Yuav ua li cas cell membrane kho cov proteins tswj transcription factor signal transduction los tswj lub raum fibrosis

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Haichang Li

Lub raumfibrosis yog txuam nrog kev loj hlob ntawm mobraumraug mob rau mob raum mob. MG53, cell membrane kho protein, tau pom tias tiv thaiv kev raug mob rau lub raum epithelial hlwb thiab mob raum raug mob. Ntawm no, peb tau soj ntsuam lub luag haujlwm ntawm MG53 hauv kev hloov kho ntawmraumfibrosis nyob rau hauv cov nas laus thiab nyob rau hauv nas nrog unilateral ureteral obstruction (UUO) ib tug paub qauv ntawm lub raum fibrosis. Cov nas nrog ablation ntawm MG53 tau tsim muaj ntau qhov sib txuam nrog fibrosis nrog lub hnub nyoog tshaj li MG53-cov nas uas muaj hnub nyoog tib yam. Ib yam li ntawd, thaum tsis muaj MG53,raumfibrosis tau exaggerated piv rau nas nrog intactMG53 nyob rau hauv obstructed ob lub raum piv rau contralateral unobstructed raum los yog ob lub raum ntawm sham-operated nas. Lub ureteral obstructedlub raumlos ntawm MG53 cov nas uas tsis muaj zog kuj pom tias muaj mob ntau dua li cov ureteral obstructed ob lub raum los ntawm MG53 cov nas tsis zoo. Hauv vitro thwmsim pom tau tias MG53 tuaj yeem nkag mus rau hauv lub nuclei ntawm cov tubular epithelial hlwb thiab cuam tshuam ncaj qha nrog p65 feem ntawm kev hloov pauv NF-kB, muab cov lus piav qhia txog kev txhim kho o thaum tsis muaj MG53. Txhawm rau kuaj qhov no, txhim kho MG53 kev qhia los ntawm cov hlwb tsim los yog kev xa cov protein ncaj qha tau muab rau cov nas uas raug UUO. Qhov no txo ​​NF-kB ua kom thiab mob thiab ua rau lub raum fibrosis. Yog li, MG53 tej zaum yuav muaj lub luag haujlwm kho mob hauv kev kho mob raum mob thiab yog li muab kev tiv thaiv tiv thaiv.fibrosisuas ua rau mob raum mob phenotype.

Cistanche tubulosa tiv thaiv kab mob raum, nyem qhov no kom tau txais cov qauv

Cov Lus Txhais Lus Txhais Kev mob mob ntev ua rau kev kho mob fibrotic uas tuaj yeem cuam tshuam kev hloov pauv ntawm lub raum raug mob mus rau mob raum mob. Kev ua kom cov protein-inflammatory transcription factor nuclear factor-kB (NF-kB) yog koom nrog hauv pathogenesis ntawm lub raum o. Ntawm no, peb muab pov thawj tias MG53, yav dhau los txheeb xyuas cov cell membrane kho cov protein, ncaj qha cuam tshuam nrog NF-kB thiab txo nws cov kev ua haujlwm transcriptional. Concomitantly, exogenously tswj MG53 tuaj yeem txo cov kev hloov kho fibrotic ntawm lub raum mob. Cov kev tshawb pom no taw qhia txog kev tiv thaiv kev sib cuam tshuam ntawm MG53 thiab NF-kB kom txo qis kev txhim kho ntawm cov mob hauv lub raum fibrosis. Pharmacological kev tswj hwm ntawm MG53 tej zaum yuav yog ib qho kev cog lus rau kev kho mob ntawm lub raum fibrosis.

Lub raum tsis ua haujlwm, uas tuaj yeem raug cais raws li mob raum raug mob (AKI) lossis kab mob raum ntev (CKD), tau loj hlob mus rau kev sib kis hauv cov neeg laus. Hauv xyoo 2017, qhov kev nthuav dav ntawm CKD tau mus txog 14.5 feem pua ​​​​ntawm cov neeg muaj hnub nyoog 65 thiab tshaj saud, uas ua rau Medicare siv nyiaj ntau dua $ 84 nphom rau kev kho mob.^1,2AKI thiab CKD feem ntau tshwm sim hauv cov neeg laus, feem ntau yog vim muaj kev raug mob ntau ntxiv ntxiv rau qhov muaj peev xwm txo qis los kho lub raum laus. Cov kev tshawb fawb soj ntsuam tau pom tias CKD yog ib qho kev pheej hmoo tseem ceeb rau AKI, thiab, ntawm qhov tsis sib xws, cov sijhawm ntawm AKI tuaj yeem ua rau kev loj hlob ntawm CKD mus rau theem kawg ntawm cov kab mob raum. Tam sim no, kev kho mob ntawm AKI feem ntau yog txhawb nqa, thiab kev kho mob ntawm CKD yog tsom rau kev ua kom qeeb qeeb los ntawm kev cuam tshuam ntawm renin-angiotensin-aldosterone system thiab tam sim no muaj peev xwm los ntawm sodium-glucose cotransporter -2 inhibition.^3,4

Raws li qhov hnyav ntawm qhov raug mob, proximal tubular cells, lub hom phiaj tseem ceeb ntawm kev raug mob hnyav, tuaj yeem hloov pauv hauv lub voj voog ntawm tes, 5,6 metabolism, 7 tso tawm ntawm cov protein-inflammatory thiab profibrotic cytokines, los yog ib feem ntawm epithelial-mesenchymal hloov, 8. uas yuav txiav txim siab seb qhov kev hloov kho / kho puas yuav ua tiav lossis tsis zoo thiab ua rau lub raum fibrosis, ib qho cim ntawm CKD.9,10 Qhov kev hloov kho no feem ntau tshwm sim ntawm keeb kwm ntawm lub raum mob,

txo qis vascular mov, thiab zus tau tej cov extracellular matrix (ECM) proteins thiab muaj xws li poob ntawm epithelial hlwb thiab tsub zuj zuj ntawm collagens, ib tug du nqaij actin, thiab fibronectin; nws kuj muaj kev sib raug zoo nrog rau deterioration ntawm lub raum kev ua haujlwm.

Kev mob ntev ua rau muaj kev hloov kho fibrotic uas tuaj yeem cuam tshuam qhov kev hloov pauv ntawm AKI mus rau CKD.11-13 Kev tshawb fawb ntau ntxiv qhia txog kev koom tes ntawm cov khoom siv hluav taws xob nuclear-kB (NF-kB) transcription factor nyob rau hauv lub pathogenesis ntawm lub raum mob tshwm sim los ntawm kab mob, raug mob, los yog transplantation.11,12,14 Ua kom txoj hauv kev canonical NF-kB pib nrog kev ua kom lub zog ntawm cov inhibitor ntawm NF-kB (IkB) kinase, uas ua rau phosphorylation thiab degradation ntawm IkBa thiab lub nuclear translocation ntawm NF-kB heterodimers.15 Kev ua kom NF-kB signaling nyob rau hauv lub raum epithelial hlwb thiab infiltrating lub cev tiv thaiv kab mob yuav elicited los ntawm pathophysiological triggers xws li raug rau lipopolysaccharides (LPSs) los yog ischemia-reperfusion raug mob.16 Molecular profiling kev tshawb fawb qhia nfkb1 raws li ib tug loj tsav ntawm lub raum fibrosis.¹⁷Ntxiv nrog rau lub raum tubular hlwb, lub cev tsis muaj zog xws li macrophages thiab dendritic cells tseem ua rau lub raum raug mob, mob, thiab kho fibrotic.^13,18–20

cistanche muaj txiaj ntsig zoo hauv kev kho raum tsis ua haujlwm

Cov pov thawj loj hlob qhia tau hais tias cov leeg-los ntawm secretory yam tseem ceeb (piv txwv li, myokines) hloov kho lub cev lub cev los ntawm cov ntaub so ntswg crosstalk los cuam tshuam kev loj hlob ntawm cov kab mob raum.²¹MG53 (tseem hu ua TRIM72) yog cov nqaij leeg-ua kom muaj txiaj ntsig tripartite motif (TRIM) cov protein nrog lub luag haujlwm tseem ceeb hauv kev kho cell membrane.22,23 TRIM tsev neeg cov proteins muaj ntau yam haujlwm xws li kev tswj hwm kev tiv thaiv kab mob rau kev kho cov ntaub so ntswg.²⁴MG53- kho kho daim nyias nyias yog koom nrog hauv kev txo qis kev raug mob rau cov leeg pob txha,²⁵lub raum,²⁶plawv,²⁷ lub ntsws, 28 lub hlwb,^29,30thiab daim tawv nqaij.³¹Peb cov kev tshawb fawb yav dhau los tau txheeb xyuas qhov qis ntawm MG53 qhia hauv lub raum, uas yog ib qho tseem ceeb hauv kev tiv thaiv AKI, thiab cov nas tsis muaj peev xwm ntawm MG53 (mg53 /) tau raug cuam tshuam rau kev ntxhov siab vim AKI.²⁶Peb kuj tsis ntev los no tau pom tias qhov nce siab ntawm MG53 nyob rau hauv txoj kev ncig txhim kho cov ntaub so ntswg raug mob thiab rov tsim dua tshiab.32 Lub luag haujlwm txheeb ze ntawm lub raum tshwj xeeb thiab extrinsic circulating MG53 hauv kev hloov kho raum fibrosis thaum lub sij hawm CKD tseem tsis tau paub.

Tsis ntev los no, lub luag haujlwm tseem ceeb ntawm MG53 hauv kev hloov kho cov ntaub so ntswg tiv thaiv los ntawm inhibition ntawm o, tshwj xeeb tshaj yog los ntawm kev hloov kho ntawm NF-kB signaling, tau hloov zuj zus. Piv txwv li, MG53 attenuates LPS-induced neurotoxicity thiab neuro-inflammation los ntawm inhibiting TLR4 / NF-kB txoj kev, 33 thiab plawv MG53 tswj KChIP2 qhia los tswj electrophysiological remodeling thaum lub sij hawm cardio hypertrophy.34 Ntxiv thiab, peb pom ib tug dual muaj nuj nqi ntawm MG53. tiv thaiv cov lus teb tsis zoo ntawm hyperinflammatory thaum lub sij hawm tus kab mob sublethal influenza A tus kab mob, tus cwj pwm los ntawm kev tsim tawm ntau dhau ntawm interferon-b, los ntawm kev tawm tsam ntawm IRF3 / NF-kB ua kom thiab inhibition ntawm aberrant intracellular Ca2þ signaling hauv macrophages.35 Tsis tas li ntawd, raws li kev ua kom tuag ntawm tus mob khaub thuas. Tus kab mob kis tau tus kab mob, MG53 tau pom tias tiv thaiv kev tuag thiab lub ntsws puas tsuaj, tsis yog los ntawm kev cuam tshuam ncaj qha rau tus kab mob titers ib se tab sis los ntawm kev txo qis cytokine cua daj cua dub (interferon-b, interleukin{19}}, thiab interleukin -1b) los ntawm kev tswj tsis zoo ntawm NLRP3 inflammasome thiab tiv thaiv pyroptosis ntawm lub ntsws.36 Tsis tas li ntawd, kev kho mob ntev ntev ntawm exogenous MG53 tau txuas rau kev tawm tsam ntawm NF-kB. - kho mob o nyob rau hauv lub hnub nyoog lub plawv.37 Cov kev tshawb fawb no qhia tias MG53 tuaj yeem hloov pauv tsis zoo NF-kB signaling thaum lub sij hawm o tab sis seb qhov no tshwm sim hauv cov ntsiab lus ntawm lub raum mob tsis tau kawm.

Peb tau tshaj tawm tias MG53 tswj lub raum mob los ntawm kev hloov pauv kev ua haujlwm ntawm NF-kB thiab ua li ntawd tuaj yeem ua rau lub raum fibrosis uas tshwm sim los ntawm qhov mob ntev. Qhov kev tshawb fawb no tau ua los daws qhov kev xav no.

cistanche muaj txiaj ntsig zoo hauv kev kho mob raum

Txoj kev

Kev tshawb fawb tsiaj

Txhua qhov kev sim nrog cov tsiaj ua raws li National Institutes of Health Guide for the Care and Use of Laboratory Tsiaj thiab ua raws li cov txheej txheem pom zoo los ntawm Ohio State University Institutional Animal Care and Use Committee. Age-matched wild type (WT) lossis mg53^-/-nonlittermates ntawm 129S1 / SvImJ strain tau siv22 los tsim lub luag haujlwm ntawm MG53 hauv kev hloov pauv ntawm hnub nyoog-dependent thiab unilateral ureteric obstruction (UUO)-induced raum fibrosis. Cov nas C57B6/J (9-10 lub lis piam) tau yuav los ntawm Jackson Labs.

Rau kev tshawb fawb txog kev raug mob raum, nas (9-12 lub lis piam) tau raug UUO los ua rau lub raum fibrosis. Cov nas raug tshuaj loog los ntawm isoflurane (1.5 feem pua ​​-2.0 feem pua ​​) kom paub meej tias lub dav hlau sib sib zog nqus ntawm cov tshuaj loog. Cov txheej txheem UUO tau ua los ntawm ligating sab laug ureter nrog 5-0 phais kab mob ob zaug raws li cov txheej txheem luam tawm.38 Sham nas tsuas muaj daim tawv nqaij mob plab. Txhawm rau ntsuas qhov cuam tshuam ntawm rhMG53 ntawm obstructive raum raug mob, 1 pawg ntawm UUO nas tau txais rhMG53 (2 mg / kg) los ntawm kev txhaj tshuaj vein tail pib tam sim tom qab UUO phais (hnub 0) thiab tom qab ntawd txhua hnub rau 7 hnub, nrog 2 koob tshuaj ntxiv rau hnub 9 thiab 11. Ib pawg tswj hwm ntawm UUO nas tau txais kev txhaj tshuaj saline raws li tib lub sijhawm. Cov nas raug tua nyob rau hnub 12 tom qab UUO phais, ob lub raum tau perfused nyob rau hauv situ nrog phosphate-buffered saline, thiab cov ntaub so ntswg los ntawm UUO raum thiab lub raum contralateral tau sau rau histology thiab Western blot tsom xam.

Hauv kev tshawb fawb cais, WT thiab mg53^-/-Cov nas tau txais kev phais UUO lossis sham phais thiab raug tua rau hnub 7 tom qab ua haujlwm. MG53 tau muab rau cov nas uas siv txoj kev kho mob ntawm tes.39 RAW 264.7 macrophages tau kis tus kab mob doxycycline (DOX)-dependent Ad tPA-MG53- cherry viral particles rau 24 teev. Cov kab mob uas muaj kab mob (ntawm qhov siab ntawm {{10}}} /100 ml saline rau nas) yog cov kab laug sab leeg txhaj rau cov nas tam sim tom qab kev phais (hnub 0 txhaj tshuaj). Cov nas tau txais 4 txhaj tshuaj ntawm Ad-tPA-MG53 transduced RAW hlwb nyob rau hnub 0, 1, 3, thiab 6. Tam sim ntawd tom qab hnub 0 kev txhaj tshuaj ntawm tes, nas tau randomized rau hauv ib pawg uas tau txais thiab ib pab pawg uas tsis tau txais DOX (2 mg/ml hauv 5 feem pua ​​sucrose tov) hauv lawv cov dej haus rau 7 hnub thiab tom qab ntawd tua. Cov ntaub so ntswg los ntawm UUO raum thiab lub raum contralateral tau sau rau histology, RNA, thiab cov tshuaj ntsuam xyuas protein.

Kev txheeb xyuas cov ntaub ntawv qhia tau hais tias txhais tau tias -SD. Kev sib piv nyob rau hauv pab pawg tau ua los ntawm Cov Tub Ntxhais Kawm Ntawv t-test thaum muab piv 2 kev sim kev tshawb fawb hauv paus H Li li al.: MG53 modulates NF-kB hauv raum fibrosis 2 Raum International (2021) -, ----pab pawg thiab 1-txoj kev tsom xam ntawm qhov sib txawv rau ntau tshaj 2 pawg (Graphpad Prism 8.2; GraphPad) ua raws los ntawm Bonferroni lossis Holm-Sidak ntau qhov sib piv kev xeem ad hoc. Tus nqi ntawm P <0.05 tau="" suav="" tias="" yog="" qhov="" tseem="">

Cov txheej txheem ntxiv tag nrho suav nrog kev soj ntsuam histologic, kev ntsuas ntshav creatinine, plasmid tsim, kab lis kev cai ntawm tes thiab thawj tubular epithelial cell cais, kev npaj adenovirus thiab kab mob ntawm tes, NF kB p65 nuclear translocation assay, immunohistochemistry thiab confocal dluab, NF-kB luciferase report, enzyme-linked immunosorbent assay, cov ntaub so ntswg fractionation, immunoblotting, coimmunoprecipitation, quantitative real-time polymerase chain reaction (Supplementary Table S1), and rhMG53 production are in the Supplementary Methods.

cistanche tubulosa extract: kho mob raum

TSEEM CEEB

Cov nas nrog ablation ntawm MG53 tsim muaj hnub nyoog raws li lub raum fibrosis

Peb piv cov txiaj ntsig ntawm kev laus ntawm lub raum ua haujlwm thiab fibrosis hauv mg53^-/-thiab WT nas. Peb tau lees paub peb qhov kev soj ntsuam yav dhau los tias txawm hais tias cov ntshav creatinine qib ntawm cov nas me (2 lub hlis) nrog thiab tsis muaj MG53 tsis txawv txav, laus dua (16-20 lub hlis) mg53.-/-Cov nas tau nthuav tawm cov ntshav creatinine ntau dua li cov hnub nyoog sib xws WT tswj (Daim duab 1a). Raws li trichrome staining, peb pom tias mg53-/-cov nas muaj ntau lub raum fibrosis piv nrog WT nas, pib thaum muaj hnub nyoog 5 hli thiab txuas ntxiv mus txog hnub nyoog 20 hli (Daim duab 1b thiab c). Immunohistochemical staining tau siv los ntsuas qhov cuam tshuam ntawm MG53 ablation ntawm kev nkag mus ntawm leukocytes, monocytes, thiab macrophages hauv 2- hli thiab 5- hli-laus nas lub raum (Daim duab 1d thiab e). Lub raum los ntawm cov tub ntxhais hluas WT thiab mg53-/- nas muaj cov qib zoo sib xws ntawm intrarenal leukocytes, tab sis cov hlwb inflammatory ntau dua tau pom nyob rau hauv mg53 / ob lub raum tshaj WT ob lub raum hauv 5-cov nas hnub nyoog hli.

Ntxiv mus, immunohistochemistry rau cov leeg nqaij du thiab fibronectin, 2 extracellular matrix proteins uas feem ntau pom nyob rau hauv cov cheeb tsam ntawm lub raum fibrosis, pom kev txhim kho (3- rau 8-fold) staining nyob rau hauv glomeruli thiab tubulointerstitium ntawm ob lub raum los ntawm { {4}}hli mg53-/- nas piv nrog 10-hli WT nas (Ntxiv daim duab S1).

UUO induces aggravated lub raum o thiab fibrosis nyob rau hauv mg53-/- nas rau raum MG53 qib kuaj pom los ntawm immunoblotting ho nce 7 hnub tom qab UUO (Daim duab 2a). Lub mg53- // UUO ob lub raum nthuav tawm exaggerated fibrosis, nrog rau 30 feem pua ​​​​ntawm qhov chaw trichrome-stained ntxiv, dua li WT UUO ob lub raum (Daim duab 2b).

MG53 modulates NF-kB ua kom thiab p65 nuclear localization

Qhov kev ua ntawm NF-kB (p65) raug soj ntsuam hauv UUO qauv. Tus naj npawb ntawm tag nrho p65 (t-p65) tau nce ntau hauv UUO ob lub raum los ntawm WT thiab mg53 / nas, tab sis kev ua kom NF-kB, ntsuas raws li phosphorylation ntawm p65, ntau dua (16-fold) hauv mg53 / UUO lub raum tshaj WT UUO lub raum (2-fold) piv nrog cov tsiaj ua sham (Daim duab 3a). Peb tau txheeb xyuas qhov txheeb ze RNA theem ntawm kev hloov pauv kev loj hlob b1 (TGF-b1), plasminogen activator inhibitor-1 (PAI-1), thiab collagen hom I alpha 1 (Col1a1) hauv obstructed raum.40 Peb kuaj pom cov qib qis ntawm cov noob qhia hauv sham tsiaj thiab zoo sib xws induction (TGF-b1, w10 quav; PAI-1, w40 quav; Col1a1, w40 quav) hauv UUO raum los ntawm WT thiab mg53 / (Daim duab 3b). Txhawm rau tshuaj xyuas qhov spatial faib ntawm MG53 hauv lub raum cortex nyob rau hauv ib txwm muaj mob, lub raum cov ntaub so ntswg tau txheeb xyuas. Peb niaj hnub tau txais 10-fold tag nrho cytosol protein ntau dua li tag nrho cov protein rho tawm nuclear. Cov qib t-p65 tau muab piv rau hauv cov cytosolic feem ntawm WT thiab mg53-/- nas tab sis ntau dua nyob rau hauv cov feem pua ​​ntawm mg53-/- nas (Daim duab 3c). MG53 tau kuaj pom hauv cytosol feem (nrog tubulin ua tus cim cytosol). Poob ceeb thawj, ib qho tseem ceeb ntawm MG53 tau kuaj pom ntawm cov feem ntawm lub nuclear (nrog rau histone H3 raws li ib tug nuclear feem cim).

Txhawm rau kom paub tseeb tias qhov kev tshawb pom no, peb kuj tau tshuaj xyuas MG53 qhov chaw nyob hauv cov leeg pob txha (cov duab ntxiv S2). Peb rov tsim dua peb cov kev tshawb fawb yav dhau los uas MG53 nyob rau hauv cytosol thiab sarcolemmal membrane (sodium-potassium adenosine triphosphatase raws li ib qho cim ntawm daim nyias nyias) 22,23,25 tab sis kuj tau kuaj pom muaj zog nuclear MG53 los ntawm cov leeg pob txha.

MG53 cuam tshuam nrog p65 los tswj cov qog necrosis factor-a- induced transcriptional kev ua kom paub meej tias peb cov ntaub so ntswg fractionation, peb tau ua cov kev tshawb fawb immunohistochemical hauv qab no. Hauv kab lis kev cai WT proximal tubular epithelial hlwb, p65 tau pom tsuas yog hauv cytosol tab sis nyob rau hauv lub nuclei thaum tsis muaj MG53 (Daim duab 4a). Thaum cov hlwb no tau kho nrog LPS (5 mg) rau 8 teev, pro-inflammatory cytokine secretion tau loj dua hauv mg53 / hlwb dua li WT hlwb (Daim duab 4b).

Vim tias cov ntaub ntawv no qhia tias MG53 tuaj yeem cuam tshuam nrog p65 los tswj cov kab mob cytokine inflammatory, kev sib cuam tshuam ncaj qha ntawm p65 thiab MG53 tau soj ntsuam los ntawm coimmunoprecipitation assay siv HEK293 hlwb cotransfected nrog Flag-p65 thiab HA-MG53. Peb pom muaj kev sib cuam tshuam tsis muaj zog (Daim duab 4c). Cotransfection ntawm GFP-p65 thiab RFP-MG53 plasmids thiab confocal tsom xam tau lees paub colocalization ntawm p65 thiab MG53 (Daim duab 4d). Tom qab ntawd peb txheeb xyuas seb MG53 puas cuam tshuam rau NF-kB (p65) kev ua haujlwm transcriptional tom qab TNF-stimulation los ntawm kev ntsuas cov luciferase kev ua los ntawm qhov ruaj khov NF-kB/293-GFP-Luc transcriptional reporter cell kab uas tau transiently transfected nrog rau pHM6 vector. lub HA-MG53. Cov xovtooj ntawm cov neeg sau xov xwm muaj cov kab mob kis tau tus kabmob kis tau tus kabmob luciferase tus kws tshaj xov xwm uas tau tsav los ntawm tus kws tshaj lij cytomegalovirus tsawg kawg nkaus nrog 4 daim ntawv pom zoo ntawm NF-kB cov lus teb cov lus teb (kB site) nce toj. Thaum tsis muaj TNF-a, muaj kev ua haujlwm tsawg ntawm cov luciferase hauv cov hlwb uas qhia txog MG53 proteins. MG53 suppressed TNF-a-elicited NF-kB (p65) kev ua haujlwm hloov pauv los ntawm kwv yees li 40 feem pua ​​(Daim duab 4e).

Overexpression ntawm MG53 tiv thaiv NF-kB p65 nuclear translocation raws li TNF-kev kho mob NF-kB p65 translocated mus rau lub nucleus raws li TNF-kev kho mob nyob rau hauv lub raum proximal tubule epithelium.41 Los tshawb xyuas seb kev kho mob nrog MG53 tuaj yeem txo qis NF-kB p65 nuclear translocation. raws li TNF-a stimulation, peb overexpressed MG53 nyob rau hauv HKC -8 hlwb los ntawm DOX-dependent Ad-tPA-MG53 kis kab mob los yog los ntawm kev kho mob nrog recombinant tib neeg MG53 (rhMG53). Siv ib qho MG53- tshwj xeeb monoclonal antibody, tom qab DOX-dependent induction MG53 tau kuaj pom los ntawm kev tiv thaiv kab mob. MG53 tam sim no nyob rau theem siab heev hauv Ad-tPA-MG53 transduced HKC-8 hlwb,

sib npaug rau 1 ng rhMG53 / mg cellular lysate. Hauv qhov sib piv, HKC-8 tau coj thiab nthuav tawm qhov tsawg dua ntawm rhMG53 (Daim duab 5a). Lub uptake ntawm FL fluorescent-labeled rhMG53 los ntawm HKC-8 tau lees paub los ntawm confocal microscopy (Cov duab ntxiv S3). Tom qab TNF-kev kho mob, lub sijhawm ua kom p65 nce siab yog 15 mus rau 30 feeb (Daim duab 5b). Nyob rau hauv cov xwm txheej basal, NF-kB p65 nyob hauv cytosol, thiab overexpression ntawm MG53 tsis hloov qhov no (Daim duab 5c thiab d). Txawm li cas los xij, tom qab TNF-a stimulation, p65 translocated mus rau lub nuclei hauv HKC -8 hlwb tsis overexpressing MG53. Nyob rau hauv kev sib piv, nuclear translocation ntawm p65 poob los ntawm 33 feem pua ​​(cov cell muab rhMG53) mus rau 50 feem pua ​​(transduced hlwb muab DOX) nyob rau hauv hlwb overexpressing MG53 (Daim duab 5c thiab d).

Kev hloov pauv ntawm kev tsim cov hlwb RAW rau hauv UUOmouse qauv tau ua nrog 1 - 106 cov hlwb uas tswj hwm los ntawm kev txhaj tshuaj rau cov leeg tom qab UUO phais. Cov nas tau muab faib ua ib pawg uas tau txais cov dej haus ib txwm (-DOX) thiab ib pawg uas tau txais DOX (2 mg / ml) hauv cov dej haus. UUO nas tau txais RAW Txhua hnub rau tag nrho 4 txhaj tshuaj. Lub raum tau sau 7 hnub tom qab kev phais.

Cov nas infused nrog engineered RAW ua raws li DOX induction tso tawm kom txo qis (los ntawm 30 feem pua) lub raum fifibrosis piv nrog cov neeg uas tsis tau txais cov hlwb lossis cov infused nrog engineered RAW hlwb tab sis tsis muaj Dox induc tion (66 feem pua ​​​​ntawm fibrotic cheeb tsam; Daim duab 6a thiab b).

Tsuas yog ib tug me me RAW / Ad-tPA-MG53 cov pej xeem tau dim ntawm kev soj ntsuam ntawm cov kab mob reticuloendothelial thaum kawg ntawm kev sim. Immunohistochemistry staining paub tseeb tias DOX kho-vim RAW-mediated MG53 qhia hauv lub raum (Daim duab 6c). Interestingly, UUO ob lub raum los ntawm RAW-infused nas thiab DOX-induced MG53 nthuav tawm qis p-65/t-p65 piv (los ntawm kwv yees li 40 feem pua) dua li cov uas tsis tau txais RAW lossis cov uas muab RAW yam tsis muaj DOX (Daim duab 6d thiab e). Qhov txo qis p-p65 tau lees paub nrog p-p65 (S536) immunohistochemistry staining ntawm UUO ob lub raum (Daim duab 6f). Peb xav tias ncig MG53 xa los ntawm Raw / Ad-tPA-MG53 / þDOX thiab uptake los ntawm lub raum proximal tubule cells tuaj yeem koom nrog txo p-p65 (S536) thiab txo UUO raum mob. Piv nrog UUO-tsuas ob lub raum, TGF-b1, PAI- 1, thiab Col1a1 qhia tau nce ntau hauv UUO ob lub raum los ntawm cov nas infused nrog RAW yam tsis muaj DOX (tsis muaj MG53 induction). Kev nce siab ntawm cov noob profibrotic no tuaj yeem raug ntaus nqi los ntawm kev ua kom muaj zog allograft cov tshuaj tiv thaiv los ntawm cov hlwb infused rau hauv cov tshuaj tiv thaiv kab mob. Txawm li cas los xij, UUO ob lub raum los ntawm cov nas infused nrog RAW thiab muab DOX (MG53 induction) qhia qis dua (60 feem pua ​​-72 feem pua ​​​​txo) PAI-1 thiab qis dua TGF-b1 thiab Col1a1 dua cov nas uas tsis muaj DOX kho (Daim duab 6 g).

Kev tswj hwm ntawm rhMG53 txo qis NF-kB kev ua haujlwm thiab kev txhim kho fibrosis hauv cov nas uas raug UUO

Peb tau sim seb rhMG53 puas pom muaj cov nyhuv antifibrotic zoo ib yam li kev kho RAW cell kho tau piav qhia ua ntej. Peb cov ntaub ntawv pharmacokinetic ua ntej tau qhia qhov luv luv, ncig ib nrab ntawm lub neej (kwv yees li 90 feeb hauv cov nas) ntawm rhMG53.42 Cov nas tau txais kev tswj hwm ntev ntawm 2 txog 6 mg / kg.

rhMG5326,37 zoo, yog li kev tswj hwm lub tswv yim qhia hauv daim duab 7a tau siv. UUO nas tau random muab lub tsheb (saline) lossis rhMG53 (2 mg / kg) txhua hnub rau thawj 7 hnub tom qab kev phais thiab tom qab ntawd txhua hnub kom txog thaum lawv raug tua rau hnub 12. Sham thiab contralateral ob lub raum nthuav tawm roughly sib npaug t-p65. thiab cov qib fibronectin qhia, nrog rau qib p-p65 nce me ntsis hauv ob lub raum sib piv nrog rau lub raum sham. Tom qab UUO, lub raum t-p65 nce ntxiv. Piv nrog rau cov tsiaj uas muaj ntsev, qhov p-65/t-p65 piv hauv UUO ob lub raum ntawm rhMG53-cov tsiaj kho tau raug txo los ntawm 37 feem pua, thaum IkBa nce li ntawm 27 feem pua ​​(Daim duab 7b thiab c). Tsis tas li ntawd, fibronectin poob li ntawm 33 feem pua ​​(Daim duab 7b thiab c). Immunohistochemistry paub tseeb tias rhMG53 muaj nyob rau hauv UUO ob lub raum tom qab kev kho mob (Daim duab 7d). Tag nrho cov fibrosis tau txo qis hauv rhMG53- kho UUO ob lub raum (Daim duab 7e). Txawm li cas los xij, RNA qhia ntawm TGF-b1, PAI-1, thiab Col1a1 zoo ib yam hauv cov nas uas tau kho nrog saline lossis rhMG53 (Daim duab 7f).

cistanche muaj txiaj ntsig zoo hauv kev kho raum tsis ua haujlwm

Kev sib tham

MG53 yog cov leeg-enriched TRIM protein pib pom tias yog ib qho tseem ceeb ntawm cov plasma membrane kho machinery.22,26,42,43 Txawm li cas los xij, cov yeeb yam ntawm MG53 tsis yog nyob rau hauv cov leeg pob txha, thiab MG53 tuaj yeem tso rau hauv cov hlab ntsha. myokine.32 yav dhau los peb tau pom tias MG53 tuaj yeem tswj tau qhov mob thaum lub sij hawm cov ntaub so ntswg puas nyob rau hauv tej thaj chaw deb plab hnyuv siab raum.27,29,33,35–37 Qhov kev tshawb nrhiav tam sim no nthuav dav ntawm MG53-kev tiv thaiv kab mob hauv lub raum. Peb xaus lus tias MG53 tuaj yeem txo qis qhov mob hauv lub raum thiab lub raum fibrosis los ntawm kev thaiv cov cytokine-induced activation ntawm proinflammatory thiab profibrotic transcription factor NF-kB. Qhov kev txiav txim siab no yog ua raws li cov kev soj ntsuam hauv qab no: (i) genetic deletion ntawm MG53 hauv cov nas ib txwm muaj.

ua rau lub raum tsis ua haujlwm raws li cov nas muaj hnub nyoog uas tau nrog rau kev nce hauv lub raum fibrosis thiab infiltration ntawm lub raum los ntawm leukocytes thiab macrophages; (ii) nyob rau hauv murine UUO raum raug mob qauv, obstructed ob lub raum ntawm MG53-cov nas uas tsis muaj peev xwm nthuav tawm ntau fibrosis thiab o ntau dua MG53- cov nas rov qab; (iii) TNF-a thiab interleukin-6kev tsim tawm los ntawm LPS-kho cov kab mob sib txuas ntawm cov tubular epithelial tau txo qis hauv qhov muaj MG53; (iv) kev kho mob los yog overexpression ntawm MG53 nyob rau hauv ib tug proximal tubular epithelial cell kab diminished TNFa-induced NF-kB activation los ntawm thaiv lub nuclear translocation ntawm p65 tivthaiv ntawm-kB (MG53 tau pom los khi ncaj qha rau p65, albeit weakly); thiab (v) phosphorylation ntawm p65, ib kauj ruam tsim nyog hauv kev ua kom NF-kB, tau txo qis hauv obstructed ob lub raum ntawm UUO nas kho nrog recombinant MG53 induced overexpress MG53 hauv macrophages. Tsis tas li ntawd, tus inhibitor ntawm NF-kB ua kom, IkBa, tau nce hauv obstructed ob lub raum ntawm UUO nas txhaj nrog recombinantMG53.

Genetic deletion ntawm MG53 nyob rau hauv cov nas ib txwm tso cai rau cov teebmeem ntawm kev laus ntawm lub raum. Qhov no qhia tau hais tias lub xub ntiag ntawm endogenous MG53 muab ib theem pib ntawm kev tiv thaiv thaum laus. Ib yam li ntawd, qhov poob ntawm endogenous MG53 ua rau AKI vim muaj kev cuam tshuam tsis zoo. UUO ob lub raum sau MG53, tejzaum nws xav txog tej thaj chaw deb myokine kev ua los ntawm cov leeg pob txha. Nws tseem tsis tau paub meej tias cov leeg MG53 tuaj yeem raug xaiv rau lub raum thaum raug mob; Txawm li cas los xij, lub raum puas feem ntau txhim kho hypoxia thiab raug oxidative kev nyuaj siab,^44,45Cov cim qhia uas tuaj yeem ua haujlwm los nrhiav MG53. Yog tias muaj, qhov "cov leeg nqaij-ob raum-lub cev tiv thaiv kab mob" axis tuaj yeem siv los ua kom lub raum puas tsuaj.46 Xwb, lub raum hlwb qhia qis qis ntawm MG53 ntawm lub hauv paus,²⁶thiab, thaum raug mob los yog mob ntev, MG53 ntau lawm tuaj yeem raug kho.

NF-kB yog ib qho tseem ceeb ntawm kev hloov pauv ntawm kev mob thiab fifibrosis hauv lub raum raug mob thiab kev laus.^14,17,47,48Peb tau txais kev tshoov siab los tshawb xyuas seb qhov teebmeem ntawm MG53 puas tuaj yeem kho los ntawm NF-kB vim tias muaj MG53 nyob rau hauv lub tshuab hluav taws xob ntawm lub raum homogenates qhia txog qhov muaj peev xwm cuam tshuam txog kev cuam tshuam. Nuclear localization ntawm MG53 tsis yog qhov ua ntej. Nyob rau hauv tus qauv transgenic overexpressing myocardial MG53, nuclear MG53 tswj kev qhia ntawm peroxisome proliferator-activated receptor-alpha ntawm qib transcriptional thiab cuam tshuam lub plawv lipid metabolism.49 Ntxiv mus, peb tsis ntev los no tau pom rhMG53 suppressed NF-kB ua kom txo tau lub hnub nyoog tsis ua haujlwm. Los ntawm kev txo cov pro-inflammatory cytokines, apoptosis, thiab oxidative kev nyuaj siab nyob rau hauv cov laus cardiomyocytes.³⁷

Lub tshuab molecular ntawm yuav ua li cas MG53 modulates p65 nuclear / cytoplasmic mobilization tseem tsis meej. MG53 tsis muaj cov teeb liab hauv cheeb tsam nuclear, tab sis nws muaj 3 putative N-terminal leucine-nplua nuj nuclear export signals, ib tug motif lees paub los ntawm nuclear export protein exportin.⁵⁰Kev sib cuam tshuam ncaj qha ntawm MG53 thiab p65 yog qhov tsis muaj zog vim yog qhov cuam tshuam loj ntawm MG53 ntawm NF-kB ua kom muaj zog. Txawm hais tias MG53 zoo li cuam tshuam rau lub zog nucleocytoplasmic shuttling ntawm NF-kB cov khoom tseem ceeb, cov txheej txheem tseeb tseem yuav raug txiav txim siab.

Cov txiaj ntsig tseem ceeb ntawm MG53-kev kho kom rov kho dua tshiab zoo li yuav txo qis kev hloov kho fibrotic los ntawm kev txo qis ntawm NF-kB-kev kho mob o. Lub raum fifibrosis yog cov txheej txheem nyuaj uas cuam tshuam nrog kev tso tawm ntawm ntau yam ECM proteins.51 MG53 zoo li muaj qhov cuam tshuam rau ECM protein fibronectin. Fibronectin nce hauv UUO ob lub raum tsis txaus hauv MG53 thiab txo qis hauv UUO nas kho nrog MG53. Kev qhia ntawm lwm cov ECM cov proteins nyob rau hauv lub xub ntiag thiab tsis muaj MG53 yog qhov nyuaj rau to taub. Peb yav dhau los qhia tau hais tias rhMG53 tsis zoo tswj TGF-b1 / Smad signaling thiab suppresses cov synthesis ntawm ECM proteins nyob rau hauv ib qho kev kawm nyob rau hauv vitro.31 Nyob rau hauv vivo, TGF b1, PAI-1, thiab Col1a1 transcripts tsis nce nyob rau hauv UUO ob lub raum los ntawm mg53 / tsiaj tsis txaus. Cov ntawv sau tseg no tau txo qis hauv UUO tsiaj txhu ua rau overexpress MG53 los ntawm kev tswj hwm tus nas kis kab mob.

macrophages, tab sis thaum recombinant MG53 tau muab, tsis pom muaj txiaj ntsig. Qhov no tsuas yog ib rooj plaub ntawm kev noj tshuaj txaus vim tias cov qib intrarenal MG53 ua tiav tau ntau dua nyob rau hauv cov tsiaj muab cov macrophage vector uas tsis yog recombinant MG53. Tsis tas li ntawd, cov qib protein tsis tau ntsuas, thiab cov xwm txheej tom qab hloov pauv tuaj yeem hloov kho ECM hauv UUO. Kev tshawb fawb ntxiv yog xav tau los piav qhia txog lub luag haujlwm ntawm MG53 hauv kev hloov kho ECM cov protein. Hauv kev xaus, txoj kev tshawb no tau pom tias myokine tuaj yeem pab tswj cov kab mob hauv nruab nrab ntawm lub cev xws li lub raum thiab, nyob rau hauv qhov chaw ntawm lub raum raug mob, tuaj yeem pab txo qis thiab tswj kev mob. Daim duab 8 nthuav tawm cov qauv ua haujlwm ntawm lub luag haujlwm ntawm MG53 hauv kev tiv thaiv kev tiv thaiv. Thaum muaj kev puas tsuaj-txuas nrog cov qauv molecular (DAMP, xws li TNF-a) thiab cov kab mob sib txuas nrog cov qauv molecular (PAMP, xws li LPS) stimulation thiab ischemic stress, NF-kB signalosome yog qhib rau hauv lub raum. MG53 tiv thaiv lub raum los ntawm inhibiting phosphorylation thiab ua kom cov NF-kB signaling, stabilizing IkBa, thiab txo p65 nuclear mobilization, thaiv kev ua kom cov kev pab cuam pro-inflammatory. Txawm hais tias endogenous MG53 tuaj yeem kho cov teebmeem no, peb xav kom muaj kev tiv thaiv zoo dua tuaj yeem siv tau siv MG53 kho, uas yog ua tau raws li lub peev xwm ntawm exogenous MG53 kom txo tau kev raug mob hauv UUO. Kev tshawb nrhiav ntxiv tau lees paub kom nkag siab tias qhov no myokine yuav raug siv los kho mob li cas.

Cov txiaj ntsig ntawm cistanche: tiv thaiv daim siab thiab tiv thaiv fibrosis

Qhia tawm

JM muaj kev ncaj ncees txaus siab rau TRIM-edicine, Inc., uas txhim khorhMG53 rau kev kho mob ntawm tib neeg cov kab mob. Patents ntawm kev siv MG53 yog tuav los ntawm Rutgers University thiab Ohio State University. Tag nrho lwm tus kws sau ntawv tshaj tawm tsis muaj kev sib tw nyiam.

TXOJ CAI

Txoj haujlwm no tau txais kev txhawb nqa los ntawm National Institutes of Health (NIH) R01-DK106394 (JM, BHR, thiab P-HL) thiab NIH R01-AG062896 (P-HL thiab BHR). Qhov project no kuj yog ib feem kev txhawb nqa los ntawm ib qho nyiaj intramuralLockwood los ntawm Ohio State University (P-HL) thiab ib qho khoom plig los ntawm National Center for Advancing Translational Sciences (NCATS; P-HL, puav pheej naj npawb UL1TR002733). Cov ntsiab lus tsuas yog lub luag haujlwm ntawm cov kws sau ntawv thiab tsis tas yuav sawv cev rau cov lus pom zoo ntawm NCATS lossis NIH.

AUTHOR CONTRIBUTIONSHL,

PD, P-HL, JM, thiab BHR tsim thiab / lossis tsim cov kev tshawb fawb; HL, PD, P-HL, ZL, thiab XZ ua qhov kev sim, tau txais cov ntaub ntawv, thiab ua cov ntaub ntawv tsom xam; PD, P-HL, thiab BHR tau txhais cov txiaj ntsig; thiab P-HL thiab BHR tau tsim, hloov kho, thiab pom zoo cov ntawv sau. Txhua tus kws sau ntawv tau nyeem thiab pom zoo rau qhov kawg version.

SUPPLEMENTARY KHOOM

Supplementary File (PDF) Supplementary Methods.

Tab S1.Primer sequences. Daim duab S1. mg53-/- nas qhia ECM protein deposition. Hnub nyoog sib tw (10 hli hnub nyoog) hom tsiaj qus (WT) thiab mg53- // raum cov ntaub so ntswg tau stained nrog 40 6-diamidino-2-phenylindole (DAPI; xiav, sab laug panels), a- cov leeg nqaij leeg (SMA; ntsuab, thib ob panels), fibronectin (liab, peb panels), thiab merged (txoj cai panels). Bar ¼ 25 mm. Kev ntsuas ntawm lub raum fibrosis cheeb tsam (n ¼ 3 ib pawg). Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias -SD. Tub kawm ntawv t-test. ***P <>

Daim duab S2.Kev faib tawm ntawm cov leeg pob txha MG53 ntawm nucleus thiab cytoplasm. Cov leeg pob txha-los ntawm cov tsiaj qus (WT) ormg53-/- nas tau fractionated thiab immunoblotted nrog kev cai-ua los tiv thaiv-MG53 antibody (3 mg lysates) thiab cell compartment markers (20 mg lysates). Sodium-potassium adenosine triphosphatase yog siv los ua cov cim cell membrane, histone H3 ua tus cim nuclear, thiab tubulin ua tus cim cytosol.

Daim duab S3.HKC-8 lub raum proximal tubule hlwb tshwj xeeb uptake AlexaFluor 647-labeled rhMG53. rhMG53 (Trimedicine) thiab bovine serum albumin (BSA; ThermoFisher Scientific) tau sau npe nrog Alexa Fluor (AF) Antibody Labeling kits (Life Technologies) raws li cov chaw tsim khoom raws tu qauv. HKC-8 hlwb raug coj los ntawm Delta TPG iav-bottomed phaj (Bioptechs Inc.) hauv Dulbecco's modified Eagle's nruab nrab (DMEM) nrog 10 feem pua ​​​​fetal bovine serum (FBS) nyob rau hauv lub xub ntiag ntawm cov proteins uas sau npe, BSA-AF647 (sab laug ) thiab rhMG53-AF647 (txoj cai), rau 24 teev. Z-pawg cov duab tau muab los ntawm LSM780 confocal microscope (Zeiss). Cov duab qhia cov hlwb hauv qab ntawm cov tais diav. Bar ¼ 25 mm.

Daim duab S4.Engineering RAW hlwb los kho kom haum xeeb inducible MG53secretion. (A) RAW hlwb tau kis nrog Ad-tPA-MG53 rau 24 teev thiab kho yam tsis muaj (doxycycline [Dox]) lossis nrog Dox (þDox, 1 mg / ml) ntxiv rau 24 teev los txhawb tPA-MG53 qhia. Confocal dluab pom qhov induction ntawm MG53 qhia (kev cai-ua anti-MG53 antibody, liab, qis panels) los ntawm macrophage-zoo li RAW hlwb (F4/80 staining, ntsuab, sab saum toj panels). Bars ¼ 25 mm. (B, C) Western blot tsom xam ntawm cell lysates (nyob rau hauv tus nqi ntawm 0.6, 3, thiab 6 mg) rau induced qhia ntawm tPA-MG53 thiab MG53 los ntawm cov kab mob RAW hlwb (B) thiab cov kab lis kev cai coj media concentrates (hauv ntim ntawm 2, 5, thiab 10ml) (C). Ntau qhov ntau ntawm rhMG53 (hauv qhov nyiaj ntawm 0.2, 1, 0.05, thiab 0.025 ng) tau thauj khoom raws li cov qauv. Mw, molecular luj markers.