Kev Tiv Thaiv Kev Tiv Thaiv Kab Mob-Associated Raum Kev Puas Tsuaj Tom Qab Hloov Siv Tus Tshiab PrC-210 Dawb radical scavenger hauv nas
Mar 18, 2022
Torsten R. Goesch 1, Nancy A. Wilson2, Weifeng Zeng2, Bret M. Verhoven2, Weixong Zhong2, Maya M. Coumbe Gitter3thiab William E. Fahl1,3,*
Abstract:
Allograftraumtransplantation, uas ua rau tus tswv tsev cellular- thiab antibody-mediated rejection ntawm lubraum, yog ib qho tseem ceeb pab rau lub raum puas thaum lub sij hawm hloov. Ntawm no, peb nug seb PRC -210 puas yuav cuam tshuam kev puas tsuaj pom nyob rau hauv allograft raum hloov pauv. Brown Norway (BN) nas ob lub raum tau perfused nyob rau hauv situ (UW Solution) nrog los yog tsis ntxiv 30 mM PrC-210 thiab ces tam sim ntawd transplanted rau hauv Lewis (LEW) nas. 20 teev tom qab ntawd, hloov BN ob lub raum thiab LEWrat plasma tau txheeb xyuas.Lub raumhistology, thiab lub raum / ntshav qib ntawm ob peb qhov mob ua rau muaj feem cuam tshuam nrog cytokines, tau ntsuas los ntsuas qhov tsis sib xws ntawm lub raum pathology thiab PrC-210 tiv thaiv kev ua tau zoo. Nees nkaum teev tom qab hloov pauv ntawm allograft: (i) tseem ceeb histologic raum tubule puas thiab mononuclear inflammatory cell infiltration tau pom nyob rau hauv allograft ob lub raum; (ii)raumKev ntsuas kev ua haujlwm (creatinine thiab BUN) tau nce siab; (iii) Cov kev hloov pauv tseem ceeb hauv cov cytokines tseem ceeb, piv txwv li, TIMP-1, TNF-alpha thiab MIP-3A/CCL20, thiab lub raum qhib cov qib caspase tau pom. Hauv PRC-210- kho lub raum thiab cov nas tau txais, (i) raum kev puas tsuaj rau lub raum (Banff Scores) thiab mononuclear infiltration tau txo qis rau qhov tsis tau kho tom qab theem; (ii) creatinine thiab BUN tau txo qis heev; thiab (iii) activated caspase thiab cytokine kev hloov pauv tau txo qis, qee qhov rau keeb kwm yav dhau. Hauv kev xaus, cov txiaj ntsig tau qhia tias PrC-210 tuaj yeem muab kev tiv thaiv lub cev dav dav rau ntau yam kev hloov pauv hloov pauv hloov pauv; Nws tuaj yeem tiv thaiv lub raum hloov pauv thaum lub sijhawm thiab tom qab txhua theem ntawm txoj kev hloov pauv - los ntawm kev pub khoom nruab nrog cev, los ntawm kev thauj mus los, rov cog qoob loo, thiab tom qab kev phais mob - kom txo qis qhov mob thiab mob ntev.
Keywords: raumallograft; lub raum tsis kam; ischemia
Hu rau:joanna.jia@wecistanche.com
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1. Taw qhia
Lub raum tsis ua haujlwm kawg ua rau ntau dua 1.2 lab tus neeg tuag txhua xyoo thoob ntiaj teb [1].Lub raumKev hloov pauv yog qhov kev kho mob zoo tshaj plaws rau cov neeg mob uas muaj kab mob raum kawg. Tshaj 90,000 hloov raum tau ua txhua xyoo thoob ntiaj teb.
Txoj kev hloov, nws tus kheej, induces tseem ceeb cellular thiab lub cev raug mob rau lubraum, uas txo cov kev muaj sia nyob ntev ntawm lub cev. Peb qhov kev thuam tseem ceeb rau lub raum thaum hloov pauv hloov pauv yog (i) cov pa oxygen thiab nitrogen reactive (ROS thiab RNS)-vim kev puas tsuaj thaum lub caij txias ischemia ('txias-cia) [2], (ii) ROS-vim kev puas tsuaj thaum cog ('re-perfusion raug mob') [3], thiab (iii) post-allograft-transplant inflammation, uas ua rau lub cev tsis muaj zog tiv thaiv kab mob thiab cov tshuaj tiv thaiv kab mob sib kho (ABMR) [4].
Neutrophils thiab macrophages tsiv mus rau hauv qhov kev hloov pauv tsis zoo nyob rau hauv 6 h ntawm reperfusion thiab txhawb cov chemokine synthesis hauv cov hlwb dendritic uas tom qab ntawd qhib T lymphocytes thiab nrhiav cov kab mob hloov pauv. Thaum cov kab mob tiv thaiv kab mob no nkag mus rau cov kab mob sib kis ntawm cov kab mob epithelial, lawv tsim cov myeloperoxidase hauv neutrophils thiab nicotinamide adenine dinucleotide phosphate (NADPH) oxidase hauv macrophages, ob qho tib si uas ua rau cov dawb radicals hauv zos. Cov txheej txheem inflammatory no ua rau muaj kev ua kom muaj txoj hauv kev ntxiv thiab ntxiv kev hloov kho ntawm tes thiab lysis hauv lub cev.raumallograft [5] .ABMR tuaj yeem tshwm sim los ntawm ob qho tib si, lossis ob qho tib si, preformed alloantibody tiv thaiv kev graft los yog los ntawm de novo txoj kev loj hlob ntawm tus neeg pub khoom tshwj xeeb antibody (dnDSA) [5–7]. Tus mob (min / hnub), hloov mus rau ntev (hnub / lub lis piam), cov lus teb o nyob rau hauv lub raum allograft, nrog tas li tsim ROS thiab inflammatory cytokines, muaj peev xwm tsim ib tug hnyav, nws tus kheej perpetuating teb uas ua rau.raumlub cev tsis ua haujlwm.
Yuav kom nkag siab zoo dua cov cellular thiab molecular txoj kev koom nrog hauv pathogenesis ntawmraumallograft o thiab tsis lees paub, peb tau tsim thiab ua tus qauv nas uas rov ua dua feem ntau ntawm cov txheej txheem kho mob ntawm lub cev tiv thaiv kab mob, ABMR, thiab lub raum poob [8]. Cov qauv no tau siv los ntsuas ntau qhov kev hloov pauv tshiab tom qab-allograft cov tswv yim.
Ob qho tam sim no tau lees paub txoj hauv kev los txo qhov mob hnyav thiab lub sijhawm ntev tiv thaiv kab mob tiv thaivraumallograft yog: (i) txhawm rau txhawm rau txhawm rau txhawm rau nrhiav tus neeg sib tw sib tw, thiab (ii) tshem tawm cov tshuaj tiv thaiv preexisting tawm tsamraumallograft siv desensitization raws tu qauv [9,10].
Hauv kev ua haujlwm tau piav qhia hauv phau ntawv no, peb tau nug seb qhov thib peb txoj hauv kev txhawm rau txhawm rau txhawm rau mob hnyav thiab mob ntev ntev yuav muaj txiaj ntsig. Peb tau tswj xyuas qhov ua haujlwm tam sim ntawd, dawb radical scavenger, PRC-210, ob qho tib si rau cov cog qoob looraumthiab rau tus neeg txais nas, txhawm rau txiav txim siab seb puas muaj kev puas tsuaj rau ROS puas tuaj yeem raug tshem tawm. Txawm hais tias lub tswv yim ntawm kev tiv thaiv kab mob ua rau muaj kev cuam tshuam nrog ROSinraumKev hloov pauv tsis yog tshiab, kev siv ntawm no ntawm tus tshiab, tam sim ntawd-ua PRC -210 ROSscavenger yog. Ob qho tib si tam sim ntawd thiab tsis ntev los no thiab tsis ua haujlwm ntawm cov kab mob ua paug, thiab tsim tawm, cov dawb radicals nyob rau hauv cov hloov tshiab allograft.raumwould significantly enhance the existing strategies to suppress allograft rejection and would provide another pathway to reduce post-transplant kidney cell damage, and with it, suppress Delayed Graft Function to improve survival of the kidney allograft.PRC-210 is a new small-molecule, aminothiol, free radical scavenger [11]; it has no measurable nausea/emesis nor hypotension side effects [12]. Unlike traditional antioxidants that act indirectly over hours to days via NrF-2 to activate the expression of protective genes [13], PRC-210 directly scavenges ROS to confer 100% protection in seconds [11]. PRC- 210 was the most potent of the 13 commonly studied "antioxidants" screened in an assay that scored the ability of molecules to prevent x-ray-induced damage to naked DNA; the majority of the tested "antioxidants" showed no protection [14,15]. In a related essay, the addition of PrC-210 30 s before a 60 s pulse of •OH to naked DNA provided complete protection against the •OH insult that induced >95 feem pua kev puas tsuaj DNA hauv kev tswj tsis tau kev tiv thaiv [16]. Hauv ob qhov kev tshawb fawb ntawm ob lub raum hloov pauv yav dhau los [16,17], PRC-210 tau pom tias txhawm rau txhawm rau ROS-induced raum puas tsuaj tshwm sim thaum (i) 30 h txias cia [17] thiab (ii) reperfusion raug mob thaum cog [16 ] mus rau theem tom qab, yog li tshem tawm ob qhov chaw tseem ceeb ntawm kev raug mob rau lub raum hloov pauv. PRC-210 molecule kuj tau pom tias yuav txwv tsis pub dawb radical-induced raug mob hauv ob peb lwm yam khoom nruab nrog cev [15,18]. Yog li, peb xav tias PrC-210 kuj tseem tuaj yeem tiv thaiv ib qho allograft tawm tsam oxidative kev nyuaj siab uas yog tsim los ntawm BOTH (i) cellular- thiab (ii) antibody-mediated rejection txheej txheem uas tsim cov dawb radicals raws li ib tug byproduct.
Txhawm rau tshawb nrhiav qhov kev xav no, peb tau tsim tus qauv nas tshiab uas zam qhov kev cuam tshuam ntawm cov xwm txheej tseem ceeb ntawm ischemic thiab reperfusion thiab tswj hwm PrC-210 ob qho tib si ua ntej thiab tom qab cog. Brown nas ob lub raum tau yaug nrog UW cov tshuaj uas muaj PrC-210 thiab tam sim ntawd hloov mus rau hauv syngeneic Lewis nas tau txais. Lub sijhawm txias ischemic tau raug tshem tawm. Tam sim ntawd cov cog hauv qab no, thiab rau 8 teev tom qabraumimplants, cov neeg tau txais nas tau txais kev txhaj tshuaj PrC -210 ntawm cov tshuaj uas yuav pab kom tsis tu ncua dawb radical-scavenging nyob rau hauv lub transplantedraum. Hloov lub raum thiab cov ntshav ntshav tau sau tseg 20 teev tom qab hloov pauv kom muaj kev ntsuas ntawm PrC-210-conferred (i) suppressing inflammatory byproducts thiab (ii)raumkev tiv thaiv.
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2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Tsiaj txhu
Cov neeg laus (200–250 g) txiv neej Lewis thiab BN nas tau yuav los ntawm Envigo (Indian-napolis, IN, USA) thiab nyob hauv lub tsev tu tsiaj hauv University of Wisconsin hauv Madison, WI, USA. Txhua tus txheej txheem tau ua raws li Txoj Cai Saib Xyuas Tsiaj thiab Siv Tsiaj ntawm University of Wisconsin. Kev saib xyuas tsiaj txhu, suav nrog tsiaj tuag, chav tsev kub, 12 teev lub teeb / tsaus ntuj, thiab kev tu lub tawb, ntawm lwm cov dej huv, tau ua txhua hnub los ntawm cov neeg saib xyuas tsiaj. Khoom noj thiab dej muaj nyob rau hauv libitum. Qhov kev tshawb fawb no tau txais kev pom zoo los ntawm Lub Tsev Kawm Ntawv Tshuaj thiab Pej Xeem Kev Noj Qab Haus Huv Institutional Animal Care and Use Committee ntawm University of Wisconsin (Tsib Cai Lij Choj #M005204). Txhua pawg muaj 4-6 tsiaj.
2.2. Khoom siv
Synthesis ntawm PrC-210 HCl aminothiol, ib tug preclinical molecule, tau piav qhia nyias [19,20]. PrC-210 HCl crystals (3-(methylamino)-2-(methyl monomethyl)propane-1- thiol) yog khaws cia nyob rau hauv ib qho chaw nitrogen ntawm −20 ◦C, thiab txawm tias niaj hnub thawing, siv, thiab rov khaws cia, crystalline PrC -210 yog ruaj khov rau ntau tshaj 4 xyoo los ntawm kev tsom xam spectrometry. Lwm cov tshuaj reagents tau txais los ntawm Sigma Aldrich (St. Louis, MO, USA). UW Organ Preservation Solution tau yuav los ntawm Choj rau Lub Neej, Columbia, SC, USA.
2.3. Kev phais thiab kev sim txheej txheem
Cov txheej txheem hloov pauv siv nyob rau hauv cov kev sim no yog pom hauv daim duab 1. Hauv BN pub nas, tom qab ob npaug ntawm lub aorta, ligation ntawm txoj cai raum cov hlab ntsha thiab cov hlab ntsha, thiab ntu phais ntawm lub raum sab laug, sab laug nas.raumtau perfused nyob rau hauv situ siv 5 mL ntawm chav tsev kub UW tov (tshaj li 15 s lub sij hawm). Lub perfusate yog UW Solution ib leeg (rau "0 h" thiab "20 h Tsis Kho" pab pawg), lossis UW Solution uas crystalline PrC-210, kom ua tiav 30 mM [17], tau ntxiv, yaj tam sim ntawd, thiab tom qab ntawd pH hloov mus rau qhov pib UW Solution pH ntawm 7.4 los ntawm kev ntxiv 0.0619 µL 5N NaOH ib µmol ntawm PrC-210 HCL ntsev (FW: 220). Lub neej ib nrab ntawm PrC-210 thiol (daim ntawv nquag) yog kwv yees li 3.5 teev nyob rau hauv physiologic pH daws xws li UW Solution thiab tib neeg cov ntshav [14]. Tom qab nyob rau hauv situ perfusion, sab laug BNraumraug phais tshem tawm thiab tom qab ntawd sutured los ntawm blunt anastomosis ntawm cov hlab ntsha thiab ureter mus rau hauv lub vacated raum qhov chaw ntawm LEW tus neeg txais nas. Lawdraumtau ligated thiab tshem tawm tam sim ua ntej. Tsib feeb tom qab kev phais kaw ntawm LEW nas, tus nas tau txais PrC {{0}} koob tshuaj (121 ug PrC-210 HCl ib gm lub cev hnyav, uas sib npaug 0.24 X Maximum Tolerated Dose) los ntawm kev txhaj tshuaj intraperitoneal. Raws li pom nyob rau hauv daim duab 1 schematic, tus nas kuj tau txais kev txhaj tshuaj intraperitoneal ntawm PrC-210 (0.24 MTD) ntawm ntxiv 4 h thiab ntxiv rau 8 h tom qab hloov pauv. Cov nas tau euthanized ntawm ntxiv rau 20 teev tom qab hloov pauv, thiablub raumthiab cov qauv plasma tau sau rau kev tshuaj xyuas. Muaj tsawg kawg ntawm tsib nas nyob rau hauv txhua pab pawg kho mob.
2.4. Serum BUN thiab creatinine ntsuas
BUN thiab creatinine tau ntsuas hauv cov qauv kuaj ntshav siv Catalyst One AnalyzerTechnology (IDEXX Laboratories, Westbrook, ME, USA).
2.5. Enzyme-Linked Immunosorbent Assays
Kev soj ntsuam tau ua raws li tau piav qhia hauv ELISA cov khoom siv raws tu qauv (Rat TIMP-1, Cat# RTM- 100; Rat MIP3-A, Cat# DY540; Rat TNF-alpha, Cat# RTA{ {5}}; R&D Systems, Minneapolis, MN, USA). Piv txwv li, dilutions ntawm nas plasma tau ntxiv rau pre-coated daim hlau, incubated rau 2 h ntawm 37 C. biotin-conjugated antibody tshwj xeeb rau cov assayed protein yog ntxiv thiab incubated rau 1 h ntawm 37 ℃, ntxuav, avidin-conjugated. horseradish peroxidase tau ntxiv, ua raws li kev ntxuav thiab TMB substrate ntxiv. Cov tshuaj tiv thaiv yog incubated rau 10-30 min, nres nrog sulfuric acid, thiab nyeem ntawm 450 nm.
2.6. Proteome Profiller Rat Cytokine Array
Kev ntsuam xyuas tau ua qhov tseem ceeb raws li tau piav qhia hauv cov khoom raws tu qauv. Luv luv, plasma tau incubated nrog nitrocellulose membranes pom nrog ntes thiab tswj cov tshuaj tiv thaiv. Tom qab incubation, daim nyias nyias tau ntxuav thiab incubated nrog streptavidin HRP. Hauv kev sib txawv ntawm cov txheej txheem, peb tau siv SuperSignal West Femto (ThermoFisher, Madison, WI, USA; Cat# 34094) rau kev tshawb nrhiav chemiluminescent, vim nws muab lub teeb liab muaj zog. Blots tau pom ntawm FotoDyne gel doc system.
2.7. Histology
Formalin-fixed (10 feem pua formalin), paraffin-embedded,lub raumtau txiav rau hauv 5 um seem. Cov swb tau deparaffinized, rehydrated los ntawm xylene los ntawm qib ethanol series rau dej, thiab tom qab ntawd kho raws li tau piav qhia hauv qab no. Slides raug tshuaj xyuas siv lub hom phiaj 20- hauv Aperio Digital Pathology Slide Scanner. Txhua H&E slides raug tshuaj xyuas los ntawm Dr. Weixiong Zhong, MD, PhD, hloov cov kab mob, thiab tau qhab nia rau PTC, glomerulitis (g), vasculitis (v) / intimal arteritis, interstitial inflammatory (I), thiab C4d staining, raws li Banff 2009 [21].
Cais, cov swb tau muab tus lej dig muag, thiab cov duab digital tsis sib tshooj ntawm lub raum tubules raug coj los ntawm qhov sib cuam tshuam ntawm medulla thiab cortex los ntawm txhua H / E swb. Kev saib xyuas kom tsis txhob suav nrog cov hlab ntsha loj lumens thiab glomeruli. Automated quantification ntawm liab thiab xiav pixels hauv txhua 10-raumduab tau ua tiav siv cov kev cai macro sau hauv ImageJ software (https://imagej.nih.gov/ij/index.html
nkag mus rau 13 Plaub Hlis 2021). Liab pixels reflected proximal tubular thickness nrog rau txhuam ciam teb. Nuclei tau txheeb xyuas nyob rau hauv xiav channel. Qhov piv ntawm xiav nuclear pixels rau cov tubules liab tau muab cov qhab nia Inflammatory Infiltration rau cov qe ntshav dawb infiltration tom qab hloov pauv.lub raum. Cov qhab nias tau nruab nrab thiab npaj siv Graphpad Prism.
2.8. Activated Caspase Enzyme Kev Ua Haujlwm
Activated caspase 3 thiab 7 kev ua hauvraumhomogenate supernates tau txiav txim siab siv Apo-ONE fluorescent substrate (Promega, Madison, WI, USA) [16]. Luv luv, thawedlub raumtau tov nrog 8-fold lysis buffer uas muaj 50 mM Na HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 10 feem pua glycerol thiab homogenized ntawm 4 OC rau 30 s nrog Omni cov ntaub so ntswg homogenizer . Lub raum homogenate tau centrifuged ntawm 4OC (16,{12}} g) hauv Eppendorf microfuge rau 20 min. Cov supernates tau soj ntsuam tam sim ntawd rau kev ua haujlwm caspase, thiab cov ntsiab lus protein los ntawm Bradford txoj kev siv bovine serum albumin raws li tus qauv. Kev ntsuam xyuas caspase tau ua raws li hauv qab no: 5 uL supernate (~ 40 ug ntawm supernate protein) tau diluted mus rau tag nrho cov ntim ntawm 50 uL nrog rau saum toj no lysis tsis, yog tov nrog 50 uL ntawm undiluted Apo-ONE substrate nyob rau hauv lub qhov dej. ntawm ib tug dub, opaque, 96 zoo phaj pib lub 60 min cov tshuaj tiv thaiv. Cov phaj tau shaken ntawm 200 RPM ntawm 37 ℃ rau 60 min. Lub DEVD caspase substrate peptide cleavage tau ntsuas los ntawm BMG Clariostar fluorescent phaj nyeem ntawv ntawm qhov excitation wavelength ntawm 499 nm thiab emission wavelength ntawm 521 nm. Ib tus qauv caspase tau suav nrog hauv txhua qhov kev sim.
2.9. Rat raum Mitochondria
Cov purified mitochondrial feem yog npaj los ntawm homogenized naslub raumlos ntawm tus txheej txheem centrifugation [22] Cov purified mitochondria raug tshem tawm hauv 0.15 M Tris HCl tsis, pH 7.4.
Txhawm rau txiav txim siab seb qhov sib ntxiv ntawm exogenous PrC {{0}}} suppresses ROS-induced fragmentation ntawm mitochondrial DNA [22], hauv 25 uL cov tshuaj tiv thaiv ntim (hauv PCR raj), peb ntxiv: 1{{29} } uL purified mitochondria, 5 uL PrC-210 dilution los yog dej (PrC-210 tau ntxiv 10 min ua ntej Fe plus plus ADP plus H2O2 ●OH generator), thiab 10 uL uas muaj FeCl2 (2.5 mM; FW: 127), adenosine 5′ -diphosphate sodium ntsev (10 mM; FW: 427) thiab H2O2 (0.003 feem pua siab kawg). Tom qab 20 min ntawm 37 О C, 10 uL ntawm cov tshuaj tiv thaiv yog tov nrog 5 uL ntawm 6- gel loading dye uas muaj 0.3 feem pua SDS; cov hlab zaum hauv 60 ℃ dej rau 1 min, 10 ul tau muab tso rau hauv qhov dej ntawm 1 feem pua agarose TAE gel, thiab tom qab 60 feeb ntawm 60 volts, gels tau stained thiab yees duab. Yam tsawg kawg ntawm peb qhov kev hloov pauv tau ua tiav rau txhua qhov kev ntsuam xyuas kom muaj kev sib piv cov lej.
2.10. Kev txheeb cais
Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ntxiv / ib STDs. Cov tub ntxhais kawm qhov kev xeem t-test tau siv los txiav txim qhov sib txawv ntawm qhov sib txawv thiab p tus nqi siv GraphPad Prism 7.03 software. p-tus nqi tsawg dua 0.05 tau suav tias yog qhov tseem ceeb.
3. Cov txiaj ntsig
3.1. PrC-210 Kev Tiv Thaiv Raum Allograft Pathology
Thaum 20 teev tom qab hloov pauv, histology ntawm transplanted BNlub raum(Daim duab 2A–D) kho nrog UW Solution ib leeg kom pom tseeb tau nce Banff cov qhab nia rau tubulitis thiab peritubular capillaritis (cov xub liab thiab daj); cov qhab nia ntawm pathology tau qhia hauv vaj huam sib luag E. BNlub raumperfused nrog PrC{{0}}} muaj UW Solution thiab tau txais cov tshuaj tom qab cog qoob loo intraperitoneal systemic koob tshuaj PrC-210 (Daim duab 2D) pom meej meej suppressed inflammatory pathology rau lub raum thiab suppressed Banff cov qhab nia rau tubulitis thiab peritubular. capillaritis sib npaug rau Banff cov qhab nia ntawm "0 h" tswj BN pawg (Daim duab 2E). Kev tswj hwm ntawm PrC-210 tau txiav txim siab txo qis hauv ob qho tib si creatine (p=0.032) thiab BUN (p=0.046) raum cov cim kev puas tsuaj.
3.2. Activated Caspase Levels hauv Post-Transplant Kidneys
Qib ntawm activated caspase hauv BNraumhomogenates tau txo qis hauv BN allograftlub raumuas tsis raug rau PrC{{0}} kev kho mob thaum 20 teev tom qab hloov pauv (Daim duab 5). Perfusion ntawm BN ob lub raum nrog PrC-210-muaj UW Solution thiab hloov mus rau hauv Lewis nas uas tau txais cov kab ke PrC-210 ua rau tib lub sijhawm ua haujlwm caspase rau qhov pom hauv "0 h" tswj lub raum.
3.3. Kab mob Cytokine theem tom qab BN raum Allograft
Hauv kev sim tshuaj ntsuam,raumhomogenate supernates los ntawm 0 h tswj thiab 20 h Tsis Kholub raumtau kuaj nrog Proteome Profiller 29 cytokine array txhawm rau txheeb xyuas qhov hloov pauv, ua rau mob sib kis, cytokine thiab chemokine qhia theem 20 h tom qab hloov pauv. Raws li pom nyob rau hauv ob daim duab 6microarray insets, kev hloov pauv tau pom hauv TIMP-1, TNF-alpha, thiab MIP-3a/CCL20. Tus kheej ELISA daim hlau tau siv los ntsuas cov kev hloov pauv hauv lub raum homogenates thiab sera, tam sim no suav nrog cov nas kho nrog PrC-210 ib yam. Ob leeg TIMP-1 thiab TNF-alpha qib tau nce 20 teev tom qab hloov pauv, thiab hauv ob qho tib si, lawv qib tau txo qis thaum muaj PrC-210 (Daim duab 6A–C). MIP-3a/CCL20 qib tau nce ntawm 20 teev, tab sis lawv tau nce ntau dua hauv PrC-210-kho nas (Daim duab 6D).
3.4. PrC-210 Tiv thaiv nas raum Mitochondria
Sustained mitochondrial muaj nuj nqi thaum lub sij hawm thiab tom qabraumKev hloov pauv yuav tsum tau ua kom muaj sia nyob ntawm lub cev hloov pauv. ROS tsim tawm thaum lub sij hawm hloov pauv-koom nrog ischemia, ischemia-reperfusion, thiab post-implant inflammation tuaj yeem cuam tshuam rau lub raum mitochondrial kev ua haujlwm thiab kev ciaj sia. Vim yog lub luag haujlwm tseem ceeb ntawm mitochondrial, peb cais mitochondria los ntawm nas raum thiab txiav txim siab seb PrC -210 ntawm cov tshuaj muaj txiaj ntsig tau tuaj yeem tiv thaiv cov kab mob no los ntawm kev thuam ROS.
Hauv daim duab 7, purified nasraummitochondria tau incubated nrog ib tug ●OH generator [23]. Tom qab cov tshuaj tiv thaiv luv luv, peb pom qhov tseem ceeb ROS fragmentation ntawm nas raum mitochondrial DNA. Tom qab ROS kev thuam, ib qho aliquot ntawm mitochondria tau solubilized hauv SDS-muaj gel-loading tsis, thiab mitochondrial DNA tau sib cais thiab "loj" siv agarose gel chromatography (Daim duab 7). Kev thuam ROS kom meej meej txo qhov loj me me ntawm nas raum mitochondrial DNA (txoj kab b), thiab ntxiv ntawm PrC-210 rau mitochondria tiv thaiv DNA tawg (kab kab c–g) hauv PrC-210 concentration-dependent yam.
maca ginseng cistanche
4. Kev sib tham
Allograftraumtransplantation, uas ua rau lub hauv paus ntawm lub cellular- thiab antibody-mediated rejection ntawm lub raum, yog ib tug tseem ceeb contributor rau luv luv thiab ntev.raumkev puas tsuaj thaum lub sij hawm hloov pauv, thiab qhov cuam tshuam ncua sij hawm Graft Function tau pom txog li 50 feem pua ntawm cov raum hloov pauv. Peb tau lees paub qhov kev tshawb fawb no los txiav txim siab seb PrC-210 puas yuav muaj txiaj ntsig zoo hauv kev tiv thaiv qhov hnyav ntawm kev puas tsuaj tshwm sim tom qab lub raum hloov pauv hauv cov qauv nas uas tshem tawm ntau lub sijhawm hloov pauv ischemic thiab nws cuam tshuam oxidative kev nyuaj siab. Peb qhov kev xav tau yog tias txoj hauv kev no yuav tsum tso cai rau peb pom qhov cuam tshuam ntawm PrC-210 ntawm kev hloov pauv tom qab hloov pauv hauv kev thuam nrog kev cuam tshuam tsawg kawg ntawm ischemia.
Qhov nce hauv TNF-alpha thiab ntau ntau mononuclear infiltration qhia tau hais tias allograft raum hloov pauv hauv lub raum ua rau muaj kev mob tshwm sim hauv 20 teev tom qab hloov pauv, thiab qhov no tau cuam tshuam nrog kev puas tsuaj ntawm lub raum tubular hlwb pom hauv lub cev.raumhistology (Banff Scores). TNF-alpha feem ntau yog tsim los ntawm cov macrophages activated thiab yog cell-signaling protein koom nrog rau mob mob. Nws yog ze ze rau lub pathogenesis ntawm mob thiab mob ntev allograft rejection [24].
Nyob rau hauv sib piv rau cov saum toj no nrhiav nyob rau hauv uas tsis yog-kho BNlub raum, PrC-210 muab los ua ib feem ntawm UW Solution thiab muab kev tswj hwm hauv cov nas tom qab hloov pauv tau txo qis TNF-alpha qib thiab lub raum infiltration los ntawm cov hlwb mononuclear, uas yog ob qho tib si qhia txog kev txo qis mob. PrC-210 txo qis lub raum puas raws li pom hauv cov qhab nia ntawm lub raum raug mob (Banff Cov qhab nia) rau qib tom qab tsis kho thiab txo qis ntawm ob lub raum pathology cov qhab nia ua haujlwm, creatinine, thiab BUN.
Kev mob hauv lub raum tsis kho BN tau cuam tshuam nrog kev nce hauv TIMP-1 thiab MIP-3A/CCL20. Los ntawm kev sib piv, peb pom tias PrC-210 kev kho mob tau txo qis TIMP-1 qib thiab nce MIP-3A/CCL20 theem.
Cov ntaub so ntswg inhibitor ntawm metalloproteinase-1 (TIMP-1) yog ib qho tseem ceeb regulator ntawm extracellular matrix (ECM) synthesis thiab degradation. Tshaj ECM tsub zuj zuj yog lub ntsiab pathological mechanism ntawm kev txhim kho fibrosis thaum lub sij hawm thiab tom qab mob raum raug mob. Nws yog qhov tseem ceeb tsis muaj kev qhia ntawm TIMP-1 li qubraumcov ntaub so ntswg [25], ib qho kev soj ntsuam uas yog corroborated nyob rau hauv peb daim duab 6A, B, tab sis TIMP-1 paub hais tias yuav tsum tau qhia nyob rau hauv lub raum raug mob, feem ntau nyob rau hauv lub raum tubular epithelial hlwb, lub raum tubular hauv qab daim nyias nyias, thiab lub cytoplasm ntawm interstitial. hlwb. Nce TIMP-1 qhia tau zoo cuam tshuam nrog kev ua haujlwm ntawm lub raum tsis zoo [26]. Cov nas kho nrog PrC-210 pom qhov txo qis hauv TIMP-1 qib (p=0.001), ob qho tib si raum homogenate thiab ntshav ntshav; qhov no txhais tau hais tias PrC-210 ua rau muaj kev tiv thaiv muaj zog tiv thaiv kev hloov pauv ntawm lub raum extracellular matrix.
Lub chemokine MIP-3a/CCL20 qhib lub CCR6 receptor, uas yog qhia tshwj xeeb tshaj yog nyob rau hauv txoj cai T-cells (Tregs). CCL20 yog qhia los ntawm tubular endothelial thiab interstitial hlwb thiab tseem upregulated nyob rau hauv ob lub raum nrog mob raum raug mob. CCL20- CCR6 txoj hauv kev ua lub luag haujlwm tseem ceeb hauv Treg-mediated T-cell nrhiav neeg rau lub raum, thiab Tregs tau piav qhia kom muaj lub luag haujlwm zoo hauvraumkho, hloov kev kam rau siab, thiab raum ciaj sia taus. Ob qho tshuaj tiv thaiv kab mob ntawm CCL20-CCR6 txoj hauv kev, nrog rau kev siv CCR6-cov nas tsis muaj zog hauv kev mob hnyavraumCov kev sim raug mob, tau pom tias yuav ua rau muaj mob raum tsis ua haujlwm thiab kev tuag [27]. Qhov no qhia tau hais tias, qhov chaw kho mob, CCL20-CCR6 txoj hauv kev txhim kho thiab kev ua kom Treg tuaj yeem yog txoj hauv kev kho mob kom txo qis mob thiab mob ntev.raumraug mob [28]. Hauv peb txoj kev tshawb fawb (Daim duab 6D), MIP-3 qib a/CCL20 tau siab dua hauv PrC-210- cov nas kho tau ntau dua li cov nas tsis kho. Peb xav tias qhov no yog ib qho laj thawj rau ob qho tib si (i) qhov tseem ceeb txo qis kev nrhiav neeg ua haujlwm ntawm cov hlwb mononuclear rau ob lub raum (Daim duab 3C) thiab (ii) txo qis raum puas tsuaj (Daim duab 2) hauv PrC-210- kho nas. .
Lub raum mitochondrial ua haujlwm li qub, thiab qhov tseem ceeb, kev thuam rau nws thaum lub raum cia, cog, thiab tom qab cog cov kab mob yog qhov tseem ceeb ntawm ROS kev raug mob thiab lub raum tsis ua haujlwm thaum hloov pauv. Nws yog li qhov tseem ceeb uas PrC-210 tau pom tias muaj kev cuam tshuam tag nrho ntawm mitochondrial DNA fragmentation (Daim duab 7) ntawm qhov ntau (2–4 mM) uas tau ua tiav hauv cov ntshav ntawm cov nas thiab nas uas tau muab rau hauv lub cev lossis qhov ncauj qhov systemic 0.5 MTD koob tshuaj PrC-210 uas tau raug zam tsis muaj tshuaj toxicity [29].
Hauv peb cov kev tshawb fawb txog kev hloov hauv lub raum ua ntej [16,17], peb pom muaj qhov nce ntxiv hauv cov kab mob hauv lub raum raug mob "txias ischemia" thiab "ischemia-reperfusion" raug mob. Cov ischemia-induced insults rau lub raum tau txo mus rau keeb kwm yav dhau los ntawm kev kho nrog PrC-210 (Daim duab 8). Hauv cov kev tshawb fawb ntawm cov ntawv sau no (Daim duab 5), nyob rau hauv uas mob khaub thuas ischemia thiab ischemia-reperfusion yog qhov tseem ceeb tshem tawm los ntawm kev hloov pauv tam sim ntawd, tsis muaj qhov nce hauv cov kab mob hauv lub raum hloov pauv. Es tsis txhob, activated caspase tau txo qhov tseem ceeb ntawm ntxiv 20 h hauv "Tsis Muaj Tshuaj Kho Mob" tswj, thiab PrC-210 kev kho mob tshem tawm tag nrho qhov txo qis caspase hauv ntxiv rau 20 h nas thiab ua kom cov qib caspase ruaj khov. Peb qhov kev txhais ntawm cov txiaj ntsig zoo no yog tias tsis muaj qhov tseem ceeb ntawm ischemia-induced dawb radical insult los ntawm ROS thiab RNS rau tom qab hloov pauv.lub raum, tsis muaj kev cuam tshuam ntawm tes tuag thiab apoptosis cov cim xws li qhib cov caspases. Es tsis txhob, nyob rau hauv cov allograft raum hloov pauv, cov kab mob tshwm sim los ntawm cov tshiab cyto- thiab chemokines tam sim no tswj cov cell metabolism, uas suav nrog cuam tshuam txoj hauv kev apoptosis. Cov ntaub ntawv piav qhia tias overexpression ntawm TIMP-1 ua rau muaj kev tawm tsam ntawm apoptosis [26]. Peb cov txiaj ntsig caspase (Daim duab 5) txhawb qhov piav qhia TIMP-1 cov nyhuv, thiab lawv txhais tau tias TIMP-1 yog ib qho tseem ceeb hauv kev tswj cov kab mob ntawm cov cell puas tom qab hloov lub raum allograft. Hauv kev lees paub ntawm qhov ua ntej PrC-210 kev tawm tsam ntawm TIMP-1 qhia (Daim duab 6A, B), PrC-210 kev kho mob tau tshem tawm qhov hloov pauv caspase, ua kom cov qib caspase nyob rau tib theem pom hauv tswj "0 h" raum. Vim tias qhov txo qis PrC-210 ntshav TIMP-1 qib ntawm ntxiv 20 h (Daim duab 6B) raug cuam tshuam qhov tseem ceeb ntawm kev tawm tsam ntawm allograft: (i) apoptosis (Daim duab 5), (ii) histologic pathology (Figures 2and 3) thiab (iii) inflammatory cell infiltration (Daim duab 3), peb cia siab tias kev soj ntsuam cov ntshav TIMP-1 qib hauv tib neeg lub raum allograft cov neeg tau txais yuav yog ib txoj hauv kev los saib xyuas PrC-210 kev soj ntsuam kev ua tau zoo hauv kev sim tshuaj tom ntej .
Hauv peb txoj haujlwm txog hnub tim [16,17], peb tau pom tias PrC-210 muaj peev xwm tiv thaiv lub raum transplanted tiv thaiv ob qho tib si txias-ischemia thiab ischemia-reperfusion insults. Hauv txoj kev tshawb no, peb tam sim no pom tias PrC-210 kuj tseem tiv thaiv lub raum allograft los ntawm cov tsis yog ischemia inflammatory insults uas tshwm sim tom qab lub raum cog. PrC-210 txo qis theem ntawm cov kab mob cytokines mob, xws li TNF-alpha, thiab inhibits qhov kev qhia ntawm TIMP-1 chemokine. Ob leeg ntawm cov xwm txheej no, thiab muaj peev xwm, txhawb ntxiv los ntawm CCL20 qhia ntxiv, yuav xav tias yuav: (i) txo qis allograft raum puas, (ii) suppress T-cell recruitment rau lub raum, thiab (iii) suppress activation ntawm innate thiab adaptive tiv thaiv kab mob. Hauv daim duab 8, peb sau cov kev tshawb pom no los txhawb lub luag haujlwm uas peb xav tias PrC- 210 tuaj yeem ua rau tib neeg lub raum hloov pauv; nws suppresses: (i) txias ischemia reactive oxygen hom (ROS) thiab reactive nitrogen hom (RNS) kev puas tsuaj rau keeb kwm yav dhau [17],
(ii) ischemia-reperfusion ROS kev puas tsuaj rau keeb kwm yav dhau [16], thiab (iii) allograft inflammatory kev puas tsuaj loj heev, qee zaum, rau keeb kwm yav dhau. Txij li thaum thawj PrC -210 mechanism ntawm kev txiav txim rau PrC-210 yog scavenging oxygen thiab nitrogen dawb radicals, qhov no txhais tau hais tias cov dawb radicals yog ib qho tseem ceeb contributor rau lub raum puas pom nyob rau hauv uas tsis yog-ischemic tej yam kev mob, piv txwv li, lub allograft-koom nrog o uas tau kawm hauv phau ntawv no.
Hauv cov ntsiab lus, qhov no qhia tias PrC-210 tuaj yeem muab kev tiv thaiv kab mob hauv nruab nrog cev rau ntau yam kev hloov pauv hloov pauv; Nws tuaj yeem tiv thaiv lub raum hloov pauv thaum lub sijhawm thiab tom qab tag nrho cov theem ntawm cov txheej txheem hloov pauv - los ntawm kev pub khoom nruab nrog cev, los ntawm kev thauj mus los, rov ua dua tshiab, thiab tom qab kev phais mob - kom txo qis kev mob hnyav thiab mob ntev.
Cistanche tubulosa tiv thaiv kab mob raum, nyem qhov no kom tau txais cov qauv
Tus sau kev koom tes:NAW, WZ (Weifeng Zeng), WZ (Weixiong Zhong), BMV thiab MMCG tau koom nrog kev ua haujlwm ntawm kev tshawb fawb thiab kev txheeb xyuas cov ntaub ntawv; TRG thiab NAW tau koom nrog kev tshawb fawb tsim thiab kev txheeb xyuas cov ntaub ntawv; WEF thiab TRG tau koom nrog hauv kev tshawb fawb tsim, kev ua haujlwm ntawm kev tshawb fawb, kev txheeb xyuas cov ntaub ntawv, thiab kev sau ntawv. Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.
Nyiaj txiag:Cov kev tshawb fawb no tau txais nyiaj los ntawm UW Lub Tsev Haujlwm Saib Xyuas Kev Kho Mob thiab Kev Txhais Lus thiab muab UL1TR002373 rau UW ICTR los ntawm NIH/NCATS, American Society of Transplant Surgeons (133 AAA1552), American College of Surgeons (133 AAB2176), thiab muab kev txhawb nqa rau WEF (# R03CA176799).
Institutional Review Board Statement: Qhov kev tshawb fawb no tau pom zoo los ntawm Lub Tsev Kawm Ntawv ntawm Tshuaj thiab Kev Noj Qab Haus Huv Hauv Lub Tsev Haujlwm Saib Xyuas Tsiaj thiab Siv Hauv Tsev Kawm Qib Siab University of Wisconsin (Tsuas Protocol #M005204).
Cov ntaub ntawv muaj nyob:Cov ntaub ntawv tshawb fawb muaj nyob rau hauv daim ntawv no. Kev tsis sib haum xeeb ntawm kev txaus siab: Cov neeg sau ntawv tshaj tawm tsis muaj teeb meem ntawm kev txaus siab.
Cov ntaub ntawv
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