Part 1: Renovascular Effects Of Inorganic Nitrate Tom Qab Ischemia-reperfusion Ntawm Lub raum
May 16, 2022
Yog xav paub ntxiv. tiv taujtina.xiang@wecistanche.com
TSAB NTAWV
Keeb kwm: Lub raum ischemia-reperfusion (IR) raug mobyog ib qho ua raumob raum raug mob(AKI), uas yog txuam nrog oxidative kev nyuaj siab thiab txo nitric oxide (NO) bioactivity, thiab muaj kev pheej hmoo ntawm kev loj hlob.mob raum mob(CKD) thiab kab mob plawv (CVD). Cov tswv yim tshiab uas ua kom rov qab redox tshuav nyiaj li cas yuav muaj kev cuam tshuam kev kho mob thaum lub sij hawm AKI thiab cov teeb meem cuam tshuam.
Hom phiaj: Txhawm rau tshawb xyuas tus nqi kho mob ntawm kev txhawb nqa cov nitrate-nitrite-NO txoj hauv kev thaum lub sij hawm kev loj hlob ntawm IR-induced lub raum thiab cov hlab plawv tsis ua hauj lwm.
Cov txheej txheem: Txiv neej C57BL / 6 J nas tau muab sodium nitrate (10 mg / kg, ip) los yog lub tsheb 2 h ua ntej sov ischemia ntawm lub raum sab laug (45 min) ua raws li sodium nitrate supplementation hauv dej haus (1 mmol / kg / hnub) rau 2 lub lis piam tom ntej. Ntshav siab thiab glomerular filtration rate tau ntsuas thiab cov ntshav thiab ob lub raum tau sau thiab siv rau kev tshuaj xyuas biochemical thiab histological nrog rau cov kev tshawb fawb lub raum reactivity. Glomerular endothelial hlwb raug rau hypoxia-reoxygenation, nrog lossis tsis muaj angiotensin Ⅱ, tau siv rau kev tshawb fawb txog kev siv tshuab.
Cov txiaj ntsig: IR tau cuam tshuam nrog txo lub raum ua haujlwm thiab nce ntshav siab me ntsis, ua ke nrog rau lub raum raug mob, o, endothelial dysfunction, nce qib Ang Ⅱ, thiab Ang II-mediated vasoreactivity, uas yog tag nrho ameliorated los ntawm nitrate. Ntxiv mus, kev kho mob nrog nitrate (hauv vivo) thiab nitrite (hauv vitro) rov qab tsis muaj bioactivity thiab txo mitochondrial oxidative kev nyuaj siab thiab raug mob.
Cov lus xaus: Kev kho mob hnyav nrog inorganic nitrate ua ntej lub raum ischemia tuaj yeem ua txoj hauv kev kho tshiab los tiv thaiv AKI thiab CKD thiab qhov cuam tshuam ntawm kev txhim kho.cardiovascular tsis ua haujlwm.
Nyem mus cuag tus neeg muag khoom rau cistanche kev paub
1. Taw qhia
Ischemia-reperfusion (IR) raug mob ntawm lub raum, cuam tshuam nrog kev hloov pauv, kev phais mob plawv, thiab kev poob siab, yog ib qho kev pheej hmoo loj rau kev tsim mob raum mob (AKI) thiab mob raum mob raum (CKD) [1]. Cov txheej txheem hauv qab yog qhov nyuaj thiab koom nrog kev sib koom ua ke oxidative kev nyuaj siab, nitric oxide deficiency, thiab kev tiv thaiv kab mob ntawm tes, uas ua ke ua rau endothelial tsis ua haujlwm [2, 3]. Cov kev tshawb fawb tsis tu ncua tau tsom mus rau kev nrhiav cov kev kho tshiab thiab muaj txiaj ntsig zoo rau kev tiv thaiv thiab kho AKI thiab nws txoj kev loj hlob mus rau CKD, xws li tej thaj chaw deb ischemic preconditioning, ib ntus hauv zos hypothermia nrog rau cov tshuaj kho mob tshiab [3,4].
Lub gaseous signaling molecule nitric oxide (NO) tseem ceeb pab rau txoj cai ntawm lub plawv thiab lub raum homeostasis thiab tau pom tias ua si lub luag hauj lwm tiv thaiv thaum raug mob IR [5,6]. Txawm li cas los xij, thaum muaj kev tshwm sim ischemic, qhov tsis muaj oxygen cuam tshuam rau kev ua haujlwm ntawm endothelial NO synthase (eNOS), uas tuaj yeem ua rau "tsis muaj qhov tshwm sim" hauv cov ntaub so ntswg ischemic thaum lub sijhawm rov ua dua. Qhov no nyob rau hauv lem propagates endothelial thiab tubular epithelial cell raug mob [7], uas pab rau txoj kev loj hlob ntawm lub raum tsis ua hauj lwm. Ntxiv nrog rau hypoxia thaum lub sij hawm ischemic, ntau ntau ntawm reactive oxygen hom (ROS) generated ntau tshaj los ntawm nicotinamide adenine dinucleotide phosphate (NADPH) oxidase thiab mitochondria thaum lub sij hawm reperfusion theem scavenges NO [8]. Cov txheej txheem tshiab uas txo cov kev ntxhov siab oxidative thiab tswj tsis tau bioactivity yuav muaj txiaj ntsig kho mob hauv kev sib ntaus sib tua tiv thaiv IR-vim lub raum tsis ua haujlwm.
Inorganic nitrate (NO3) thiab nitrite (NOZ) yav dhau los tau pom tias yog cov khoom lag luam tsis zoo uas tau los ntawm NO metabolism. Txawm li cas los xij, kev tshawb fawb tau ua rau ntau tshaj li ob xyoo caum tau pom tias cov anions tuaj yeem dhau mus bioconversion, los ntawm kev txo qis, thiab rov ua NO thiab lwm yam bioactive nitrogen oxide hom [9]. Cov kev ua ntawm no lwm txoj kev nitrate-nitrite-NO yog txhim kho thaum lub sij hawm tej yam kev mob nrog hypoxia thiab pH qis thaum lub classical NOS system tuaj yeem ua tsis zoo [10].
Kev noj zaub mov, ntxiv rau NOS system, yog qhov tseem ceeb ntawm kev ncig ntawm nitrate thiab nitrite, vim muaj nitrate ntau ntau piv txwv li hauv cov nplooj ntsuab ntsuab. Kev ntxiv nrog inorganic nitrate thiab nitrite tau cuam tshuam nrog cov txiaj ntsig zoo ntawm cov hlab plawv, nrog rau kev txo cov ntshav siab, hauv kev sim thiab kev tshawb fawb soj ntsuam [11]. Lub cev tiv thaiv kab mob kuj tau pom nyob rau hauv kev sim IR raug mob qauv ntawm daim siab [12], hlwb [13], ntsws [14], thiab plawv [12,15]. Txawm li cas los xij, qhov muaj peev xwm kho tus nqi ntawm inorganic nitrate thiab nitrite hauv raum IR raug mob tseem muaj teeb meem. Raws li tsis ntev los no tau tshuaj xyuas [5], cov txiaj ntsig sib txawv hauv IR qauv, siv nitrite therapy, tej zaum vim qhov sib txawv ntawm qhov siv ntau npaum li cas, txoj kev tswj hwm, kev kho mob ntev, nrog rau cov qauv kev sim nws tus kheej [16,17]. Ntau cov kev tshawb fawb sim tau pom tias muaj kev tiv thaiv los ntawm kev noj zaub mov nitrate ntxiv hauv cov qauv ntawm cov kab mob cardiorenal, los ntawm cov txheej txheem uas cuam tshuam nrog kev cuam tshuam ntawm oxidative kev nyuaj siab thiab kev txhim kho tsis muaj bioactivity nrog rau kev hloov kho.inflammatory teb[5]. Txog niaj hnub no, tsis muaj kev paub txog qhov cuam tshuam ntawm inorganic nitrate therapy hauv lub raum IR raug mob. Peb tau dhau los ua pov thawj tias kev noj zaub mov tsis tu ncua nrog nitrate attenuated tom qab IR-induced raum raug mob [18]. Txawm li cas los xij, tsis muaj kev paub txog qhov muaj peev xwm ntawm kev txhawb nqa cov nitrate-nitrite-NO txoj hauv kev thaum pib ntawm IR kom txo tau txoj kev pheej hmoo ntawm kev tsim AKI thiab CKD. Yog tias muaj kev tiv thaiv, qhov no tuaj yeem muaj kev siv tshuaj kho mob dav dav hauv cov neeg mob uas muaj kev pheej hmoo ntawm kev tsim AKI piv txwv li cov uas tau phais loj.
Hauv txoj kev tshawb no, peb siv tus qauv nas ntawm lub raum IR raug mob los tshawb xyuas cov txiaj ntsig zoo ntawm kev kho mob nitrate tam sim ua ntej qhov tshwm sim ischemic. Qhov no tau ua ke nrog kev tshawb fawb txog kev siv tshuab ex vivo thiab hauv vitro cell kab lis kev cai sim nrog kev kho nitrite. Peb xav tias kev txhawb nqa cov nitrate-nitrite-NO txoj hauv kev yuav txhawb lub raum tiv thaiv IR raug mob los ntawm kev hloov pauv ntawm oxidative kev nyuaj siab thiab tsis muaj bioactivity nrog rau kev ua haujlwm mitochondrial.
2. Cov txheej txheem
2.1.Reagents
Tshwj tsis yog hais tias, tag nrho cov reagents tau txais los ntawm Sigma-Aldrich, Sweden.
2.2. Tsiaj txhu thiab lub raum ischemia-reperfusion qauv
C57BL/6 J nas (Txiv neej, 10 lub lis piam) tau txais los ntawm Janvier Lab-oratories (Fabkis) thiab nyob hauv peb cov chaw tsiaj ntawm Karolinska Institutet nyob rau hauv kev tswj hwm huab cua nrog lub sijhawm 12-h lub teeb / tsaus ntuj thiab muab nrog tus qauv nas chow thiab kais dej. Tag nrho cov txheej txheem tsiaj tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees Hauv Cheeb Tsam rau Tsiaj Txhaj Tshuaj (Txoj Cai ID: N139/15) thiab tau ua raws li National Institutes of Health (NIH) cov lus qhia thiab nrog EU Cov Lus Qhia 2010/63 / EU rau kev coj ua ntawm kev sim hauv cov tsiaj.
Tom qab 1 lub lis piam ntawm acclimatization, nas tau muab cov placebo (NaCl) los yog sodium nitrate (NaNO3) intraperitoneally (10 mg / kg) 2 h ua ntej unilateral ischemia ntawm lub raum sab laug. Lub raum sab xis raug tshuaj xyuas tab sis lwm yam sab laug tsis zoo. Sham phais tau ua tib yam, tab sis tsis muaj clamping lub raum sab laug. Cov nas tau muab tshuaj loog nrog isoflurane thiab lub cev kub tau khaws cia ntawm 37 ± 0.5 degree siv lub teeb cua sov thiab lub ntsuas kub ntawm tus kheej thaum lub sijhawm phais. Tom qab kev phais, cov nas raug kho nrog sodium nitrate (1 mmol / kg / hnub) lossis placebo rau 2 lub lis piam kom txog thaum txiav.
2.3. Kev ntsuas ntshav siab
Cov ntshav siab tau ntsuas hauv cov nas uas siv cov txheej txheem uas tsis yog-tus Tsov tus tw-cuff ua ntej txiav.Mice tau txias rau ntawm lub phaj sov ntawm 35 ° C rau 10 min thiab muab tso rau hauv cov yas tuav. Lub cuff nrog pneumatic mem tes sensor tau txuas rau tus Tsov tus tw, thiab cov txiaj ntsig ntshav siab tau sau tseg nrog CODA system (Kent Scientific, Torrington, CT, USA). Tsiaj txhu tau txais kev cob qhia nrog cov tuav tuav ntawm lub phaj ua kom sov tsawg kawg peb hnub ua ntej kev ntsuas ntshav siab. Systolic, diastolic, thiab txhais tau tias cov hlab ntsha siab tau sau ntau dua 3 hnub sib law liag thiab tus neeg nruab nrab ntawm txhua qhov kev lees paub tau muab tso ua ke rau kev soj ntsuam.
2.4.Cov ntaub so ntswg
Ua ntej txiav tawm, cov nas tau muab tso rau hauv cov kab mob metabolic kom khaws cov zis rau kev suav cov glomerular filtration rate (GFR). Tsiaj txhu tau muab tshuaj loog nrog isoflurane thiab kuaj ntshav tau sau los ntawm qhov tsis zoo vena cava. Tag nrho cov qauv raug centrifuged tam sim ntawd ntawm 6000 × g, 4 degree C rau 5min nyob rau hauv lub xub ntiag ntawm EDTA (Sigma-Aldrich, Stockholm, Sweden), qhov kawg concentration ntawm 2 mM. Cov plasma kuaj tau raug xa mus rau hauv cov raj tshiab thiab tam sim ntawd snap-khov hauv cov dej khov qhuav thiab thaum kawg khaws cia ntawm -80 degree .Kidneys tau muab rho tawm thiab txiav mus rau ntau qhov chaw rau ex vivo vascular function thwmsim (interlobar arteries thiab afferent arterioles), histology ( HE thiab PAS staining), thiab kom muaj nuj nqis ntawm apoptosis thiab o (Tissue-Tek optimum cutting tempera-ture(OCT) compound embedding and frozen for TUNEL and F4/80 staining).
2.5. Kev ntsuas ntawm nitrate, nitrite, cGMP, creatinine, ntshav urea nitrogen (BUN), thiab angiotensin II
Nitrite thiab nitrate tau txheeb xyuas los ntawm HPLC(ENO-20) raws li tau piav ua ntej 【19】. Plasma kuaj (10 ul tau txhaj rau hauv HPLC system nrog Hamilton syringe. Tom qab ntawd, nitrite thiab nitrate tau sib cais los ntawm thim rov qab-theem / ion pauv chromatography tom qab nitrate txo rau nitrite los ntawm cadmium thiab txo cov tooj liab. Nitrite yog derivatized siv Griess reagent los tsim diazo tebchaw thiab kuaj pom ntawm 540 nm. Txhawm rau tiv thaiv kev degradation ntawm cGMP, plasma raug xa mus rau cov hlab ntsha uas muaj PDE inhibitor, BMX(3-Isobutyl-1 methylxanthine; Sigma-Aldrich #I5879) kom muab qhov kawg concentration (10 μM). Tom qab ntawd khov thiab khaws cia ntawm -80 degree ua ntej tshuaj ntsuam cGMP nrog cov khoom siv ELISA (GE Healthcare #RPN226), raws li cov tuam txhab cov lus qhia.
Cov qib ntshav plasma thiab zis creatinine tau kuaj pom los ntawm kev siv Creatinine Colorimetric Assay Kits (Cayman Chemical, # 700460 thiab # 500701). Creatinine clearance formula (CLcr =Urinecrx Urine flow/Plasmacr) yog siv los kwv yees GFR. Cov qib plasma BUN tau kuaj pom los ntawm kev siv BUN Colorimetric Detection Kit (Invitrogen, #EIA-BUN). Cov theem ntawm Ang hauv cov ntshav thiab lub raum cov ntaub so ntswg tau ntsuas los ntawm Ang ⅡI ELISA cov khoom siv (Cloud-Clone Corp., # CEA005Mu) raws li cov tuam txhab cov lus qhia. Txawm li cas los xij, kev npaj cov ntaub so ntswg txawv me ntsis ntawm qhov kev pom zoo.10 vol 50 mM HCl hauv 50 feem pua EtOH tau ntxiv rau cov ntaub so ntswg thiab ua kom sov li 10 min ntawm 100 degree, centrifuged 10 000 × g 10 min. Tom qab ntawd cov qauv tau khov thiab khaws cia ntawm -80 degree ua ntej yuav tshuaj xyuas. Txhua qhov kev nyeem ntawv nqus tau ua tiav hauv SpextraMax iD3 los ntawm Molecular Devices.
2.6. Kev rho tawm thiab perfusion ntawm cov hlab ntsha interlobar
Lub raum tau sau tom qab kev txi thiab khaws cia rau hauv cov dej khov-txias physiological ntsev tov (PSS) kom txog thaum kev sib cais ntawm cov hlab ntsha interlobar. Lub raum interlobar cov hlab ntsha tau muab tshem tawm (2 hli ntev) thiab muab tso rau hauv a
myograph chamber (Model 620 M, Danish Myo Technology, Denmark) nrog PSs raws li tau piav ua ntej [20]. Isometric nro tau kaw nrog Powerlab system (Powerlab 4/30, AD Instruments, Australia). Tom qab mounted, cov hlab ntsha tau sib npaug rau 20 min hauv PSs bubbling nrog carbogen (95 feem pua O2; 5 feem pua CO2) ntawm 37 degree, pH7.4.Tom qab ntawd cov leeg nro ntawm cov hlab ntsha tau teeb tsa raws li tau piav qhia yav dhau los [21], uas yog raws li Mulvany tus txheej txheem normalization [22]. Tom qab qhov sib npaug thib ob, 0.1 mol / L KCl tau raug siv los ntsuas cov hlab ntsha ntawm lub nplhaib.
2.7. Kev rho tawm thiab micro-perfusion ntawm afferent arterioles
Cov nas C57BL/6 J tau txhaj tshuaj loog nrog isoflurane thiab cov menyuam yaus raug tshem tawm thiab hlais sai sai raws li tau piav qhia dhau los [23-25]. Lub raum slices tau muab tso rau hauv dej khov-txias Dulbecco's modified Eagle's medium (DMEM). Ib qho afferent arteriole nrog txuas nrog glomerulus tau micro-dissected nyob rau hauv lub stereomicroscope thiab pauv mus rau ib tug
kub tswj chamber nyob rau theem ntawm inverted microscope. Lub glomerulus tau kho nrog lub micropipette tuav thiab cov afferent arteriole tau cannulated thiab perfused nrog ib txheej ntawm micropipettes. Lub siab intraluminal ntawm perfused afferent arteriole tau khaws cia ntawm 60 mmHg thaum kev sim. Lub sijhawm rau kev txiav tawm thiab cov micro-perfusion hauv qab no tau txwv rau 120 min tom qab tus nas raug txi raws li tau piav qhia yav dhau los [26]. Tom qab 30 feeb sib npaug ntawm 37 degree, tsub zuj zuj ntawm koob tshuaj tiv thaiv kab mob rau Ang II (10-12 rau 10-8 mol / L) tau txais. Txhua qhov concentration ntawm Ang II tau perfused rau 2 min, cov lus teb constrictive tau sau tseg thiab tom qab ntawd ntsuas lub cheeb ntawm afferent arteriole.
2.8. Kev kuaj mob histological
Lub raum kuaj tau raug sau thiab kho hauv 4 feem pua ntawm cov tshuaj paraformaldehyde. Cov ntaub so ntswg tsau tau muab tso rau hauv paraffin thiab tom qab ntawd hlais thiab pleev xim nrog Hematoxylin-Eosin (H&E) lossis Periodic Acid Schiff (PAS) rau kev soj ntsuam histological. Plaub tus tsiaj uas raug xaiv los ntawm txhua pawg sim tau siv los tshuaj xyuas. Kaum qhov chaw xaiv xaiv los ntawm txhua ntu tau raug ntes nyob rau hauv 400 × magnification. Txhua qhov kev ntsuam xyuas morphometric tau ua nyob rau hauv qhov muag tsis pom.
2.9. TUNEL staining
OCT-embedded raum kuaj tau siv rau TUNEL staining. 6-μm tuab ntu tau tsim nrog 20ug/mL proteinase K rau 15 feeb ntawm chav tsev kub, thiab TUNEL cov tshuaj tiv thaiv tau ua los ntawm kev siv Click-iTTM Plus TUNEL Assay (Invitrogen C10618, USA) ua raws li cov chaw tsim khoom cov lus qhia. Thaum kawg ntawm txoj cai, DAPIstaining tau ua. Rau qhov kom muaj nuj nqis, peb tus tsiaj thiab peb thaj chaw ntawm daim duab (200 ×) tau raug xaiv los ntawm randomly thiab yees duab siv Zeiss LSM800-Airy confocal microscope, cov cim zoo tau suav nrog siv ImageJ/Fiji software, thiab tau suav raws li qhov piv ntawm cov cim zoo rau DAPI.
2.10. Tissue immunofluorescence staining thiab confocal microscopy (F4/80)
Lub raum tau khov hauv OCT (Tissue-Tek, Torrence, CA, USA) thiab ntu (6 μm) tau npaj thiab ua tiav ntxiv. Frozen seem raug ntxuav nrog 1 × PBS thiab thaiv nrog 2 feem pua BSA hauv PBS rau 30 min thiab tom qab ntawd incubated nrog anti-F4/80 (1:50, BIO-RAD Cl: A3-1) hmo ntuj hauv 4 degree chav txias, thiab tom qab ntawd nrog cov tshuaj tiv thaiv kab mob thib ob (1: 200 Cell signaling Tech #4417) ntawm chav tsev kub rau 1 h, ua raws li counterstaining nrog 300 nmol / L DAPI (4'6-diamidino-2-phenyl -indole, dihydrochloride, Invitrogen) rau 3 feeb. Cov kab mob immunofluorescence tau pom nyob rau hauv Zeiss LSM800-Airy confocal microscope. peb tus tsiaj thiab peb thaj tsam ntawm daim duab (200 ×) raug xaiv random. F4/80 cov teeb liab zoo tau suav nrog siv ImageJ / Fiji software thiab tau suav ua qhov piv ntawm cov cim zoo rau DAPI.
2.11. Cell kab lis kev cai
Rat glomerular endothelial cells (GECs) (DSMZ ACC262, Lub teb chaws Yelemees) tau coj mus kuaj hauv RPMI1640 (Gibco 21870-076) nruab nrab nrog 10 feem pua fetal bovine serum thiab 2 m ML-Glutamine (Gibco 25030-082), penicillin thiab streptomycin (50 mg/L, Gibco 15140-122). Cells tau khaws cia ntawm 37 degree nyob rau hauv ib qho chaw humidified ntawm 5 feem pua CO2. Hauv kev sim hypoxia, cov hlwb raug tsim nyob rau hauv HBSS (Gibco 14025-092) es tsis txhob siv cov tshuaj dawb dawb hauv chav nrog 1 feem pua oxygen ntawm 37 degree C rau 2 teev. Cells tau pre-incubated nrog nitrite (10 μM, nrog / tsis muaj xanthine oxidoreductase (XOR) inhibitor febuxostat (100 nM), rau 30 min ua ntej hyp-oxia. Cell viability raug soj ntsuam los ntawm PrestoBlue⑧ assay (Invitrogen, A13261, thiab A13262), thiab cell mortality raug soj ntsuam los ntawm Trypan Blue exclusion assay (Sigma, T8154-20 ML).
2.12. Kev kuaj pom ntawm mitochondrial ROS
Mitochondrial superoxide qib tau kuaj pom nrog fluorogenic dye (MitoSOXTM, Invitrogen M36008). Tom qab kev kho mob, cov hlwb endothelial tau incubated nrog 2 μmol / L MitoSox hauv kab lis kev cai nruab nrab ntawm 37 degree rau 10 min. Tom qab ntawd cov hlwb raug ntxuav nrog 1 × HBSS ob zaug thiab ntsuas cov teeb liab fluorescent (SpectraMax iD3 nyeem ntawv, Molecular Devices, USA) thiab normalized rau cell viability.
2.13. Immunoblotting tsom xam
Cov kab mob raum khov los yog cov hlwb endothelial tau lysed hauv qhov tsis muaj 10 mmol / L Tris-HCl pH 8, 150 mmol / L NaCl, 5 mmol / L EDTA, 60 mmol / L N-octyl-glucoside, 1 feem pua Triton X{{9 }}, protease inhibitor cocktail, thiab protein phosphatase inhibitors. Cov cell lossis cov ntaub so ntswg lysates tau centrifuged rau 5 min ntawm 10, 000 × g ntawm 4 degree . Cov supernatants / proteins tau pauv mus rau hauv cov hlab tshiab thiab ntsuas los ntawm kev siv Bio-Rad
kev ntsuam xyuas protein. Protein extracts uas muaj qhov sib npaug ntawm cov proteins tau txheeb xyuas los ntawm kev siv {{{0}}} feem pua sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Cov proteins raug xa mus rau immobilon polyvinylidene difluoride membrane rau 1 h ntawm 300 mA.Membranes raug thaiv hauv 20mM Tris-HCl (pH7.4), 150 mM NaCl, thiab 0.1 feem pua Tween 20 (TBS-T) uas muaj 5 feem pua tsis muaj roj. mis hmoov rau 1 h thiab immunodetected nrog antibodies rau anti-TFAM (1: 2000, ab131607), anti-OXPHOS (1: 1000, ab110413), anti-vinculin (1: 5000, ab129002) (Abcam Cambridge, UK) thiab tshuaj tiv thaiv -Cleaved caspase-3 (1:1000,#9664)(Cell Signaling Technology, Beverly, MA, USA). Tag nrho cov tshuaj tiv thaiv kab mob thib ob rau kev tshuaj ntsuam immunoblot yog los ntawm Cell Signaling Technology. Rau Western blots, uncropped, annotated, full-length im-hnub nyoog nrog MW markers yog qhia nyob rau hauv cov ntaub ntawv ntxiv.
2.14. Kev txheeb cais
Ntau qhov kev sib piv ntawm cov pab pawg tau txheeb xyuas los ntawm ib- lossis ob-txoj kev ANOVA ua raws li kev pom zoo tom qab kev xeem. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± SEM. Txhua qhov kev suav suav suav tau siv Graphpad Prism 8.0. Statistical tseem ceeb tau txhais raws li p<>
