Exogenous Wnt1 tiv thaiv mob raum mob thiab nws qhov kev loj hlob ntxiv mus rau mob raum mob

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

Xu Hong^1†, Yanni Zhou^2†, Dedong Vang^3†, Fuping Liu⁴, Tianjun Guan³, Youhua Liu^1,5thiab Liangxiang Xiao^3*

¹State Key Laboratory ntawm Organ Failure Research, National Clinical Research Center of Kidney Disease, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, Suav teb,²Department of Nephrology, XiamenHospital Affiliated rau Beijing University of suav tshuaj, Xiamen, Suav teb,³Department of Nephrology, ZhongshanHospital Affiliated to Xiamen University, Xiamen, Suav teb,⁴Department of Endocrinology thiab Diabetes, Thawj Lub Tsev Kho Mob ntawm Xiamen University, Xiamen, Suav,⁵Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, Tebchaws Asmeskas

Cov kev tshawb fawb qhia tias Wnt / -catenin agonists tau txais txiaj ntsig zoo hauv kev kho mobraug mob acutekidney(AKI); Txawm li cas los xij, nws tseem tsis tau paub txog nws lub luag haujlwm hauv kev tiv thaiv AKI thiab nws txoj kev loj hlob mus rau kab mob raum ntev (CKD). Nyob rau hauv txoj kev tshawb no, lub raum Wnt / -cateninsignaling yog qhib los ntawm overexpression ntawm exogenous Wnt1 los yog inhibited byadministration nrog ICG-001, ib tug me me molecule inhibitor ntawm -catenin signaling, ua ntejmice raug rau ischemia / reperfusion raug mob (IRI) induce AKI thiab tom qab CKD. Peb cov txiaj ntsig tau pom tias nyob rau hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR tiv thaiv nas tiv thaiv AKI, thiab cuam tshuam qhov kev loj hlob ntawm AKI rau CKD hauv cov nas, ua pov thawj los ntawm ob qho tib si ntshav biochemical thiab lub raum histological analyses. Hauv qhov sib piv, kev kho ua ntej ntawm ICG-001 ua ntej IR tsis cuam tshuam rau lub raum Wnt/ -catenin signaling orthe progression of AKI to CKD. Mechanistically, nyob rau hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR suppressed qhov kev qhia ntawm proapoptotic proteins nyob rau hauv AKI nas thiab txo cov inflammatory teb nyob rau hauv ob qho tib si AKI thiab CKD nas. Tsis tas li ntawd, exogenous Wnt1inhibited apoptosis ntawm tubular hlwb induced los ntawm hypoxia-reoxygenation (H / R) kev kho mob hauv vitro. Txhawm rau xaus, qhov kev tshawb fawb tam sim no muab pov thawj los txhawb kev tiv thaiv kev tiv thaiv ntawm Wnt / -catenin ua rau ntawm IR-txog AKI thiab nws cov kev nce qib tom ntej mus rau CKD.

Keywords: mob raum raug mob, mob raum mob, Wnt1, -catenin, ischemia-reperfusion raug mob

Kev kho mob ntawmmob raum raug mob(AKI): cistanche

Taw qhia

Mob raum mob (AKI)yog ib qho kev kho mob vim tias lub raum tsis ua haujlwm sai los ntawm kev raug mob ntau yam, xws li tshuaj, ischemia, sepsis, thiab co toxins (Chawla and Kimmel, 2012; Scholz et al., 2021).AKI yog txuam nrog kev mob hnyav heev thiab Kev tuag, lub luag haujlwm rau kwv yees li 2 lab tus neeg tuag txhua xyoo thoob ntiaj teb (Belayev thiab Palevsky, 2014). Tshwj xeeb tshaj yog, AKI yog ib tus kws paub txog kev ua haujlwm ywj pheej rau kev txhim kho cov kab mob raum tsis zoo (CKD) lossis kab mob raum kawg (ESRD), uas tau muab lub nra hnyav rau kev noj qab haus huv (Leung et al., 2013; Belayev thiab Palevsky, 2014; Kurzhagenet al., 2020). Hmoov tsis zoo, tsis muaj cov tshuaj zoo rau kev tiv thaiv thiab kho AKI.

Wnt/ -catenin signaling plays lub luag haujlwm tseem ceeb hauv kev ua haujlwm tsis zoo, cov ntaub so ntswg homeostasis, thiab kev loj hlob ntawm ntau yam kab mob, suav nrog cov kab mob raum (Soni, 2021). -Catenin muaj ob lub luag haujlwm los ntawm kev ua haujlwm raws li ob qho tib si structuralprotein thiab tus tswj hwm kev hloov pauv (MacDonald li al.,2009). Lub membrane-bound -catenin yog ib feem ntawm adherensjunction complex thiab pab txhawb rau cell-cell kev sib cuam tshuam, whereas lub cytosolic -catenin yog phosphorylated los ntawm glycogensynthase kinase -3 (GSK-3 ) thiab tsom rau ubiquitination thiab proteasomal degradation. Txawm li cas los xij, xws li degradation ntawm -catenin tuaj yeem cawm tau los ntawm Wnt ligands, tsev neeg ntawm glycoproteins zais cia nrog ntau yam kev ua haujlwm lom neeg.Qhov kev khi ntawm Wnt stabilizes -catenin, uas translocatesinto lub nucleus thiab ua raws li kev hloov pauv rau kev tsim cov noob caj noob ces. Qhov tseem ceeb, Wnt / -catenin signaling kuj tseem nyob hauv cov neeg laus lub raum tsis raug mob tab sis rov ua haujlwm thaum lub raum mob hnyav thiab mob ntev (Huffstater li al., 2020). Ua kom muaj Wnt / -catenin thaum ntxov ntawm AKI txo qis kev raug mob thiab txhawb kev rov zoo. ntawm lub raum ua haujlwm, qhov kev txhawb nqa Wnt / -catenin activation tuaj yeem tsav kev loj hlob ntawm AKI mus rau CKD (Xiao li al., 2016; Huffstater li al., 2020). Yog li ntawd, Wnt / -catenin signaling yog ob-edgedsword nyob rau hauv lub raum raug mob thiab warrants ib tug zoo dua kev nkag siab.Ntawv qhia zaub mov, nws tseem elusive txog lub luag hauj lwm ntawm Wnt / -cateninactivation nyob rau hauv kev tiv thaiv ntawm AKI thiab nws tom ntej AKI-CKD kev loj hlob.

Lub raum ischemia-reperfusion raug mob (IRI) yog suav tias yog qhov ua rau AKI. Yog li ntawd, tus qauv tsiaj lub raum IRI tau nquag siv rau kev tshawb fawb ntawm AKI thiab AKI-CKDprogression (Bonventre and Yang, 2011). Ob txoj kev tshuaj tshuaj thiab caj ces tau siv los piav qhia lub luag haujlwm ntawm Wnt / -catenin activation hauv IRI qauv ntawm AKI thiab CKD, feem. Lithium thiab GSK-3 inhibitors feem ntau yog siv los ua pharmacologically qhib Wnt / -cateninsignaling hauv preclinical AKI qauv, tab sis nws yuav tsum tau sau tseg txog lawv cov -catenin-kev ywj pheej cov teebmeem (Bao et al., 2014).Genetic modifications of GSK{{9 }} , -catenin, los yog Wnt ligands (Wnt1 thiab Wnt9a) tau siv los qhia lub luag haujlwm ntawm Wnt/ -catenin ua kom nyob rau hauv AKI thiab CKD (Bonventre andYang, 2011; Bao et al., 2014; Zhou et al., 2013, 2018; Liuet al., 2020). Txawm li cas los xij, kev tshawb fawb txog lub luag haujlwm ntawm Wnt / -cateninsignaling hauv kev tiv thaiv AKI yog qhov tsawg heev. Cov kev tshawb fawb tam sim no tsom los piav qhia lub luag haujlwm ntawm Wnt / -catenin signaling hauv kev tiv thaiv ntawm AKI thiab nws qhov kev loj hlob ntxiv rau CKD.

FIGURE1 | In vivo expression of exogenous Wnt1 before IR prevents AKI and activates renal β-catenin in mice

DAIM 1|Hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR tiv thaiv AKI thiab ua kom lub raum -catenin hauv cov nas.

(A)Cov pab nas kho mob. Cov nas raug kho nrog pcDNA3 (IRI ntxiv rau pcDNA3) lossis PHA-Wnt1 (IRI ntxiv rau PHA-Wnt1) plasmid ntawm hydrodynamic tail vein txhaj ntawm 2 hnub ua ntej BIRI. Cov qib creatinine hauv cov ntshav(B)thiab ntshav urea nitrogen (BUN) qib(C) raug ntsuas. (D) Cov neeg sawv cev micrographs ntawm lub raum morphology ntawm 1 hnub tom qab BIRI. Cov kab mob raum tau raug rau Periodic acid-Schiff staining. Boxed thaj chaw yog loj. Cov xub qhia txog lub raum tubules. Scale bar, 200 µm.(E, F)Western blot tsom xam ntawm active -catenin nyob rau hauv lub raum ntawm nas. *P < 0.05="" tiv="" thaiv="" sham="" (n="5)." †p="">< 0.05="" versus="" iri="" txhaj="" nrog="" pcdna3="" plasmid="" (n="">

Cistanche txhim kho lub raum ua haujlwm

Cov khoom siv thiab cov txheej txheem

Kev Kawm Tsiaj

Txiv neej C57BL / 6 nas hnyav kwv yees li 22-25 g tau yuav los ntawm Vital River Laboratory Tsiaj Technology (Beijing, Tuam Tshoj), thiab khaws cia hauv cov chaw tsiaj (kub: 22 ± 2◦C, av noo: 60 ± 5 feem pua, 12h tsaus / lub teeb ci ) ntawm Southern Medical University (Guangzhou, Tuam Tshoj) nrog dej thiab zaub mov ad libitum. Cov kev tshawb fawb tsiaj tau pom zoo los ntawm Animal Ethical and Welfare Committee ntawm Southern MedicalUniversity. Txhua tus tsiaj raug kho raws li Phau Ntawv Qhia Txog Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Txhaum Cai.

Qhov induction ntawm AKI thiab CKD hauv nas tau piav qhia peb cov kev tshawb fawb yav dhau los (Xiao li al., 2016; Zhou et al., 2018). Ob sab lub raum ischemia/reperfusion raug mob (BIRI) tau siv dav los ntxias AKI hauv cov nas, whereasit nce kev tuag thaum lub sij hawm kev loj hlob ntawm AKI toCKD. Yog li, unilateral lub raum ischemia / reperfusioninjury (UIRI) yog siv rau kev tshawb fawb txog kev loj hlob ntawm AKI rau CKD. Luv luv, ib qho kev phais plab hauv nruab nrab tau ua rau tus nas nyob rau hauv cov tshuaj loog, thiab ob sab laug lub raum pedicles raug txiav rau 30 min siv microaneurysm clamps (khoom tsis. 18051-35; Fine ScienceTools, Cambridge, United Kingdom). Thaum lub sij hawm phais, lub cev kub tau khaws cia ntawm kwv yees li 37-38 ◦C siv qhov ntsuas kub tswj qhov cua sov.

Txhawm rau ntsuas qhov kev tiv thaiv ntawm Wnt / -catenin activationon AKI, nas tau txhaj nrog 1 mg / kg hemagglutinin (HA)-tagged Wnt1 qhia vector (PHA-Wnt1, UpstateBiotechnology) lossis khoob vector (pcDNA3) 2 hnub ua ntejBIRI siv hydrodynamic- raws li cov txheej txheem hloov noob caj noob ces tau tshaj tawm yav dhau los (Xiao li al., 2016). Cov ntshav thiab lub raum cov ntaub so ntswg tau sau 1 hnub tom qab lub raum BIRI.

Txhawm rau ntsuas qhov tshwm sim ntawm Wnt / -catenin thaum ntxov ntawm kev nce qib ntawm AKI rau CKD, cov nas tau raug rau txhua hnub hydrodynamic tail vein txhaj ntawm Wnt1 expressionplasmids ntawm 1 mg / kg lossis niaj hnub intraperitoneal txhaj ntawm ICG-001({{4 }}, Chembest, Shanghai, Suav teb) ntawm 5 mg / kg rau 3 hnub ua ntej UIRI. Cov ntshav thiab lub raum cov ntaub so ntswg tau sau 11 hnub tom qab UIRI. Hauv peb qhov kev tshawb fawb ua ntej, 2 hnub ntawm kev txhaj tshuaj Wnt1 tuaj yeem ua rau muaj kev qhia ntawm Wnt1 hauv AKI nas (cov ntaub ntawv tsis qhia). Nws yuav tsum raug sau tseg tias 3 hnub ntawm Wnt1 txhaj tshuaj raug xaiv los xyuas kom meej thiab ua kom ntev ntawm Wnt1 thaum lub sijhawm kev loj hlob ntawm AKI rau CKD.

Acute kidney injury is a clinical condition due to a rapid loss of renal function

Mob raum raug mob (AKI) yog ib qho kev kho mob vim yog lub raum tsis ua haujlwm sai

Cell Kab lis kev cai thiab kev kho mob

Human proximal tubular epithelial cell line (HKC-8) tau muab los ntawm Dr. L. Racusen ntawm Johns Hopkins University (Baltimore, MD, United States). Cell kab lis kev cai tau ua raws li tau piav qhia (Zhou et al., 2013). HKC-8 hlwb raug kho nrog tib neeg recombinant Wnt1 (SRP4754; Sigma-Aldrich, St.Louis, MO, United States) ntawm 100 ng/mL los yog ICG-001 (847591-62-2, Chembest, Shanghai , Tuam Tshoj) ntawm 10 µM rau 4 h. Cov hlwb raug muab tso rau hauv qhov chaw ua haujlwm hypoxia (X3-CK, Biospherix) ntawm 1 feem pua pO2 rau 48 teev. Tom qab ntawd, cov hlwb tau rov ua oxygen rau 2 teev ua ntej sau rau ntau yam kev tshuaj ntsuam.

FIGURE2| In vivo expression of exogenous Wnt1 before IR reduces tubular cell apoptosis and NF-κB activation in AKI mice

DAIM 2|Hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR txo cov tubular cell apoptosis thiab NF-κB ua kom muaj zog hauv AKI nas.

(A) Cov neeg sawv cev micrographs qhia TUNEL-zoo hlwb hauv ntau pawg raws li qhia. Xub qhia qhov zoo staining. Scale bar, 100 µm.(B)Daim duab nthuav qhia qhia tau hais tias TUNEL-zoo cellper high power field (HPF) nyob rau hauv ntau pab pawg raws li qhia.(C–F)Cov neeg sawv cev Western blot tsom xam qhia lub raum qhia ntawm FasL, p53, thiab Bax hauv pawg sib txawv raws li qhia. *P < 0="" 05="" tiv="" thaiv="" sham="" tswj;="" †p="">< 0.05="" versus="" iri="" txhaj="" nrog="" pcdna="" plasmids="" (n="">(G) Tus neeg sawv cev Western blot ntawm p65 thiabp-p65 proteins nyob rau hauv ntau pawg raws li qhia.

Apoptosis Assay

Cells tau trypsinized, sau, thiab ntxuav nrog PBS.PE Annexin V staining tau ua raws li tus neeg tsim khoom raws tu qauv. Kev soj ntsuam cytometric tau ua nrog lub ntsuas cytometer (BD FACSCanto II, San Jose, CA, United States) los ntsuas cov apoptosis tus nqi los ntawm kev txheeb xyuas cov txheeb ze ntawm PE Annexin V zoo thiab 7-AAD negativecells. Txhua qhov kev ntsuam xyuas tau ua hauv triplicate.

Creatinine thiab ntshav Urea NitrogenAssay

Cov qib creatinine hauv cov ntshav thiab cov zis nrog rau cov ntshav ureanitrogen (BUN) qib tau ntsuas los ntawm kev ntsuas tsis siv neeg biochemical analyzer (AU480, Beckman-Coulter Inc., Brea, CA, United States).

Histology thiab ImmunohistochemicalStaining

Cov kab mob raum thiab cov tshuaj tiv thaiv kab mob tau ua raws li tau piav qhia yav dhau los (Liu li al., 2020). Cov tshuaj tiv thaiv tseem ceeb suav nrog luav polyclonal anti-Wnt1(ab15251; Abcam, Inc.), luav polyclonal anti- -catenin (ab15180; Abcam, Inc. .), thiab luav polyclonal anti-fibroectin (F3648; Sigma-Aldrich).

Western Blot Analysis

Western blot tsom xam tau ua raws li tau piav qhia yav dhau los (Zhou et al., 2019). Cov tshuaj tiv thaiv tseem ceeb suav nrog rabbitpolyclonal anti-fibronectin (F3648; Sigma-Aldrich), mousemonoclonal anti- -catenin antibody (610154; BD TransductionLaboratories), nas monoclonal anti- -SMA antibody (A2547; Sigma-Aldrich), polyclonal anti-Wnt1 (ab15251; Abcam), nas anti- -tubulin (T9026; Sigma-Aldrich), nas anti PAI-1 antibody (AF3828; R&D Systems), anti-active -catenin (#05 –665; EMD Millipore), anti-Fas ligand (FasL) (SC -6237; Santa Cruz, CA, United States), p53 (#2524S; CST), Bax (SC 20067; Santa Cruz), p65 ( #8242S; CST), p-p65 (#3033S; CST), PCNA (#2586S; CST).

RNA Extraction thiab qPCR Analysis

Tag nrho RNA cais thiab qPCR tau ua raws li tau piav qhia dhau los. Cov qib mRNA ntawm ntau cov noob tau normalized nrog -actin. Cov txheej txheem tseem ceeb ntawm cov noob tau teev nyob rau hauv Daim Ntawv Ntxiv 1.

FIGURE 3 | In vivo expression of exogenous Wnt1 before IR prevents the progression of AKI to CKD in mice

DAIM 3|Hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR tiv thaiv kev loj hlob ntawm AKI rau CKD hauv cov nas.

(A)Cov pab nas kho mob. Cov nas tau raug txhaj tshuaj hydrodynamic tail vein txhaj ntawm 1 mg/kg pcDNA3 (IRI plus pcDNA3) los yog PHA-Wnt1 (IRI plus PHA-Wnt1) plasmid, los yog intraperitoneal txhaj ntawm 5 mg/kg ICG-001 ntawm 3 hnub ua ntej UIRI . Cov ntshav thiab cov ntaub so ntswg tau sau 11 hnub tom qab UIRI. Cov qib creatinine hauv cov ntshav(B) thiab ntshav urea nitrogen (BUN) qib(C) raug ntsuas.(D) Cov neeg sawv cev micrographs ntawm lub raum morphology ntawm 11 hnub tom qab UIRI. Cov kab mob raum tau raug rau Periodic acid-Schiff staining. Boxed thaj chaw yog nthuav. Cov xub qhia txog lub raum tubules. Scale bar, 100 µm. *P <0.05; **p=""><0.01. n="">

Kev txheeb cais

Tag nrho cov ntaub ntawv tau hais tias txhais tau tias ± SEM. Cov ntaub ntawv raug txheeb xyuas siv Sigma Stat software (Jandel ScientificSoftware, San Rafael, CA, United States). Kev sib piv ntawm cov pab pawg tau tsim los siv ib txoj kev ANOVA, ua raws li Kev Xeem Tub Ntxhais Kawm-Newman-Keuls. P < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="">

Cistanche benefit

CISTANCHE HERB BENEFIT KIDNEY

TSEEM CEEB

Nyob rau hauv vivo Kev nthuav qhia ntawm Exogenous Wnt1Ua ntej IR Tiv Thaiv Mob Raum Mob thiab Ua kom lub raum -Catenin hauv nas

Txhawm rau txiav txim siab lub luag haujlwm ntawm Wnt / -catenin activation hauv AKIprevention, nas tau muab tshuaj nrog HA-taggedWnt1 qhia vector (PHA-Wnt1) lossis khoob vector (pcDNA3) ntawm hydrodynamic tail vein txhaj ntawm 2 hnub ua ntej BIRI(Daim duab 1A). Cov qib ntshav ntawm creatinine thiab BUN, ob lub cim ntawm lub raum raug mob, tau nce ntau hauv cov nas 1 hnub tom qab BIRI, qhia txog qhov muaj nyob ntawm AKI (Figures 1B, C). Tsis tu ncua, lub raum tseem ceeb morphologic lesions, xws li kev raug mob astubular nyob rau hauv corticomedullary junction, yog soj ntsuam AKI nas(Daim duab 1D). Qhov tshwj xeeb, cov kev hloov pauv no hauv cov kab mob hauv lub cev raug mob biomarkers thiab morphologic lesions hauv AKI cov nas tau pom zoo los ntawm exogenous Wnt1. Western blotanalyses qhia tias exogenous Wnt1 txhawb kev nthuav qhia ntawm lub raum -catenin hauv AKI nas.(Daim duab 1E, F). Ua ke, cov kev tshawb pom no qhia tias kev kho ua ntej ntawm exogenous Wnt1activates raum -catenin thiab tiv thaiv AKI hauv cov nas.

Hauv vivo Kev nthuav qhia ntawm Exogenous Wnt1 Ua ntej IR Txo Tubular CellApoptosis thiab Inhibits NuclearFactor-Kappa B Ua Haujlwm

Txhawm rau tshawb xyuas seb qhov kev cai ntawm apoptosis yog lub hauv paus ntawm kev tiv thaiv kev tiv thaiv ntawm exogenous Wnt1 ntawm AKI, TUNEL staining tau ua los txheeb xyuas qhov apoptosis hauv lub raum cov ntaub so ntswg ntawm AKI nas. Piv nrog rau Sham nas, IRImarkedly nce tus nqi apoptosis nrog rau qib ntawm apoptosis cov proteins uas muaj feem xyuam nrog, xws li FasL, p53, thiab Bax, hauv plab nas.(Daim duab 2A–F). Xws li kev hloov pauv hauv lub raum ntawm AKI nas tau txo qis hauv vivo nthuav tawm ntawm exogenous Wnt1 ua ntej IR. Nuclear factor-kappa B (NF κB), tshwj xeeb tshaj yog nws daim ntawv heterodimer p65 / p50, ua lub luag haujlwm tseem ceeb hauv kev tswj cov kab mob hauv lub raum ntawm AKI nas. Western blot tsom xam pom tias ob qho tib si p65 thiab nws cov phosphorylated daim ntawv (p-p65) tau nce inkidneys ntawm nas tom qab IRI, qhov kev hloov pauv no tau tiv thaiv los ntawm exogenous Wnt1.(Daim duab 2G). Cov kev tshawb pom no pom zoo tias hauv vivo qhia ntawm exogenous Wnt1 ua ntejIR tiv thaiv apoptosis thiab inhibits NF-κB ua kom lub plab hnyuv ntawm AKI nas.

FIGURE 4 | In vivo expression of exogenous Wnt1 before IR down-regulates endogenous Wnt1 and β-catenin in kidneys of mice after AKI-CKD progression

DAIM 4|Hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR down-regulates endogenous Wnt1 thiab -catenin hauv ob lub raum ntawm nas tom qab AKI-CKD kev loj hlob

(A)Cov neeg sawv cev micrographs qhia Wnt thiab -catenin protein qhia nyob rau hauv ntau pawg raws li qhia. Scale bar, 50 µm.(B–E)Cov neeg sawv cev Western blotanalyses ntawm Wnt1, -catenin, thiab nquag -catenin protein ntau hauv ntau pawg raws li qhia.(F) mRNA qhia ntawm TGF- nyob rau hauv ntau pawg raws li qhia.*P < 0.05;="" **p=""><0.01. n="">

Hauv vivo Kev nthuav qhia ntawm Exogenous Wnt1Ua ntej IR Tiv Thaiv Kev Loj Hlob ntawm Kev Mob Raum Raum Rau Mob Raum Mob

Tom ntej no peb soj ntsuam seb puas ua kom Wnt / -catenin ua ntej IR tuaj yeem tiv thaiv kev nce qib ntawm AKI rau CKD. Qhia hauvDaim duab 3A, cov nas tau txais kev txhaj tshuaj hydrodynamictail leeg ntawm Wnt1 qhia plasmid (PHA-Wnt1) lossis intraperitoneal txhaj ntawm ICG-001 ntawm 3 hnub ua ntej UIRI. Ob leeg ntshav creatinine thiab BUN qib tau nce hauv cov nas ntawm 11 hnub tom qab UIRI(Daim duab 3B, C). Tsis tas li ntawd, Masson trichrome staining tau qhia txog qhov tseem ceeb ntawm collagen deposition thiab fibrotic lesions nyob rau hauv cov nas 11 hnub tom qab UIRI.(Daim duab 3D). Cov kev soj ntsuam no qhia txog kev nce qib ntawm AKI rau CKD hauv cov nas tom qab UIRI. Qhov tseem ceeb, cov kev hloov pauv ntawm cov ntshav raum raum biomarkers thiab lub raum fibrosis hauv CKD nas yuav luag tag nrho tiv thaiv los ntawm kev kho ua ntej ntawm exogenousWnt1, tab sis tsis yog ICG-001. Yog li ntawd, hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR tiv thaiv kev nce qib ntawm AKI rau CKD hauv cov nas.

Hauv vivo Kev nthuav qhia ntawm Exogenous Wnt1 Ua ntej IR Down-Regulates EndogenousWnt1 thiab -Catenin hauv raum

Peb tau tshawb xyuas ntxiv txog cov txiaj ntsig ntawm exogenous Wnt1 onendogenous Wnt / -catenin signaling nyob rau hauv ob lub raum ntawm CKD nas.Raws li pom nyob rau hauv daim duab 4A, immunohistochemical staining qhia tau hais tias ob endogenous Wnt1 thiab -catenin tau markedly up-regulated nyob rau hauv lub raum ntawm nas ntawm 11 hnub tom qab IRI. Lawv cov protein ntau tau txo qis heev los ntawm kev kho ua ntej ntawm exogenousWnt1, tab sis tsis yog ICG-001. Western blot tsom xam tau lees paub qhov cuam tshuam ntawm exogenous Wnt1, tab sis tsis yog ICG-001, kev qhia ntawm onendogenous ntawm Wnt1, -catenin, thiab nquag -cateninin ob lub raum ntawm CKD nas (Figures 4B-E). Wnt/ -cateninand TGF- signaling pathways crosstalk nyob rau hauv lub raum fibrosis.Qhov mRNA qhia ntawm TGF- yog induced nyob rau hauv ob lub raum ntawm CKD nas, thiab xws induction tau ploj zuj zus los ntawm exogenousWnt1, tab sis tsis yog ICG-001 (Daim duab 4F). Yog li, kev kho ua ntej ntawm exogenous Wnt1 inhibits endogenous lub raum Wnt / -cateninsignaling hauv CKD nas.

FIGURE 5 | In vivo expression of exogenous Wnt1 before IR downregulates renal Wnt/β-catenin target genes in mice after AKI-CKD progression

DAIM 5|Hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR downregulates raum Wnt / -catenin hom phiaj noob hauv nas tom qab AKI-CKD kev loj hlob

(A, B) mRNAexpression of PAI-1 thiab MMP-7 nyob rau hauv ntau pab pawg raws li qhia.(C–E)Tus neeg sawv cev Western blot tsom xam ntawm PAI-1 thiab Klotho protein ntau ntau.(F)mRNAexpression of Klotho. *P < {{0}}.05;="" **p=""><0.01. n="">

Hauv vivo Kev nthuav qhia ntawm Exogenous Wnt1Ua ntej IR Downregulates RenalWnt / -Catenin Target Genes hauv nas

Piv nrog rau Sham nas, mRNA qhia ntawm PAI-1thiab MMP-7, ob lub hom phiaj ncaj qha downstream ntawm Wnt/ -cateninsignaling, tau nce siab- tswj hauv lub raum ntawm cov nas 11 hnub tom qab UIRI(Daim duab 5A, B). Kev kho ua ntej ntawm exogenous Wnt1, tab sis tsis yog ICG-001, inhibited PAI-1 thiab MMP-7 mRNA qhia hauv CKD nas. Western blot tsom xam pom tias PAI-1 qib protein ntau tau nce hauv lub raum ntawm CKD nas, qhov twg raug tshem tawm los ntawm kev ua ntej ntawm exogenous Wnt1, tab sis tsis yog ICG-001(Daim duab 5C, D). Klotho yog anendogenous Wnt antagonist los ntawm kev khi thiab sequesteringWnt ligands. Piv nrog rau Sham nas, ob qho tib si mRNA thiab cov protein ntau ntawm Klotho tau txo qis hauv ob lub raum ntawm 11 hnub tom qab UIR(Daim duab 5E, F). Kev kho ua ntej ntawm exogenous Wnt1, tab sis tsis yog ICG-001, yuav luag tag Klotho mRNA thiab cov protein qhia hauv ob lub raum ntawm CKD nas. Cov kev tshawb pom no qhia ntxiv txog qhov inhibitory nyhuv ntawm exogenous Wnt1 ntawm endogenous Wnt / -catenin signaling inkidneys ntawm CKD nas.

Nyob rau hauv vivo Kev nthuav qhia ntawm Exogenous Wnt1Ua ntej IR Txo Lub Raum Fibrosis hauvMice Tom Qab Mob Raum Kev Kho Mob-ChronicKidney Disease Progression

Peb tom ntej no tau soj ntsuam cov nyhuv ntawm Wnt / -catenin activation ua ntej IR ntawm lub raum matrix noob thiab fibrotic lesions nyob rau hauv CKD nas.Piv nrog Sham nas, mRNA qhia ntawm fibronectin (FN), collagen I, collagen III, thiab -SMA tau markedly inducedin lub raum nas ntawm 11 hnub tom qab UIR(Daim duab 6A–D).Qhov kev hloov pauv no tau txo qis los ntawm kev kho ua ntej ntawm exogenousWnt1, tab sis tsis yog ICG-001. Western blot tsom xam pom tias cov protein ntau ntawm FN thiab -SMA tau nce inkidneys ntawm CKD nas, thiab cov kev hloov pauv no tau raug tshem tawm los ntawm kev kho ua ntej ntawm exogenous Wnt1, tab sis tsis yog ICG-001(Daim duab 6E–G). Cov teebmeem ntawm exogenous Wnt1 thiab ICG-001 ntawm FNprotein qhia hauv CKD nas tau raug lees paub los ntawm lub raum immunostaining nrog FN antibody(Daim duab 6H). Cov kev tshawb pom no qhia tias kev kho ua ntej ntawm exogenous Wnt1 tiv thaiv renalfibrogenesis hauv IR-induced CKD nas.

FIGURE 6 | In vivo expression of exogenous Wnt1 before IR reduces renal fibrosis in mice after AKI-CKD progression

DAIM 6|Hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR txo lub raum fibrosis hauv nas tom qab AKI-CKD kev loj hlob

(A–D)mRNA qhia txog fibronectin, collagen I, collagen III, thiab -SMA.(E–G)Tus sawv cev Western blot tsom xam ntawm fibronectin thiab -SMA-SMA protein ntau.(H)Immunostaining ntawm fibronectin inkidney seem. *P < {{0}}="" 05;="" **p=""><0.01; †p="">< 0.05.="" n="5." scale="" bar,="" 50="">

Nyob rau hauv vivo Kev nthuav qhia ntawm Exogenous Wnt1Ua ntej IR Attenuates Raum InflammationAfter mob raum mob-mob raum kab mob

Piv nrog rau Sham nas, mRNA qhia txog cov noob caj noob ces, xws li IL-1, IL-6, thiab TNF-, yog qhov tseem ceeb tau tswj hwm hauv lub raum ntawm nas ntawm 11 hnub tom qab UIR(Daim duab 7A–C). Cov kev hloov pauv no yuav luag tag nrho los ntawm kev kho mob ua ntej ntawm exogenous Wnt1, tab sis tsis yog ICG-001.Western blot tsom pom tias cov protein ntau ntawm P65 thiab nws daim ntawv phosphorylated (p-p65) tau nce inkidneys ntawm nas ntawm 11 hnub. tom qab UIRI, qhia txog kev ua haujlwm ntawm NF-κB signaling hauv CKD nas(Daim duab 7D–F). Qhov tseem ceeb, kev kho ua ntej ntawm exogenous Wnt1, tab sis tsis yog ICG-001, txo qis p65 thiab p-p65 protein ntau hauv lub raum ntawm CKD nas.Cov kev tshawb pom no qhia tias kev kho ua ntej ntawm exogenous Wnt1 tiv thaiv lub raum inflammatory teb hauv CKD nas.

FIGURE 7 | In vivo expression of exogenous Wnt1 before IR attenuates renal inflammation in mice after AKI-CKD progression

DAIM 7|Hauv vivo qhia ntawm exogenous Wnt1 ua ntej IR attenuates lub raum mob hauv nas tom qab AKI-CKD kev loj hlob

(A–C) mRNA qhia ntawm IL-1, IL-6, thiab TNF- nyob rau hauv ob lub raum ntawm nas. (D–F)Tus sawv cev Western blot tsom xam p65 thiab p-p65 protein ntau. *P < 0="" 05;="" **p=""><0.01. n="">

Exogenous Wnt1 tiv thaiv Tubular Cells tiv thaiv Apoptosis Induced byHypoxia-Reoxygenation Injury hauv vitro

Txhawm rau txhawm rau txheeb xyuas lub luag haujlwm ntawm Wnt1 hauv AKI, peb tsimHKC-8 tubular cell qauv ntawm AKI siv kev kho mob hypoxia-reoxygenation (H / R). Flow cytometry tau ua hauj lwm los kuaj apoptosis thiab qhia txog qhov nce ntawm apoptosis tus nqi hauv HCK 8 hlwb tom qab H / R kho.(Daim duab 8A, B). Exogenous Wnt1markedly txo apoptosis hauv HCK-8 hlwb tom qab kev kho H/R.Western blot tsom xam pom tias kev kho H/R tau nce qib ntawm apoptosis-txog cov proteins, xws li FasL, p54, Bax, thiab Parp{{4} } hauv HCK-8 hlwb(Daim duab 8C–G). Cov kev hloov pauv tau txo qis los ntawm exogenous Wnt1. Cov ntaub ntawv no qhia tias exogenous Wnt1 tiv thaiv H / R-inducedapoptosis hauv tubular hlwb.

FIGURE 8 | Exogenous Wnt1 protects tubular cells against apoptosis induced by hypoxia-reoxygenation (H/R) injury in vitro

DAIM 8|Exogenous Wnt1 tiv thaiv tubular hlwb tiv thaiv apoptosis induced los ntawm hypoxia-reoxygenation (H / R) raug mob hauv vitro

(A)Tus neeg sawv cev FACS tsom xam qhia tias Wnt1 inhibited hypoxia / reoxygenation (H / R)-induced cell apoptosis. HKC-8 hlwb tau nthuav tawm ua ntej nrog exogenous Wnt1, ua raws li incubation inhypoxic mob rau 48 h thiab tom qab ntawd reoxygenation rau 2 h.(B) Daim duabkev nthuav qhia qhia qhov feem pua ntawm cov hlwb apoptotic hauv ntau pawg raws li tau hais tseg. ThePE-labeled Annexin V-zoo hlwb raug suav los ntawm kev ntws cytometry. *P < 0="" 05="" tiv="" thaiv="" kev="" tswj="" hwm;="" †p="">< 0.05="" versus="" h/r="" (n="">(C) Cov neeg sawv cev Western blotanalyses qhia pom cov protein ntawm FasL, p53, Bax, thiab Parp-1 hauv HKC-8 hlwb.(D–G)Cov duab nthuav qhia qhia txog cov txheeb ze ntawm FasL, p53, Bax, thiabParp-1 hauv HKC-8 hlwb. *P < 0="" 05="" tiv="" thaiv="" kev="" tswj;="" †p="">< 0.05="" versus="" h/r="" (n="">

Kev sib tham

Txawm hais tias AKI nce hauv qhov xwm txheej thiab yog qhov pheej hmoo loj rau kev loj hlob mus rau CKD, tsis muaj cov kev kho mob zoo rau AKI tiv thaiv thiab kev kho mob. Ntau qhov xwm txheej, xws li kev phais plawv, kev tso nyiaj hauv tubular, thiab kev tswj hwm cov tshuaj nephrotoxic, tuaj yeem ua rau kev txhim kho ntawm AKI (Mas-Font li al., 2017). Qhov no qhia txog qhov xav tau rau kev tiv thaiv ntawm AKI.

Txawm hais tias ntau cov kev tshawb fawb txhawb nqa cov txiaj ntsig zoo ntawm Wnt / -catenin activation hauv pathogenesis ntawm AKI, nws tseem muaj txiaj ntsig txog kev tiv thaiv ntawm Wnt / -catenin agoniston AKI. Ib qho kev tshawb nrhiav tshiab hauv txoj kev tshawb fawb tam sim no yog tias hauv vivoexpression ntawm exogenous Wnt1 ua ntej IR tuaj yeem tiv thaiv miceagainst AKI thiab tiv thaiv kev loj hlob ntawm AKI rau CKD. Hauv vivoexpression ntawm exogenous Wnt1 ntawm 2 hnub ua ntej lub raum bilateralIR tuaj yeem ua kom lub raum -catenin, ua rau txo qis apoptosis thiab mob hauv AKI nas (1 hnub tom qab IR). Tsis tas li ntawd, hauv vivo qhia ntawm exogenous Wnt1 ntawm 3 hnub ua ntej lub raum unilateral IR inhibited. endogenous Wnt / -cateninsignaling thiab tiv thaiv kev txhim kho ntawm lub raum fibrosis hauv CKD nas (11 hnub tom qab IR). Nyob rau hauv sib piv, nyob rau hauv vivo qhia ntawm exogenous Wnt1 ntawm 5 hnub tom qab lub raum IR tau pom tias induce -catenin activation thiab accelerate AKI rau CKD kev loj hlob (Xiao li al., 2016). Cov kev tshawb fawb no qhia tias lub sij hawm ntawm Wnt / -catenin agonists yog qhov tseem ceeb thaum lawv siv rau kev tiv thaiv thiab kho AKI.

Cov kev tshawb fawb qhia tias kev ua kom ruaj khov ntawm Wnt / -cateninsignaling tuaj yeem tsav qhov kev loj hlob ntawm AKI rau CKD. ICG 001 selectively inhibits Wnt / -catenin / CREB binding protein (CBP) signaling thiab tam sim no nyob rau hauv kev sim tshuaj rau ntau yam mob qog noj ntshav. Peb yav dhau los tau qhia tias kev tswj hwm ntawm ICG-011 ntawm 5 hnub tom qab IR rov ua haujlwm rau lub raum thiab cuam tshuam kev loj hlob ntawm AKI rau CKD, raws li pom los ntawm kev txo qis renalfibrosis (Xiao li al., 2016). Tsis tas li ntawd, ICG-001 kev tswj hwm pib 3 hnub tom qab unilateral ureteral obstruction (UUO) tuaj yeem ua rau lub raum fibrotic lesions hauv UUO qauv ntawm CKDmice (Hao li al., 2011). Txawm li cas los xij, txoj kev tshawb fawb tam sim no tau pom tias kev kho mob ua ntej ntawm ICG-001 ntawm 3 hnub ua ntej IR tsis muaj qhov cuam tshuam rau lub raum Wnt/ -catenin signaling lossis kev nce qib ntawm AKI toCKD. Qhov no tsis yog qhov xav tsis thoob vim hais tias Wnt / -catenin signalingis qhia nyob rau theem qis heev hauv cov neeg laus lub raum tsis raug mob (Tanet al., 2014). Txawm li cas los xij, peb qhov kev tshawb pom qhia tias lub sijhawm thiab lub sijhawm ntawm ICG{15}} kev tswj hwm yog qhov tseem ceeb rau kev txiav txim siab txog qhov txiaj ntsig kho mob.

Mechanisms hauv qab cov nyhuv tiv thaiv ntawm Wnt / -cateninmay koom nrog kev hloov pauv ntawm apoptosis thiab txoj hauv kev ciaj sia.Genetic ablation ntawm tubule -catenin txhawb nqa tubular apoptosisafter IRI los yog folic acid kev kho mob, whereas activation ntawm Wnt / -catenin suppresses pro-apoptotic proteinsal. , 2009; Zhou et al., 2012). Hauv kev tshawb fawb tam sim no, exogenousWnt1 tau ua rau muaj kev cuam tshuam cov tubular cell apoptosis nyob rau hauv ob qho tib si IRImouse qauv ntawm AKI thiab H / R-induced cell qauv ntawm AKI.Qhov no qhia tias apoptosis tuaj yeem yog ib txoj hauv kev tiv thaiv kev tiv thaiv ntawm exogenous Wnt1 ntawm AKI thiab AKI rau CKD kev loj hlob. Txoj kev tshawb fawb tam sim no kuj tau qhia txog cov nyhuv inhibitory ntawm exogenous Wnt1 ntawm NF-κB ua kom nyob rau hauv ob qho tib si AKI thiab CKD, qhia txog kev tawm tsam ntawm inflammatory teb. Txawm li cas los xij, nws yuav tsum raug sau tseg tias seb qhov txo qis yog qhov cuam tshuam rau kev tiv thaiv lossis cov txheej txheem ntawm kev tiv thaiv kev sib kho los ntawm exogenous Wnt1 tseem yuav txiav txim siab. Cov kev tshawb fawb ntxiv yog xav tau los piav qhia qhov tseeb mechanism rau exogenous Wnt 1-kev sib haum xeeb tiv thaiv AKI thiab kev tiv thaiv ntawm AKI mus rau CKD kev loj hlob.

effects of cistanche

Siv Wnt/ -catenin modulators los tiv thaiv thiab kho AKI thiab CKD

XAIV

Hauv kev xaus, qhov kev tshawb fawb tam sim no muab cov pov thawj los txhawb kev tiv thaiv kev tiv thaiv ntawm Wnt / -catenin ua rau ntawm IR-txog AKI thiab CKD, txawm tias xav tau kev ua haujlwm ntau ntxiv los piav qhia qhov tseeb ntawm cov txheej txheem uas exogenous Wnt1 tiv thaiv AKI thiab CKD. Muab lub luag haujlwm ntawm ob lub luag haujlwm ntawm Wnt / -catenin signaling hauv AKIand CKD, txoj kev tshawb no qhia txog qhov tseem ceeb ntawm lub sijhawm thaum siv Wnt / -catenin modulators rau kev tiv thaiv thiab kho ntawm AKI. Cov kev tshawb fawb yav tom ntej uas siv ntau cov tshuaj pharmacologic thiab caj ces nrog rau lub sijhawm kho kom zoo thiab lub sijhawm raug lees paub los tshawb xyuas cov txiaj ntsig ntawm Wnt / -cateninagonists hauv AKI tiv thaiv thiab kho.

LOS NTAWM: Exogenous Wnt1 Tiv Thaiv Mob Raum Raum Mob thiab Nws Cov Kev Loj Hlob Tom Qab Mus Rau Raum Kab Mob, Xu Hong^1†, Yanni Zhou^2†, Dedong Vang^3†, Fuping Liu⁴, Tianjun Guan³, Youhua Liu^1,5thiab Liangxiang Xiao^{3 *}

Pem hauv ntej. Physiol., 08 Nov 2021|https://doi.org/10.3389/fphys.2021.745816

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