Pinocembrin Ameliorates Post-infarct Lub Plawv Tsis Ua Haujlwm Los Ntawm Kev Ua Haujlwm Ntawm Nrf2/HO-1 Kev Qhia Txog Txoj Kev 1

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Abstract

Keeb kwm:Oxidative stress yog ib qho tseem ceeb uas cuam tshuam rau kev ua haujlwm ntawm lub plawv tsis ua haujlwm. Txoj kev tshawb fawb tam sim no tau ua los tshawb xyuas seb pinocembrin puas tuaj yeem kho lub plawv tsis ua haujlwm tom qab mob plawv (PIHF) thiab cov txheej txheem hauv qab.

Thov nias ntawm no kom paub ntxiv

Txoj kev:Cov nas tsuag tau ua rau sab laug anterior descending artery ligation los ntxias myocardial infarction thiab tom qab ntawd tsa rau 6 lub lis piam los ua lub plawv tsis ua hauj lwm. Tom qab ntawd pinocembrin tau muab tshuaj txhua hnub rau 2 lub lis piam. Cov teebmeem tau raug soj ntsuam los ntawm echocardiography, western blot, Masson's staining, biochemical kuaj, immunohistochemistry, thiab fluorescence. Hauv vitro, peb kuj tau kuaj H9c2 cardiomyocytes thiab cardiac myofibroblasts los ua pov thawj ntxiv rau cov txheej txheem.

Cov txiaj ntsig:Peb pom tias PIHF-vim kev puas tsuaj ntawm lub plawv ua haujlwm tau ua kom zoo dua los ntawm kev tswj hwm pinocembrin. Tsis tas li ntawd, kev kho mob pinocembrin kuj txo qis collagen deposition thiab augmented vascular endothelial kev loj hlob yam tseem ceeb receptor 2 nyob rau hauv infarct ciam teb nrog rau attenuated apoptosis, uas yog muaj feem xyuam rau ib tug amelioration ntawm oxidative kev nyuaj siab proven los ntawm txo cov reactive oxygen hom (ROS) nyob rau hauv lub plawv cov ntaub so ntswg. thiab malondialdehyde (MDA) hauv cov ntshav, thiab nce ntawm superoxide dismutase (SOD). Qhov no tau nrog los ntawm upregulation ntawm lub nuclear factor erythroid 2-txog yam 2 (Nrf2)/heme oxygenase-1(HO-1) txoj kev. Hauv kev sim vitro peb pom tias cov Nrf2 inhibitor tshwj xeeb thim rov qab cov teebmeem tshwm sim los ntawm pinocembrin nrog rau cov tshuaj tiv thaiv kab mob antioxidant, tiv thaiv apoptosis, tiv thaiv fibrosis, thiab neovascularization, uas tau qhia ntxiv tias qhov amelioration ntawm PIHF los ntawm pinocembrin yog nyob rau hauv Nrf2 / HO-1 txoj kev nyob ntawm txoj kev.

Cistanche tuaj yeem txhim kho kev tiv thaiv

Xaus:Pinocembrin ameliorated cardiac functions thiab remodeling uas tshwm sim los ntawm PIHF los ntawm ROS scavenging thiab Nrf2 / HO-1 txoj kev ua kom ua ntxiv uas ntxiv attenuated collagen fibers deposition thiab apoptosis, thiab txhawb angiogenesis.

Ntsiab lus:Pinocembrin, Lub plawv tsis ua hauj lwm, Oxidative stress, Nrf2/HO-1

Taw qhia

Lub plawv tsis ua hauj lwm (HF), tus cwj pwm los ntawm ntau yam ntawm cov chaw kho mob uas tshwm sim los ntawm cov qauv thiab / lossis kev ua haujlwm tsis zoo ntawm lub plawv, tseem yog lub nra hnyav thoob ntiaj teb, nrog rau myocardial infarction (MI) ua qhov ua rau (Ziaeian thiab Fonarow 2016; Galli thiab Lombardi 2016; Rengo et al.2013). Txawm hais tias muaj kev txhim kho zoo uas tau ua tiav hauv kev kho mob thiab kev saib xyuas zoo hauv HF, muaj kwv yees li ntawm 37.7 lab tus tib neeg txhua xyoo thoob plaws ntiaj teb, nrog rau 50 feem pua ​​​​ntawm kev tuag ntawm 5 xyoo tom qab kuaj mob HF (Go et al. .2014). Qhov teeb meem tam sim no tuaj yeem sau ib feem rau qhov tom qab-infarct HF (PIHF) muaj ntau yam ntawm cov txheej txheem pathophysiological uas tam sim no cov kev kho mob tsis tuaj yeem xa mus.

Cov kev tshawb fawb sib sau tau pom tias oxidative kev nyuaj siab (OS) feem ntau koom nrog hauv cov kab mob pathological ntawm PIHF (Dubois-Deruy li al.2020; Ma et al. 2019; Bubb et al.2017; Habtemariam 2019).OS, feem ntau nthuav tawm los ntawm ib qho Kev sib sau ntau dhau ntawm cov dawb radicals / reactive oxygen hom (ROS) uas muaj hydroxyl radical, superoxide, nitric oxide, thiab lwm yam.(Dubois-Deruy li al. 2020; Habtemariam 2019), tau ua pov thawj kom muaj peev xwm ua kom muaj kev hloov pauv oxidative lossis kev puas tsuaj ntawm lipid. , proteins, thiab DNA, nrog rau cov kab mob organelle, o, apoptosis hauv myocytes thiab interstitial fibrosis supervening (Dubois-Deruy li al.2020; Bubb et al.2017; Hermida et al. 2018). Los ntawm kev tsim txiaj ntawm ntau yam kev puas tsuaj ntawm ROS, cov tshuaj tua kab mob antioxidant tau raug coj los txiav txim siab hauv PIHF cov kev kho mob uas cov txheej txheem muaj xws li ROS scavenging ncaj qha thiab / lossis stimulation ntawm antioxidant tiv thaiv muaj glutathione, superoxide dismutase (SOD), catalase, thiab lwm yam. Habtemar-iam 2019). Txawm li cas los xij, nws tau tu siab tias kev siv qee cov tshuaj antioxidants, tsis muaj vitamin E (Lee et al.2005) lossis resveratrol (Made et al. 2015; Olesen et al. 2014), ua tsis tau raws li kev txaus siab hauv kev kho mob plawv, ua pov thawj los ntawm kev ua tsis tiav hauv kev txhim kho plawv. Yog li ntawd, yuav tsum muaj cov tshuaj tua kab mob antioxidant ntau dua hauv kev kho PIHF.

Pinocembrin (Pino) ​​(cov qauv tshuaj muaj nyob rau hauv Fig.1A) yog ib tug natural flavonoid compound, uas yuav muab rho tawm los ntawm zib mu, propolis, thiab ob peb lwm yam nroj tsuag (Lan li al.2015; Rasul li al.2013) ntxiv rau Artificial synthesis (Costa and Leitao 2011), exerting anti-inflammatory, antibacterial, anti thiab anticancer teebmeem (Rasul li al.2013; Ye et al.2019), nrog rau cov teebmeem antioxidant (Kim et al.2020; Sangweni li al. .2020). Raws li yav dhau los ua qauv qhia, phenolic qauv ntawm Pino lub luag haujlwm rau kev tshem tawm ROS ncaj qha nrog rau ob qho tib si phenolic thiab nonphenolic cov qauv uas ua rau muaj zog tiv thaiv antioxidant (Habtemariam 2019) account rau cov nyhuv antioxidant ntawm Pino, uas Pino tau kawm thiab siv dav hauv ntau yam kab mob, xws li cov kab mob neurodegenerative, atherosclerosis, doxorubicin-induced cardiomyocyte toxicity (Sangweni li al. 2020; Sang et al.2012; Liu et al.2014). Txawm li cas los xij, muab ntau cov tshuaj antioxidants rau myocardial infarction thiab HF tsis raug lossis tsis txaus los hloov mus rau kev kho mob, txawm tias cov nyhuv antioxidant ntawm Pino tuaj yeem ua tiav hauv kev kho mob ntawm PIHF tseem tsis to taub. Qhov kev tshawb fawb no tau ua li no los tshawb xyuas seb qhov kev tswj hwm ntawm Pino puas tuaj yeem ua rau muaj txiaj ntsig zoo rau PIHE, tuav cov lus cog tseg los ua ib qho tshiab, lossis yog tias tsis yog qhov tseem ceeb tab sis tsawg kawg yog pab kho.

Cov txheej txheem thiab cov khoom siv

Tsiaj qauv thiab kev sim raws tu qauv

56 tus txiv neej Sprague-Dawley nas, yuav los ntawm qhov chaw sim tsiaj ntawm Tuam Tshoj Peb Gorges University,

tau loj hlob hauv Wuhan No.3 Tsev Kho Mob Kev Tswj Xyuas tsiaj. Txhua qhov kev sim tau raug tso cai thiab ua nyob rau hauv Hubei Key Laboratory of Cardiology, ua raws li Cov Lus Qhia rau Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Txhaum Cai uas luam tawm los ntawm US National Institutes of Health (NIH Publication No.82-23, kho dua xyoo 1996).

Cov nas pib tau txais sab laug anterior descending coronary artery (LAD) ligation lossis phais phais. Luv luv, nas raug tshuaj loog los ntawm 3 feem pua ​​pentobarbital (2 mL / kg; Sigma-Aldridge, St.Louis, USA, intraperitoneal), tom qab ntawd los ntawm tracheal intubation thiab kev ua pa (tidal vol-ume: 5-10} mL, tus nqi: 70 zaug / feeb). Tom qab lub plawv raug tshwm sim thiab LAD tau ligated los ntawm 7-0 monofilament nylon sutures. Qhov tshwm sim ntawm qhov daj ntseg ntawm apexcordis lossis ST-segment nce siab ntawm I lead ntawm electrocardiogram qhia tias induction ntawm MI. Kev lag luam sham tau ua cov txheej txheem zoo ib yam tshwj tsis yog rau LAD ligation. Penicillin (200,{9}} IU) tau txhaj tshuaj intramuscularly ib hnub ib zaug rau ib lub lis piam tom qab ua haujlwm.

Afterward, the rats were carefully raised for 6 weeks to produce PIHF. Rats were then randomly split into 4 groups:(i) sham+saline (sham group);(i)sham+Pino group;(ii)LAD ligation+saline(HF group);(iv)LAD ligation+Pino(HF+Pino group). Pinocembrin(5 mg/kg, purity>98 feem pua; Sigma-Aldridge) lossis tib lub ntim ntawm saline tau txhaj rau hauv cov hlab ntsha ntawm tus Tsov tus tw txhua hnub rau 2 lub lis piam. Cov koob tshuaj xa mus rau qhov kev kawm dhau los (Lan et al.2017). Cov txheej txheem tau piav qhia hauv Fig.1B.

Cov tswj kev tswj tau zoo muaj 3 pawg: sham plus saline(sham group); LAD ligation plus saline (HF pab pawg); LAD ligation plus enalapril (HF ntxiv rau ACEI pawg). Cov nas tau ua tiav cov haujlwm sib txuas tshwj tsis yog tias cov nas ntawm HF ntxiv rau ACEI pawg tau noj ntawm qhov ncauj los ntawm 20 mg / kg enalapril.

Echocardiography ntsuas

Lub plawv ua haujlwm tau raug soj ntsuam los ntawm transthoracic echocardiography siv lub tshuab ntsuas ultrasound (Vevo 2100, Visual Sonics Inc., Toronto, Canada), raws li tau tshaj tawm yav dhau los hauv peb pab pawg (Fo et al.2020). Sab laug ventricular puab qhov ntev ntawm qhov kawg-diastolic theem (LVIDd) thiab kawg-systolic theem (LVIDs), sab laug ventricular end-systolic thiab kawg-diastolic ntim (LVESV thiab LVEDV) tau kaw los ntawm ob-dimensional M-mode echocardiograms tsawg kawg 3. sib law liag rau txhua tus nas. Sab laug ventricular ejection fraction (LVEF) thiab fractional shortening (FS), uas qhia txog kev ua haujlwm systolic, tau suav tom qab.

Kev npaj ua qauv

Tom qab kev tswj hwm ntawm Pino thiab echocardiography tests, nas raug tshuaj loog dua. Ntau tshaj 2 mL venous ntshav tau raug ntes los ntawm cov leeg leeg mus rau hauv ethylenediaminetetraacetic acid (EDTA) - uas muaj cov hlab ntsha nqus ntshav nrog kev ua tiav centrifugation ntawm qhov ceev ntawm 3000 r / min rau 15 min thiab ntawm qhov kub ntawm 4 ° C, tom qab ntawd los ntawm kev sib cais ntawm serum supernatant thiab preservation ntawm -80 "C. Ces lub siab twb apace sau. Ib feem ntawm infarct ciam teb cheeb tsam ntawm txhua lub plawv raug txiav tawm thiab khov nyob rau hauv kua nitrogen rau western blot tsom xam, nrog rau lwm lub plawv cov ntaub so ntswg tsau nyob rau hauv 4 feem pua ​​paraformaldehyde rau ntau tshaj 24 teev.

Masson txoj kev staining

Peb tau ua Masson txoj kev staining kom pom cov collagen deposition nyob rau hauv infarct ciam teb. Paraformaldehyde-fixed ventricles tau embedded los ntawm paraffin thiab txiav mus rau hauv 5-μm seem. Cov seem tau tom qab stained nrog Masson trichrome qhov twg cov collagen tau dyed nyob rau hauv xiav, lub myocardium nyob rau hauv liab, thiab lub nucleus dub. Lub collagen deposition feem yog xam los ntawm Image J software (NIH, Bethesda, MD) ntawm × 200 magnification.

Immunohistochemical tsom xam

Immunohistochemical tsom xam tau ua los ntsuas qhov ceev ntawm vascular endothelial kev loj hlob yam tseem ceeb receptor 2 (VEGFR2) uas nws upregulation txhawb angiogenesis uas yog salutary thiab tseem ceeb hauv attenuation ntawm remodeling (Rengo li al. 2013). Cov seem ventricular tau tsim zoo li cov uas tau ua nyob rau hauv Masson staining, ua raws li deparaffinized thiab rehydrated. Tom qab ntawd cov ntu tau txais kev kho cua sov los tiv thaiv gen thiab kho nrog 3 feem pua ​​​​H, O, los thaiv cov kab mob endogenous peroxidase, nrog incubation ntawm 1 ug / mL VEGFR2 antibody (Abcam, ab9530) rau 12h ntawm 4 degree ua tiav. Tom qab ntawd, cov seem tau incubated los ntawm nees-radish peroxidase (HRP)-labeled Secondary antibody (1:200, Service, G1213) rau 50 min ntawm 37 degree . Tsis tas li ntawd, 3,3'-diaminobenzidine tau siv los tsim qhov zoo staining uas yog qhia nyob rau hauv liab. Thaum kawg, qhov nruab nrab qhov muag qhov ntom ntom ntawm VEGFR2 tau txheeb xyuas thiab suav los ntawm Duab-Pro Plus 6.0 software (Media Cybernetics, USA).

Terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) kev soj ntsuam

Apoptosis tau kuaj pom tom qab los ntawm TUNEL kev soj ntsuam. Yav dhau los tsim cov khoom siv paraffin uas embed ventricular cov ntaub so ntswg raug txiav rau hauv lwm daim rau TUNEL kev soj ntsuam, ua raws li TUNEL staining raws li cov chaw tsim tshuaj paus cov lus qhia ntawm cov khoom lag luam soj ntsuam (Roche, Switzerland; 11684817910). Luv luv, cov ventricular seem tau raug rau TUNEL reagent, qhov twg TdT thiab dUTP tau tov nrog ib feem ntawm 1: 9, rau 1 h nyob rau hauv chav tsev kub, tom qab incubation ntawm proteinase K thiab rupture ntawm cell membrane. Thaum kawg, cov duab fluorescent tau pom ntawm lub wavelength ntawm 530 nm uas tau tshwm sim los ntawm emission wavelength ntawm 450 nm (× 400 magnification). Tus apoptotic tus nqi ntawm cov ntaub so ntswg ventricular nyob rau hauv txhua pab pawg tau soj ntsuam thiab xam los ntawm Image Software.

Western blot

Peb siv western blot los qhia txog cov txheej txheem hauv qab uas Pino ameliorated PIHF. Cov txheej txheem ntawm kev rho tawm cov qauv protein thiab kev npaj zoo ib yam li cov uas tau tshaj tawm yav dhau los hauv peb txoj kev tshawb fawb (Chen et al.2020a).Cov ntaub so ntswg tau soj ntsuam nrog cov tshuaj tiv thaiv kab mob tiv thaiv cov khoom siv hluav taws xob nuclear erythroid 2-txog yam 2(Nrf2), heme oxygenase{{ 4}}(HO-1; 1:2000, Abcam, ab189491), p53(1:1000, cell signaling technology,2524S), BCL-2 (1:1000, cell signaling technology,2870P) ,bax (1:1000, Abcam, ab32503), cleaved caspase-3(1:1000, cell signaling technology, 9664 T), collagen I(1:1000, Affinity, AF7001), glyceraldehyde-3- phosphate dehydrogenase (GAPDH, Kev Pabcuam, GB12002), nrog rau kev pom tom qab los ntawm HRP-labeled lwm cov tshuaj tiv thaiv kab mob (Kev Pabcuam, G1213 thiab G1214) thiab chemiluminescence nrhiav kom tau reagents. Cov duab blot thaum kawg tau txheeb xyuas thiab ntsuas los ntawm Image J software.

H9c2 cardiomyocytes kab lis kev cai thiab pov tseg hauv vitro Cov nas plawv ventricular-derived H9c2 cardiomyocytes, uas tau yuav los ntawm American Type Culture Collection (CRL-1446), tau coj mus kuaj ntxiv los txheeb xyuas qhov cuam tshuam los ntawm Pino hauv vitro. H9c2 cardiomyocytes tau coj mus kuaj hauv e Dulbecco's modified Eagle's nruab nrab (DMEM) / F12 nruab nrab uas muaj 10 feem pua ​​​​fetal bovine serum (Sijiqing, Hangzhou, Suav teb) nyob rau hauv qhov kub ntawm 37 degree thiab cua ntawm 95 feem pua ​​​​ntawm huab cua thiab 5 feem pua ​​CO. tau cog rau 6- cov phiaj zoo tom qab loj hlob mus rau 90 feem pua ​​​​, ntxiv rau, cov kua dej nruab nrab uas muaj 25 uM isoprenaline (ISO) tom qab tau hloov pauv rau qhov nruab nrab hauv 3 qhov dej kom incubate cov hlwb rau 48 h kom ua raws li HF xeev txij li thaum HF kuj yog tus cwj pwm los ntawm kev ua haujlwm dhau ntawm lub suab siab thiab txhawb nqa ntawm -adrenergic receptor generates ROS (Velusamy li al.2020). Tom qab ntawd cov hlwb raug faib ua 6 pawg: (i) tswj pawg; (ii) tswj nruab nrab ntxiv nrog 25 μM Pino rau 4 h (tswj ntxiv rau Pino pawg); (i) tswj nruab nrab co-incubated los ntawm 25 μM Pino thiab 5 μM ML385 rau 4 h (Nrf2 inhibitor; MCE, New Jersey, USA)(tswj ntxiv Pino ntxiv rau ML385 pawg);(iv)ISO ​​pawg; v. ISO nruab nrab nrog 4-h incubation los ntawm 25 μM Pino (ISO ntxiv rau Pino pab pawg); vi. ISO plus 25 μM Pino ntxiv 5 μM ML385 pawg. Lub concentration thiab incubating lub sij hawm ntawm cov tshuaj xa mus rau yav dhau los kev tshawb fawb los yog cov lus qhia (Jin et al.2015; Oliveira li al.2017).

Neonatal nas cardiac myofibroblasts kab lis kev cai thiab kev soj ntsuam kev loj hlob

Neonatal nas plawv myofibroblasts (CMFs) tau coj los soj ntsuam cov teebmeem ntawm Pino ntawm collagen secretion. Cov CMFs tau sib cais los ntawm kev siv cov nas me me uas muaj hnub nyoog 3 hnub, xa mus rau cov kev qhia yav dhau los (Cui li al. 2021). Lub siab cais thiab minced tau zom los ntawm 0.125 feem pua ​​​​ trypsin tom qab los ntawm ib qho dej cawv uas muaj 0.25 feem pua ​​trypsin thiab 0.08 feem pua ​​collagenase ob peb zaug, nrog rau centrifugation thiab hloov mus rau petri phaj tom qab ua los mus sau cov cell los ntawm supernatant. Tom qab ntawd cov xov xwm tau hloov los ntawm cov kua nruab nrab zoo ib yam li siv rau H9c2 cardiomyocytes kab lis kev cai tom qab adhesion ntawm 90 min. CMFs tau txais cov khoom pov tseg zoo ib yam li cov ntawm H9c2 cardiomyocytes. Peb kuj tau siv cov khoom siv suav ntawm tes-8(CCK-8) kev soj ntsuam (Dojindo, Nyiv) txhawm rau txheeb xyuas qhov kev loj hlob ntawm CMFs thaum ncav cuag qhov ceev ntawm 1.5 × 10 degree nyob rau hauv txhua qhov dej, los ntawm kev siv lub microplate nyeem ntawv ntawm lub wavelength ntawm 450 nm, qhov twg tus nqi siab dua qhia tau hais tias muaj kev ua haujlwm loj hlob ntxiv.

ROS nrhiav pom

Lub peev xwm ntawm Pino txhawm rau txhawm rau ROS tau ntsuas los ntawm fluorescent sojntsuam dihydroethidium (DHE; Beyotime, Shanghai, Suav). DHL, uas tau yaj nyob rau hauv DMSO, tau diluted rau 5 μM rau cov seem khov los yog ntxiv rau cov kua dej nruab nrab kom diluted mus rau tib qhov concentration raws li cov lus qhia. Paraformaldehyde-taw lub siab tau khov ua raws li raug txiav mus rau 4-μm ntu. Cov ntu lossis H9c2 cardiomyocytes tau koom ua ke nrog DHE tov rau 30 min ntawm qhov kub ntawm 37 degree, duab tom qab los ntawm lub tshuab hluav taws xob fluorescent inverted (IX70; Olympus, Tokyo, Nyiv). Luv luv, lub nucleus uas tau sau nrog DNA thiab RNA tau dyed nyob rau hauv txawv liab vim muaj kev sib raug zoo ntawm nucleic acid thiab DHE. Tsis tas li ntawd, qhov muaj zog fluorescence siv tau qhia tias muaj ntau dua ntawm ROS.

Kev kuaj mob biochemical

Kev tshuaj xyuas biochemical tau ua los soj ntsuam cov kev hloov pauv ntawm cov ntshav BNP, SOD, thiab malondialdehyde (MDA) hauv cov ntshav thiab cellular supernatant uas qhia tias HF phenotype thiab oxidant tiv thaiv, raws li. Cov concentration tau ntsuas los ntawm kev siv cov khoom siv tshuaj biochemical raws li cov chaw tsim khoom cov lus qhia (Jiancheng, Nanjing, Suav).

Kab lus no yog muab rho tawm los ntawm Chen li al. Mol Med (2021) 27:100 https://doi.org/10.1186/s10020-021-00363-7