Macrophage Activation Los Ntawm Tsawg Molecular Luj Saccharides Los Ntawm Cistanche Deserticola
Mar 09, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
AB Abstract
Txhawm rau tshawb xyuas cov tshuaj tiv thaiv kab mob thiab cov txheej txheem ntawm qhov hnyav molecular saccharides los ntawmCistanche deserticola(LMSC) ntawm nas peritoneal macrophages, RAW264. 7 cev. RAW 264. 7 lub hlwb tau muab faib ua pawg tswj hwm ib txwm muaj, pawg tswj hwm zoo LPS, thiab LMSC pawg kho mob. RAW 264. 7 lub hlwb raug kho nrog ntau qhov sib txawv ntawm LMSC los ntawm 3. 91 txog 62. 5 g·L － 1. Kev kuaj liab nruab nrab tau ua haujlwm los kuaj xyuas cov haujlwm phagocytic ntawm macrophages. Tsis muaj kev tso tawm tau kuaj pom los ntawm kev siv NO kit, thiab macrophage activation txuam nrog cov protein qhia theem (TNF-, IL-6, IKK, p-IKK, IκB, p-IκB, NF-κB, thiab p-NF-κB) tau kuaj pom los ntawm Western blot. Cov txiaj ntsig tau pom tias LMSC muaj qhov ua kom muaj txiaj ntsig ntawm macrophages; nws tuaj yeem nce qhov tso tawm ntawm NO hauv RAW264. 7 hlwb thiab txhawb kev qhia ntawm cytokines TNF- thiab IL-6. Ntxiv mus, LMSC tau nce qhov phosphorylation ntawm IKK , IκB , Thiab NF-κB p65. Tsis tas li ntawd, mannitol yog ib qho ntawm cov khoom tseem ceeb hauv LMSC tau txhim kho phagocytic kev ua ntawm macrophages. Cov txiaj ntsig no tau pom tias LMSC tuaj yeem qhib cov macrophages los ntawm kev tswj hwm txoj hauv kev NF-κB, thiab mannitol yuav yog ib qho ntawm cov khoom tseem ceeb hauv LMSC.
［ Cov ntsiab lus ］ Tsawg molecular hnyav saccharides; Cistanche deserticola; kev tiv thaiv kab mob; macrophage; inflammatory yam; NF- IB
Cistanche deserticola YC Mayog ib tsob nroj teej tug mus rau lub genusCistanche deserticolaYC Ma. Nws cov tawv nqaij tuaj yeem siv los ua tshuaj. Nws muaj lub luag haujlwm ntawm nourishing raum yang, nourishing essence thiab ntshav, thiab moisturizing cov hnyuv. Nws yog lub npe hu ua "desert ginseng". Cov tshuaj Cheebtsam ntawm Cistanche muaj xws li phenylethanoid glycosides, iridoid glycosides, suab thaj, lignans, thiab ntau lwm yam. Cov kev tshawb fawb tshuaj niaj hnub no tau pom tias Cistanche muaj ntau yam kev ua ub no xws li neuroprotection, tiv thaiv qog, tiv thaiv kab mob, tiv thaiv kab mob, thiab antioxidant [1]. Cov kev tshawb fawb tau pom tias decoction ntawm Cistanche thiab polysaccharides tuaj yeem txhim kho lub peev xwm phagocytic thiab secretion ua haujlwm ntawm macrophages [2-3]. Txawm li cas los xij, tsis muaj ntawv tshaj tawm txog kev tshawb fawb txog kev ua haujlwm ntawm nws cov carbohydrates qis-molecular-yuag. Yog li, qhov kev tshawb fawb no tau siv cov kab ke hauv vitro kab lis kev cai ntawm RAW264. 7 nas peritoneal macrophages los tshawb txog cov haujlwm immunomodulatory thiab cov txheej txheem cuam tshuam ntawm Cistanche low-molecular-weight carbohydrates. Qhov kev tshawb fawb no tuaj yeem muab pov thawj kev sim ntau ntxiv rau kev nkag siab zooCistanche's immunomodulatory nyhuvnyob rau hauv lub neej yav tom ntej, thiaj li mus ntxiv qhia nws daim ntawv thov kev kho mob.
Cistanche deserticola muaj ntau yam teebmeem, nyem qhov no kom paub ntau ntxiv
1. Khoom siv
RAW 264. 7 lub hlwb tau yuav los ntawm Cell Center ntawm Suav Academy ntawm Kev Tshawb Fawb Txog Kev Kho Mob; fetal bovine serum (FBS) tau yuav los ntawm PAN Biotech, Lub teb chaws Yelemees; Cov tshuaj tua kab mob thiab DMEM nruab nrab tau yuav los ntawm Zhongke Machen Technology Co., Ltd. Lipopolysaccharide los ntawm Escherichia coli O55:B5 (LPS) tau yuav los ntawm Sigma Company, Tebchaws USA. Cov khoom siv Nitric oxide (NO) tau yuav los ntawm Nanjing Jiancheng Institute of Bioengineering. Cov khoom siv ECL chemiluminescence tau yuav los ntawm Thermo Fisher, Tebchaws Asmeskas; tshuaj tiv thaiv tau yuav los ntawm Cell Signaling Technology, USA; Cistanche cistanche tag nrho glycosides, cistanche qis-molecular-yuag suab thaj, thiab cistanche polysaccharides yog tus kheej tsim los ntawm peb pawg tshawb fawb. Qhov loj feem ntawm cistanche tag nrho glycosides yog 78.76 feem pua. Qhov loj feem ntawm cov suab thaj qis-molecular-yuag ntawmCistanche deserticolayog 58. 00 feem pua thiab cov feem loj ntawmCistanche deserticolapolysaccharides yog 65. 24 feem pua. Tanon-5200Multi gel imaging split system tau yuav los ntawm Shanghai Tianneng Technology Co., Ltd.; Sunrise-Basic microplate nyeem ntawv tau yuav los ntawm TECAN tuam txhab.
2. Txoj kev
2. 1 Neutral liab txoj kev ntsuas phagocytic kev ua ntawm macrophages
Noj cov nas peritoneal macrophages nyob rau hauv lub logarithmic kev loj hlob theem, tom qab trypsinization, suav cov hlwb, kho cov xov tooj ntawm tes mus rau 5 × 104 hlwb / mL, thiab inoculate lawv nyob rau hauv ib tug 96-zoo cell kab lis kev cai phaj, 100 μL/zoo , ntawm 37 degree Cultured nyob rau hauv 5 feem pua CO2 tej yam kev mob, tom qab lub hlwb ua raws li phab ntsa, muab pov tseg cov kab lis kev cai supernatant, ntxiv DMEM nruab nrab (10 feem pua FBS ntxiv rau 1 feem pua P /S), LPS (1 mg·L － 1) thiab Cistanche cistanche feem ntau glycosides,Cistanchetag nrho cov polysaccharides, thiab Cistanche cistanche qis-molecular-yuag suab thaj (3.91 ~ 62.5 g·L -1, raws li, lim nrog lub lim membrane ua ntej kev tswj hwm) tau coj mus kuaj. Tom qab culturing rau 48 teev, ntxiv 100 μL ntawm 0.075 feem pua ntawm nruab nrab liab physiological saline tov rau txhua lub qhov dej, txuas ntxiv culturing rau 4 teev, aspirate lub supernatant, ntxuav 3 zaug nrog PBS, ntxiv cell lysate (glacial acetic acid. -ethanol 1: 1) 100 μL, muab tso rau ntawm 4 degree rau 2 h, thiab ntsuas qhov nqus (A540 nm) tom qab cov hlwb raug lysed.
2. 2 Kev kuaj pom tsis muaj qhov tso tawm hauv cell supernatant
Inoculate RAW 264. 7 hlwb nyob rau hauv ib tug 48-zoo phaj (1 × 105 hlwb/mL, 500 μL/well), aspirate lub cell kab lis kev cai nruab nrab tom qab ib hmo incubation, ntxiv DMEM nruab nrab (10 feem pua FBS ntxiv rau 1 feem pua P/S), 500 μL txhua ntawm LPS (1 mg·L -1) thiab Cistanche cistanche tag nrho glycosides, tag nrho cov polysaccharides, thiab cov suab thaj qis molecular (3.91 ~ 62.5 g·L -1, feem). Tom qab 24 teev ntawm induction kab lis kev cai, lub supernatant tau sau. Txiav txim siab qhov concentration ntawm NO hauv cell supernatant ntawm txhua pab pawg nrog NO cov khoom siv, ua raws li cov lus qhia, thiab thaum kawg kuaj pom qhov nqus (A570 nm) nrog tus nyeem ntawv microplate
2. 3. Western blot
Inoculate RAW 264. 7 lub hlwb hauv 6-phaj zoo, nqus cov cell kab lis kev cai nruab nrab tom qab thaum hmo ntuj incubation, ntxiv DMEM nruab nrab (10 feem pua FBS ntxiv rau 1 feem pua P /S), LPS (1 mg·L － 1), thiabCistancheTag nrho cov Polysaccharides thiab cov suab thaj qis molecular (3.91 ~ 62.5 g·L -1) yog txhua 2 mL, thiab cov supernatant raug muab pov tseg tom qab 24 teev ntawm induction kab lis kev cai. Cov hlwb raug ntxuav ob zaug nrog PBS thiab muab pov tseg. Tom qab trypsinization, lub hlwb yog pipetted. Centrifuge, tshem tawm cov supernatant, ntxiv 100 μL NP40 Lysis Buffer, thiab lyse on dej khov rau 40 min. Tom qab centrifugation, lub supernatant raug coj mus, thiab cov protein concentration tau txiav txim siab los ntawm txoj kev BCA, thiab cov protein concentration ntawm txhua histone raug kho sib npaug. Siv tib cov qauv ntawm cov protein thiab cais lawv ntawm 12 feem pua SDS-PAGE nyob rau hauv lub siab tas li. Tom qab ntawd siv Bio-Rad hloov electrophoresis tank hloov cov membrane nrog qhov tam sim no ntawm 350 mA rau 1 teev, thiab hloov cov protein mus rau PVDF daim nyias nyias (pretreatment ntawm PVDF daim nyias nyias: soak hauv methanol thiab sib npaug nrog cov hloov tsis tau). Tom qab thaiv nrog 5 feem pua ntawm cov mis nyuj skimmed (configured nrog TBST muaj 0.1 feem pua Tween-20) rau 1 h ntawm chav tsev kub, daim nyias nyias tau ntxuav 3 zaug nrog TBST, thiab daim nyias nyias tau incubated nyob rau hauv cov protein thawj antibody diluent ntawm 4. degree thaum hmo ntuj. Cov daim nyias nyias tau ntxuav 3 zaug nrog TBST txhua hnub, thiab cov khoom sib txuas tau muab ntxiv rau incubate rau 1 teev. Cov daim nyias nyias tau ntxuav 3 zaug nrog TBST, thiab cov strips tau tsim nrog ECL chemiluminescence tus tsim tawm.
2. 4 cov ntaub ntawv tsom xam
Cov ntaub ntawv tau qhia raws li x ± s, thiab qhov tseem ceeb ntawm qhov sib txawv yog sim siv GraphPad Prism 6 statistical software rau ib txoj kev tsom xam ntawm qhov sib txawv. Coj P<0.01 as="" a="" very="" significant="" difference,="" and=""><0.05 as="" a="" significant="">
3. Cov txiaj ntsig
3. 1. Cov teebmeem ntawm Cistanche cistanche qis-molecular-hnyav qab zib, tag nrho glycosides, thiab tag nrho cov polysaccharides ntawm phagocytic kev ua si ntawm RAW264. 7 cev
Kev tshawb fawb ntawm cov tshuaj lom neeg ntawm Cistanche qhia tau hais tiasCistanchefeem ntau muajphenylethanoid glycosidesiridoid glycosides, thiab carbohydrates. Hauv qhov kev sim no, tag nrho glycosides ntawm cistanche (xws li phenethyl cawv glycosides thiab iridoid glycosides), cov suab thaj qis molecular, thiab cov suab thaj raug xaiv. Peb qhov chaw nquag ntawm tag nrho cov polysaccharides tau tshawb xyuas rau lawv cov teebmeem ntawm kev ua kom RAW264. 7 cev. Ob qho tib si Cistanche cistanche polysaccharide thiab cistanche qis-molecular-hnyav qab zib tuaj yeem ua rau phagocytic kev ua si ntawm RAW264. 7 hlwb, tab sis phagocytic kev ua ntawm tag nrho cov glycoside tswj pawg tsis hloov pauv loj. Tsis tas li ntawd, cov tshuaj tswj tau zoo LPS kuj tseem txhim kho phagocytic kev ua haujlwm ntawm RAW264. 7 hlwb, raws li qhia hauv daim duab 1. Cov txiaj ntsig saum toj no qhia tias cov suab thaj qis-molecular-yuag thiab polysaccharides ntawm Cistanche tuaj yeem ua rau muaj kev ua kom RAW264. 7 cev.
3. 2. Cov nyhuv ntawm Cistanche tag nrho glycosides, qis molecular qab zib, thiab tag nrho cov polysaccharides ntawm cov ntsiab lus ntawm nitric oxide (NO) hauv cell supernatant
Txhawm rau txhawm rau tshawb xyuas qhov ua kom muaj txiaj ntsig ntawm peb qhov chaw nquag ntawm Cistanche ntawm RAW264. 7 hlwb, cov ntsiab lus ntawm NO nyob rau hauv lub cell supernatants stimulated los ntawm cov tshuaj sib txawv tau txiav txim siab. Cov txiaj ntsig tau pom tias kev tso tawm NO hauv pawg LPS tau nce siab dua li hauv pawg tswj hwm dawb paug (P<0.01); the="" release="" of="" no="" in="" the="" 5="" doses="" of="" low="" molecular="" sugar="" and="" polysaccharides="" in="" the="" cistanche="" cistanche="" group="" was="" significantly="" higher="" than="" that="" in="" the="" control="" group="" (p=""><0.01) ),="" and="" was="" dose-dependent;="" the="" total="" glycosides="" of="">Cistanchetsis muaj qhov cuam tshuam loj. Cov txiaj ntsig saum toj no qhia tau hais tias ob qho tib si polysaccharides thiab cov suab thaj qis molecular-yuag ntawm Cistanche tuaj yeem txhawb kev tso tawm NO los ntawm RAW264. 7 cev. Ntawm lawv, cov nyhuv ntawm cov suab thaj qis-molecular-yuag ntawm Cistanche yog qhov tseem ceeb, raws li pom hauv daim duab 2.
3. 3. Cov nyhuv ntawm Cistanche cistanche qis-molecular-hnyav qab zib ntawm kev qhia ntawm TNF- thiab IL-6 protein nyob rau hauv RAW264. 7 cev
TNF- yog ib qho tseem ceeb inflammatory mediator uas tshwm nyob rau hauv cov txheej txheem ua kom RAW264. 7 hlwb, uas tuaj yeem txhawb kev sib txuas thiab tso tawm ntawm lwm cov cytokines (xws li IL-6). Cov txiaj ntsig tau pom tias qhov kev qhia ntawm TNF- thiab IL-6 tau nce ntxiv tom qab cov suab thaj qis-molecular-yuag ntawm Cistanche cistanche tau txhawb nqa, qhia tias cov suab thaj qis-molecular-yuag ntawm Cistanche tuaj yeem txhawb kev qhia ntawm TNF. - thiab IL-6 hauv RAW264. 7 hlwb, yog li ua kom lub cev tiv thaiv kab mob Phagocytosis, saib daim duab 3.
3. 4. Kev tswj cov nyhuv ntawm Cistanche cistanche qis-molecular-hnyav qab zib ntawm IKK / IκB / NF-κB kev taw qhia
Txhawm rau tshawb nrhiav lub peev xwm txheej txheem ntawm Cistanche cistanche qis-molecular-hnyav qab zib hauv kev ua kom RAW264. 7 lub hlwb, cov kws sau ntawv tau tshawb xyuas nws cov kev tswj hwm ntawm IKK / IκB / NF-κB classical signaling pathway. Cistanche cistanche qis-molecular-yuag qab zib tuaj yeem ua rau cov phosphorylation ntawm NF-κB thiab phosphorylation ntawm nws cov teeb liab tseem ceeb ntawm cov proteins IKK thiab IκB, thiab txo qhov kev qhia ntawm tag nrho cov protein IκB, qhia tias nws muaj kev ua kom muaj zog ntawm IKK / IB/NF-κB signaling pathway. Cov tshuaj tswj qhov zoo LPS kuj muaj cov kev cai zoo sib xws. Hauv cov ntsiab lus, cov suab thaj qis-molecular-yuag ntawm Cistanche tuaj yeem ua rau IKK / IκB / NF-κB teeb liab txoj hauv kev, yog li ua rau cov synthesis thiab tso tawm ntawm cytokines xws li TNF- thiab IL-6, thiab thaum kawg ua rau lub cev tsis muaj zog. Kev ua haujlwm ntawm RAW264. 7 hlwb, saib daim duab 4.
3. 5. Kev tshuaj xyuas cov khoom xyaw nquag nrog macrophage activating nyhuv hauv Cistanche qis-molecular-yuag qab zib
Peb lub chaw kuaj mob tau txheeb xyuas cov suab thaj qis molecular uas muaj nyob hauvCistancheThaum ntxov thiab pom tias nws tsuas yog muaj mannitol (16.53%), sucrose (8.34%), fructose (25.38%), thiab qabzib (7. 75%), thiab lwm yam khoom xyaw. Yog li tus kws sau ntawv siv txoj hauv kev nruab nrab liab los tshuaj xyuas qhov ua kom muaj txiaj ntsig ntawm cov khoom saum toj no ntawm macrophages. Mannitol tuaj yeem ua kom cov phagocytic kev ua si ntawm macrophages nyob rau hauv ib tug npaum li cas ntawm cov tshuaj, thaum cov teebmeem ntawm lwm yam Cheebtsam tsis tseem ceeb. Qhov no qhia tias mannitol yuav yog ib qho ntawm cov khoom tseem ceeb hauv cov suab thaj qis-molecular-yuag ntawm Cistanche cistanche kom qhib cov macrophages, raws li qhia hauv daim duab 5.
4. Kev sib tham
Cistanche cistanche, tsob nroj kab mob ntawm tsev neeg Lydanaceae, tau tshaj tawm tias muaj cov tshuaj tiv thaiv kab mob, yog li nws yuav tsum muaj cov tshuaj tiv thaiv kab mob. Hauv qhov kev sim no, tus kws sau ntawv tau tshuaj xyuas cov haujlwm phagocytic ntawm nas peritoneal macrophages ntawm ntau qhov chaw huv ntawmCistanchethiab pom tias cov suab thaj qis-molecular-yuag thiab polysaccharides ntawm Cistanche muaj cov nyhuv ntawm macrophage activation, tab sis tag nrho glycosides ntawm Cistanche tsis pom tseeb. Nyob rau tib lub sijhawm, tus kws sau ntawv pom tias cov suab thaj qis-molecular-yuag tuaj yeem qhib cov macrophages ntawm qis dua, yog li cov kws sau ntawv xaivCistancheqis-molecular-hnyav qab zib raws li kev tsom mus rau kev tshawb fawb tom ntej.
Los ntawm kev tshuaj xyuas cov txiaj ntsig ntawm qhov hnyav molecular yuag cistanche qab zib ntawm kev qhia ntawm cytokines TNF- thiab IL-6, cov kws sau ntawv pom tias qhov hnyav molecular qis cistanche qab zib tau nce qhov kev qhia ntawm TNF- thiab IL-6, uas ua pov thawj ntxiv tias qhov hnyav molecular qis cistanche qab zib muaj qhov cuam tshuam rau cov nas. Peritoneal macrophages muaj cov nyhuv activating. Yog li ntawd, tus kws sau ntawv ntxiv tshawb nrhiav nws lub peev xwm ntawm kev ua haujlwm. Muaj kev tshawb fawb
Nws tau pom tias cov kab mob lipopolysaccharide (LPS) tuaj yeem ua rau cov nas macrophages thiab nas glial hlwb los ntawm txoj kev teeb liab xws li JAK2/STAT3, NF-κB, p38/ERK, thiab MAPK [4-8]. Txoj kev NF-κB tau lees paub tias yog txoj hauv kev classic, yog li cov teeb liab proteins ntawm txoj kev no tau raug sim hauv qhov kev sim no. Cov txiaj ntsig tau pom tias cov suab thaj qis-molecular-yuag ntawm Cistanche tuaj yeem nce phosphorylation ntawm NF-κB thiab phosphorylation ntawm cov ntsiab lus tseem ceeb tshaj tawm cov proteins IKK thiab IκB, qhia tias cov suab thaj qis molecular-yuag ntawmCistanchetej zaum yuav qhib nas peritoneal giants los ntawm IKK / IκB / NF-κB signaling pathway. Phage hlwb tso cov inflammatory cytokines thiab tom qab ntawd ua lub luag haujlwm hauv kev ua kom lub cev tiv thaiv kab mob.
Nyob rau hauv thaum ntxov theem ntawm lub chaw soj nstuam, cov tshuaj muaj pes tsawg leeg ntawm cov uas tsis muaj-molecular-hnyav qab zib extract ntawmCistanche deserticolatau soj ntsuam systematically. Nws tau pom tias cov suab thaj qis-molecular-yuag feem ntau muaj cov monomers xws li mannitol, sucrose, fructose, thiab qabzib. Kev tshawb fawb ntxiv pom tias mannitol tuaj yeem txhim kho phagocytic kev ua haujlwm ntawm macrophages. Yog li, nws tau kwv yees tias mannitol yuav yog ib feem tseem ceeb ntawm cov suab thaj qis-molecular-yuag uas ua rau lub cev tiv thaiv kab mob.
Hauv cov ntsiab lus, txoj kev tshawb no tau siv cov macrophage hauv Vitro kab lis kev cai thiab pom tias cov suab thaj qis-molecular-yuag.Cistanchemuaj qhov ua kom muaj txiaj ntsig tseem ceeb ntawm macrophages, thiab tuaj yeem qhib nas peritoneal macrophages los ntawm NF-κB txoj kev taw qhia, thiab tom qab ntawd ua lub luag haujlwm. Mannitol hauv cov suab thaj qis-molecular-yuag tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev ua kom phagocytic kev ua haujlwm ntawm macrophages.
Cov ntaub ntawv
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