Cov nyhuv ntawm Tanshinone IIA Ntawm LPS-induced Inflammatory Response hauv ROS-NLRP3 Inflammasome Dependent Manner hauv RAW264.7 Cells
Mar 10, 2022
Yog xav paub ntxiv:emily.li@wecistanche.com
Qhov tseem ceeb
Tan IIA tuaj yeem cuam tshuam cov lus teb inflammatory thiab NLRP3 qhia tawm los ntawm LPS lossis LPS ntxiv rau ATP. Acetylcysteine (N-acetyl-l-cysteine, NAC), ROS inhibitor, tuaj yeem inhibit LPS ntxiv rau ATP-induced nce qib NLRP3. Cov txheej txheem hauv qab cov teebmeem ntawm Tan IIA tuaj yeem koom nrog kev tswj hwm ntawm ROS-NF-κB / P38-NLRP3 txoj hauv kev. Txoj kev tshawb no ntxiv tus cwj pwm ntawm cov txheej txheem molecular ntawm Tan IIA thiab muab cov kev xav tshiab rau nws cov kev kho mob.
Abstract
Cov pov thawj tshiab tau pom tias Tanshinone IIA (Tan IIA) tiv thaiv kev raug mob cardiomyocytes, plawv fibroblasts, thiab atherosclerosis. Txawm li cas los xij, cov txheej txheem molecular hauv qab cov teebmeem ntawm Tan lA tseem tsis paub meej. Txhawm rau tshawb xyuas lub luag haujlwm ntawm Tan ll A hauv cov lus teb inflammatory hauv ROS-NLRP3 inflammasome-dependent yam. RAW264.7 hlwb txhawb nrog LPS tau nrhiav los tsim cov qauv ntawm tes ntawm cov lus teb inflammatory. Peb cov txiaj ntsig tau qhia tias kev tsim cov NO tau nce ntxiv tom qab tau txhawb nqa los ntawm LPS. thiab Tan IIA kho tau txo qis qib ntawm NO. mRNA expression of NLRP3. IL-1B thiab TNF-a tau cuam tshuam loj heev los ntawm Tan LLA piv nrog LPS kho cov hlwb. Protein expression of NLRP3. IKBa, pp65/p65, thiab pp38/p38 tau kos npe txo qis los ntawm Tan IIA, piv nrog LPS lossis LPS ntxiv rau ATP cov pab pawg txhawb nqa. Lub caij no. Tan IA ho inhibited theem ntawm ROS induced los ntawm LPS ntxiv rau ATP. Thiab NAC, ROS inhibitor, tuaj yeem cuam tshuam cov protein qhia ntawm NLRP3. Raws li cov kev tshawb pom no nws tuaj yeem kwv yees tias cov txheej txheem hauv qab ntawm Tan IIA tuaj yeem koom nrog kev tswj hwm ntawm ROS-NF-kB / P38-NLRP3 txoj hauv kev. Txoj kev tshawb no ntxiv tus cwj pwm ntawm cov txheej txheem molecular ntawm Tan IA thiab muab cov kev xav tshiab rau nws cov kev kho mob.
Ntsiab lus: Tanshinone IIA,Kev mob, NLRP3 inflammasome, ROS
Cov txiaj ntsig ntawm Cistanche cov khoom
Keeb kwm
Macrophages ua lub luag haujlwm tseem ceeb hauv kev ua kom lub plawv homeostasis thiab orchestrating cov ntaub so ntswg kho nrog rau kev kho dua tshiab. Hauvplawvua tsis tiavCov neeg mob, nce ntshav siab proinflammatory cytokines yuav tsum tau ua pov thawj biomarkers ntawm cov txiaj ntsig kho mob tsis zoo, thiabmobtuaj yeem ua rau kev mob plawv tsis ua haujlwm [1]. Txawm li cas losmobtau tshwm sim los ua lub hom phiaj kho mob los kho cov kab mob plawv, nws, txawm li cas los xij, zoo li kev tiv thaiv kab mob tsis tuaj yeem txhim kho cov txiaj ntsig kho mob tom qab myocardial infarction (MI) lossis hauv plawv tsis ua haujlwm [2,3].
Ntau cov kev tshawb fawb soj ntsuam tau pom tias inhibition ntawm ib qhomobtxoj kev, zoo li lubmobcytokine, IL-1 , tuaj yeem txo cov xwm txheej ntawm cov hlab plawv [4, 5]. Nws tuaj yeem kwv yees tias kev tiv thaiv kab mob ntawm cov kab mob inflammatory muaj qhov tseem ceeb rau kev kuaj mob thiab kho qhov tseem ceeb hauv cov txheej txheem ntawm cov kab mob plawv.
NLRP3 inflammasomes yog qhov kev tshawb fawb ntau tshaj plaws ntawm txhua yam ntawm inflammasomes. Nws tau raug sau tseg tias NLRP3 inflammasome plays lub luag haujlwm tseem ceeb hauv ntau yam kab mob, xws li ntau yam sclerosis (MS), Alzheimer disease (AD), atherosclerosis, rog, thiab hom 2 mob ntshav qab zib mellitus [6], thiab ntau thiab ntau cov pov thawj qhia tias NLRP3 inflammasome tseem koom nrog hauv myocardial ischemia / reperfusion raug mob (MI / RI) [7,8]. Cov txheej txheem ntawm NLRP3 kev ua kom muaj xws li kev tsim cov pa oxygen reactive (ROS), potassium efflux, thiab tso tawm cathepsins rau hauv cytosol tom qab lysosomal destabilization. Thaum cov hlwb raug txhawb los ntawm cov kab mob lossis cov cim phom sij, NLRP3 tau qhib thiab nrhiav cov pro-caspase-1 los ntawm adapter protein ASC. NLRP3. ASC thiab pro-caspase{13}} ua NLRP3 inflammasome complex sib dhos. Raws li qhov kev txiav txim ntawm NLRP3 inflammasome, pro-caspase{17}} tau qhib kom ua caspase-1, nws thiaj li ua rau kev zais ntawm IL-1 thiab IL-18, thiab ua rau series ntawm cov tshuaj tiv thaiv inflammatory [9,10].
Tan IIA yog ib qho liposoluble muaj nyob hauv Danshen (Sahia miltiorrhiza Bge), muaj ntau yam khoom lom thiab koom nrog hauv cov tshuaj biochemical. Tan IA muajtiv thaiv-mob, antioxidantthiab txhawb cov nyhuv angiogenesis, thiab kuj tau dav effused rau kev kho mob ntawm cov kab mob cardio-cerebrovascular [1, 12]. Cov pov thawj tshiab tau pom tias Tan IIA tiv thaiv cardiomyocytes raug mob, mob plawv fibroblasts, atherosclerosis, thiabneuroinflamation[13-16]. Txawm li cas los xij, cov txheej txheem molecular hauv qab cov teebmeem ntawm Tan IIA tseem tsis meej.
Thaum lub sij hawm ischemia / reperfusion raug mob, monocyte / macrophage, neutrophils, thiab endothelial hlwb pib ua haujlwm.mobteb, inducing tso tawm ntau yammobcytokines, thiab ua rau muaj zog vascular permeability, cov ntaub so ntswg edema, thiab puas. Macrophage yog ib qho tseem ceeb inflammatory kab mob ntawm tes ntawm tag nrho covmobcell hom thiab plays lub luag haujlwm tseem ceeb hauv cov txheej txheem ntawm myocardial infarction [17]. Monocyte / macrophage recruited rau lub puas myocardium tuaj yeem tso tawm ROS ntxiv, cov neeg kho mob inflammatory, thiab proteases uas ua rau mob myocardial raug mob [18].
Vim yog vim li cas macrophages ua lub luag haujlwm tseem ceeb hauv kev ua kom lub plawv homeostasis thiab orchestrating cov ntaub so ntswg kho, peb nrhiav RAW264.7 hlwb txhawb nrog LPS los tsim cov qauv ntawm tes ntawm inflammatory teb. Txoj kev tshawb fawb tam sim no tau tshawb xyuas seb Tan IIA puas muaj kev cuam tshuam rau LPS txhawb nqa macrophages, thiab cov txheej txheem hauv qab ntawm Tan ⅡA ntawm macrophage hauv ROS-NLRP3 inflammasome-dependent yam.
Cov ntaub ntawv thiab cov txheej txheem
Cell Kab lis kev cai thiab kev kho mob
RAW264.7 cell tau yuav los ntawm Cell Culture Center ntawm Suav Academy ntawm Kev Kho Mob Sciences (Beijing, Suav). Cells tau coj mus kuaj hauv DMEM nruab nrab (cov piam thaj siab), ntxiv 10 feem pua fetal bovine serum (FBS), 100 U/mL penicillin, thiab 100 ug/mL streptomycin. Cells raug coj los ntawm 37 degree nyob rau hauv ib tug siab humidified huab cua nrog 5 feem pua CO2. Rau txhua qhov kev sim, cov hlwb raug kho ntawm 80-90 feem pua ntawm tes sib txuas. Cov hlwb raug kho nrog ntau qhov sib txawv ntawm Tan IIA (ntawm 10,1, thiab 0.1 uM) rau lub sijhawm sib txawv, nrog lossis tsis muaj 1 ug / mL LPS. Lub caij no, cov hlwb hauv pawg tswj tau raug kho nrog qhov sib npaug ntawm 0.1 feem pua DMSO.
Kev ntsuas ntawm NO ntau lawm
TSIS MUAJ Assay Kit (Beyotime, Tuam Tshoj) tau siv los ntsuas qhov tsim ntawm NO raws li cov chaw tsim khoom cov lus qhia. Luv luv, cov qauv tau diluted nrog DMEM ntxiv rau 10 feem pua FBS thiab 50μL tus qauv lossis cell culture supernatant tau ntxiv rau txhua qhov dej ntawm 96-zoo phaj. Tom qab ntawd 50uL ntawm Griess Reagent I thiab II tau muab tso rau hauv txhua qhov dej ntawm lub phaj thiab qhov pom kev ntom ntom (OD) tau kuaj pom los ntawm Varioskan LUX multimode microplate nyeem ntawv ntawm 540 nm.
RNA Extraction
RAW264.7 hlwb raug plated ntawm qhov ceev ntawm 1 × 106 hlwb/zoo nrog 10 feem pua FBS DMEM hauv rau-zoo daim hlau. 24 teev tom qab ntawd, qhov nruab nrab raug tshem tawm thiab ntxiv nrog cov tshuaj tsis muaj tshuaj tua kab mob, tsis muaj tshuaj tua kab mob DMEM. RAW264.7 hlwb raug kho nrog DMSO (tswj), LPS (1 ug / mL) thiab LPS ntxiv rau Tan IIA (10,1, thiab 0.1uM rau 6h. Tom qab ntawd tag nrho RNA raug rho tawm los kuaj xyuas qib mRNA ntawm NLRP3, IL{ {18}} , thiab TNF- . Tag Nrho RNA tau muab rho tawm los ntawm kev siv TRIzol reagent (Ambion). RNA raug ntsuas los ntawm qhov ntau ntawm Va ib qho ultramicrofluorescence spectrophotometer (DeNovix). Cov chaw tsim khoom cov lus qhia 1 ug ntawm RNA cov qauv tau thim rov qab, thiab cov txheej txheem tshuaj tiv thaiv tau ua raws li hauv qab no: 42 degree C rau 50 min, 85 degree C rau 5 min, thiab tom qab ntawd khaws cia ntawm 4 degree.
RT-PCR
Ouantitative PCR (OPCR) kev soj ntsuam tau ua nrog O-PCR ntsuas (Lightcycler 96, Roche), siv UltraSYBR sib tov (cwbiotech). PCR raws tu qauv yog 95 degree rau 10min, ua raws li 40 cycles ntawm 95 degree rau 10s, 56 rau 30s, thiab 72 rau 32s. Cov primers siv rau qRT-PCR yog qhia raws li nram no: GADPH-Forward: 5'-AACTTTGGCATTGTGGAAGG-3';GADPH-Rov qab:5'-ACACATTGGGGGTAGGAACA-3',NLRP3-Forward: 5'-ATGCTGCTTCGACATCTCCT-3'NLRP3-Reverse:5'-AACCAATGCGAGATCCTGAC-3';IL-1 -Forward:5'-GCCCATCCTGTGACTCAT-3';IL{ {32}}Reverse:5'-AGGCCACAGGTATTTTGTCG-3':TNF- -Forward:5'-GCAGATGGGGCTGTACCTTATC-3'TNF-a-Reverse:5'-GCAAGATGGGCTGTACCTTATC-3 ! Qib qhia cov noob tau suav nrog siv 2-AACt txoj kev.
Western blotting
Western blotting tau ua nrog cov hauv qab notshuaj tiv thaiv: anti-NLRP3 antibody (CST), anti- pp38 antibody (CST), anti-p38 antibody (CST), anti-pp65 antibody (CST), anti-GADPH antibody (protein tech), anti - -tubulin antibody (protein tech), anti- HMGB1 antibody (protein tech), anti- p65 antibody (protein tech), anti- IKB- antibody (protein tech). Cov feem pua ntawm cov protein tau muab rho tawm los ntawm cov lysates ntawm cov kab lis kev cai thiab cov khoom siv Enhanced BCA Protein Assay (Beyotime, Tuam Tshoj) tau siv los ntsuas cov protein ntau. Proteins tau denatured thiab daws ntawm SDS-PAGE, ces pauv mus rau PVDF daim nyias nyias. Tom qab thaiv nrog QuickBlockTMBlocking Buffer (Beyotime, Tuam Tshoj) rau 1 h, PVDF membranes ces incubated nrog thawj cov tshuaj tiv thaiv thaum hmo ntuj ntawm 4 degree degree. Tom qab ntawd cov membranes tau ntxuav nrog Western Wash Buffer (Beyotime, Tuam Tshoj) peb zaug, thiab incubated nrog HRP conjugated thib ob antibodies rau 1 h ntawm chav tsev kub. Cov khoom siv BeyoECL Plus (Beyotime, Tuam Tshoj) tau siv los kuaj xyuas cov proteins raws li cov chaw tsim khoom cov lus qhia. Lub C-DiGit 3600 (Li-Cor, USA) tau ua haujlwm rau cov duab thiab cov qhab nia tau suav nrog los ntawm Image J Software.
Kev ntsuas ntawm ROS tsub zuj zuj
Lub DCFH-DA sojntsuam Reactive Oxygen Species Assay Kit (Beyotime, Tuam Tshoj) tau siv los ntsuas intracellular ROS qib hauv RAW264.7 hlwb raws li cov chaw tsim khoom cov lus qhia. Luv luv, DCFH-DA tau diluted nrog DMEM tsis muaj ntshav qab zib thiab qhov kawg concentration tau hloov kho rau 10μM / L. Tom qab incubation nrog 10 μM DCFH-DA dye rau 30 min ntawm 37 degree C, RAW264.7 hlwb raug ntxuav peb zaug nrog DMEM-dawb cov kua nruab nrab, thiaj li yuav tshem tawm qhov tshaj DCFH-DA. Rau kev tswj kom zoo, zoo, lμL ntawm Rosup tau ntxiv rau cov hlwb. Cov fluorescence tau suav nrog siv Nikon IX73 inverted microscope. Cov duab ntawm cov hlwb raug ntsuas siv Image J Software.
Kev txheeb cais
Txhua qhov kev sim tau rov ua dua tsawg kawg peb zaug. SPSS software tau siv rau txhua qhov kev tshuaj ntsuam, thiab GraphPad Prism 5 tau ua rau cov duab kos duab. Tag nrho cov txiaj ntsig tau muab raws li tus nqi nruab nrab ± SD. Ib-txoj kev ANOVA tau siv los txheeb xyuas cov ntaub ntawv ntawm cov pab pawg thiab ua ke sib piv siv SNK-q xeem. Tus nqi P< 0.05="" was="" considered="" statistically="" significant="" in="" all="">
Daim duab 1. Tan IIA muaj kev tswj xyuas cov txiaj ntsig ntawm TSIS MUAJ ntau lawm, NLRP3, IL-1 , TNF- mRNA
kev qhia
A. RAW264.7 Cells TSIS MUAJ ntau pawg sib txawv. Cov hlwb raug pretreated nrog Tan IIA 10µM, 1µM, thiab
{{0}}.1µM rau 30 min thiab txhawb nqa nrog LPS (1 ug/mL) rau 24 teev. * P < 0.05="" vs="" ctrl,="" #="" p="">< 0.05="" vs="" lps.="" bd.="" tan="">
muaj kev tswj xyuas cov nyhuv ntawm mRNA qhia ntawm TNF- , NLRP3, thiab IL-1 . Cov hlwb raug pretreated nrog Tan IIA
Cov txiaj ntsig
Qhov cuam tshuam ntawm RAW264.7 Cells TSIS TAU ntawm Tan IIA Kev Kho Mob
Tsis yog, tus tub txib thib ob, ua haujlwm ntau yam hauv cell physiological thiab pathological txheej txheem. Tsis pub zais los ntawm macrophages ua lub luag haujlwm tseem ceeb hauv inflammatory, tiv thaiv kab mob thiab tiv thaiv kab mob [19, 20]. Vim li no, TSIS tuaj yeem siv los kwv yees qhov cuam tshuam ntawm LPS ntawm kev tiv thaiv kab mob thiab kab mob. Cov hlwb raug kho nrog qhov sib txawv ntawm Tan IIA (ntawm 10, 1, thiab 0.1 μM) nrog lossis tsis muaj 1 ug / mL LPS rau 24h, thiab cov hlwb hauv pawg tswj tau raug kho nrog qhov sib npaug ntawm 0.1% DMSO. Raws li pom hauv daim duab 1A, piv nrog rau pawg tswj hwm, hauv pawg qauv txhawb nqa nrog LPS rau 24h, kev tsim tawm NO tau nce ntxiv (P<0.05); and="" various="" concentrations="" of="" taniia="" treatment="" (at="" 10,="" 1,="" and="" 0.1="" μm)="" significantly="" decreased="" the="" no="" production=""><0.05) of="" the="" cells="" stimulated="" with="">
Cov nyhuv ntawm NLRP3, IL-1 thiab TNF- mRNA qhia txog Tan IIA Kev Kho Mob
RAW264.7 hlwb stimulated nrog LPS tuaj yeem ua rau lub zog ntawm cov lus teb inflammatory, thiab nws tuaj yeem ua kom zoo dua los ntawm Tan IIA, suav nrog mRNA qhia ntawm NLRP3, IL-1 , thiab TNF- . Raws li pom hauv daim duab 1B-D, piv nrog rau pawg tswj hwm, hauv pawg qauv txhawb nqa nrog LPS, mRNA qhia ntawm NLRP3, IL-1 thiab TNF- tau nce ntau ntxiv (P < {{10="" }}}="" 05);="" thiab="" ntau="" qhov="" ntau="" ntawm="" tan="" iia="" kev="" kho="" mob="" (ntawm="" 10,="" 1,="" thiab="" 0.1="" μm)="" txo="" qis="" mrna="" qhia="" ntawm="" nlrp3,="" il-1="" thiab="" tnf-="" (p="">< 0.05)="" hauv="" hlwb="" txhawb="" nrog="">
Cov nyhuv ntawm NLRP3 protein qhia ntawm Tan IIA Kev Kho Mob
ATP, qhov thib ob lub teeb liab molecule, yog tus activator xav tau rau NLRP3 inflammasome activation. Yog li, ATP tau siv los txhawb kev ua haujlwm ntawm NLRP3 [21, 22]. Peb cov txiaj ntsig tau pom tias LPS ntxiv rau ATP-induced ua kom NLRP3 protein qhia thiab nws tuaj yeem raug kho los ntawm Tan IIA. Raws li tau pom hauv daim duab 2, piv nrog rau pawg tswj hwm, hauv pawg qauv tsim los ntawm LPS ntxiv rau ATP, qib protein qhia ntawm NLRP3 tau nce ntxiv (P < 0.05);="" thiab="" taniia="" kev="" kho="" mob="" (ntawm="" 10μm)="" txo="" qis="" cov="" protein="" qhia="" ntawm="" nlrp3="" (p="">< 0.05)="" hauv="" hlwb="" txhawb="" nrog="" lps="" ntxiv="" rau="">
LPS-induced activation ntawm NF-κB thiab p38 txoj hauv kev tuaj yeem cuam tshuam los ntawm Tan IIA
Raws li pom nyob rau hauv daim duab 2, piv nrog rau cov tswj pab pawg, nyob rau hauv lub hlwb stimulated nrog LPS los yog LPS ntxiv rau ATP, cov protein qhia ntawm IKB, pp65/p65 tau nce ntau (P < 0.05);="" thiab="" tan="" iia="" kev="" kho="" mob="" (ntawm="" 10μm)="" txo="" qis="" kev="" txhim="" kho.="" cov="" qib="" protein="" ntawm="" pp38="" p38="" tau="" nce="" siab="" hauv="" cov="" hlwb="" txhawb="" nrog="" lps="" ntxiv="" rau="" atp,="" thiab="" kev="" tswj="" hwm="" tau="" raug="" txwv="" los="" ntawm="" tan="" iia="" (10μm)="" (daim="" duab="" 2).="" txawm="" li="" cas="" los="" xij,="" qib="" protein="" ntawm="" pp38="" p38="" tsis="" hloov="" pauv="" loj="" hauv="" cov="" hlwb="" txhawb="" nrog="" lps.="" tsis="" tas="" li="" ntawd,="" cov="" lus="" qhia="" protein="" ntawm="" hmgb1="" kuj="" tau="" txhim="" kho="" hauv="" cov="" hlwb="" uas="" raug="" cuam="" tshuam="" nrog="" lps="" lossis="" lps="" ntxiv="" rau="" atp="" tab="" sis="" tsis="" pom="" qhov="" sib="" txawv.="" cov="" txiaj="" ntsig="" no="" qhia="" tias="" lps="" lossis="" lps="" ntxiv="" rau="" atp="" induced="" activation="" ntawm="" nf-κb="" thiab="" p38="" txoj="" kev="" tuaj="" yeem="" cuam="" tshuam="" los="" ntawm="" tan="">
Tan IIA muaj kev tswj xyuas cov txiaj ntsig ntawm ROS
Txhawm rau paub meej tias Tan IIA puas tuaj yeem tshem tawm cov kab mob hauv lub cev ROS, kev tsim khoom ntawm intracellular ROS tau soj ntsuam los ntawm fluorescence microscopy siv Reactive Oxygen Species Assay Kit. Lub hlwb RAW264.7 tau raug LPS rau 6 h, thiab 1 mM ATP tau ntxiv 30min ua ntej cov hlwb raug sau. ROS-zoo hlwb tau pom zoo li cov pob ntsuab ntsuab, thiab LPS ntxiv rau ATP kev kho mob nce qib ntawm intracellular ROS ntau, piv nrog rau hauv pawg tswj hwm (Daim duab 3). Lub caij no, kev kho mob nrog Tan IIA inhibited LPS ntxiv rau ATP-induced nce hauv intracellular ROS loj (Daim duab 3). Cov txiaj ntsig no qhia tias Tan IIA muaj peev xwm antioxidant.
Daim duab 3. Tan IIA muaj kev tswj xyuas cov nyhuv ntawm ROS qhia ( nplai bar 50μm)
Cov hlwb raug pretreated nrog Tan IIA 10µM rau 30 min thiab txhawb nqa nrog LPS (1 ug / mL) rau 5.5 teev. ATP tau ntxiv 30 feeb ua ntej cov hlwb raug sau. A. Cov duab sawv cev fluorescence ntawm cell ROS qhia. B. Cov ntaub ntawv ntau ntawm qhov nruab nrab ntawm peb qhov kev sim cais. * P < 0.05="" vs="" ctrl,="" #="" p="">< 0.05="" vs="" lps="" lps="" ntxiv="" rau="">
Daim duab 4. NAC muaj kev tswj xyuas cov txiaj ntsig ntawm cov protein qhia ntawm NLRP3.
Cov hlwb raug pretreated nrog Tan IIA 10µM rau 30 min thiab txhawb nqa nrog LPS (1 ug/mL) / LPS (1 ug/mL) ntxiv rau NAC (1mM) rau 5.5 teev. ATP tau ntxiv 30 feeb ua ntej cov hlwb raug sau. A. Tus sawv cev Western blot ntawm NLRP3. B. Cov ntaub ntawv ntau ntawm qhov nruab nrab ntawm peb qhov kev sim cais. * P < 0.05="" vs="" ctrl,="" #="" p="">< 0.05="" vs="" lps="" ntxiv="" rau="">
NAC muaj txoj cai tswjfwm ntawm NLRP3
Acetylcysteine (N-acetyl-l-cysteine, NAC), ib tug scavenger ntawm ROS, tuaj yeem cuam tshuam cov protein qhia ntawm NLRP3. Raws li tau hais dhau los, cov lus qhia protein ntawm NLRP3 tau nce ntxiv tom qab txhawb nqa nrog LPS ntxiv rau ATP rau 6h, thiab kev kho mob nrog Tan IIA inhibited LPS ntxiv rau ATP-induced nce qib ntawm NLRP3 protein. Tsis tas li ntawd, kev kho mob nrog NAC kuj tseem tuaj yeem cuam tshuam LPS ntxiv rau ATP-induced nce qib ntawm NLRP3 protein ntau (Daim duab 4). Cov txiaj ntsig no tau pom tias kev tswj hwm cov txiaj ntsig ntawm Tan IIA ntawm NLRP3 tuaj yeem nyob ntawm txoj hauv kev ROS-NLRP3.
Kev sib tham
Macrophages koom nrog cov txheej txheem ntawm cov lus teb inflammatory thiab ua ib feem tseem ceeb hauv cov txheej txheem ntawm myocardial infarction. Monocyte / macrophage recruited mus rau lub puas myocardium tuaj yeem tso tawm ROS, inflammatory mediators, thiab protease, uas aggravates myocardial raug mob [23]. RAW264.7 hlwb tau raug suav hais tias yog tus thawj coj ntawm kev sim macrophage tus qauv, thiab ua lub luag haujlwm tseem ceeb hauv kev tshawb fawb ntawm macrophage cuam tshuam cov teeb liab qhia txoj hauv kev [24]. Macrophages ua lub luag haujlwm tseem ceeb hauv kev ua kom lub plawv homeostasis thiab kho cov ntaub so ntswg thiab kho dua tshiab. LPS-stimulated RAW264.7 hlwb tau siv dav los kawm cov tshuaj uas muaj cov tshuaj tiv thaiv kab mob thiab cov txheej txheem hauv qab [25,26].
NLRP3 inflammasome yog ib qho tseem ceeb ntawm kev sib haum xeeb ntawm cov lus teb inflammatory, thiab tau muaj pov thawj tias NLRP3 inflammasome plays ib qho tseem ceeb hauv cov kab mob plawv. Hauv tib neeg cov hlab ntsha, NLRP3 siab qhia tau cuam tshuam nrog kev pheej hmoo ntawm cov kab mob atherosclerotic thiab qhov hnyav ntawm cov kab mob plawv [27], thiab qib ntawm NLRP3 inflammasome-txog cov noob tau cuam tshuam nrog atherosclerotic plaque [28]. Kev ua kom NLRP3 inflammasome kuj tau cuam tshuam nrog kev kho mob plawv. NLRP3 tau qhib rau hauv cardiomyocytes hauv transverse aortic constriction (TAC) induced nas adverse cardiac remodeling model, and the activation of NLRP3 inflammasome brings about the transformation of pro-caspase-1 to its active form [29]. MCC950.a NLRP3 inflammasome inhibitor tuaj yeem cuam tshuam cov nyhuv atherosclerotic, tej zaum nyob rau hauv ib txoj kev ntawm inhibiting adhesion ntawm monocytes thiab txo intraplaque macrophages tsub zuj zuj [30].
Tan IIA yog ib qho loj monomer rho tawm los ntawm Danshen, muaj ntau yam khoom lom neeg. Tan ⅡA muajtiv thaiv-mob, antioxidantthiab txhawb nqa cov nyhuv angiogenesis, thiab tau siv dav rau kev kho mob ntawm cov kab mob plawv-cerebrovascular [11]. Cov pov thawj tshiab tau pom tias Tan A tiv thaiv kev raug mob cardiomyocytes, plawv fibroblasts, atherosclerosis, thiabneuroinflamation[13-16]. Peb cov kev tshawb fawb yav dhau los tau pom tias Tan IIA tau siv cov tshuaj tiv thaiv kab mob los ntawm inhibiting TLR4-MyD88-NF-KB kev taw qhia txoj hauv kev thiab kho cov kev qhia txog miRNA hauv LPS-stimulated RAW264.7 hlwb [31] . Tsis tas li ntawd, Tan IIA tuaj yeem txhawb nqa macrophage polarization los ntawm pro-inflammatory M1 phenotype mus rau anti-inflammatory M2 phenotype [32]. Txawm li cas los xij, cov txheej txheem molecular hauv qab cov teebmeem ntawm Tan IIA tseem tsis meej.
Peb cov txiaj ntsig tau pom tias kev tsim cov NO tau nce ntau ntxiv hauv RAW264.7 hlwb txhawb nqa nrog LPS, thiab Tan IIA kev kho mob (ntawm 10,1, thiab 0.1 uM) txo qis qhov NO ntau lawm ntawm cov hlwb. RAW264.7 hlwb stimulated nrog LPS tuaj yeem ua rau lub zog ntawm cov lus teb inflammatory, thiab nws tuaj yeem ua kom zoo dua los ntawm Tan IIA, suav nrog mRNA qhia ntawm NLRP3, IL-1 , thiab TNF- .ATP yog lub teeb liab thib ob ntawm NLRP3 inflammasome activation. Hauv txoj kev tshawb no, ATP tau siv los txhawb kev ua kom NLRP3. Cov txiaj ntsig tau pom tias LPS ntxiv rau ATP-induced ua kom NLRP3 protein qhia thiab nws tuaj yeem ua kom zoo dua los ntawm Tan IIA. Hauv cov hlwb txhawb nrog LPS lossis LPS ntxiv rau ATP cov protein qhia ntawm IKBa, pp65 / p65 tau nce ntau ntxiv; thiab Tan IA kev kho mob (ntawm 10μM) txo qis kev txhim kho. Cov qib protein ntawm pp38 / p38 tau nce siab hauv cov hlwb txhawb nrog LPS ntxiv rau ATP, thiab kev tswj hwm tau raug txwv los ntawm Tan IA (10uM). Tsis tas li ntawd, cov lus qhia protein ntawm HMGB1 kuj tau txhim kho hauv cov hlwb uas raug cuam tshuam nrog LPS lossis LPS ntxiv rau ATP tab sis tsis pom qhov sib txawv. Cov txiaj ntsig no tau qhia tias LPS lossis LPS ntxiv rau ATP induced activation ntawm NF-kB thiab p38 txoj hauv kev tuaj yeem cuam tshuam los ntawm Tan IIA.
LPS ntxiv rau ATP kev kho mob tau nce qib ntawm intracellular ROS ntau heev, thiab Tan IIA inhibited LPS ntxiv rau ATP-induced nce hauv intracellular ROS ho. Acetylcysteine (N-acetyl-I-cysteine, NAC) yog ib qho khoom pov tseg ntawm ROS. NAC tuaj yeem cuam tshuam IPS ntxiv rau ATP-induced nce hauv NLRP3 protein ntau. Cov txiaj ntsig no tau qhia tias Tan IIA muaj peev xwm tiv thaiv kab mob antioxidant thiab tias kev tswj hwm ntawm Tan IIA ntawm NLRP3 yuav nyob ntawm txoj kev ROS-NLRP3.
Los xaus. Tan IIA tuaj yeem cuam tshuam cov lus teb inflammatory thiab NLRP3 qhia tawm los ntawm LPS lossis LPS ntxiv rau ATP, thiab NAC tuaj yeem cuam tshuam LPS ntxiv rau ATP-induced nce hauv qib NLRP3. Ntawm no, nws tuaj yeem kwv yees tias cov txheej txheem hauv qab ntawm Tan IIA tuaj yeem koom nrog kev tswj hwm ntawm ROS-NF-kB / P38-NLRP3 txoj hauv kev. Txoj kev tshawb no ntxiv tus cwj pwm ntawm cov txheej txheem molecular ntawm Tan IA thiab muab cov kev xav tshiab rau nws cov kev kho mob.
Cov ntaub ntawv
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