tshwj xeeb tau ua raws li tau piav qhia dhau los (32). Cov poj niam sim sim tau muab faib ua plaub pawg: pawg tswj (tswj), pawg intrauterine hypoxia (Hpx), intrauterine hypoxia ntxiv rau spermidine pawg (Hpx-Spd), thiab intrauterine hypoxia ntxiv rau spermidine plus inhibitor pawg (Hpx-Spd-DFMO). ). Rau nas rau ib pab pawg tau txhaj tshuaj intraperitoneally rau 15th - 21hnub ntawm cev xeeb tub. Cov nas tsuag tau muab 0.9 feem pua ​​​​ saline (1 mL / kg / d) hauv kev tswj hwm thiab Hpx pawg, SPD (5 mg / kg / d) hauv pawg Hpx-Spd, thiab SPD (5 mg / kg / d) thiab difuoromethy -L-ornithine (DFMO, ib qho inhibitor ntawm cov enzyme tseem ceeb ntawm polyamine synthesis ODC) (5 mg / kg / d) hauv pawg Hpx-Spd-DFMO, feem. Tom qab yug me nyuam, 1-cov me nyuam yug tshiab hnub nyoog tau muab txi, thiab lawv lub siab raug muab rho tawm rau kev soj ntsuam kev soj ntsuam.

3.3. Histological Analysis

Sab laug ventricular cov ntaub so ntswg ntawm nas raug txiav mus rau hauv ib tug tuab ntawm <1 cm, tsau nrog 4 feem pua ​​paraformaldehyde, dehydrated nrog cawv, thiab embedded nyob rau hauv paraffin. Cov embedded paraffin raug txiav rau hauv 5 hli tuab slices, ces qhuav ntawm qhov cub tas li ntawm 60 degree, dewaxed nrog xylene, thiab tom qab ntawd los ntawm staining hematoxylin-eosin (HE). Cov ntaub so ntswg tau raug soj ntsuam los ntsuas cov kev hloov pauv hauv plawv morphology thiab cov qauv nrog lub tshuab ntsuas qhov muag (Eclipse E200; Nikon, Tokyo, Nyiv).

3.4. Immunofluorescence Analysis

Ki67 immunofluorescence staining tau ua raws li tau piav qhia yav dhau los (33). Luv luv, sab laug ventricular cov ntaub so ntswg ntawm nas raug kho nyob rau hauv 4 feem pua ​​formalin, embedded nyob rau hauv paraffin; dewaxing, hydration, thiab kho antigen ua ntej ua tiav. Cov ntaub so ntswg raug thaiv nrog 0.5 feem pua ​​​​bovine serum albumin rau 2 teev thiab tom qab ntawd incubated nrog Ki67 Luav Monoclonal Antibody (1: 100, AF1738, Beyotime, Tuam Tshoj) ntawm 4 degree thaum hmo ntuj. Tom qab ntxuav nrog PBS, cov ntaub so ntswg tau incubated nrog Alexa fluor sau ntawv Tshis los tiv thaiv luav IgG (1: 500, A 0468, Beyotime, Tuam Tshoj) thiab counterstained nrog DAPI rau nuclei. Cov duab tau saib thiab luam tawm hauv laser confocal microscopy (OLYMPUS, FV1000, Nyiv). Lub software tau siv los txheeb xyuas cov colocalization ntawm cov duab merged.

3.5. Quantification ntawm Fibrosis

Cardiac fibrosis tau soj ntsuam los ntawm Masson's trichrome staining. Raws li tau piav qhia saum toj no, cov kab mob plawv los ntawm cov nas me nyuam mos tau dewaxed thiab hydrated los ntawm cov qauv txheej txheem thiab tom qab ntawd stained nrog Masson's trichrome raws li kev cai. Ob qhov chaw tsis sib xws tau siv rau txhua lub siab. Qhov feem pua ​​​​ntawm thaj chaw fibrotic hauv tag nrho sab laug ventricular myocardial cheeb tsam tau txheeb xyuas siv ImageJ software, vl.52 (NIH, Bethesda, MD).

3.6. TdT-Mediated dUTP Nick End Labeling thiab Apoptotic Cell Measurements

Kev ntsuas TdT-mediated dUTP nick end labeling (TUNEL) tau siv los txiav txim seb tus naj npawb ntawm cov hlwb apoptotic nyob rau hauv ib hnub-laus cov menyuam lub siab lub ntsws. Cov ntaub so ntswg hauv plawv tau raug kho raws li peb cov txheej txheem uas tau piav qhia dhau los siv Cell Detection Kit (Roche, Germany) raws li cov chaw tsim khoom cov lus qhia. Peb qhov swb los ntawm txhua qhov thaiv tau raug tshuaj xyuas rau feem pua ​​​​ntawm cov hlwb apoptotic. Plaub slides teb tau random kuaj nrog 200 × magnification. Nyob rau hauv tag nrho, 100 hlwb raug suav nyob rau hauv txhua qhov chaw.

3.7. Kev ntsuas ntawm Antioxidant Enzyme Kev Ua Haujlwm

Superoxide dismutase (SOD) thiab catalase (CAT) kev ua haujlwm tau ntsuas los ntawm cov khoom siv coj mus muag (SOD: A001-3- 1 thiab CAT: A007-1-1; Jiancheng Bio. Institute, Nanjing, Tuam Tshoj) nrog lub spectrophotometer (Perkin- Elmer, Norwalk, CT, Tebchaws Asmeskas). Raws li cov chaw tsim khoom cov lus qhia, kev ua haujlwm tau ua tiav, thiab cov protein ntau tau ntsuas los ntawm cov txheej txheem bicinchoninic acid (Pierce, Rockford, United States) nrog bovine serum albumin (BSA) raws li tus qauv (34).

3.8. Kev ntsuas ntawm Adenosine 5'-Triphosphate Cov ntsiab lus

Cov ntsiab lus ATP hauv cov ntaub so ntswg plawv tau ntsuas siv cov khoom siv ATP (S0026B, Beyotime, Bio. Institute, Suav). Raws li cov chaw tsim khoom cov lus qhia, lysate tau ntxiv rau hauv kev faib ua feem raws li cov ntaub so ntswg hnyav, homogenized nrog lub khob homogenizer, thiab ces centrifuged ntawm 4 degree 12000 g. Lub supernatant raug coj mus, thiab cov ntsiab lus ntawm ATP hauv txhua tus qauv tau kuaj pom nrog lub luminometer (NanoDrop, Nanodrop2000, Thermo, USA) BCA cov khoom siv (P0012s, Beyotime, Bio. Institute, Suav). Txoj kev bicinchoninic acid tau siv los txiav txim siab cov protein ntau thiab tom qab ntawd hloov cov concentration ntawm ATP rau nmol / g protein.

3.9. Kis Electron Microscopy

Lub plawv apical cov ntaub so ntswg tau muab faib ua kwv yees li 1 hli × 1 hli × 1 hli me me thiab tom qab ntawd tsau rau hauv glutaraldehyde phosphate tsis nyob ntawm 4 degree. Tom qab niaj hnub lub cev qhuav dej, soaking, embedding, thiab staining, lub ultrathin seem ntawm 50 - 70 nm tau ua. Lub ultrastructure ntawm lub plawv cov ntaub so ntswg tau pom nyob rau hauv ib tug kis tau tus mob electron microscope (TEM) thiab yees duab (H600 Hitachi, Tokyo, Nyiv). Ib leeg mitochondria thiab myofilaments tau mapped nyob rau hauv cov xwm txheej ntawm 10000 lub sij hawm magnification nrog Image J software version 1.80 (National Institutes of Health), thiab lawv cheeb tsam tau ntsuas los ntawm txhua lub plawv (35). Lub sijhawm no, mitochondrial fragments <1 µm3, uas tsis tau muab faib (feem ntau yog puag ncig), tau txheeb xyuas, thiab qhov nruab nrab feem pua ​​​​ntawm cov mitochondrial fragments nyob rau hauv qhov chaw saib tau suav nrog Mitochondrial Fragmentation Index (MFI).

3.10. Mitochondrial Isolation

Mitochondria raug cais tawm ntawm 4 degree siv qhov sib txawv centrifugation nrog Mitochondria Isolation Kit (Beyotime Biotechnology, Shanghai, Suav). Luv luv, cov ntaub so ntswg tshiab tau txiav rau hauv cov ntaub so ntswg thiab centrifuged nrog 10 ntim ntawm pre-chilled PBS, lub supernatant raug muab pov tseg, cov precipitate tau zom nrog trypsin rau 20 min, qhov ntxiv kev sib cais tsis pub A yog centrifuged, tus supernatant raug xa mus rau lwm tus. raj, centrifuged dua, thiab cov feem ntau precipitated yog cov mitochondria cais. Qhov kawg ntawm lub plawv mitochondrial pellet tau raug tshem tawm hauv homogenizing tsis, khaws cia rau hauv dej khov, thiab siv rau kev sim ntawm mitochondrial ua pa ua haujlwm hauv 4h.

3.11. Kev ntsuas ntawm Mitochondrial Oxygen Consumption

Mitochondrial oxygen noj tau ntsuas los ntawm Clark-hom oxygen electrode (Hansatech Instruments, Norfolk, UK) hauv mitochondrial respiration buffer. Pyruvate (5 mM) thiab malate (5 mM) tau siv los ua substrate rau complex I-muaj mitochondria ntawm qhov kawg concentration ntawm 500 µg protein / mL. ADP-stimulated oxygen noj (lub xeev 3 ua pa) tau ntsuas nyob rau hauv lub xub ntiag ntawm 200 µM ADP, thiab ADP-yooj yim oxygen noj (lub xeev 4 ua pa) tau soj ntsuam. Kev tswj kev ua pa (RCR, xeev 3 faib los ntawm lub xeev 4) qhia txog kev siv oxygen los ntawm phosphorylation (coupling). Cov txheej txheem txuas ntxiv, raws li tau piav qhia dhau los (36).

3.12. Western Blot Analysis

Cov qauv ntawm cov ntaub so ntswg plawv tau sau thiab khaws cia ntawm -80 degree . Frozen tshuav ventricular cardiac cov ntaub so ntswg tau homogenized nyob rau hauv ice txias RIPA lysis tsis (Beyotime Inc., Shanghai, Tuam Tshoj P0013B). Cov protein ntau ntau tau suav nrog siv cov khoom kuaj BCA protein (Beyotime Inc., Shanghai, Tuam Tshoj P0006C). Cov qauv uas muaj tag nrho cov protein tau sib cais los ntawm 10 feem pua ​​(w/v) SDS-PAGE thiab xa mus rau PVDF membrane (Millipore, Bedford, MA, United States). Cov tshuaj tiv thaiv hauv qab no tau siv: Antibodies rau GAPDH (1:2000,10494-1-AP), MFN2 (1:1000,12186-1- AP), SIRT-1 (1:1000,{{16 }}AP), NRF-2 (1:600,16396-1-AP), TFAM (1:1000,19998-1-AP), and BAX (1.1000,{26}}Ig) tau muas los ntawm Proteintech (WuHan, Tuam Tshoj), BCL2 (1: 2000, sc-7382) thiab DRP1 (1:1000, sc-271583) tau yuav los ntawm Santa Cruz Biotechnology (Dallas, TX) thiab PGC -1 (1:1000, ab106814, Abcam, Cambridge, MA, UK). Cov tshuaj tiv thaiv kab mob thib ob (horseradish peroxidase-labeled tshis tiv thaiv luav IgG) yog los ntawm Beyotime Corporation (Shanghai, Suav). Cov kev siv ntawm cov protein bands tau txiav txim siab siv Fluor Chem Chemiluminescence gel imaging system (Protein Simple, USA). Qhov optical ceev ntawm cov protein bands tau soj ntsuam nrog Image J software version l.52 (NIH, Bethesda, MD).

3.13. Hypoxic Cardiomyocyte Model

Neonatal nas cardiomyocytes (NRMCs) raug cais thiab coj los ntawm cov txheej txheem, raws li tau piav qhia yav dhau los (32). Luv luv, lub siab ntawm peb-hnub neonatal nas raug muab rho tawm, minced, kab lis kev cai, thiab tom qab ntawd zom hauv 0.25 feem pua ​​trypsin thiab 0.02 feem pua ​​​​EDTA (Beyotime Biotechnology, Shanghai, Tuam Tshoj). Tom qab centrifugation, cov precipitates tau pauv mus rau DMEM ntxiv nrog 10 feem pua ​​​​ntawm fetal calf serum (Biolot, Russia) thiab incubated nyob rau hauv cov huab cua ntub uas muaj 5 feem pua ​​2. Peb hnub tom qab tau noob, cov hlwb tau muab tso rau hauv ib lub khob hypoxic chamber (Biospherix OxyCycler C42. , Redfield, NY), thiab ntim nrog nitrogen rau 8 min kom tso tawm cov pa seem. Cov cardiomyocytes no tau muab faib ua pawg hauv qab no: (1) Pab pawg tswj hwm (tswj), cov kab ke kab lis kev cai nyob rau hauv cov xwm txheej incubation; (2) pab pawg hypoxia (Hpx), cov hlwb muab tso rau hauv chav tsev hypoxic rau 24 teev thiab tom qab ntawd coj mus kuaj ib txwm rau 24 teev; (3) Hpx-Spd pawg, cov hlwb muab tso rau hauv lub chav hypoxic thiab incubated nrog 10 µmol / L SPD rau 24 h; (4) Hpx-SpdDFMO pab pawg, cov hlwb muab tso rau hauv ib chav hypoxic thiab incubated nrog 10 µmol / L SPD ntxiv rau 2 mmol / L DFMO rau 24h.

3.14. Kev ntsuas ntawm Reactive Oxygen Hom

ROS tiam yog ntsuas los ntawm dihydroethidium (DHE) staining assay (Cat No. S0063, Beyotime, Suav). Luv luv, thawj cardiomyocytes tau incubated nrog 5 µmol / L DHE ntawm 37 degree rau 30 min, ntxuav nrog PBS, thiab tom qab ntawd tsiv mus nyob rau hauv lub microscope los soj ntsuam cov kev hloov hauv fluorescence siv. Cov duab tau muab coj los siv nrog Olympus FluoView FV1000 fluorescence microscope (Olympus Optical Co., Ltd., Takachiho, Nyiv) ntawm qhov excitation wavelength ntawm 535 nm, thiab qhov siab tshaj plaws emission wavelength yog 610 nm, n> 20 hlwb ib pab.

3.15. Kev txiav txim siab ntawm Mitochondrial Membrane Potential

Cov xim tetramethylrhodamine ethyl ester (TMRE) yog qhov zoo thiab tuaj yeem xaiv nyob hauv mitochondria. Nws yog dav siv los txiav txim siab mitochondrial membrane peev xwm (∆Ψm). Nyob rau hauv luv luv, TMRE staining ua hauj lwm kev daws teeb meem ntawm ib tug concentration ntawm 200 µmol / L tau ntxiv rau cov pab pawg neeg kho cardiomyocytes, sib tov kom huv si, thiab muab tso rau hauv lub cell incubator ntawm 37 degree rau 20 min. Lub supernatant tau aspirated, thiab cov hlwb raug ntxuav nrog PBS thiab tsiv mus rau ib qho inverted microscope los soj ntsuam qhov kev hloov ntawm fluorescence siv. Lub excitation wavelength yog 549 nm, thiab lub teeb emission yog 579 nm. Kev siv tshuaj fluorescence liab qhia tau hais tias qhov kev hloov pauv ntawm ∆Ψm, n> 20 hlwb rau ib pawg.

3.16. Mitochondrial thiab Lysosomal Localization Experiment

Raws li kev qhia, Mito-Tracker Green (NO.C1048, Beyotime, Tuam Tshoj) thiab Lyso-Tracker Liab (NO.C1046, Beyotime, Tuam Tshoj) tau siv mitochondrial thiab lysosomal colocalization tsom xam. NRCMs tau ntim nrog 200 nM MitoTracker Green FM thiab 50 nM LysoTracker Liab hauv HBSS rau 30 feeb ua ntej kev sim, cov hlwb raug ntxuav nrog PBS, thiab tom qab ntawd cov duab ntawm tes tau txais los ntawm Olympus FluoView FV1000 fluorescence microscope. Lub 488 nm laser kab tau siv los ua kom muaj zog MitoTracker Green fluorescence, ntsuas ntawm 505 thiab 515 nm. Rau LysoTracker Liab, 577 nm laser kab tau siv nrog kev ntsuas ntawm 590 nm. Liab thiab ntsuab pixel siv zog overlay tau txiav txim siab siv software quantification ntawm Nikon Eclipse Microscope, n> 20 hlwb rau ib pawg.

3.17 ib. Kev txheeb cais

Tag nrho cov ntaub ntawv los ntawm cov pab pawg sim tau muab piv nrog ib txoj kev ANOVA ua raws Bonferroni post hoc test nrog GraphPad Prism software version 8 (GraphPad Software Inc., La, Jolla.CA) thiab SPSS software version 17.1 (SPSS, Chicago, IL, United. Xeev). Cov ntaub ntawv tau qhia raws li qhov txhais tau tias ± SEM, thiab theem tseem ceeb tau teeb tsa los ua P < 0.05.

4. Cov txiaj ntsig

4.1. Cov xeeb ntxwv thiab lub plawv yam ntxwv

Lub cev hnyav (BW) thiab lub plawv qhov hnyav (HW) tau ntsuas, thiab HW rau BW piv (HW / BW) raug xam (Daim duab 1). Cov txiaj ntsig tau pom tias cov nas me nyuam mos 'BW thiab HW poob qis, thiab lawv HW / BW tau nce ntxiv vim yog intrauterine hypoxia. Piv rau pawg IUH, BW thiab HW ntawm cov nas me me hauv pawg SPD tau nce (P < 0 05), thiab HW / BW poob qis (P < 0 . 05); Piv nrog rau pawg SPD, ob qho tib si BW thiab HW ntawm DFMO pab pawg kho mob poob qis (P <0.05), thiab HW / BW nce ntau (P <0.05).

4.2. Kev cuam tshuam ntawm SPD ntawm Myocardial Morphological Structure, Cell Proliferation, thiab Fibrosisin Newborn Disposed

Lub plawv HE staining cov txiaj ntsig tau pom tias lub siab ntawm ib hnub cov xeeb ntxwv raug rau IUH pom o thiab xoob txheej txheej ntawm myocardial fibers. Txawm li cas los xij, IUH lub siab kho nrog SPD khaws cov qauv ntawm cov ntaub so ntswg myocardial (Daim duab 2A). Tom ntej no, peb ntsuas tus naj npawb ntawm binuclear cardiomyocytes nrog HE-stained cov ntaub so ntswg; tus naj npawb ntawm binuclear cardiomyocytes loj dua hauv IUH pawg dua li hauv pawg tswj hwm (P < 0.05). Piv rau pawg IUH, qhov kev faib ua feem ntawm binuclear cardiomyocytes hauv pab pawg kho SPD poob qis (P < 0.05); DFMO tau txo cov txiaj ntsig ntawm SPD (P < 0 05) (Daim duab 2C). Peb tau tshawb pom ntxiv txog qhov qhia ntawm Ki67 (ib qho cim ntawm cell proliferating) hauv nas myocardium siv txoj kev immunofluorescence. Qhov siab dua qhov qhia ntawm Ki67, qhov muaj zog ntawm cov fluorescence liab los ntawm kev sib xyaw xim liab thiab xiav (Daim duab 2E). Cov txiaj ntsig tau pom tias piv rau pawg tswj hwm, kev qhia ntawm Ki67 hauv IUH pawg tau txo qis (P < 0.05), Ki67 qhia tau nce ntxiv tom qab kev tswj hwm SPD (P < { {28}}.05), thiab cov nyhuv ntawm SPD raug tshem tawm los ntawm DFMO (P < 0.05) (Daim duab 2F). Cov txiaj ntsig no qhia tau hais tias SPD tuaj yeem cuam tshuam qhov kev tshem tawm ntxov ntawm cardiomyocytes los ntawm lub voj voog ntawm tes raug ntxias los ntawm IUH thiab txhawb kev loj hlob ntawm cardiomyocytes hauv cov xeeb ntxwv tshiab raug rau IUH. Tom qab ntawd peb siv Masson staining txhawm rau txheeb xyuas cov kev hloov pauv hauv myocardial collagen cov ntsiab lus thiab ntsuas myocardial fibrosis los ntawm kev ntsuas thaj tsam collagen (Daim duab 2BandD). Peb pom tias myocardial collagen deposition nyob rau hauv lub siab lub ntsws ntawm cov me nyuam mos nas raug IUH nce (P < 0.05), qib uas yog siab dua piv rau pawg tswj. Ntawm qhov tsis sib xws, thaj tsam ntawm myocardial fibrosis tom qab kev kho SPD tau txo qis (P <0.05). Txawm li cas los xij, piv rau pab pawg kho mob SPD, thaj tsam fibrosis tau nce ntxiv hauv HpxSpd-DFMO pawg (P <0.05).

4.3. Kev cuam tshuam ntawm Spermidine ntawm Myocardial Mitochondrial Structure, Kev ua pa ua pa, thiab Adenosine 5'-Triphosphate Cov ntsiab lus hauv cov menyuam yug tshiab raug rau Intrauterine Hypoxia

Cov qauv kev hloov pauv ntawm cov qauv ntawm lub plawv thiab cov yam ntxwv mitochondrial tau txheeb xyuas los ntawm TEM (Daim duab 3A). Duab J software tau siv los ntsuas qhov feem pua ​​​​ntawm cov ntsiab lus mitochondrial ( cheeb tsam mitochondrial nyob rau hauv tag nrho cov cell cheeb tsam) thiab mitochondrial cheeb tsam (Figures 3B thiab C). Cov txiaj ntsig tau pom tias nyob rau hauv pawg tswj hwm, cov myocardial myofilaments tau raug teeb tsa, cov qauv sarcomere yog qhov tseeb, mitochondria tau compact, matrix yog denser, thiab mitochondrial cristae tau raug teeb tsa. Txawm li cas los xij, mitochondria o o, thiab cov xoos matrix thiab qhov ntom ntom tau pom hauv qee qhov cardiomyocytes ntawm IUH pawg. Piv nrog rau pawg tswj hwm, qhov kev faib ua feem ntawm mitochondria hauv cardiomyocytes thiab thaj tsam ntawm mitochondria raug txo qis (P < 0.05). Txawm li cas los xij, nyob rau hauv cov nas lub siab ntawm SPD kev kho mob, myocardial myofilaments tau zoo huv si, cov qauv sarcomere meej, mitochondrial matrix yog compact, thiab mitochondrial o o poob. Piv nrog rau pawg IUH, qhov feem ntawm mitochondria hauv cardiomyocytes nce (P < 0 05), thiab thaj tsam ntawm mitochondria nce (P < 0.05). DFMO inhibited cov teebmeem SPD-induced (P <0.05).

Peb siv pyruvate/malate ua lub substrate los ntsuam xyuas lub mitochondrial ua pa muaj nuj nqi, nrog rau cov xeev 3 thiab 4 respiratory rates thiab RCR (Daim duab 3D - F). Peb pom tias piv rau pawg tswj hwm, qhov ua pa ntawm lub xeev 3 thiab 4 thiab RCR ntawm IUH pawg tau qis dua (P < 0.{{10}}5). Qhov zoo siab, RCR ntawm lub xeev 3 thiab 4 tau rov qab los tom qab kev kho SPD (P < 0.05). Hauv qhov sib piv, cov teebmeem SPD no tau cuam tshuam loj heev hauv DFMO pab pawg kho mob (P < 0.05). Ib yam li ntawd, piv rau pawg tswj hwm, cov ntsiab lus myocardial ATP hauv IUH pawg tau txo qis (P <0.05). Piv nrog rau pawg IUH, ATP cov ntsiab lus tau nce siab heev hauv SPD-kho pab pawg (P < 0.05), thiab DFMO txo qis cov teebmeem ntawm SPD (P < 0.05) (Daim duab 3G). Cov kev tshawb pom no qhia tias SPD tuaj yeem tiv thaiv cov qauv myocardial mitochondrial thiab ua haujlwm puas tsuaj thiab tiv thaiv kev poob qis ntawm ATP qib hauv cov menyuam yug menyuam nas uas raug IUH.

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