Novel Effects of Combination Therapy Los Ntawm Inhibition of Caspase-1/ Gasdermin D Induced-Pyroptosis in Lupus Nephritis

Yog xav paub ntxiv:ali.ma@wecistanche.com

Heng Cao, Junyu Liang, Jing Liu, Ye He, Yini Ke, Yiduo Sun, Song Jiang thiab Jin Lin

TSAB NTAWV

Lub Hom Phiaj: Kev sib xyaw nrog kev kho mob nrog mycophenolate mofetil, tacrolimus, thiab steroids tau txais txiaj ntsig zoo hauv kev ua tiav kev tshem tawm hauvmob lupus nephritis(LN). Kev sib xyaw ua ke tshwj xeeb txo qis kev tswj hwm caspase-1 piv nrog monotherapy, uas tuaj yeem tshem tawm gasdermin D (GSDMD) thiab tsis ntev los no tau txheeb xyuas tias yogpyroptosistus tua neeg. Yog li ntawd, peb tau tshawb xyuas seb qhov kev kho mob sib xyaw ua ke puas muaj kev cuam tshuam ntawm caspase-1/GSDMD-mediatedpyroptosishauv LN (mob lupus nephritis).

Nyem rau cistanche deserticola ma rau nephritis

Txoj Kev: Kev nthuav qhia thiab ua kom muaj GSDMD tau kuaj pom hauv lub raum qauv ntawm tib neeg thiab nas nrog LN (mob lupus nephritis) siv immunohistochemical staining thiab immunoblotting. Thawj podocytes cais los ntawm MRLlpr nas tau incubated nrog LPS ntxiv rau ATP thiab pretreated nrog monotherapy los yog kev kho mob ua ke. Inhibition ntawm caspase-1/GSDMD-inducedpyroptosislos ntawm kev sib xyaw ua ke tau soj ntsuam hauv MRLlpr nas thiab tib neeg cov qauv. Kuv raug tshuaj xyuas siv FAM caspase-1 cov khoom siv thiab ntws cytometry. Kev sib raug zoo ntawmpyroptosisHauv cov ntshav peripheral thiab qhov systemic lupus erythematosus kab mob kev ua index (SLEDAl) tau soj ntsuam.

Cov txiaj ntsig: raum cov ntaub so ntswg los ntawm LN (mob lupus nephritis) Cov neeg mob thiab cov nas tau nthuav tawm ntau ntau ntxiv thiab kev sib cais ntawm GSDMD. Hauv kab lis kev cai podocytes, kev kho mob ua ke ua rau txo qis kev ua kom NLRP3 thiab caspase-1 thiab txo GSDMD N-terminal theem. Kev sib xyaw ua ke tau txwv cov kab mob kev loj hlob los ntawm inhibition ntawm caspase-1/GSDMD-mediatedpyroptosishauv tib neeg thiab MRL/LPR nas. Caspase-1/PI cov xov tooj ntawm tes zoo hauv cov ntshav peripheral tau sib raug zoo nrog SLE-Dal. LN (mob lupus nephritis) cov neeg mob uas tau txais kev tso cai ua tiav thiab kev tso tawm ib nrab tau txo qis cov caspase zoo kawg nkaus -1/PI cov xov tooj ntawm tes zoo piv rau cov kab hauv qab. Ac-FLTD-CMK, GSDMD-derived inhibitor, tiv thaiv kev txhim kho ntawm LN (mob lupus nephritis).

Xaus: Kev sib xyaw ua ke kho tau txo qis caspase-1/GSDMD-mediatedpyroptosishauv vitro thiab hauv vivo thiab txo tus kab mob.

Ntsiab lus:mob lupus nephritis, pyroptosis, caspase-1, gasdermin D, kho

Kev kho mob raupyroptosis

Taw qhia

Lupus nephritis(LN) yog lub raum mob tshwm sim raws li qhov tshwm sim ntawm tus kab mob autoimmune systemic lupus erythematosus (SLE).LN (mob lupus nephritis) yog qhov tshwm sim loj hauv cov neeg mob uas raug kev txom nyem los ntawm SLE. Kwv yees li ntawm 60 feem pua ​​​​ntawm cov neeg mob tsim tus kab mob no, nrog rau kwv yees li 20 feem pua ​​​​nce mus rau theem kawg kab mob raum (ESRD)(1). Kev sib xyaw ua ke nrog mycophenolate mofetil (MMF), calcineurin inhibitors (CNI), thiab cov tshuaj steroids ua rau muaj kev cuam tshuam ntxiv thiab txo cov tshuaj toxicity (2). Txoj kev kho no raug pom zoo raws li kev kho thawj zaug rau LN (mob lupus nephritis) thiab ua kom muaj txiaj ntsig zoo dua thiab ua tiav kev tshem tawm ntau dua piv nrog cov tshuaj tua kab mob hauv cov hlab ntsha cyclophosphamide (IVCY) thiab steroids (3-5).

Txhawm rau tshawb xyuas cov molecular thiab cellular mechanisms ntawm kev sib xyaw ua ke, kev txheeb xyuas kev sib txuas lus tau ua haujlwm hauv murine qauv ntawm LN. (mob lupus nephritis). MRL/LPR nas tau muab ib leeg MMF, tacrolimus, lossis prednisone ua monotherapy, lossis triple-drug ua ke. Profiles ntawm kev qhia cov noob nyob rau hauv lub raum ntawm nas nrog LN (mob lupus nephritis)tau nthuav tawm cov txheej txheem muaj peev xwm uas yuav suav tau rau qhov ntxiv thiab kev sib koom ua ke ntawm kev kho ua ke piv nrog monotherapy. Ib qho ntawm 45 cov noob caj noob ces uas tau hloov kho tshwj xeeb los ntawm kev kho ua ke yog caspase-1 (6). Nws yog qhib los ntawm inflammasomes, uas yog multiprotein oligomers uas muaj NLRP3, pro-caspase-1, thiab apoptosis-associated speck-zoo li protein (ASC), nrog rau kev sib koom ua ke caspase affinity domain. NLRP3/ASC/caspase-1 inflammasome tau raug lees paub tias yog ib qho tseem ceeb contributor rau pathogenesis ntawm LN (mob lupus nephritis). NLRP3 inflammasome drives autoinflammatory ntshawv siab los ntawm activating caspase-1, pib tsim cov pro-inflammatory cytokines xws li IL-1 , uas yog paub los ua rau podocyte dysfunction nyob rau hauv LN(7-9).

Active caspase-1 cleaves gasdermin D (GSDMD) nyob rau hauv kev sib txuas ntawm nws cov N-thiab C-terminal domains. Tom qab ntawd, N-terminal domain pab txhawb qhov pore tsim nyob rau hauv cell membrane, stimulates cellpyroptosisthiab cytokine IL-1 thiab IL-18 ntau lawm(10). Cov kev tshawb fawb tshiab tau pom tias GSDMD-mediatedpyroptosiskoom nrog hauv kev loj hlob ntawm cov kab mob xws li autoimmune encephalomyelitis (11), ntau yam sclerosis (12), cawv kab mob siab (13), thiab kab mob plab hnyuv (14). Tsis tas li ntawd, genetic lossis pharmacological inhibition ntawm caspase-1/GSDMD muaj tau pom los tiv thaiv ob lub raum los ntawm kev raug mob hauv murine qauv ntawm cov kab mob raum mob ntshav qab zib (15, 16) thiab tom qab mob raum mob (17-19). Txawm li cas los xij,pyroptosishauv LN (mob lupus nephritis)tseem hais tsis zoo rau hnub tim (20). Peb xav tias kev sib xyaw ua ke tau muab cov txiaj ntsig zoo hauv LN (mob lupus nephritis)) los ntawm inhibitingpyroptosisinduced los ntawm caspase-1/GSDMD.

Hauv txoj kev tshawb fawb tam sim no, kev ua kom cov caspase-1 thiab GSDMD hauv lub raum cov ntaub so ntswg tau tshawb xyuas nrog rau cov teebmeem ntawm kev sib xyaw ua ke ntawm caspase-1/GSDMD-dependentpyroptosisinhibition hauv MRL / LPR nas thiab cov neeg mob LN (mob lupus nephritis). Muaj peev xwm sib txheeb ntawmpyroptosisnrog rau SLE kab mob kev ua index (SLEDAI) thiab cov lus teb kho mob kuj tau soj ntsuam.

Kev kho mob raupyroptosis

Cov khoom siv thiab cov txheej txheem

Kawm Pej Xeem

Cov kws tshawb fawb tau muaj cov neeg mob kuaj pom los ntawm biopsy nrog LN (lupusnephritis) Tsawg dua lossis sib npaug li 6 lub hlis ua ntej tso npe. Tus neeg mob lub hnub nyoog muaj hnub nyoog yog 18-65 xyoo thiab txhua tus ua tau raws li kev faib tawm ntawm American College of Rheumatology rau SLE (2l, 22) thiab International Society of Nephrology/Renal Pathology Society (2003). Cov neeg mob tau raug xaiv los ntawm Zhejiang University School of Medicine thiab Nanjing University Tsev Kawm Ntawv Tshuaj Kho Mob uas muaj proteinuria (Ntau dua lossis sib npaug li 1.5 g / d) thiab cov ntshav creatinine ntau dua lossis sib npaug li 265.2 umol / L. Cov txheej txheem cais tawm suav nrog: tus neeg mob yav dhau los tau raug kho nrog tshuaj ntau ntawm methylprednisolone; biologics (txhim kho cov kab mob tshwj xeeb) lossis kev kho ua ke; plasmapheresis tam sim no lossis kev kho mob nrog globulin intravenous hauv 12 lub lis piam tom qab kuaj xyuas; lub siab ua haujlwm tsis zoo; kab mob tam sim no; thiab mob qog nqaij hlav hauv 4 lub lis piam ntawm kev pib kawm. LN (lupusnephritis) cov neeg mob tau txais kev kho mem tes nrog methylprednisolone rau 3 hnub ntawm koob tshuaj 0.5 g / d. Thaum methylprednisolone pulse therapy tiav lawm, pawg ua ke nrog prednisone (0.6 mg/kg/d), MMF (0.5 g, ob koob/d), thiab tacrolimus (2 mg, ob koob tshuaj / d) tau muab rau tag nrho 4 lub lis piam. Tom qab ntawd cov koob tshuaj ntawm prednisone, uas tseem ua ke nrog MMF (0.5 g, ob koob / d) thiab tacrolimus (2 mg, ob koob / d), raug txo los ntawm 5 mg / d txhua 2 lub lis piam mus txog thaum ua tiav koob tshuaj 10 mg/d (4). Kev tshem tawm kom tiav (CR) raug coj los ua 24-h cov zis protein ntau dua lossis sib npaug li 0.4 g, tsis muaj cov zis tso quav, cov ntshav albumin concentration ntau dua lossis sib npaug li 35 g / L, thiab cov creatinine ib txwm muaj. (23). Kev tshem tawm ib nrab (PR) raug coj los ua ntau dua lossis sib npaug li 50 feem pua ​​​​ntawm qhov txo qis hauv 24-h cov zis protein ntau kom tsawg dua lossis sib npaug li 3.5 g, cov ntshav albumin concentration ntau dua lossis sib npaug li 30 g / L, thiab ib txwm lossis tsawg dua lossis sib npaug li 25 feem pua ​​​​nce hauv cov ntshav creatinine concentration los ntawm lub hauv paus. Peb Pawg Saib Xyuas Kev Tshawb Fawb tau pom zoo txoj kev tshawb no (No. IIT20200410A) thiab txhua tus neeg mob tau sau ntawv tso cai ua ntej tso npe kawm.

Tsiaj Qauv

Poj niam MRL/emoji-FasPr(MRL/LPR) nas tau muab los ntawm SLAC Laboratory Animal Co.Ltd (Shanghai, Tuam Tshoj), tag nrho cov nas tau muab faib ua ke nrog kev pab los ntawm tus lej random generator, 5 nas nyob rau hauv txhua pab pawg thiab txhua lub tawb. . Lawv raug kho thaum muaj hnub nyoog 8 lub lis piam, nrog kev sib xyaw ntawm 0.5 mg/kg tacrolimus (MCE HY-13756), 50 mg/kg MMF (MCE HY-B0199), thiab 1 mg/ kg prednisone (MCE HY-B0214) lossis tsheb txhua hnub rau tag nrho 8 lub lis piam raws li tau piav qhia dhau los (6). Ac-FLTD-CMK tau tsim los ntawm MCE thiab 10 mg / kg tau txhaj ip txhua hnub. Txhua qhov kev kho mob tau ua rau qhov muag tsis pom kev rau cov neeg koom nrog lub luag haujlwm rau kev tshawb pom hauv qab no. Cov zis albumin-to-creatinine piv (UACR) tau txiav txim siab siv Albuwell M thiab Creatinine Companion (Excell, Phil, US). Lub raum cov qauv raug kho nyob rau hauv 4 feem pua ​​​​ntawm paraformaldehyde ua ntej histological thiab Immunohistochemical staining thiab raum cortices muab rho tawm rau immunoblotting tsom xam. Kev siv nas tau pom zoo los ntawm peb Pawg Saib Xyuas Kev Saib Xyuas Tsiaj thiab Siv Tsiaj (Tsis yog.2019-883).

Mice Glomeruli cais thiab Podocyte Culture

Glomeruli raug ntxuav tawm ntawm ob lub raum ntawm 12- poj niam hnub nyoog C57 thiab MRL/LPR nas raws li tau piav ua ntej (24). Cov nas tsuag anesthetized tau perfused nrog 37 degree C preheated magnetic hlaws. Lub raum raug tshem tawm thiab minced rau dej khov rau hauv 1 mm³. Tom qab ntawd cov ntaub so ntswg raum tau zom ntawm 37 degree nrog 1 mg / ml collagenase thiab 100 U / ml DNase I rau 30 feeb, tom qab ntawd lim ob zaug nrog 100-μm Falcon cell strainers (431752, BD). Tom qab ob peb ntxuav nrog 4 degree HBSS tov (H1025, Solarbio) thiab maj mam centrifugation ntawm 200g rau 5 feeb, glomeruli uas muaj cov hlaws dai tau sau los ntawm kev siv cov khoom sib nqus (HY-K0200, MCE) thiab ntxuav nrog HBSS daws peb zaug. Cov glomeruli cais tau los ntawm hom I collagen (C3867, Sigma-Aldrich) coated cultured tais diav hauv 5 feem pua ​​​​CO, ntawm 37 degree raws li txoj kev piav qhia los ntawm Jeffrey (25). Txoj Kev 2 podocytes tau siv hauv qhov kev sim hauv vitro.PyroptosisInduction, Cytotoxicity, thiab IL-1 Kev Tshawb Nrhiav zais cia

Podocytes los ntawm MRL / LPR nas tau primed nrog 1 mg / ml LPS (qhia-pb5lps, Hauv InvivoGen) daws hauv Opti-MEM rau 4h, tom qab ntawd ua raws li 5mM ATP (tlrlatpl, Hauv Vivo gene) stimulation. Podocytes los ntawm C57 nas raug txiav raws li ib txwm tswj. Monotherapies tacrolimus (10μM), MMF (luM), thiab prednisone (10μM), los yog kev kho ua ke tau ua ntej incubated nrog cov hlwb rau 1 teev ua ntej LPS priming. Lub cytotoxicity tom qab stimulation tau txiav txim los ntawm kev siv cov khoom siv cytotoxicity kuaj pom (11644793001, Roche) raws li cov chaw tsim khoom cov lus qhia. Lub supernatant tom qab stimulation tau sau thiab ntsuas siv IL -1 nas ELISA Kit (BMS6002, Invitrogen) raws li cov chaw tsim khoom cov lus qhia.

Antibodies thiab Reagents

Caspase-1(sc-398715, Santa Cruz Biotech), GSDMDC1 (sc-81868, Santa Cruz Biotech) qib raug soj ntsuam raws li cov chaw tsim khoom cov lus qhia.PyroptosisHauv cov ntshav peripheral tau ntsuas siv FAM FLICAMCaspase-1 Cov Khoom Siv (Bio-Rad, USA) los ntawm kev ntws cytometry.

Kev txheeb cais

Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism(vers. 8.0) thiab cov ntaub ntawv muab raws li txhais tau tias ± SD. Ob pawg kev sib piv tau ua los ntawm Tub Ntxhais Kawm T-test thiab kev txheeb xyuas kev sib raug zoo siv Pearson qhov sib txheeb. Ob tug p<0.05 was="" considered="" statistically="">

Kev kho mob raupyroptosis hauvlupus nephritis (LN) kab mob

TSEEM CEEB

GSDMD-MediatedPyroptosisYog Activated nyob rau hauv lub raum cuam tshuam los ntawm LN (lupusnephritis)

Txhawm rau tshawb xyuas seb GSDMD tau pab txhawb rau cov kab mob ntawm LN (lupusnephritis), peb thawj zaug kuaj cov protein qhia theem ntawm GSDMD hauv raum cov qauv coj los ntawm LN (lupusnephritis) cov neeg mob. Immunohistochemical tsom xam tau nthuav tawm qhov qhia tau nce qib ntawm GSDMD hauv raum biopsy cov qauv coj los ntawm LN (lupusnephritis) cov neeg mob uas muaj hom IV, V, I ntxiv rau Vand IV ntxiv rau V, thiab piv rau cov kev noj qab haus huv. GSDMD tau qhia thoob plaws hauv glomerular podocytes, tubular cells, thiab infiltrated interstitial hlwb hauv LN (lupusnephritis) cov neeg mob. Qhov chaw uas muaj qhov zoo (Xub xub liab) thiab qhov tsis zoo (Dub xub) tau nthuav dav (Daim duab 1A). Qhov feem pua ​​​​ntawm GSDMD-zoo thaj chaw hauv lub raum tau kwv yees (Daim duab 1B). Cov txiaj ntsig zoo sib xws tau pom nyob rau hauv ob lub raum ntawm MRL / LPR nas thiab kev tswj hwm tsiaj qus. Kev soj ntsuam Immunoblotting tau lees paub tias muaj kev qhia ntau ntxiv thiab kev sib cais ntawm GSDMD hauv lub raum cov qauv ntawm MRL / LPR nas (Daim duab 1C). GSDMD hauv glomerular podocytes, tubular cells thiab infiltrated interstitial hlwb tau nce siab hauv MRL / LPR nas piv rau cov nas tswj (Daim duab 1D). Cov ntaub ntawv no tau qhia tias GSDMD-mediated tau qhib rau hauv lub raum cov ntaub so ntswg cuam tshuam los ntawm LN (lupusnephritis).

DAIM 1|GSDMD tau nce siab thiab cleaved nyob rau hauv lub raum ntawm cov neeg mob thiab nas cuam tshuam los ntawm LN (lupus nephritis).

(A) Immunochemical staining ntawm GSDMD hauv lub raum cov qauv coj los ntawm cov khoom noj qab haus huv thiab LN (lupusnephritis) cov neeg mob, qhov zoo (Liab xub) thiab qhov tsis zoo staining (Dub xub) tau nthuav dav.

(B) Quantification ntawm GSDMD thaj chaw zoo yog pom nyob rau hauv hom III ntxiv rau V, IV, IV ntxiv rau V, V LN (lupusnephritis) cov neeg mob thiab cov kev noj qab haus huv.

(C) Immunoblot tsom xam ntawm tag nrho-ntev, cleaved N-terminal domain thiab C-terminal domain GSDMD hauv lub raum cortex los ntawm peb khub tswj thiab MRL / LPR nas.

(D) Immunochemical staining ntawm GSDMD hauv lub raum cov qauv coj los ntawm kev tswj thiab MRL / LPR nas.

*p < {{0}}.05,="" **p="">< 0.01.="" ln="">lupusnephritis), lupusnephritis; FL, tag nrho-ntev; NT, N-terminal sau npe; CT, C-terminal sau npe.

Kev sib xyaw ua ke txo qis GSDMD-MediatedPyroptosisUa kom muaj zog hauv Primary Podocytes Hauv Vitro

Txhawm rau tshawb xyuas qhov cuam tshuam inhibitory ntawm kev kho ua ke ntawmpyroptosis, peb siv LPS ntxiv rau ATP stimulation txoj kev los ntxiaspyroptosishauv podocytes los ntawm MRL / LPR nas, noj podocytes los ntawm cov nas qus-hom C57 raws li kev tswj hwm ib txwm muaj. Podocytes los ntawm MRLlpr nas tau primed rau 4h nrog LPS thiab tom qab ntawd kho rau 4h nrog ATP, ib qho kev txhawb nqa rau GSDMD-mediatedpyroptosiskev ua haujlwm (26). Piv nrog rau kev tswj hwm ib txwm muaj, podocytes tom qab ntawd nthuav tawm pyroptotic morphological nta xws li cell o thiab bubbling. Pretreatment nrog kev sib xyaw ua ke ua kom muaj txiaj ntsig zoo ntawm pyroptotic morphological nta thiab cytotoxicity (Daim duab 2A). LPS ntxiv rau ATP induced podocytes cytotoxicity. Piv txwv li, kev sib xyaw ua ke tau txo qis cytotoxicity thiab IL -1 tso tawm hauv supernatant induced los ntawm LPS ntxiv rau ATP stimulation (Figures 2B, C). Txhawm rau txheeb xyuas ntxiv qhov ua kom GSDMD-mediatedpyroptosis, peb soj ntsuam canonical inflammasomes thiab GSDMD ua kom siv immunoblotting. Raws li kev hloov pauv ntawm pyroptotic morphological thiab cytotoxicity, LPS ntxiv rau ATP ua rau NLRP3, caspase-1, thiab cleaved GSDMD hauv podocytes (Daim duab 2D). Kev kho mob sib xyaw ua ke tau txwv tsis pub ua kom NLRP3 thiab caspase-1 thiab txo qib ntawm GSDMD N-terminal. Qhov tseem ceeb, kev kho mob monotherapy nrog tacrolimus, MMF, los yog prednisone suppressed LPS ntxiv rau ATP stimulated cell cytotoxicity thiab L-1 secretion, tab sis kev sib xyaw ua ke tau pom tias muaj kev cuam tshuam loj dua ntawm Caspase-1 thiab GSDMD. Cov txiaj ntsig no tau muab pov thawj uas txhawb nqa qhov kev pom tias kev kho mob sib xyaw ua ke ameliorated GSDMD-mediatedpyroptosislos ntawm kev txwv kev ua kom NLRP3 thiab caspase-1.

DAIM 2|Kev sib xyaw ua ke ntawm kev kho mob ameliorates pyroptosis hauv podocytes hauv vitro.

(A) Pyroptotic nta tau nthuav tawm hauv kab lis kev cai thawj podocytes tom qab incubated nrog LPS ntxiv rau ATP thiab pretreated nrog kev kho mob ua ke. Cov xib xub qhia cov hlwb pom cov yam ntxwv zoo li pyroptotic.

(B) Supernatants los ntawm cov hlwb sib tw tau soj ntsuam rau kev tuag ntawm tes, raws li ntsuas los ntawm LDH secretion.

(C) Supernatants los ntawm cov hlwb sib tw tau txheeb xyuas rau IL-1b tso tawm.

(D) Immunoblotting tsom xam ntawm NLRP3, caspase-1, GSDMD-NT, thiab ASC qhia tom qab tacrolimus, MMF, prednisone, los yog kev kho mob ua ke tom qab NLRP3 inflammasome activation.

*p < {0}}05;="" **p="">< 0.01;="" ***p=""><0.001; ****p=""><0.0001. ldh,="" lactate="">

DSMO, Dimethyl sulfoxide, FK506, tacrolimus; MMF, mycophenolate mofetil; PNS, prednisone; COM, kev sib xyaw ua ke; ASC, apoptosis-associated speck-zoo li protein.

Combination Treatment Suppressed Caspase-1/GSDMD-InducedPyroptosishauv MRL/LPR nas

Yim-lub lim tiam MRL / LPR nas tau muab kev sib xyaw ua ke ntawm MMF, tacrolimus, thiab prednisone lossis tswj tsheb. Raws li kev tshawb fawb yav dhau los (6), tom qab kev kho mob rau 8 lub lis piam, tsis muaj qhov sib txawv ntawm kev tuag lossis lub cev hnyav ntawm lub tsheb thiab cov pab pawg kho mob sib xyaw. Nyob rau hauv sib piv rau lub tsheb pab pawg neeg, kev kho mob ua ke tau zoo heev nyob rau hauv kev tiv thaiv proteinuria, glomerulosclerosis, thiab lub raum interstitial immune hlwb infiltration (Ntxiv daim duab 1).

Tom ntej no, peb tau tshuaj xyuas caspase-1 thiab GSDMD cov theem qhia hauv kev kho ua ke thiab pab pawg tswj tsheb. Immunohistochemical tsom xam tau qhia tias glomerular thiab lub raum interstitial caspase-1 thiab GSDMD qhia tau txo qis hauv pawg kho mob ua ke (Daim duab 3A). Kev tshuaj ntsuam xyuas immunoblotting kuj pom qhov txo qis thiab qhov sib cais ntawm caspase-1 thiab GSDMD hauv lub raum cortices ntawm pawg kho mob ua ke (Daim duab 3B). Ntxiv mus, caspase-1/propidium iodide (PI) ob-zoo hlwb raug suav nrog cov flow cytometry thiab apyroptosisFAM Caspase-1kit. Cov txiaj ntsig tau nthuav tawm qhov kev txo qis hauv caspase-1*/PI' cov xov tooj ntawm tes hauv nruab nrab hauv MRL / LPR nas uas tau txais kev kho mob sib xyaw ua ke piv nrog MRL / LPR nas kho nrog lub tsheb.(Figures 3C,D). Tsis tas li ntawd, kev kho mob ua ke kuj tseem txo qis cov ntshav ntshav Ii concentration (Daim duab 3E). Yog li peb tau txiav txim siab tias kev kho mob sib xyaw ua ke txo qis caspase-1/GSDMD-inducedpyroptosisnyob rau hauv lub raum thiab cov qe ntshav ntawm LN (lupusnephritis) mus.

DAIM 3|Kev sib xyaw ua ke kev kho mob txo qis caspase-1/GSDMD-mediated pyroptosis hauv MRL/LPR nas.

(A) Immunochemical staining ntawm GSDMD thiab caspase-1 hauv MRL/LPR nas cov kab mob raum. Piv nrog rau kev tswj lub tsheb, caspase-1 thiab GSDMD kev qhia tau raug txwv tsis pub muaj kev cuam tshuam hauv pawg kho mob ua ke.

(B) Immunoblot tsom xam ntawm tag nrho-ntev thiab cleaved GSDMD thiab caspase-1 nyob rau hauv lub raum cortex specimens ntawm MRL/LPR nas; kev sib piv ntawm cov txiaj ntsig tau los ntawm cov tsheb thiab cov pab pawg kho mob ua ke.

(C) FACS ntawm caspase-1/PI ob-zoo hlwb nyob rau hauv peripheral ntshav los ntawm MRL/LPR nas uas tau muab los yog tsis tau muab kev kho mob ua ke.

(D) Quantification of caspase-1/PI double-positive cells in peripheral blood of MRL/LPR nas muab los yog tsis muab kev kho ua ke.

(E) Qhov ntau npaum ntawm cov ntshav IL{{0}}b concentrations hauv MRL/LPR nas muab los yog tsis muab kev kho mob ua ke. ####P <0.0001. facs,="" fluorescence-activated="" cell="" sorting;="" ct,="" kev="" sib="" xyaw="" ua="">

Combination Therapy Suppressed Caspase-1/GSDMD-InducedPyroptosishauv LN (lupusnephritis) Cov neeg mob

Nyob nruab nrab ntawm Lub Ib Hlis 2020 thiab Lub Rau Hli 2020, 47 cov neeg mob uas muaj LN (lupusnephritis) tau cuv npe thiab 43 ua tiav 24-lub lim tiam ntawm kev kho ua ke. Cov kab mob hauv paus thiab cov pej xeem ntawm pawg neeg sau npe tau teev nyob rau hauv Daim Ntawv Ntxiv 1. Quantification of caspase-1/propidium iodide(PI ob-zoo hlwb tau ua rau cov neeg mob LN (lupusnephritis) hom III, IV, V, III ntxiv V, IV ntxiv rau V thiab hauv kev noj qab haus huv uas siv apyroptosisFAM Caspase-1 cov khoom siv thiab ntws cytometry. Piv nrog rau kev tswj hwm kev noj qab haus huv, cov naj npawb ntawm caspase-1 / PI ob lub hlwb zoo hauv cov ntshav peripheral tau nce (Figures 4A, B). Peb kuj tau tshuaj xyuas cov kab mob caspase-1/PI ob-zoo hlwb tom qab 24-lub lim tiam ntawm kev kho mob thiab nyob rau hauv lub hauv paus. Tom qab 24-lub lim tiam ntawm kev kho induction, qhov tshwm sim ntawm CR thiab PR yog 39.5 feem pua ​​(17/43) thiab 32.6 feem pua ​​(14/43), feem. Lub caspase-1/PI ob-zoo xov tooj ntawm tes nyob rau hauv cov ntshav peripheral ntawm CR thiab PR cov neeg mob tau txo qis heev piv rau cov hauv paus (Daim duab 4C). Peb kuj tau txheeb xyuas qhov sib cuam tshuam ntawm caspase -1 inducedpyroptosisthiab SLEDA; cov ntaub ntawv qhia meej meej tias caspase-1/PI qhov zoo piv rau hauv cov ntshav peripheral tau zoo sib raug zoo nrog SLEDAI (Daim duab 4D). Ac-FLTD-CMK, GSDMD-Derived Inhibitor, Tiv Thaiv Kev Txhim Kho ntawm LN (lupusnephritis).

DAIM 4|Kev sib xyaw ua ke ntawm kev kho mob txo qis caspase-1-kev kho mob pyroptosis hauv LN (lupus nephritis) cov neeg mob.

(A) FACS ntawm caspase-1/PI ob-zoo hlwb hauv peripheral ntshav ntawm LN (lupusnephritis) cov neeg mob tau kho nrog lossis tsis muaj kev sib xyaw ua ke.

(B) Quantification of caspase-1/PI ob-positive hlwb nyob rau hauv peripheral ntshav ntawm hom III, IV, V, III plus V, thiab IV plus V LN (lupusnephritis) cov neeg mob thiab cov kev noj qab haus huv.

(C) Quantification of caspase-1/PI ob-positive cells hauv peripheral ntshav ntawm LN (lupusnephritis) Cov neeg mob ntawm lub hauv paus, CR, PR, thiab kev tsis tso cai tom qab kev sib xyaw ua ke.

(D) Kev txheeb xyuas kev sib txheeb ntawm caspase-1/PI ob-zoo hlwb piv nrog SLE-DAI.

**p < 0.01.="" facs,="" fluorescence-activated="" cell="" sorting;="" ln="">lupusnephritis), lupusnephritis; CR, ua kom tiav kev zam txim; PR, kev tso cai ib nrab; SLE-DAI, systemic lupus erythematosus kab mob ua haujlwm Performance index.

Ib txoj kev tshawb fawb tsis ntev los no tau pom tias GSDMDhmice txhim kho lub raum C3 thiab IgG deposition, mob raum mob hnyav dua, thiab txhim kho kev tuag hauv tus qauv imiquimod-induced ntawm SLE.17 Los qhia ntxiv txog lub luag haujlwm ntawm GSDMD hauv LN (lupusnephritis), MRL/LPR nas tau kho nrog Ac-FLTD-CMK21 pib ntawm 8-lub lim tiam, nrog lub tsheb raws li kev tswj hwm. Tom qab 8- lub lis piam ntawm kev kho mob, tsis muaj qhov sib txawv ntawm cov neeg tuag lossis lub cev hnyav ntawm lub tsheb thiab Ac-FLTD-CMK kho pab pawg. Piv nrog rau cov nas tswj lub tsheb, Ac-FLTD-CMK-kho MRL / LPR nas muaj cov zis qis dua albumin-creatinine piv (Daim duab 5A), qis dua cov ntshav creatinine concentration (Daim duab 5B), txo cov glomerulosclerosis (Daim duab 5C), thiab CD3. , CD4, thiab CD68 positive cell infiltration (Daim duab 5D). Immunohistochemistry cov txiaj ntsig tau pom qhov txo qis ntawm GSDMD (Daim duab 5D) thiab inhibited serum IL-1 secretion txiav txim siab los ntawm ELISA (Daim duab 5E). Ua ke, cov ntaub ntawv qhia tias Ac-FLTD-CMK inhibitedpyroptosishauv lub raum cuam tshuam los ntawm LN (lupusnephritis).

DAIM 5|Ac-FLTD-CMK, GSDMD-derived inhibitor, tiv thaiv kev txhim kho ntawm LN (lupus nephritis).

(A) Cov zis albumin-rau-creatinine piv tau suav nrog hauv MRL / LPR nas muab lossis tsis tau muab GSDMD inhibitor.

(B) Serum creatinine hauv MRL / LPR nas muab lossis tsis tau muab GSDMD inhibitor.

(C) Cov duab sawv cev ntawm lub raum cov qauv stained nrog Periodic acid-Schiff (PAS) thiab quantification glomerulosclerosis feem pua.

(D) Immunochemical staining ntawm CD3, CD4, F4/80, thiab GSDMD. (E) Quantification of serum IL-1b concentrations in MRL/LPR nas muab los tsis muab GSDMD inhibitor.

Cov ntaub ntawv qhia raws li qhov nruab nrab ± SEM. #p < {{0}}.05,="" ##p=""><>

Kev sib tham

Kev sib xyaw ua ke yog ib lub tswv yim zoo vim tias muaj CR ntau dua thiab tag nrho cov lus teb, lub sijhawm luv luv rau kev tshem tawm, thiab cov kev mob tshwm sim tsawg dua. Txog tam sim no, lub hauv paus molecular mechanisms ntawm kev sib xyaw ua ke tsis tau paub. Tsis ntev los no, Fu et al.(6) kho MRL / LPR nas nrog kev kho mob monotherapy lossis kev sib xyaw ua ke thiab ua tiav kev tshuaj xyuas cov ntaub ntawv ntawm tag nrho ob lub raum. Interestingly, lawv pom tias kev kho mob sib xyaw ua ke tab sis tsis yog monotherapy tshwj xeeb thiab tshwj xeeb suppressed caspase-1 qhia.

NLRP3/ASC/caspase-1 inflammasome tam sim no tau lees paub tias yog ib qho tseem ceeb pab txhawb rau cov kab mob ntawm LN (lupusnephritis). NLRP3 inflammasomes hauv podocytes tau qhib rau hauv cov nas uas muaj kab mob lupus thiab tseem nyob hauv cov neeg mob LN (lupusnephritis). Thaum NLRP3 inflammasomes raug txwv nrog ib qho caspase -1 inhibitor, proteinuria, lub raum histological lesions, thiab podocyte ko taw txheej txheem effacement tau pom tias yuav ameliorated hauv lupus-ntau nas (8). Huang et al. (27) tshaj tawm tias NLRP3 tau nthuav tawm hauv tubular hlwb ntawm LN (lupusnephritis) chav kawm IV thiab qhov kev ua kom NLRP3 tau zoo cuam tshuam nrog cov qhab nia ua haujlwm rau cov neeg mob LN. Caspase-1 tau raug pov thawj los ua lub luag haujlwm tseem ceeb hauv lupus thiab vascular dysfunction, thiab yog li ntawd yog lub hom phiaj rau kev kho mob tshiab (28). Kev sim tshuaj tshiab xws li RIP3(29), P2X7 receptor antagonists (30), GSK-3 inhibitors (31) thiab Nrf2 agonist (32) inhibited txoj kev loj hlob ntawm LN (lupusnephritis) hauv MRL/LPR nas los ntawm kev hloov kho NLRP3/ASC/caspase-1 inflammasome kev ua, uas qhia txog qhov tseem ceeb thiab kev thuam ntawm NLRP3/ASC/caspase-1 inflammasome hauv LN. Kev nce ntshav siab ntau ntawm IL-18 cuam tshuam nrog cov kab mob hnyav thiab qib ntawm lub raum raug mob / kev koom tes hauv cov neeg mob SLE (33). Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, nws zoo nkaus li tias tsev neeg gasdermin yuav yog qhov tseem ceeb rau kev loj hlob ntawmpyroptosis, tab sis tau muaj pov thawj tsis txaus rau qhov muaj GSDMD / GSDME hauv cov neeg mob SLE.

Zuag qhia tag nrho, peb tau tshuaj xyuas qhov ua kom thiab kev qhia ntawm GSDMD hauv lub raum biopsy cov qauv coj los ntawm LN (lupusnephritis) cov neeg mob thiab MRL/LPR nas. GSDMD tau ua kom muaj zog thiab cleaved hauv glomerular podocytes thiab tubular hlwb thiab infiltrated interstitial hlwb. Tom qab ntawd, MRL / LPR nas tau muab kev sib xyaw ua ke ntawm MMF, tacrolimus, thiab prednisone lossis tsheb. Piv nrog rau pawg tswj hwm lub tsheb, kev ua kom cov caspase-1 thiab GSDMD raug txo qis hauv kev sib xyaw ua ke pab pawg. Qhov degree ntawmpyroptosisHauv cov ntshav peripheral kuj tseem qis dua hauv LN (lupusnephritis) cov neeg mob tom qab kev tswj hwm kev sib xyaw ua ke. Ntxiv mus,pyroptosisnyob rau hauv cov ntshav peripheral tau zoo correlated nrog SLEDAI, uas qhia tiaspyroptosishauv cov ntshav peripheral tuaj yeem ua tus cim tseem ceeb rau kev ua SLE. Yog li peb xav tias kev kho ua ke tuaj yeem ua rau muaj txiaj ntsig zoo hauv LN (lupusnephritis) los ntawm inhibitingpyroptosisinduced los ntawm caspase-1/GSDMD. Yav dhau los crystal structural kev tshawb fawb ntawm caspase-1 nyob rau hauv ib tug complex nrog Ac-FLTD-CMK qhia ntau enzyme-inhibitor kev sib cuam tshuam uas tiv thaiv GSDMD paub los ntawm caspase-1. Txawm li cas los xij, peb pom tias Ac-FLTD-CMK, uas ua raws li GSDMD-derived inhibitor, inhibitedpyroptosishauv LN (lupusnephritis), uas tej zaum yuav induced los ntawm ib tug txawv mechanism.

Tsis ntev los no, hauv TLR7-induced murine qauv ntawm SLE(34), GSDMD-/- nas tau tsim muaj ntau dua ntawm lub raum kev puas tsuaj thiab kev tuag ntau ntxiv, uas pib qhia tias GSDMD tshem tawm yuav muaj kev tiv thaiv thiab cuam tshuam nrog systemic o thiab zus tau tej cov autoantibodies uas txuam nrog nce kev tuag. GSDMD-cov nas uas tsis muaj peev xwm txhim kho qhov muaj txiaj ntsig zoo ntawm cov kab mob glomerular hauv cov kab mob hauv lub raum thiab cov proteinuria ntau dua piv nrog cov nas qus. Enhanced secretion ntawm extracellular HMGB1 kuj pom nyob rau hauv GSDMD-/lymphocytes qhia tias necrosis/pyroptosistshwm sim thaum GSDMD tsis tuaj.

Caspase-1 ua raupyroptosislos ntawm GSDMD cleavage. Hauv qhov sib piv, hauv GSDMD-tsis muaj lossis GSDMD-tsawg hlwb, caspase-1 ua rau apoptosis thiab tom qab ntawd GSDME-nyob ntawm theem nrab necrosis thiabpyroptosis. Thaum GSDMD tsis ua haujlwm tom qab kho, GSDME tuaj yeem hloov pauv caspase-3- kho apoptosis raupyroptosis(35,36). Tsis tas li ntawd, kev ua kom cov caspase-8 tau txhim kho ntau yam hauv macrophages uas tsis muaj GSDMD. Kev ua kom muaj txiaj ntsig tseem ceeb ntawm caspase-8 kuj pom muaj nyob hauv TLR7-induced GSDMD-deficient LN (lupusnephritis) nas(34). Yog li, thaum tsis muaj GSDMD, nws zoo li caspase-1 koom tes nrog caspase-8 ua rau caspase-3-GSDME-mediated apoptosis hloov mus raupyroptosishauv LN (lupusnephritis), ib qho kev xav uas xav tau kev tshawb nrhiav ntxiv.

Muaj ntau qhov kev txwv hauv txoj kev tshawb fawb tam sim no. Ua ntej, raws li cov ntaub ntawv tam sim no, peb tau txheeb xyuas tias kev sib xyaw ua ke tuaj yeem cuam tshuampyroptosis, tab sis lwm yam kev kho mob xws li ob txoj kev kho mob ntawm steroids thiab MMF, steroids thiab tacrolimus, los yog anti-CD20 antibody kuj tseem muaj qhov sib piv. Qhov thib ob, qhov tseeb molecular mechanism ntawm inhibition nyhuv ntawm kev kho ua ke ntawm caspase -1 tsis zoo yuav raug tshawb nrhiav. Qhov kawg tab sis tsis kawg, peb soj ntsuam kev sib raug zoo ntawm caspase-1/PI ob lub hlwb zoo hauv cov ntshav peripheral nrog SLE-DAI thiab kev hloov pauv ua ntej thiab tom qab kev kho mob ua ke hauv kev rov qab los LN (lupusnephritis) neeg mob cohort. Tus nqi kho mob qhov kev kwv yees ntawm caspase-1/PI ob lub hlwb zoo rau kev ua haujlwm ntawm cov kab mob thiab kev kho mob yuav tsum tau tshawb xyuas ntxiv nyob rau hauv pawg loj dua yav tom ntej.

Zuag qhia tag nrho, peb tau txheeb xyuas tias kev kho mob ua ke tau muab nws cov txiaj ntsig zoo los ntawm kev txo qis caspase-1/GSDMD-inducedpyroptosishauv LN (lupusnephritis), uas tau muab ib qho kev pom tshiab rau hauv cellular mechanisms koom nrog. Tseeb tiag, kev sib xyaw ua ke kho tau txo qis caspase-1 qhia tshwj xeeb thiab tshwj xeeb. Pharmacological lossis genetic inhibition ntawm NLRP3 thiab caspase -1 txhim kho lub raum ua haujlwm thiab txo qis autoimmunity hauv LN (lupusnephritis). Lub luag haujlwm pathophysiological ntawm caspase-1 hauv LN (lupusnephritis) tsim nyog rau kev kawm ntxiv, thiab cov kev kho mob tsom rau GSDMD hauv cov neeg mob LN (lupusnephritis) yuav tsum tau ua tib zoo soj ntsuam.

Txoj kev kho raupyroptosis

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