Kev Tiv Thaiv Cov Teeb Meem Ntawm Wogonin Ntawm Lipopolysaccharide-induced Infammation Thiab Apoptosis Ntawm Lub ntsws Epithelial Cells Thiab Nws Cov Txheej Txheem Ua Tau

Hu rautina.xiang@wecistanche.com

Abstract

Keeb kwm: Wogonin (5,7-dihydroxy-8-}methoxyflavone) yog lub ntuj di-hydroxyl flavonoid rho tawm los ntawm cov hauv paus hniav ntawm Scutellaria baicalensis Georgi. Daim ntawv no tau npaj los tshawb xyuas cov txheej txheem ntawm kev ua ntawm wogonin hauv kev txo qismobthiabapoptosisnyob rau hauv mob ntsws raug mob (ALI).

Cov ntaub ntawv thiab cov txheej txheem: Lipopolysaccharide (LPS) tau siv los tsim cov qauv hauv vitro ntawm ALL. Tom qab kev kho wogonin, cell viability thiab apoptosis ntawm LPS-induced A549 hlwb tau, ntsig txog, ntsuas los ntawm CCK-8, TUNELassays, thiab acridine txiv kab ntxwv / ethidium bromide dual staining, thaum cov ntsiab lus ntawm inflammatory cytokines thiaboxidative kev nyuaj siabCov cim tau kwv yees los ntawm RT-qPCR, ELISA assay, western blot tsom, thiab cov khoom siv coj mus muag. Western blot kuj tau ua los ntsuas qhov kev qhia ntawm cov proteins koom nrog. Tom qab ntawd, cov txiaj ntsig ntawm wogonin ntawm sirtuin 1 (SIRT1)-mediated high-mobility group box 1 protein (HMGB1) deacetylation tau tshawb xyuas. SIRT1 inhibitor EX527 tau siv los ntsuas qhov kev tswj xyuas cov teebmeem ntawm wogonin ntawm SIRT1-kev kho HMGB1 deacetylation hauv A549 hlwb hauv qab LPS stimulation.

Cov txiaj ntsig: LPS induced o, oxidative stress, thiab apoptosis ntawm A549 hlwb, uas tau tshem tawm los ntawm wogonin. Nws kuj tseem pom tau hais tias wogonin txhawb nqa HMGB1 deacetylation, nrog rau kev qhia txog SIRT1. Txawm li cas los xij, SIRT1 inhibitor EX527 ib nrab thim rov qab cov kev tiv thaiv ntawm wogonin ntawm qhov mob thiab apoptosis ntawm LPS-induced A549 hlwb.

Xaus: Wogonin alleviated the o thiab apoptosis in LPS-induced A549 cells by SIRT1-mediated HMGB1 deacetylation, uas yuav sawv cev rau kev txheeb xyuas cov txheej txheem tshiab uas wogonin tawm tswv yim tiv thaiv ntawm ALI thiab muab tswv yim rau kev siv wogonin rau Txhua yam kev kho mob.

Ntsiab lus: Mob ntsws mob, Wogonin, Inflammation, SIRT1, HMGB1 deacetylation

Nyem qhov no kom tau txais cov ntaub ntawv ntxiv

Keeb kwm

Mob ntsws raug mob (AL) raug soj ntsuam raws li qhov tshwm sim ntawm cov kab mob pathological xws li sepsis, pneumonia, raug mob, thiab mob pancreatitis [1]. Nws feem ntau pom nyob rau hauv cov neeg mob uas tau txais mus rau hauv cov chaw kho mob hnyav thiab qhia tias muaj kev tuag ntau [2]. Cov uas muaj sia nyob los ntawm ALI feem ntau ntsib nrog kev ua neej tsis zoo [3]. Txawm hais tias kev ua tiav tau ua tiav los ntawm kev nkag siab txog pathophysiology ntawm AL, kev kho mob ntawm cov kev kho tam sim no tseem txwv, uas tsis tuaj yeem ua tiav qhov kev cia siab ntawm cov neeg mob thiab lawv tsev neeg [4]. Yog li, nws yog qhov tseem ceeb heev los txheeb xyuas cov kev kho tshiab lossis lub hom phiaj rau kev kho mob TAG NRHO. Wogonin(5,7-dihydroxy-8-methoxyflavone), cov qauv uas tau qhia hauv Fig.1A, yog lub ntuj di-hydroxyl flavonoid rho tawm los ntawm cov hauv paus hniav ntawm Scutellaria baicalensis Georgi [5]. Nws cov khoom tseem ceeb los tiv thaiv inflammatory thiab nws daim ntawv thov nyob rau hauv kev kho mob ntawm cov kab mob inflammatory tau nthuav qhia los ntawm ntau cov kev tshawb fawb. Piv txwv li, wogonin muaj peev xwm txhim kho lub ntsws edema hauv murine thiab tiv thaiv lawv los ntawm lipopolysaccharide (LPS)-induced ALI los ntawm kev thaiv p38 mitogen-activated protein kinase (MAPK) thiab c-Jun NH(2)-terminal kinase (INK phosphorylation [6] Wogonin kuj txo LPS-induced neutrophil infiltration, zus tau tej cov proinflammatory cytokines, qhia cov adhesion molecules, thiab suppressed LPS-induced AL nyob rau hauv nas [7]. Cov pov thawj tam sim no qhia tau hais tias cov inflammatory teb thiab ALI induced los ntawm LPS yuav tsum attenuated raws li ib tug Qhov tshwm sim ntawm kev kho wogonin los ntawm peroxisome proliferator-activated receptor gamma (PPARy)-mediated NF-KB txoj hauv kev [8]. ntau lawm thiab HMGB1-nyob ntawm qhov mob, thiab txo qis kev mob sepsis-txog kev mob thiab kev pheej hmoo ntawm lub ntsws raug mob [9].Nyob rau hauv LPS-induced hauv vitro thiab hauv vivo ALI qauv, lwm daim ntawv tshaj tawm qhia tias inhibition ntawm HMGB1 thiab Lwm cov inflammatory mediators ntawm ulinastatin pom tau tias alleviated qhov tshwm sim ntawm ALI [10]. Thiab cov kev tshawb fawb yav dhau los tau lees paub tias LPS-vim tib neeg lub ntsws epithelial hlwb (A549) tau siv dav los ua tus qauv hauv vitro rau ALI [11-13]. Sirtuin 1 (SIRT1), tus downstream effector ntawm adenosine monophosphate-activated protein kinase (AMPK), tau raug qhia los ua lub luag haujlwm tseem ceeb hauv kev tswj hwm ntawm adiponectin tso tawm [14]. Wogonin supplementation zoo kawg li nce AMPK phosphorylation thiab SIRT1 kev qhia, thaum inhibition ntawm AMPK lossis SIRT1 txo cov teebmeem ntawm wogonin ntawm kev tsim lossis tso tawm ntawm adiponectin [14]. Nws tau tshaj tawm yav dhau los tias SIRT1- hloov kho HMGB1 deacetylation inhibited mob raum raug mob los ntawm sepsis [15].

Hauv kev tshawb fawb tam sim no, peb tsom los tshawb xyuas cov txheej txheem kev ua haujlwm ntawm wogonin hauv kev txo qismobthiabapoptosisntawm LPS-induced tib neeg lub ntsws epithelial hlwb, uas tej zaum yuav muab ib tug ntau yam kev pom rau kev kho mob ntawm ALI los ntawm wogonin.

Cov txiaj ntsig

Cov teebmeem ntawm wogonin ntawm kev muaj peev xwm thiab apoptosis ntawm LPS-induced A549 hlwb

Txhawm rau soj ntsuam cov txiaj ntsig ntawm wogonin ntawm ALI, peb thawj zaug kwv yees yog tias qhov muaj peev xwm ntawm A549 lub hlwb tuaj yeem raug puas los ntawm wogonin. Raws li tau nthuav tawm hauv daim duab 1B, wogonin tsis muaj teeb meem rau A549 hlwb yam tsis tau txais kev kho mob, qhia txog kev nyab xeeb ntawm kev kho wogonin hauv A549 hlwb ntawm qhov ntau ntawm O,5,10, thiab 20 μM. Tom qab LPS stimulation rau 24 teev, qhov muaj peev xwm ntawm A549 hlwb tau raug puas tsuaj heev, uas tau tawm tsam los ntawm wogonin nyob rau hauv ib tug concentration-dependent yam (Fig. 1C). Tsis tas li ntawd, cov txiaj ntsig tau los ntawm LDH qhov kev ntsuam xyuas pom tau tias qhov nce ntxiv ntawm LDH kev ua haujlwm hauv LPS-kho A549 hlwb raug txo qis tom qab siv tshuaj wogonin nce ntxiv (Fig.1D). Tom ntej no, peb tau sim cov nyhuv ntawm wogonin ntawm apoptosis ntawm LPS-induced A549 hlwb. Raws li tau nthuav tawm hauv Fig.2A, TUNEL staining pom tau tias LPS raug cuam tshuam loj heev rau apoptosis ntawm A549 hlwb piv nrog cov pab pawg tswj, uas tau txo los ntawm wogonin nyob rau hauv ib koob tshuaj. Dhau li ntawd, pom meej downregulation nyob rau hauv Bcl -2 qhia thiab upregulation nyob rau hauv Bax, Cyto-C, thiab cleaved caspase 3 kab lus tau pom nyob rau hauv cov LPS-exposed pab pawg neeg thaum piv rau cov tswj pab pawg, uas tau rov qab los ntawm kev kho mob wogonin (Fig. 2B). Raws li qhov tshwm sim, cov txiaj ntsig ntawm AO / EB ob chav fluorescence kev soj ntsuam tau nthuav tawm hauv Fig.3 qhia tias LPS coj mus rau qhov siab ntawm cov cell apoptosis raws li kev sib piv rau pawg tswj hwm, thaum EB-zoo hlwb tau txo qis tom qab wogonin ntxiv thaum piv rau LPS. pab pawg. Yog li, wogonin rov qab muaj peev xwm thiab txo cov apoptosis ntawm A549 hlwb raug ntxias los ntawm LPS.

Cov teebmeem ntawm wogonin rau qhov mob thiab oxidative kev nyuaj siab ntawm LPS-induced A549 hlwb

Txhawm rau tshawb nrhiav cov tshuaj tiv thaiv kab mob thiab tiv thaiv oxidant muaj peev xwm ntawm wogonin hauv ALI, lubmobthiaboxidative kev nyuaj siabntawm LPS-induced A549 hlwb kho nrog wogonin, feem, kuaj pom. Raws li pom hauv daim duab 4A-C, mRNA theem ntawm inflammatory cytokines (TNF- , IL-6, thiab IL-1) ntsuas los ntawm RT-qPCR tau nce mus rau qhov tseem ceeb tshaj plaws raws li LPS stimulation, whereas wogonin kev kho mob coj mus rau qhov txo qis hauv lawv cov lus qhia. Raws li qhov tshwm sim, cov txiaj ntsig ntawm ELISA tau qhia tias LPSinduction ua rau muaj kev nce ntxiv hauv TNF- , IL-6, thiab IL-1 cov ntsiab lus piv nrog cov pab pawg tsis tau kho, uas yog koob tshuaj txo qis tom qab kho wogonin (Daim duab . 4D-F). Qhov tseem ceeb, cyclooxygenase-2(Cox-2) thiab phospho-nuclear factor (NF)-KB p65 (p-NF-KB p65), uas yog cov cim kev mob ntsig txog mob, raug txo qis los ntawm LPS thaum upregulated los ntawm wogonin (Fig.4G). Tsis tas li ntawd, LPS ua rau txo qis kev ua haujlwm ntawm SOD thiab GSH-Px thiab nce cov ntsiab lus ntawm MDA thiab ROS, uas tau thim rov qab los ntawm wogonin (Fig. 4H-K). Ua ke, wogonin alleviates o thiab oxidative kev nyuaj siab ntawm LPS-induced A549 hlwb.

Kev cai ntawm wogonin hauv SIRT1-mediated HMGB1 deacetylation

Txhawm rau paub meej tias peb qhov kev xav tias wogonin tau tawm tsam kev tiv thaiv ntawm LPS-induced A549 cell raug mob los ntawm SIRT1- kho HMGB1 deacetylation, peb tau ua rau sab hnub poob blot txhawm rau txheeb xyuas cov qib protein ntawm cov khoom cuam tshuam. Pom tau tias, LPS txwv tsis pub cov lus qhia ntawm SIRT1 thiab txhawb nqa kev hloov pauv ntawm HMGB1 los ntawm cov nucleus mus rau cytoplasm, nrog rau HMGB1 acetylation. Txawm li cas los xij, cov qauv no tau thim rov qab los ntawm wogonin raug rau LPS-induced A549 hlwb (Fig.5). Yog li, wogonin kev kho mob tswj SIRT1-mediated HMGB1 deacetylation hauv LPS-induced A549 hlwb.

Kev tswj hwm ntawm SIRT1 inhibitor EX527 ntawm kev coj cwj pwm ntawm tes ntawm LPS-induced A549 hlwb kho nrog wogonin

Txhawm rau txheeb xyuas lub luag haujlwm ntawm SIRT1 hauv cov txheej txheem hauv qab uas wogonin cuam tshuam qhov mob thiab apoptosis ntawm LPS-induced A549 hlwb, peb siv EX527, SIRT1 inhibitor, los kho LPS-induced A549 hlwb rau 24 teev. Raws li pom hauv Fig.6A, qhov sib ntxiv ntawm EX527 txo qis SIRT1 qhia, cuam tshuam cov nucleo-cytoplasmic thauj ntawm HMGB1, thiab txhawb nqa HMGB1 deacetylation, piv rau LPS ntxiv rau Wogonin pawg. Ntxiv rau CCK-8 thiab LDH kev soj ntsuam tau pom tias EX527 puas ua rau cov nyhuv rov qab los ntawm wogonin ntawm qhov muaj peev xwm ntawm LPS-induced A549 hlwb (Fig.6B). Tsis tas li ntawd, cov teebmeem ntawm wogonin ntawm qhov kev qhia ntawm Bcl-2, Bax, Cyto-C, thiab cleaved caspase 3 qhia tau ib feem los ntawm EX527 hauv LPS-induced A549 hlwb (Fig.6C). Thaum kawg, qhov kev hloov pauv ntawm cov kab mob inflammatory (TNF- , IL-6, thiab IL-1 ), cov kab mob ntsig txog kab mob (Cox{30}} thiab p-NF-kB p65), thiab oxidative stress markers (SOD, GSH-Px, thiab MDA) nyob rau hauv LPS-induced A549 hlwb co-kho nrog wogonin thiab EX527 qhia tau hais tias EX527 puas inhibitory cuam tshuam ntawm wogonin ntawm o thiab oxidative kev nyuaj siab nyob rau hauv LPS-induced A549 hlwb (Fig. 7 A-K). Yog li, SIRT1 inhibitor EX527 thim rov qab cov teebmeem tiv thaiv ntawm wogonin ntawm cov kab mob phem ntawm LPS-induced A549 hlwb.

Kev sib tham

Tam sim no, ntau tus kws tshaj lij tau lees paub wogonin ua tus neeg sawv cev muaj zog rau kev kho cov kab mob inflammatory. Nws qhov ua tau zoo hauv kev txo cov lus teb inflammatory tau raug tshaj tawm los ntawm kev tawm tsam ntawm NF-kB thiab kev ua kom muaj zog ntawm cov khoom siv hluav taws xob erythroid 2-txog yam 2(Nrf2) qhia txoj hauv kev[16]. Qhov zoo siab, cov ntawv tshaj tawm yav dhau los cuam tshuam txog kev tiv thaiv kab mob nrog kev sib haum xeeb ntawm iNOS thiab Cox-2 kev qhia lossis kev ua kom muaj cov pa oxygen reactive (ROS) / ERK / Nrf2 txoj hauv kev [5,17]. Raws li qhov kev tshawb pom yav dhau los uas qhia tias tsis muaj kev cuam tshuam ntawm wogonin ntawm chondrocyte kev muaj peev xwm, peb pom zoo tsis muaj qhov cuam tshuam cytotoxic ntawm wogonin ntawm ib txwm A549 hlwb, txhawb peb kev tshawb nrhiav ntxiv rau hauv cov txheej txheem uas wogonin cuam tshuam hauv vitro ALI cell qauv [17]. Oxidative stress thiab o yog ob lub ntsiab lus tseem ceeb hauv pathogenesis ntawm AL, thiab yog li peb tau kuaj pom cov kev hloov pauv hauv oxidative stress indicators thiab inflammatory cytokines thaum kho wogonin [18]. Intriguingly, o thiab oxidative kev nyuaj siab tau ob leeg alleviated los ntawm wogonin raug nyob rau hauv LPS-kho A549 cell qauv, raws li muaj pov thawj los ntawm blockade ntawm pro-inflammatory yam tso tawm thiab txhim kho zus tau tej cov SOD thiab GSH-Px, raws li zoo raws li txo zus tau tej cov MDA tom qab. wogonin kev kho mob HMGB1 tshwm sim raws li ib qho kev txuag protein ntau uas tau pom thawj zaug los hloov kho sepsis hauv murine qauv [19]. Targeting HMGB1 tau raug suav hais tias yog qhov kev xaiv kho tau zoo rau kev kho mob sepsis vim qhov tseeb tias cov neeg mob uas tau tshwm sim raws li cov lus teb ntev ntev tau tshwm sim txuas ntxiv siab HMGB1 lev-els [10]. Cov ntawv ceeb toom tsis ntev los no thiab kev tshuaj xyuas tau them nyiaj ntau rau kev kho mob ntawm HMGB1 ntawm cov kab mob inflammatory, tsis txwv rau sepsis. Piv txwv li, yav dhau los tau pom tias inhibition ntawm HMGB1 los ntawm ulinastatin ameliorated LPS-induced ALI raug mob hauv cov nas [10,20]. Tom qab raug tso tawm los ntawm activated monocytes / macrophages, HMGB1 tau txais kev pab raws li ib tug proinflammatory yam ntawm ntau yam stimulants [20]. Cov tsos mob tshwm sim zoo li LPS tuaj yeem ua rau acetylation-txog kev hloov pauv ntawm HMGB1 los ntawm cov nucleus mus rau cytoplasm [21]. Ntawm no, qhov kev tshawb pom tias kev hloov pauv ntawm HMGB1 los ntawm lub nucleus mus rau cytoplasm raws li LPS stimulation yog counteracted los ntawm wogonin kev kho mob indirectly elucidated hais tias qhov mob ntawm A549 hlwb tshwm sim los ntawm LPS yog impeded potently los ntawm wogonin.

SIRT1 tau raug qhia kom ua lub luag haujlwm tseem ceeb hauv ntau yam txheej txheem lom neeg [22]. Nyob ntawm nws lub hom phiaj deacetylation, kev hloov pauv hauv kev ua ntawm SIRT1 tau xav tias yuav muaj kev txhawj xeeb nrog oxidative kev nyuaj siab thiab mob [23]. HMGB1 tsis ntev los no tau lees paub tias yog lub hom phiaj deacetylation ntawm SIRT1 [20]. Cov ntaub ntawv pov thawj tseem ceeb tau qhia txog SIRT1- hloov kho deacetylation ntawm HMGB1 txo qhov mob, rov ua haujlwm rau lub raum, thiab ua rau lub sijhawm muaj sia nyob ntev ntawm cov nas uas muaj mob sepsis ntsig txog mob raum [15]. Hauv kev tshawb nrhiav kev puas tsuaj rau lub hlwb, Omega{11}} polyunsaturated fatty acid alleviated qhov mob los ntawm kev hloov kho microglia polarization los ntawm SIRT1-modulated deacetylation ntawm HMGB1/NF-KB txoj hauv kev [24]. Tsis tas li ntawd, oleanolic acid tiv thaiv tus qauv nas los ntawm subarachnoid hemorrhage los ntawm SIRT1-modulated HMGB1 deacetylation [25]. Inhibition ntawm NLRP3 / NF-kB los ntawm Aloin los ntawm kev ua kom SIRT1 attenuated LPS-tshaj ALI hauv nas [26]. Los ntawm kev tswj SIRT1 / HMGB1 / NF-KB signaling, kaempferol txhim kho lub ntsws raug mob los ntawm ischemia-reperfusion ntawm kev tiv thaiv kab mob thiab kev tiv thaiv oxidative kev nyuaj siab 27I. Raws li ib qho tseem ceeb molecule downstream ntawm HMGB1, NF-kB yog ib qho tseem ceeb qhia txoj kev koom tes nyob rau hauv cov kev cai ntawm inflammatory mediators los ntawm activating proinflammatory yam noob caj noob ces thiab tso tawm ib tug loj tus naj npawb ntawm inflammatory yam, xws li TNF-, IL-6, thiab TNF -a[28,29].Tsis tas li ntawd, NF-KBcan kuj koom nrog cov txheej txheem oxidative kev ntxhov siab los ntawm kev tswj cov khoom tsim ntawm ROS thiab cuam tshuam rau qib ntawm SOD, MDA, thiab GSH-Px [30].Nyob rau hauv txoj kev tshawb no, cov txo qis p-NF-kB p65 qhia tau pom tom qab wogonin ntxiv, uas tau rov qab los ntawm SIRT1 inhibitor, EX527. Tsis tas li ntawd, peb pom tias deacetylated HMGB1 los ntawm wogonin tau nrog los ntawm kev nthuav qhia SIRT1, uas ua rau peb xav tias wogonin tuaj yeem tiv thaiv kev tiv thaiv ntawm LPS-induced o ntawm A549 hlwb los ntawm SIRT1-mediated HMGB1 deacetylation. Kev siv EX527 hauv cov kev sim hauv qab no ntxiv qhia tau tias inhibition ntawm SIRTl thim rov qab cov kev tiv thaiv ntawm wogonin ntawm qhov mob ntawm LPS-induced A549 hlwb.

Xaus

Hauv kev xaus, peb pom tau hais tias wogonin alleviated o, oxidative kev nyuaj siab, thiab apoptosis ntawm LPS-induced ALI cell qauv. Cov txheej txheem ntawm kev ua haujlwm no tshwm sim vim yog kev tswj hwm ntawm SIRT1- kho HMGB1 deacetylation tshwm sim los ntawm wogonin. Cov kev tshawb pom no tuaj yeem txheeb xyuas cov txheej txheem tshiab uas wogonin ua haujlwm tiv thaiv kev tiv thaiv ntawm ALI thiab muab kev sim hauv paus rau kev siv wogonin rau kev kho mob ntawm ALI.

Cov ntaub ntawv thiab cov txheej txheem

Cell kab lis kev cai thiab tshuaj

Human lung epithelial cells(A549) were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Paisley, UK) containing 10% heat-inactivated fetal bovine serum(Gibco, Paisley, UK), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ℃ with 5% CO. The cells were collected after 10 μg/mL LPS（Sigma Chemical Co., St Louis, MO）stimulation for 24 h. Wogonin (purity>99 feem pua), cais los ntawm Scutellaria baicalensis Georgi, tau yuav los ntawm Biotic Chemical (Taipei, Taiwan). Nws tau yaj hauv dimethylsulfoxide (DMSO), khaws cia ntawm -20 degree C, thiab diluted los ntawm DMEM. SIRT1 inhibitor EX527 (10 μM; MedChemExpress, Shanghai, Tuam Tshoj) tau siv los kho cov hlwb rau 24 teev ua ntej kev tswj hwm raws li kev tshawb fawb dhau los [31].

Cell Counting Kit-8 (CCK-8) assay

Tom qab qhov stimulation ntawm LPS rau 24 teev thiab ntau qhov ntau ntawm wogonin raug rau lwm 4 h, cov hlwb tau muab cog rau hauv 96-zoo phaj thiab cell viability assay tau ua los ntawm kev ntxiv 10μL ntawm CCK-8 reagent (Dojindo , Kumamoto, Nyiv) rau hauv txhua qhov dej ntawm 37 degree rau 4 h. Lub absorbance ntawm 450 nm tau saib xyuas los ntawm kev nyeem ntawv microplate (Bio-Rad Laboratories, Inc.).

Lactate dehydrogenase (LDH) tso tshuaj ntsuam xyuas

Lub cell supernatant kho nrog wogonin tau noob hauv 96- cov kab lis kev cai zoo thiab incubated rau 24 h ntawm 37 degree nrog 5 feem pua ​​CO, ua ntej 100 μL ntawm Cytotoxicity Detection Kit LDH tov (Roche Diagnostics, Fabkis) tau ntxiv rau txhua tus. zoo.15 feeb tom qab, cell supernatant tau diluted, nrog optical ceev tus nqi saib xyuas ntawm 450 nm.

Terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining TUNEL assay tau ua los ntsuas cov apoptosis ntawm LPS-induced A549 hlwb nrog lossis tsis muaj wogonin kho los ntawm Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, Tuam Tshoj) raws li kev ua haujlwm cov lus qhia. Tom qab phosphate buffer saline (PBS) ntxuav ob zaug thiab kho los ntawm 4 feem pua ​​paraformaldehyde rau 0.5 h,0.3 feem pua ​​hydrogen peroxide hauv PBS tau siv los tsim cov hlwb rau lwm 20 min ntawm chav tsev kub. Cells raug kho nrog diaminobenzene (DAB) rau 10 min thiab counterstained los ntawm hematoxylin (Solarbio, Beijing, Tuam Tshoj) rau 30 s. Cov hlwb apoptotic-zoo tau pom los ntawm fluorescence microscope (Olympus Corporation) thiab tus nqi apoptotic tau tsim nyog los ntawm Image-J software (NIH, Bethesda, MD, USA).

Acridine txiv kab ntxwv / ethidium bromide dual staining

Acridine txiv kab ntxwv / ethidium bromide (AO / EB) ob chav tshuaj fluorescence tau ua los ntsuas kev hloov pauv ntawm cell apoptosis. Hauv luv luv, A549 hlwb tau loj hlob ntawm iav npog hauv 24- cov phiaj xwm zoo. Cov hlwb tau ua ntej incubated rau 24 teev nrog cov ntshav dawb nruab nrab thiab tom qab ntawd incubated nrog LPS thiab ntau qhov ntau ntawm wogonin ntawm 37C. Cov hlwb raug ntxuav nrog PBS thiab muab nrog 5μL AO / EB tov tshuaj (100 ug / ml ntawm AO thiab 100 ug / ml ntawm EB sib xyaw hauv PBS, Aladdin, Tuam Tshoj) hauv 3 min. Cov hlwb raug soj ntsuam thiab thaij duab ntawm 510 nm excitation wavelength nyob rau hauv lub fluorescence microscope (Olympus Corporation).

Western blot tsom xam

Cov proteins tau muab rho tawm los ntawm A549 hlwb siv RIPA lysis buffer (Beyotime, Shanghai, Tuam Tshoj) thiab cov concentrations ntawm cov proteins tau ntsuas los ntawm ib tug bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, USA). Tom qab ntawd, 10 feem pua ​​​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis tau ua kom cais 40 ug cov proteins thiab cov proteins tau raug xa mus rau polyvinylidene difluoride (PVDF) membranes. Cov daim nyias nyias tau raug thaiv los ntawm cov mis nyuj tsis muaj rog thiab tom qab ntawd incubated ntawm 4 degree C thaum hmo ntuj nrog thawj cov tshuaj tiv thaiv. Tom qab ntawd, HRP-conjugated Secondary Antibody tau siv los ua kom cov kab mob sib kis rau 1.5 teev ntawm chav tsev kub. Thaum kawg, bands tau pom los ntawm kev txhim kho chemiluminescence (ECL) cov khoom siv (Amersham Biosciences, Buckinghamshire, UK), thaum band siv tau saib xyuas siv Image-J software (NIH, Bethesda, MD, USA). GAPDH raug suav hais tias yog ib qho kev siv sab hauv.

Reverse transcription-quantitative PCR (RT-gPCR)

A549 hlwb tau lysed hauv 1 ml TRIzol ~ reagent (Invitrogen, Carlsbad, CA, USA) rau kev rho tawm tag nrho RNA. Tom qab sau tag nrho RNA, thim rov qab tau ua rau kev sib txuas DNA (cDNA) synthesis los ntawm PrimeScript RT reagent kit (Takara Bio, Inc.).Cov xwm txheej tau ua raws li hauv qab no: 50 degree C rau 15 min, 85 degree C rau 5s, thiab kev khaws cia ntawm 4 degree .SYBR Premix Ex Taq TM(TaKaRa, Nyiv) tau siv los teeb tsa PCR cov xwm txheej thiab cov tshuaj tiv thaiv raws li cov lus pom zoo ntawm cov chaw tsim khoom. Ib qho cuab yeej ABI 7500 (AB-4351107; Applied Biosystems; Thermo Fisher Scientific, Inc.) tau siv rau qPCR. Cov txheej txheem thermocycling hauv qab no tau siv: thawj zaug denaturation ntawm 95 degree rau 10 min; ua raws li 40 cycles ntawm denaturation ntawm 95 degree C rau 15 s thiab annealing ntawm 60 degree rau 1 min; thiab qhov kawg txuas ntxiv ntawm 10 min ntawm 72 degree C. GAPDH tau txais raws li kev tswj hwm ntawm cov txheeb ze cov noob qhia. Kev suav cov txheeb ze ntawm cov noob caj noob ces yog ua los ntawm 2-Act method.

Enzyme-linked immunosorbent assay (ELISA)

Cov qib cellular ntawm interleukin-6(IL-6), IL-1 , thiab qog necrosis factor- (TNF- ) hauv cell supernatant tau ntsuas siv IL-6 ELISA Cov Khoom Siv , IL-1 ELISA Kit, thiab TNF- ELISA Cov Khoom Siv raws li cov lus pom zoo ntawm cov chaw tsim khoom (Shanghai Xi Tang Biotechnology, Shanghai, Suav).

Kev tshawb nrhiav oxidative kev nyuaj siab

Rau kev tshawb pom ntawm oxidative kev nyuaj siab, cov kev ua ub no ntawm oxidative kev nyuaj siab markers xws li malondialdehyde (MDA), reactive oxygen hom (ROS), glutathione peroxidase (GSH-Px), thiab superoxide dismutase (SOD) tau kuaj pom raws li cov chaw tsim tshuaj paus cov lus qhia (Nanjing Jiancheng Biotechnology Institute, Suav).

Kev txheeb cais

Tag nrho cov ntaub ntawv tau qhia raws li qhov txhais tau tias ± tus qauv sib txawv (SD) ntawm tsawg kawg peb qhov kev sim ywj pheej. Rau kev sib piv ntawm ntau pab pawg, ib-txoj kev tsom xam ntawm qhov sib txawv (ANOVA) nrog Tukey's post hoc xeem tau ua, thaum cov ntawm ob pawg tau ua los ntawm Tub Ntxhais Kawm T-test. P<0.05 was="" deemed="" as="" statistically="">

Cov ntawv luv

AL: mob ntsws mob; LPS: lipopolysaccharide; HMGB1: High-mobility pawg thawv 1protein; PPARy. Peroxisome proliferator-activated receptor-gamma; MAPK: Mitogen-Activated Protein Kinase; JNK: C-Jun NH(2)-terminal kinase; CLP: Cecal ligation thiab puncture; DMEM: Dulbecco's modified Eagle medium; DMSO: Dimethylsulfoxide; CCK-8:Cell count-ingkit-8; LDH: Lactate dehydrogenase; TUNEL: Terminal-deoxynucleotidyl transferase-mediated nick-end labeling; PBS: Phosphate buffer saline; DAB: Diaminobenzene; AO/EB: Acridine txiv kab ntxwv/ethidium bromide; BCA: Bicinchoninic acid; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF: Polyvinylidene difluoride; ECL: Txhim kho chemiluminescence; cDNA: Ntxiv DNA; ELSA: Enzyme-linked immunosorbent assay; IL: Interleukin; TNF-a: qog necrosis factor-a; MDA: Malondialdehyde; GSH-Px Glutathione peroxidase; SOD: Superoxide dismutase; SD: Txheem sib txawv; ANOVA: Kev txheeb xyuas qhov sib txawv; Cox-2:Cyclooxygenase-2; Nrf2: Nuclear factor erythroid 2-related factor 2; ROS: Reactive oxygen hom.

Cov ntaub ntawv

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