Echinacoside Reverses Myocardial Remodeling Thiab Txhim Kho Lub Plawv Ua Haujlwm

Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Yajuan Ni1|Jie Deng1|Xin Liu 1|Qing Li1|Juanli Zhang1|Hongyuan Bai1|Jingwen Zhang 2

Abstract

Myocardial remodeling yog ib qho tseem ceeb pathological hauv paus ntawm HF, mitochondrial oxidative kev nyuaj siab yog txhawb nqa ntawm myocardial hypertrophy, fibrosis, thiab apoptosis. ECH yog qhov tseem ceeb ntawm cov tshuaj suav tshuaj Cistanches Herba, ntau cov kev tshawb fawb qhia tias nws muaj peev xwm antioxidant muaj zog hauv cov paj hlwb thiab cov qog, nws inhibits mitochondrial oxidative kev nyuaj siab, tiv thaiv mitochondrial muaj nuj nqi, tab sis cov txheej txheem tshwj xeeb tsis meej. SIRT1/FOXO3a/MnSOD yog ib qho tseem ceeb antioxidant axis, txoj kev tshawb fawb pom tias ECH khi covalently rau SIRT1 raws li ib tug ligand thiab up-regulates cov kev qhia ntawm SIRT1 nyob rau hauv lub hlwb hlwb. Peb xav tias ECH tuaj yeem thim rov qab myocardial remodeling thiab txhim kho lub plawv ua haujlwm ntawm HF los ntawm kev tswj hwm SIRT1 / FOXO3a / MnSOD teeb liab axis thiab inhibiting mitochondrial oxidative kev nyuaj siab hauv cardiomyocytes. Ntawm no, peb ua ntej tsim cov qauv cellular ntawm oxidative kev nyuaj siab los ntawm ISO nrog AC -16 hlwb thiab ua ntej kho nrog ECH, qib ntawm mitochondrial ROS, mtDNA oxidative raug mob, MMP, carbonylated protein, lipid peroxidation, intracellular ROS, thiab apoptosis raug kuaj pom, paub meej tias cov nyhuv ntawm ECH hauv mitochondrial oxidative kev nyuaj siab thiab ua haujlwm hauv vitro. Tom qab ntawd, peb tsim ib qho HF nas qauv induced los ntawm ISO thiab pre-kho nrog ECH. Kev ntsuas ntawm lub plawv ua haujlwm, kho myocardial remodeling, mitochondrial oxidative kev nyuaj siab thiab kev ua haujlwm, kev qhia ntawm SIRT1 / FOXO3a / MnSOD signaling axis raug ntsuas, cov ntaub ntawv qhia tias ECH txhim kho lub plawv ua haujlwm, inhibits myocardial hypertrophy, fibrosis thiab apoptosis nce kev qhia ntawm SIRTa / FOXO3. MnSOD signaling axis, txo cov mitochondrial oxidative puas tsuaj, tiv thaiv mitochondrial muaj nuj nqi. Peb xaus lus tias ECH thim rov qab myocardial remodeling thiab txhim kho lub plawv ua haujlwm los ntawm kev tswj hwm SIRT1 / FOXO3a / MnSOD axis thiab inhibiting mitochondrial oxidative stress hauv HF nas.

KEYWORDSechinacoside, lub plawv tsis ua hauj lwm, mitochondrial oxidative stress, myocardial remodeling

tshiab tshuaj ntsuab cistancheraumitochondrial oxidative kev nyuaj siab

1|Taw qhia

Lub plawv tsis ua hauj lwm (HF) yog qhov kawg tshwm sim ntawm feem ntau cov kab mob plawv nrog cov kab mob siab heev thiab cov tsos mob tsis zoo, thiab nrog cov neeg laus zuj zus, kev mus pw hauv tsev kho mob thiab kev tuag vim mob plawv tsis ua hauj lwm (CHF) tau nce siab thoob plaws ntiaj teb.1 Lub sijhawm 20 xyoo dhau los, kev kho tshuaj rau HF tau ua tiav zoo, tab sis cov txiaj ntsig muaj tsawg, yog li cov tshuaj tshiab yuav tsum tau ceev. Myocardial remodeling yog ib qho tseem ceeb pathological hauv paus ntawm HF thiab yog tus cwj pwm los ntawm myocardial hypertrophy, apoptosis, interstitial fibrosis, myocardial hlwb yog disorganized, thiab ua rau txo qis ntawm ejection feem (LVEF), nce ntawm sab laug ventricular kawg-diastolic thiab systolic qhov ntev. (LVIDd thiab LVIDs), thiab txo qis ntawm sab laug ventricular fractional shortening (LVFS).2 Tab sis cov txheej txheem hauv qab tseem tsis tau nkag siab. Nyob rau hauv xyoo tas los no, ntau cov kev tshawb fawb tau lees paub tias oxidative kev nyuaj siab yog ib qho tseem ceeb txhawb kev kho mob myocardial.3-7

Oxidative stress yog ib yam mob uas reactive oxygen hom (ROS) los yog dawb radicals yog generated ntau heev thiab ua rau oxidative puas ntawm cell Cheebtsam, qhov no, nyob rau hauv lem, ua rau cell dysfunction.8Mitochondria yog lub ntsiab qhov chaw ntawm intracellular ROS, vim hais tias ntawm Kev xau los ntawm txoj hlab pa hluav taws xob thauj khoom, thiab mitochondria yog txoj haujlwm rhiab tshaj plaws rau ROS teebmeem.9 Kev tshawb fawb hauv HF tau txheeb xyuas ROS ntau zuj zus los ntawm txoj haujlwm I ntawm txoj hlab pa.8 Nce ROS ntau lawm tawm tsam mitochondrial Cheebtsam ua ntej thiab ua rau muaj kev ntxhov siab oxidative ntau dhau. , manifests li carbonylation ntawm cov proteins, lipid peroxidation, thiab DNA puas, 10 thiab dysfunction ntawm mitochondria, uas yog li ua rau ntau heev tsim ntawm ROS, 8 ua ib tug vicious vajvoog. Kev txhawb nqa oxidative kev ntxhov siab tuaj yeem ua rau myocardial hypertrophy thiab myocardial fibrosis, 11-13 thiab ua rau apoptosis ntawm myocardial hlwb los ntawm kev ua kom cov mitochondria-dependent apoptosis los ntawm carbonylation ntawm mitochondrial membrane proteins thiab poob ntawm mitochondrial membrane muaj peev xwm. , nws tau txiav txim siab tias inhibition ntawm mitochondrial oxidative kev nyuaj siab tej zaum yuav yog lub tswv yim tshiab los tiv thaiv myocardial remodeling thiab txawm HF.15-17

Echinacoside (ECH), lub ntuj phenylethanoid glycoside, yog lub luag haujlwm tseem ceeb ntawm Cistanches Herba, uas yog cov tshuaj suav tshuaj. Nyob rau hauv xyoo tas los no, ECH tau raug tshawb fawb ntau hauv cov hlab ntsha thiab cov qog, thiab nws tau raug tshaj tawm tias muaj ntau yam tshuaj muaj txiaj ntsig, xws li tshuaj tua kab mob antioxidant, tiv thaiv kab mob, tiv thaiv apoptosis, thiab tiv thaiv qog nqaij hlav.18,19In. paj hlwb, ECH ho txhim kho antioxidant muaj peev xwm, txo ROS ntau lawm, txhim kho mitochondrial membrane muaj peev xwm (MMP), thiab tsub kom cov cell viability, 20 qhia txog kev tiv thaiv los ntawm mitochondrial muaj nuj nqi thiab inhibits mitochondrial oxidative kev nyuaj siab, nws tiv thaiv oxidative raug mob thiab txhim kho kev nco, nws thiaj li tiv thaiv. neurodegeneration.21,22 ECH kuj tseem ua rau kev ua haujlwm ntawm SOD thiab ua lub luag haujlwm tiv thaiv hauv lub hlwb thiab cov ntaub so ntswg retina, 19,23 tab sis cov txheej txheem tshwj xeeb rau nws cov nyhuv antioxidant tsis meej.

SIRT1 muaj feem xyuam rau oxidative kev nyuaj siab, thiab SIRT1 / FOXO3a / MnSOD signaling axis yog qhov tseem ceeb tshaj plaws antioxidant axis thiab yog lub luag hauj lwm rau kev tawm tsam ntawm mitochondrial oxidative kev nyuaj siab, MnSOD faib dav hauv mitochondria thiab feem ntau yog lub luag hauj lwm rau kev tshem tawm ntawm mitochondrial ROS. inhibiting oxidative kev nyuaj siab, cov tebchaw uas ua kom cov kab mob antioxidant no tuaj yeem cuam tshuam oxidative kev nyuaj siab.24,25 Cov kev tshawb fawb tsis ntev los no tau pom tias ECH tau siv nws cov tshuaj pharmacological los ntawm kev tswj ntau txoj kev qhia, xws li p-AKT, mTOR/STAT3, TGF-beta 1/Smads. , thiab SIRT1 kev taw qhia txoj hauv kev. kho dua tshiab. Txawm li cas los xij, kev tshawb fawb txog cov teebmeem ntawm ECH tau tsom mus rau lub paj hlwb lossis qog nqaij hlav, cov teebmeem thiab cov txheej txheem ntawm ECH ntawm mitochondrial oxidative stress hauv cardiomyocytes thiab myocardial remodeling tseem tsis paub. Ntawm no, peb ua ntej tsim cov qauv cellular ntawm oxidative kev nyuaj siab los ntawm ISO thiab paub meej tias cov nyhuv ntawm ECH ntawm mitochondrial oxidative kev nyuaj siab thiab kev ua haujlwm hauv vitro. Tom qab ntawd, peb tsim ib qho HF nas qauv induced los ntawm ISO thiab ua kom pom tias ECH thim rov qab myocardial remodeling thiab txhim kho lub plawv muaj nuj nqi ntawm up-regulating SIRT1 / FOXO3a / MnSOD signaling axis thiab inhibiting mitochondrial oxidative kev nyuaj siab nyob rau hauv HF nas. Cov txiaj ntsig tau npaj los muab cov pov thawj kev sim rau kev tsim cov tshuaj tshiab los tiv thaiv lub plawv tsis ua haujlwm.

2|Cov khoom siv thiab cov txheej txheem

2.1|Cell kab lis kev cai thiab kev kho mob

AC16 hlwb yog yuav los ntawm Bena Culture Collection. Cov hlwb raug khaws cia hauv cov piam thaj siab Dulbecco's modified Eagle's nruab nrab (DMEM; Hyclone, GE Healthcare, USA) ntxiv nrog 10 feem pua ​​​​fetal bovine serum yog (10 feem pua ​​FBS; Gibco) hauv T-75 flasks (Corning Glassworks, Corning, NY, USA). Cov xwm txheej tau khaws cia ntawm 37 degree hauv qhov chaw noo noo uas muaj 5 feem pua ​​​​CO2. Thaum cov hlwb ncav cuag 70 feem pua ​​-80 feem pua ​​confluence, ISO (Sigma, USA) pab pawg hlwb yog incubated nrog ib tug nruab nrab uas muaj 10uM ISO (Sigma, USA) rau 24h, ECH pawg hlwb yog pre-kho nrog 50uM ECH (MCE. , USA) rau 30min ua ntej ISO raug. Tswj hlwb (Ctrl) yog kho tsis muaj dab tsi.

cistanche muagrho tawm

2.2|Kev kuaj pom ntawm mitochondrial ROS theem siv luminol chemiluminescence

Mitochondria raug rho tawm thiab lim los ntawm AC-16 hlwb siv Mitochondria Isolation Kit (Sigma, USA) raws li cov chaw tsim khoom cov lus qhia. Protein concentrations yog txiav txim los ntawm BCA protein assay kit (Biotech). Mitochondrial ROS qib yog kuaj pom los ntawm luminal (3-aminophthalic hydrazide 5-amino-2,3-dihydro-1,4-phthalazinedione) siv cov khoom siv Elisa (GERMED SCIENTIFIC INC, USA), nws reacts nrog ROS thiab ua rau lub emission ntawm photons, uas yog ib tug band spectrum muaj ib tug ncov ntawm txog 460nm, thiab txheeb ze lub teeb Unit (RLU) yog kuaj los ntawm chemiluminescence ntsuas ntsuas thiab hloov mus rau hauv RLU / ug. mitochondrial protein ntau.

2.3|8-OHdG yog siv los ntsuas qhov kev puas tsuaj oxidative ntawm mitochondrial DNA

Mitochondrial DNA oxidative puas tsuaj yog soj ntsuam los ntawm kev kuaj xyuas cov ntsiab lus ntawm 8-Hydroxy-2-deoxyguanosine (8-OHG). Purified mitochondria hauv hlwb raug cais siv cov khoom siv mitochondria cais raws li saum toj no (Sigma). Protein concentrations yog txiav txim los ntawm BCA protein assay kit (Biotech). Tom qab ntawd, lawv tau sib cais los txiav txim siab qib ntawm 8-OHdG siv cov khoom siv Elisa (GERMED SCIENTIFIC INC, USA) raws li cov chaw tsim khoom cov lus qhia. Lub absorbance ntawm kev hloov xim yog ntsuas hauv spectrophotometer ntawm lub wavelength ntawm 450 nm. Qhov siab ntawm 8-OHdG hauv cov qauv yog txiav txim siab los ntawm kev sib piv OD ntawm cov qauv mus rau tus qauv nkhaus thiab hloov mus rau ng / ml.

2.4|Kev ntsuas ntawm MMP los ntawm JC-1

Mitochondria raug cais tawm thiab ua kom huv ntawm AC -16 thiab cov protein ntau yog txiav txim siab raws li tau hais los saum no. Tom qab ntawd cov mitochondria tau rov ua dua ntawm 1mg / ml concentration, MMP yog ntsuas los ntawm JC-1 cov khoom ntsuas raws li cov chaw tsim khoom cov lus qhia. Qhov kev siv fluorescence yog ntsuas ntawm excitation wavelength: 490nm, slit 5nm, emission wavelength: 590 nm, slit 5nm nrog lub ntsuas spectrophotometer, 30 cov txiaj ntsig tau sawv cev ua FLU / ugP.

2.5|Kev kuaj pom ntawm apoptotic cells los ntawm kev ntws cytometry hauv AC-16 Cells

Flow cytometry yog siv los ntsuas cellular apoptosis. Ib qho Annexin V-FITC / PI apoptosis detection kit (CWBIO, China) yog siv los kuaj apoptotic thiab necrotic hlwb raws li tus neeg muag khoom cov lus qhia. Thaum ntxov-theem apoptosis, late-stage apoptosis lossis necrosis, cellular khib nyiab, thiab cov hlwb siv tau raug txheeb xyuas raws li annexin V (ntxiv ) / PI (−), annexin V (ntxiv) / PI (ntxiv), annexin V (−) / PI ( plus ) and annexin V (−)/PI (−), feem. Fluorescence intensities yog soj ntsuam siv lub flow cytometer (BD FACSVerse™ Flow Cytometer, BD Biosciences, San Jose, CA, USA). Txhua qhov kev sim rov ua dua 3 zaug.

2.6|Kev ntsuam xyuas qhov intracellular ROS los ntawm flow cytometer

Tom qab kev kho mob, cov hlwb raug trypsinized, ntxuav ob zaug nrog PBS. Tom qab ntawd cov hlwb raug tshem tawm hauv PBS, stained nrog DCFH-DA (Beyotime, Tuam Tshoj) ntawm qhov kawg ntawm 1 μM thiab incubated rau 1 h ntawm 37 degree nyob rau hauv lub incubator nrog CO2, thiab ces sim nyob rau hauv ib tug flow cytometer ntawm 488 nm excitation. . Qhov kev siv fluorescence sawv cev rau theem ntawm intracellular ROS.

2.7|Test carbonyl protein cov ntsiab lus nyob rau hauv mitochondria ntawm AC-16 hlwb

Tom qab kev kho mob, purified mitochondria raug rho tawm thiab sonicated ntawm cov dej khov thiab kho nrog 1 feem pua ​​​​streptomycin sulfate rau precipitate mitochondrial nucleic acids, protein concentration yog txiav txim siab. Cov ntsiab lus carbonyl protein yog kuaj los ntawm cov khoom siv Elisa (GERMED SCIENTIFIC INC, USA) raws li derivatization ntawm pawg carbonyl nrog dinitrophenylhydrazine (DNPH), raws li cov lus qhia ntawm cov khoom siv, OD yog ntsuas los ntawm microplate nyeem ntawv thiab hloov mus rau nmol / mg protein los ntawm kev sib piv rau tus qauv nkhaus.

2.8|Kev txiav txim siab ntawm lipid peroxidation theem hauv mitochondria ntawm AC-16 hlwb

Tom qab kev kho mob, cov hlwb raug sau thiab lysed, mitochondria raug cais thiab ntxuav, thiab tawg los ntawm ultrasound, protein concentration yog txiav txim los ntawm BCA protein assay kit (Biotech), lipid peroxidation yog txiav txim los ntawm theem ntawm malondialdehyde (MDA) ntawm TBARS kev soj ntsuam. , siv cov khoom siv (Nan jing Jian cheng, Suav teb) thiab qib MDA yog ntsuas raws li cov chaw tsim khoom raws tu qauv. OD tau kuaj pom ntawm 586 nm los ntawm tus nyeem ntawv microplate thiab hloov mus rau μmol / g protein los ntawm kev sib piv rau tus qauv nkhaus.

2.9|Tsiaj txhu thiab kev kho mob

Cov txiv neej hluas Sprague-Dawley nas hnyav 120-140g tau los ntawm Xi'an Jiao Tong University Laboratorial Animal Center (Shaanxi, Suav). Kev tshawb nrhiav ua raws li Phau Ntawv Qhia Txog Kev Saib Xyuas thiab Kev Siv Tsiaj Tsiaj, luam tawm los ntawm Tebchaws Meskas Lub Tsev Haujlwm Saib Xyuas Kev Noj Qab Haus Huv (NIH tsab ntawv no.85-23, kho dua xyoo 1996), thiab cov txheej txheem kev sim tau pom zoo los ntawm cov tub ceev xwm hauv cheeb tsam ( Biomedical Ethics Committee ntawm Medical Department ntawm Xi 'a Jiao Tong University). Tus qauv nas ntawm HF yog tsim raws li tau piav qhia yav dhau los los ntawm peb.31 Luv luv, Isoproterenol (10} mg / kg) yog siv ib hnub ib zaug los ntawm kev txhaj tshuaj intraperitoneal rau 2 lub lis piam (txhais tau tias yog pab pawg HF), ECH yog siv nrog 20 ug / g ib hnub ib zaug los ntawm kev txhaj tshuaj intraperitoneal ntawm 30min ua ntej ISO raug kho (txhais tias yog pawg ECH), thiab kev tswj hwm kav ntev li 2 lub lis piam. Cov tsiaj tswj tau tswj hwm nrog 0.9 feem pua ​​NaCl (txhais tau tias yog pawg Ctrl). Echocardiography ntsuas yog ua los ntsuas lub plawv ua haujlwm.

2.10|Nws Echocardiographic ntsuas

Tsiaj txhu raug tshuaj loog los ntawm kev txhaj tshuaj intraperitoneal ntawm 300 mg / kg ntawm chloral hydrate. Lub SONOS-2500 ultrasound system nrog ib qho ultrasound transducer ntawm 7.5 MHz (HP, USA) yog siv los kuaj echocardiographic. Sab laug ventricular ejection feem (LVEF), sab laug ventricular fractional shortening (LVFS), sab laug ventricular end-diastolic qhov ntev (LVIDd), sab laug ventricular kawg-systolic qhov ntev (LVIDs), lub plawv dhia (HR), interventricular septal thickness hauv diastole (IVSTd). ), sab laug ventricular posterior phab ntsa thickness nyob rau hauv diastole (LVPWTd) yog ntsuas raws li tau piav yav tas los los ntawm peb.32

FIGURE 1 50μmol ECH zoo inhibits mitochondrial oxidative kev nyuaj siab ntawm AC-16 hlwb induced los ntawm 10μmol/L ISO thiab tiv thaiv mitochondrial muaj nuj nqi thiab txo apoptosis.

2.11|Lub plawv hnyav rau lub cev qhov hnyav piv (HW / BW) yog ntsuas

Rat yog hnyav ua ntej anaesthetization, lub plawv tshiab raug cais tawm thiab ntxuav tawm cov ntshav nrog 0.1mol/L PBS, txiav tawm cov hlab ntsha thiab lwm cov ntaub so ntswg rov qab thiab lub plawv yuav hnyav, ces HW/BW (mg/100g) yog xam.

2.12|Histological staining

Lub siab ntawm nas raug muab tshem tawm thiab kho hauv 4 feem pua ​​​​ntawm paraformaldehyde, kos rau hauv paraffin, thiab muab faib ua 5 μm-thick slices. Haematoxylin-Eosin (HE) staining yog siv los soj ntsuam cov kev hloov pauv pathological thiab Masson's trichrome yog siv los ntsuas collagen fibers ntawm nas myocardium cov ntaub so ntswg. Lub plawv collagen ntim feem (CVF) thiab cardiomyocytes cross-sectional cheeb tsam yog ntsuas nrog Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA).

2.13|TUNEL staining

Cov ntaub so ntswg myocardial los ntawm sab laug ventricle tau sau thiab TUNEL kev soj ntsuam yog ua raws li tau piav qhia dhau los ntawm peb32.

2.14|Transmission electron microscope

Cov ntaub so ntswg tshiab tau kho hauv 2.5 feem pua ​​​​glutaraldehyde ntawm 4 degree, cov ntaub so ntswg yog txiav mus rau hauv 1mm3 hauv ntim thiab kho dua hauv 2.5 feem pua ​​glutaraldehyde ntawm 4 degree rau 2h, ces ntxuav nrog 0.1mol/L PBS, tsau nyob rau hauv 1 feem pua ​​osmic acid, lub cev qhuav dej nrog acetone, embedded nrog epoxy resin, ultrathin slices uas nyias txog 800nm yog ua, stained nrog uranyl acetate thiab lead citrate, thiab ces raug soj ntsuam nyob rau hauv ib tug kis tau tus mob electron microscope (H{ {13}}, Hitachi Limited, Nyiv). Txhawm rau kom muaj nuj nqis tiv thaiv ECH hauv mitochondrial morphology, qhov nruab nrab mitochondrial ncig, txhais tau tias mitochondrial cross-sectional cheeb tsam, feem pua ​​​​ntawm degeneration, thiab feem pua ​​​​ntawm vacuolization hauv mitochondria raug txheeb xyuas nrog Image-Pro Plus 6.0 (Media Cybernetics, USA).

2.15 Nws Quantitative PCR

Tag Nrho RNA yog npaj los ntawm sab laug ventricular ntaub so ntswg nrog Trizol reagent (Invitrogen, Carlsbad, USA). cDNA yog synthesized siv TUREscript 1st Strand cDNA Synthesis Kit (Takara, Nyiv). Tshwj xeeb primer sequences ntawm collagen kuv siv rau real-time PCR yog raws li nram no: pem hauv ntej: 5′-GTCGTATCCAGTCGGTGTC-3′, revers: 5′-GTGGAGTCGGCAATTGCA-3′. GAPDH rau pem hauv ntej 5'-GGC AAG GTC ATC CCA GAG CT-3', rov qab 5'-CGC CTG CTT CAC CAC CTT CT-3'.Real-time quantitative PCR (qPCR) yog ua nrog SYBRPremix Ex Taq (Perfect Real Time) (Takara, Nyiv). Tus txheeb ze qib ntawm mRNA yog xam los ntawm normalizing rau GAPDH, raws li txoj kev 2-ΔΔCT.

2.16|Western blot tsom xam ntawm SIRT1, FOXO3a, MnSOD protein qhia

Tag nrho cov protein ntawm sab laug ventricular cov ntaub so ntswg yog muab rho tawm ntawm cov dej khov siv txias RIPA Buffer (BioRad, USA) nrog Protease Inhibitor Cocktail (Sigma-Aldrich). Qhov concentration yog txiav txim los ntawm txoj kev BCA. Cov txheej txheem ntawm Western blot tau piav qhia yav dhau los.33 Luv luv, cov qauv protein yog fractionated ntawm 15 feem pua ​​​​sodium dodecyl sulfate-polyacrylamide gels, xa mus rau PVDF membranes (BioRad, USA), thaiv los ntawm 5 feem pua ​​​​skim mis nyuj, thiab incubated nrog MYH antibody los ntawm nas. (diluted 1:200, Santa Cruz, USA). Tom qab ntawd, incubated nrog horseradish peroxidase-conjugated tshis nas antibody (diluted 1: 2, 000, Santa Cruz, USA). Tus txheeb ze qib ntawm cov protein yog xam los ntawm normalizing qhov nruab nrab ray nqi rau -actin. Protein bands raug kuaj pom los ntawm chemiluminescence system (ChemiDoc XRS, BioRad).

2.17|Kev ntsuas ntawm mitochondrial oxidative kev nyuaj siab hauv nas myocardium

Purified mitochondria ntawm nas myocardium raug cais siv Mitochondria Isolation Kit (Sigma, USA) raws li cov chaw tsim khoom cov lus qhia, mitochondrial ROS, MMP, oxidative puas ntawm mtDNA, carbonylated protein cov ntsiab lus, thiab lipid peroxidation theem nyob rau hauv mitochondria yog tag nrho cov kuaj pom siv tib txoj kev li. cov kev sim ntawm tes piav qhia saum toj no. Nco ntsoov, ROS nyob rau hauv cov ntaub so ntswg myocardial tau kuaj pom nrog DCFH-DA fluorescent probes, fluorescence siv tau kuaj pom nrog cov enzyme-labeling ntsuas ntsuas, cov txiaj ntsig tau sawv cev raws li RLU / ug mitochondrial protein.

2.18|Kev txheeb cais

Tag nrho cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± SEM. Ib txoj kev ANOVA yog siv los sib piv qhov sib txawv ntawm ntau pab pawg, ua raws li Tukey post hoc xeem rau qhov tseem ceeb. Tag nrho cov txheeb cais raug txiav txim siv SPSS15.0 software (SPSS Inc, IL, USA). Qhov tshwm sim tus nqi ntawm P < .05="" yog="" suav="" tias="" yog="" qhov="" tseem="">

3|TSEEM CEEB

3.1|50 μM ECH zoo inhibits mitochondrial oxidative puas thiab apoptosis hauv AC -16 hlwb

Cov hlwb tsis kho yog teem ua pawg Ctrl. Ua ntej, raws li daim duab 1A qhia, ECH inhibits mitochondrial ROS induced los ntawm ISO. ISO kho hlwb qhia tias muaj kev nce ntxiv hauv mitochondrial ROS piv nrog Ctrl thiab qhov kev nce no tau txo qis los ntawm kev kho ua ntej nrog 50 μM ECH. Qhov thib ob, ECH tuaj yeem cuam tshuam oxidative puas ntawm mtDNA ntxias los ntawm ISO. 8-OHdG ntseeg tau tias yog ib qho ntawm cov kab mob DNA ntau tshaj plaws uas tshwm sim los ntawm oxidative kev nyuaj siab thiab yog biomarker ntawm oxidative DNA puas thiab kho. Raws li daim duab 1B qhia, cov ntsiab lus ntawm 8-OHdG hauv purified mitochondria los ntawm AC-16 hlwb raug kuaj pom, cov txiaj ntsig tau pom tias qib ntawm 8-OHdG hauv mitochondria los ntawm ISO kho cov hlwb tau nce ntau, tab sis qhov kev hloov pauv tau thim rov qab los ntawm 50 μM ECH. Thib peb, ECH muaj txiaj ntsig los tiv thaiv MMP, uas cuam tshuam los ntawm ISO. Qhov kev siv fluorescence tau txo qis hauv cov hlwb uas kho nrog ISO piv nrog Ctrl hlwb, qhov kev kho ua ntej nrog 50 μM ECH remarkably attenuates qhov txo ntawm MMP, raws li daim duab 1C qhia. Plaub, ECH tiv thaiv oxidative kev puas tsuaj ntawm mitochondrial protein nyob rau hauv AC{12}} hlwb. Raws li daim duab 1D qhia, cov ntsiab lus carbonyl protein nyob rau hauv mitochondria ntawm ISO kho hlwb yog nce thiab ECH attenuates cov nyhuv. Fifthly, ECH kuj inhibits mitochondrial lipid peroxidation nyob rau hauv AC{14}} hlwb induced los ntawm ISO. Raws li daim duab 1E qhia tau hais tias, qib ntawm lipid peroxides hauv mitochondria ntawm ISO hlwb yog pom tseeb siab dua li ntawm Ctrl hlwb, thaum qhov nce no txo ​​qis los ntawm 50μM ECH ua ntej kho. Thaum kawg, ECH inhibits lub tsub zuj zuj ntawm intracellular ROS. Raws li daim duab 1F qhia, theem ntawm intracellular ROS nyob rau hauv ISO hlwb tau nce ntxiv thiab tseem ceeb txo qis hauv ECH cov hlwb ua ntej kho. Daim duab 1G qhia tau hais tias apoptosis tus nqi ntawm AC-16 hlwb tau nce ntxiv tom qab 24h incubations nrog 10μM ISO, piv rau Ctrl hlwb, thaum kho cov hlwb ua ntej nrog 50 μM ECH txo qis ISO-induced apoptosis.

FIGURE 2 Lub plawv hypertrophy ntawm HF nas induced los ntawm ISO yog relieved thiab lub plawv muaj nuj nqi yog ho txhim kho los ntawm ECH kev kho mob.

3.2|ECH thim rov qab myocardial remodeling thiab txhim kho lub plawv ua haujlwm ntawm ISO-induced HF nas

Raws li pom nyob rau hauv daim duab 2A, lub siab nyob rau hauv HF nas yog pom tseeb hypertrophy thiab thim rov qab los ntawm kev kho mob nrog ECH. Raws li daim duab 2B-D thiab L qhia, ECH txo HW/BW. Cov txiaj ntsig Echocardiography qhia tau tias LVEF thiab LVFS tau txo qis hauv HF pawg, LVIDd, LVIDs, thiab IVSTd tau nce ntau hauv HF pawg. Txawm li cas los xij, ECH txo qis LVIDd, LVIDs, IVSTd thiab txhim kho LVEF thiab LVFS, tsis muaj qhov sib txawv loj hauv HR ntawm peb pawg, LVPWTd tau nce hauv HF, tab sis tsis muaj qhov sib txawv ntawm ECH thiab Ctrl, lossis ntawm ECH thiab HF, tag nrho raws li Daim duab 2E-K qhia tau hais tias HE staining qhia tias myocardial hypertrophy, teeb meem tsis meej lossis degeneration necrosis, inflammatory cell infiltration thiab interstitial hyperemia thiab edema hauv HF nas, thiab ECH txo cov kev puas tsuaj no, myocardial hypertrophy thiab cov teeb meem kev teeb tsa tau daws, degeneration necrosis thiab inflammatory infiltrate raug txo, raws li qhia hauv daim duab 3A. Lub cardiomyocyte's cross-sectional cheeb tsam raug ntsuas, nws tau nce ntxiv tom qab ISO thiab txo qis hauv ECH cov pab pawg kho mob, raws li daim duab 3E qhia. Kev ntsuam xyuas ntawm collagen deposition los ntawm Masson staining qhia tau hais tias sab laug ventricular collagen ntsiab lus nyob rau hauv HF nas yog nce obviously thiab pom tseeb txo qis tom qab kho nrog ECH, uas pom tseeb txo CVF. Kev tshawb xyuas PCR tiag tiag qhia tau hais tias procollagen hom Ⅰ cov ntawv sau tseg tau tswj hwm hauv ISO nas thiab txo qis hauv ECH nas, cov txiaj ntsig tau zoo ib yam, raws li daim duab 3B thiab FG qhia, cov ntaub ntawv qhia tias ECH zoo txo ​​cov collagen deposition. . Tus naj npawb ntawm TUNEL-zoo hlwb hauv cardiomyocytes tau nce ntau hauv HF nas piv nrog Ctrl, ECH ua rau txo qis myocardial cell apoptosis, thiab daim duab 3C-D qhia cov duab sawv cev ntawm TUNEL-zoo hlwb hauv qis thiab siab zog teb ntsig txog, thiab daim duab. 3H qhia qhov apoptosis tus nqi ntawm cov hlwb myocardial. Cov txiaj ntsig no qhia tau tias ECH muaj txiaj ntsig zoo thim rov qab myocardial hypertrophy, myocardial interstitial fibrosis, thiab apoptosis, thiab txhim kho lub plawv ua haujlwm hauv HF nas vim yog ISO.

Daim duab 3 Cov txiaj ntsig ntawm histological staining qhia tau hais tias ECH thim rov qab ventricular remodeling ntawm HF nas induced los ntawm ISO.

3.3|ECH tiv thaiv myocardial ultrastructure hauv HF nas

Cov txiaj ntsig ntawm kev xa hluav taws xob micrographs qhia tau hais tias kev puas tsuaj rau myocardial ultrastructure tuaj yeem txo qis los ntawm ECH, raws li daim duab 4A-B qhia. Myofibrils yog kev sib dhos ntsug, cov qauv sarcomere pom tseeb, cov myofilaments tau teeb tsa zoo, thiab Z kab yog qhov tseeb. Mitochondria tau ua kom zoo nkauj thiab cov qauv pom tseeb hauv Ctrl nas. Txawm li cas los xij, myofibrils tsis sib haum xeeb, tawg, thiab yaj, cov qauv sarcomere yog fuzzy, tsis muaj Z kab, thaj tsam myocardial contraction yog pom, tus naj npawb thiab ntim ntawm mitochondria tau nce, mitochondria yog cov duab sib txawv thiab ntau thiab tsawg, teeb tsa hauv kev tsis sib haum xeeb, cov qauv tsis meej, o thiab crista fragmentation, vau los yog clumping thiab yaj mus rau hauv vacuole hauv HF nas. ECH pom tseeb txo qis cov kev hloov pauv no, feem ntau ntawm cov hlab plawv cov nqaij fibers yog cov txheej txheem zoo thiab cov kab mob myocardial yog qhov tseeb, Z kab muaj nyob, txawm hais tias qee qhov kev tawg ntawm filaments pom tseeb, cov qauv sarcomere yog qhov tseeb, qhov ntom ntawm mitochondrial yog txo, cov duab. thiab qhov ntau thiab tsawg tau yooj yim, npaj kom zoo zoo, mitochondria o thiab crista fragmentation, vau los yog clumping, thiab vacuolization pom tseeb txo los ntawm ECH piv nrog HF. Mitochondria o thiab crista fragmentation, vau los yog clumping, thiab ib nrab vacuolization raug txheeb xyuas raws li mitochondrial degeneration. Cov txiaj ntsig tau pom tias ECH zoo tiv thaiv myocardial ultrastructure, thiab tshwj xeeb tshaj yog tiv thaiv mitochondrial morphology hauv HF nas, raws li daim duab 4C-F qhia.

Daim duab 4 ECH tiv thaiv ultrastructure ntawm cardiomyocytes thiab mitochondrial morphology ntawm HF nas induced los ntawm ISO.

3.4|ECH up-regulates protein qhia ntawm SIRT1, FOXO3a, thiab MnSOD

Western Blot tsom xam pom tau hais tias cov protein qhia theem ntawm SIRT1, FOXO3a, thiab MnSOD yog qhov txo qis hauv HF nas, txawm li cas los xij, ECH pom tseeb nce- tswj cov lus qhia txo qis. Cov neeg sawv cev ntawm cov protein ntau ntawm cov tshuaj tiv thaiv kab mob thiab kev txheeb xyuas cov txiaj ntsig tau pom nyob rau hauv daim duab 5. Ib feem ntawm cov ntaub ntawv qhia tias ECH nce cov protein qhia ntawm SIRT1, FOXO3a, thiab MnSOD.

3.5|ECH inhibits mitochondrial oxidative kev nyuaj siab nyob rau hauv sab laug ventricular myocardial cov ntaub so ntswg

Hauv qhov no, mitochondrial ROS, MMP, oxidative puas tsuaj ntawm mitochondrial DNA, carbonyl protein cov ntsiab lus, thiab lipid peroxidation theem nyob rau hauv mitochondria nyob rau hauv sab laug ventricular myocardial cov ntaub so ntswg ntawm nas yog tag nrho cov ntsuas, thiab cov txiaj ntsig tau zoo ib yam nrog kev sim ntawm tes, uas yog hais tias, ECH txo qis mitochondrial ROS, tiv thaiv MMP, txo qhov kev puas tsuaj ntawm mtDNA, txo cov ntsiab lus carbonyl protein thiab lipid peroxidation hauv mitochondrial, raws li daim duab 6 qhia.

FIGURE 5 ECH up-regulates protein expression level of SIRT1, FOXO3a, thiab MnSOD uas yog down-regulated hauv HF nas.

4|Kev sib tham

Txoj kev tshawb no qhia tau hais tias ECH ua haujlwm zoo hauv kev tiv thaiv myocardial remodeling thiab txhim kho lub plawv ua haujlwm los ntawm kev tswj hwm SIRT1 / FOXO3a / MnSOD signaling axis thiab inhibiting mitochondrial oxidative stress.

Mitochondria yog qhov chaw tseem ceeb ntawm kev tsim ROS raws li cov khoom lag luam ntawm oxidative phosphorylation, los ntawm lawv qhov sib thooj rau ROS, mitochondrial proteins, lipids, thiab mtDNA ntseeg tau tias yog lub hom phiaj tseem ceeb ntawm kev puas tsuaj oxidative tom qab ntau dhau emission ntawm mitochondrial ROS, 34,35. qhov kev puas tsuaj no txhawb kev tsim cov ROS ntxiv, lub voj voog vicious txuas ntxiv, ROS thiab oxidative kev nyuaj siab tuaj yeem ua rau myocardial hypertrophy, apoptosis, interstitial fibrosis, thiab HF los ntawm kev ua kom muaj ntau yam kev taw qhia.35 Yog li, nws zoo li cov tshuaj uas tsom inhibition ntawm mitochondrial oxidative Kev ntxhov siab yog qhov zoo rau kev tiv thaiv ntawm myocardial remodeling. ECH yog ib qho ntuj phenylethanoid glycoside, ib feem ntawm cov tshuaj suav tshuaj Cistanche tubulosa. Nws tau kawm dav thiab tau ua pov thawj tias muaj ntau hom tshuaj ua haujlwm hauv xyoo tas los no, xws li tshuaj tiv thaiv qog, tiv thaiv apoptosis, tiv thaiv kev laus, kev tiv thaiv neuroprotective, thiab lwm yam. Cov kev tshawb fawb tau ua pov thawj tias cov tshuaj pharmacological ntawm ECH feem ntau yog nyob ntawm nws cov cuab yeej ntawm antioxidant thiab tiv thaiv kev ua haujlwm, 18,21 thiab ECH txo mitochondrial ROS thiab tiv thaiv mitochondrial muaj nuj nqi tiv thaiv oxidative puas hlwb hauv paj hlwb.20,22 Ntawm no, peb xub pom tias ECH txo mitochondrial ROS ntawm cardiomyocytes, li no, txhim kho MMP, txo oxidative puas ntawm mtDNA, protein, thiab lipids, zoo inhibiting mitochondrial oxidative kev puas tsuaj thiab txo apoptosis nyob rau hauv vitro thiab vivo, yog li, nws thim rov qab myocardial remodeling thiab txhim kho lub plawv dhia. Nws txhais tau hais tias ECH yog ib qho tshuaj tshiab rau kev tiv thaiv myocardial remodeling thiab HF.

Hauv cov xwm txheej ib txwm muaj, mitochondrial ROS feem ntau yog tshem tawm los ntawm MnSOD uas faib hauv mitochondria thiab tiv thaiv mitochondrial oxidative puas. Ang II txo qis MnSOD kev qhia ntawm cardiomyocytes thiab induces myocardial hypertrophy, txawm li cas los xij, resveratrol tuaj yeem nce qhov kev qhia ntawm MnSOD thiab inhibit myocardial hypertrophy induced los ntawm Ang II.36 Hauv HF, kev ua ntawm MnSOD txo qis thiab tiam ntawm mitochondrial ROS yog nce siab. .37,38 Hauv cov kab mob plawv tshwj xeeb MnSOD gene knockout nas, mitochondrial ROS tsis tuaj yeem raug tshem tawm thiab ROS tau nce hauv cardiomyocytes, qhov no ua rau mitochondrial dysfunction thiab kev loj hlob ntawm lub plawv, nws thiaj li ua rau lub plawv tsis ua haujlwm.39 Hauv kev tshawb fawb tam sim no, peb paub tseeb tias. hais tias qhov kev qhia ntawm MnSOD yog txo qis, uas yog attenuated los ntawm ECH kev kho mob, thiab yog li, mitochondrial ROS yog txo thiab mitochondrial oxidative kev nyuaj siab yog inhibited. Nws txhais tau hais tias ECH tiv thaiv cardiomyocytes tiv thaiv oxidative kev puas tsuaj los ntawm kev tswj hwm ntawm kev qhia ntawm MnSOD, peb ntxiv tshawb xyuas cov txheej txheem molecular hauv qab cov teebmeem no.

Kev nthuav qhia ntawm MnSOD yog tswj hwm los ntawm FOXO3a, uas yog ib qho kev hloov pauv thiab khi rau hauv thaj av txhawb nqa noob ntawm MnSOD thiab ua rau kev txhawb nqa ntawm cov noob hloov pauv. SIRT1/FOXO3a/MnSOD signaling axis plays lub luag hauj lwm tseem ceeb hauv kev tiv thaiv oxidative kev nyuaj siab nyob rau hauv hom cell hom, ua kom lub signaling axis los ntawm estradiol inhibits oxidative kev nyuaj siab thiab exerts lub plawv tiv thaiv24; Ua kom lub teeb liab axis los ntawm resveratrol inhibits oxidative kev nyuaj siab vim cell tuag40; Txwv tsis pub, angiotensin II inhibits SIRT1/FoxO3a/MnSOD txoj kev thiab induces mitochondrial oxidative kev nyuaj siab, mtDNA puas nyob rau hauv osteoblasts, 41types 5 adenylyl cyclase tsub kom oxidative kev nyuaj siab los ntawm inhibition ntawm kev qhia ntawm MnSOD ntawm lub Sirt1/FoxO42 txoj kev tshawb no. Kev nthuav qhia ntawm SIRT1, FoxO3a, thiab MnSOD txhua tus tau txo qis hauv HF nas thiab mitochondrial ROS tau nce siab thiab oxidative kev nyuaj siab tau txhim kho, txawm li cas los xij, cov kev hloov pauv tau thim rov qab los ntawm ECH. Kev tshawb fawb tshiab qhia tau hais tias ECH ncaj qha khi SIRT1 raws li ib tug ligand thiab tsim ib tug complex nrog 9 covalent bonds thiab up-regulates cov kev qhia ntawm SIRT1 nyob rau hauv paj hlwb.26 Raws li txoj kev tshawb no, txoj kev tshawb no qhia tau hais tias ECH kuj up-regulates cov kev qhia ntawm SIRT1 nyob rau hauv cardiomyocytes, nws ces up-regulates noob qhia ntawm MnSOD los ntawm FoxO3a thiab inhibits mitochondrial ROS thiab oxidative kev nyuaj siab.

Lub sij hawm ntev ischemia tuaj yeem ua rau myocardial hibernation, cov hlwb hibernating myocardial feem ntau pom qhov poob ntawm cov qog nqaij hlav, muaj ntau tshaj glycogen granules nyob rau hauv cytosol, sarcoplasmic reticulum, thiab transverse tubules poob, 43 kuj, muaj qhov txawv txav ntawm qhov loj thiab me me. , reactive myocardial hypertrophy thiab interstitial fibrosis, uas thib ob rau myocyte loss.44 Peb kuj tau pom cov kev hloov pauv saum toj no hauv HF nas hauv peb txoj kev tshawb fawb, whereas ECH txo cov kev hloov pauv no, raws li txoj kev tshawb fawb tshiab tshaj tawm qhia txog kev rov ua haujlwm ntawm lub plawv tom qab ischemic kev nyuaj siab yuav yog feature ntawm myocardial hibernation, 45 lub myocardial glycogen theem kuj tau ntsuas, cov txiaj ntsig tau qhia tias tsis muaj qhov sib txawv ntawm peb pawg, raws li qhia hauv daim duab 7J.

Cov kev tshawb fawb yav dhau los kuj tau kuaj pom myocardial hibernation nyob rau hauv tus qauv uas tsis yog-ischaemic plawv tsis ua hauj lwm npua los ntawm pacing LV dawb phab ntsa nrog high-frequency, 46 vim microvascular obstruction plays lub luag hauj lwm tseem ceeb nyob rau hauv myocardial hibernation thiab lub resulting contractile dysfunction, thiab uas yog markedly txhim kho thaum lub sij hawm. microvascular obstruction yog thim rov qab los ntawm ib qho tshuaj.47 Tam sim no, qee qhov kev tshawb fawb pom tau tias kev kho revascularization tau zoo nyob rau hauv hibernating myocardium, 43 los ntawm kev siv HE thiab Masson staining, peb pom tias myocardial capillary thiab arteriolar density raug txo kom pom tseeb hauv HF nas thiab txo qis. los ntawm ECH, raws li qhia hauv daim duab 7A-H. Cov kev tshawb fawb yav dhau los tau ua pov thawj tias lwm cov khoom siv ntuj tsim, xws li barley beta-glucan siv cov teebmeem cardioprotective los ntawm kev nthuav tawm ntawm MnSOD, 48,49, thiab nce ntawm MnSOD yog cuam tshuam nrog kev nce myocardial capillary ntom thiab VEGF qhia. Peb tau ua RT-PCR txhawm rau txheeb xyuas qhov kev qhia ntawm VEGF, cov ntaub ntawv zoo ib yam nrog kev tshawb nrhiav histological staining, uas txhais tau hais tias ECH tuaj yeem txhawb kev tsim cov tshuaj angiogenesis thiab nce myocardial capillary ntom ntawm kev tswj hwm VEGF qhia, raws li qhia hauv daim duab 7I. Txoj kev tshawb no pom tias ECH tuaj yeem tiv thaiv thiab nce myocardial capillary thiab arteriolar los ntawm kev tswj hwm SIRTI / FOXO3a / MnSOD txoj hauv kev thiab kev qhia ntawm VEGF, yog li inhibiting hibernating myocardium thiab pab txhim kho lub plawv dhia.

Cistanche pab txhawbrautxhim kho lub plawv ua haujlwm.

Yog xav paub ntxiv, thov nyem qhov no.

Nco ntsoov, cardiomyocyte apoptosis paub zoo tias yog ib qho tseem ceeb ntawm kev hloov pauv hauv lub plawv tsis ua haujlwm, thiab qhov kev tshawb fawb tam sim no tau lees paub tias muaj ntau cov hlwb apoptotic hauv HF nas thiab ECH txo cov apoptosis ntawm kev tswj SIRTI / FOXO3a / MnSOD txoj hauv kev thiab inhibiting. mitochondrial oxidative kev nyuaj siab, raws li tau hais los saum no.

Qhov tseem ceeb tshaj plaws, ECH thim rov qab myocardial remodeling thiab txhim kho lub plawv ua haujlwm los ntawm kev tswj hwm SIRT1 / FoxO3a / MnSOD teeb liab axis, yog li inhibiting mitochondrial oxidative kev nyuaj siab thiab txo hibernating myocardium. Yog li, nws tau pom zoo tias ECH yog cov tshuaj muaj peev xwm rau kev tiv thaiv lossis kho myocardial remodeling thiab HF.