Kev cob qhia lub cev inhibits Fibrosis tsim nyob rau hauv Alzheimer tus kab mob raum cuam tshuam rau TGFB Signaling Pathways
Mar 15, 2022
Taw qhia
Alzheimer's disease (AD) yog hom kev dementia ntau tshaj plaws, tab sis nws cov tsos mob tau pib sib tham hauv ntau xyoo dhau los. Txawm hais tias muaj kev siv zog loj los tsim tshuaj thiab kho cov kab mob hloov kho, tsis muaj cov txheej txheem kho kom zoo rau hnub tim [1–3]. Kev tsim ntawm amyloid plaques thiab kev ua haujlwm tsis zoo ntawm amyloid-protein precursors (APP) yog suav tias yog thawj cov tsos mob ntawm tus kab mob tsis yog nyob rau hauv lub hauv nruab nrab paj hlwb (CNS) tab sis kuj nyob rau hauv periphery [4]. Cov ntaub ntawv pov thawj txhawb nqa lub luag haujlwm tseem ceeb ntawm amyloid-B (AB) nyob rau hauv pathogenesis ntawm AD [5] taw qhia tias qhov txo qis ntawm AB yog ib qho ntawm cov txheej txheem tseem ceeb hauv pathomechanism. Nyob rau hauv AD, ob peb lub cev peripheral koom nrog xws li testis [5], pancreas [6], thiabraum[7], qhia txog kev txhim kho ntawm cov kab mob nyuaj nrog ntau lub hom phiaj [8]. Nws tau raug pov thawj tias lubraumkoom nrog AB clearance [9]. Ntxiv mus, cov ntshav concentration ntawm AB yog nce siab nyob rau hauv ntevmob raum (CKD) cov neeg mob [10, 11]. AB tsub zuj zuj kuj tau tshwm sim nyob rau hauvraum[12], ua rau pom tsis meej. Kev pom zoo yog ib feem tswj hwm los ntawm kev hloov pauv kev loj hlob B (TGFB), uas yog tus tswv cytokine hauv kev tsim cov kab mob.lub raumo thiab fibrosis [13], thiab nws tseem paub tias yog ib qho tseem ceeb hauv AD pathogenesis [14]. Hauv AD, TGFB signaling plays lub luag haujlwm hauv microglial activation thiab astrocyte-mediated neuroprotection [15]. Tsis tas li ntawd, kev hloov pauv ntawm TGFB tseem cuam tshuam rau qhov mob hauv CNS thiab cov ntaub so ntswg peripheral [13, 16].
CISTANCHE yuav txhim kho rau lub raum/ raum mob
TGFB muaj peb isoforms: TGFB1, TGFB2, thiab TGFB3 [17]. TGFB1 koom nrog ntau yam txheej txheem xws li kev loj hlob hauv plab, tswj kev loj hlob ntawm tes thiab kev sib txawv, fibrosis thiab caws pliav nqaij tsim, qog loj hlob, thiab kev tiv thaiv kab mob [18]. Nws yog ib qho tseem ceeb cytokine hauv fibrosis tsim nyob rau hauv ntau yam kabmob xws li lub siab thiabraum[19]. TGFB1 cov ntaub ntawv dimers, uas thawj khi rau TGFB hom II receptor (TGFRII), ces qhib TGFB receptor hom I (TGFRI) Ser / Thr kinase, pib intracellular signaling complex-tsim ntawm Smad transcription yam. Nyob rau hauv canonical TGFB signaling pathway, Smad2/3 transcription yam yog phosphorylated thiab, tom qab complex tsim nrog Smad4, translocate mus rau hauv lub nucleus [20]. Ob peb lub noob tuaj yeem cuam tshuam los ntawm TGFB1 kev ua kom muaj zog vim nws ua rau muaj qhov sib txawv ntawm collagens, tswj kev loj hlob ntawm tes, cuam tshuam rau apoptosis [20] thiab tswj cov matrix metalloproteinase (MMP) qhia [21]. Txoj kev uas tsis yog-canonical TGFB signaling txoj kev pib ua kom cov MAPKes los ntawm phosphorylation uas nyob rau hauv lem yuav induce ntxiv TGFB qhia [22]. JNK thiab p38 kinases raws li TGFB lub hom phiaj raug txheeb xyuas hauvlub raumfibrosis thiab tswj apoptosis [23]
Lub cev ua si tau pom tias muaj txiaj ntsig zoo rau tib neeg cov neeg mob. Kev tawm dag zog tuaj yeem tswj hwm thiab ncua qhov kev tshwm sim ntawm AD [24, 25] thiab txo qis ntawm AD biomarkers xws li tau thiab AB hauv ntshav kuj tau tshaj tawm [25]. Kev ua haujlwm ntawm lub cev tau pom tias txo qis qhov tshwm sim ntawm AD, AD-koom nrog cov teeb meem neuropathological, thiab kev txawj ntse poob [26]. Txawm hais tias muaj ntau cov kev tshawb fawb tau luam tawm txog cov txiaj ntsig ntawm kev ua si ntawm lub cev ntawm AD tsim [27], cov txheej txheem intracellular koom nrog cov txiaj ntsig zoo no tsis tau tham txog meej. Muaj ob peb tus qauv tsiaj uas hloov caj ces nrog tau thiab AB overexpression tau raug sim thaum lub sij hawm qoj ib ce. Hauv cov qauv no, nws tau raug pov thawj tias kev ua kom lub cev muaj zog tuaj yeem ncua qhov kev tshwm sim ntawm AD [28, 29]. Tsis tas li ntawd, nws tau pom tias kev cob qhia mus sij hawm ntev ntawm cov nas no muaj txiaj ntsig zoo rau kev ua haujlwm ntawm lub cev hauv nruab nrog cev hauv AD. Kev ua lub cev hloov pauv PACAP-BMP crosstalk hauv ADlub raumntawm normalizing qhov qhia ntawm collagen hom IV thiab A tsub zuj zuj [30]. Ntxiv mus, cov ncauj lus kom ntxaws tsom xam ntawm kev tiv thaiv kev ua haujlwm ntawm Sox9 qhia hauv AD testis tau luam tawm [31]. Kev cob qhia nquag kuj tseem cuam tshuam rau TGFB signaling raws li nws suppresseslub raumo thiab fibrosis tom qab [32] Tsis tas li ntawd, TGFB thiab nws cov kev taw qhia yog qhov tseem ceeb rau cov neeg laus kho cov pob txha uas cuam tshuam rau hauvraumfibrosis [33]. Tom qab ntawd, peb qhov kev xav tseem ceeb yog qhov kev cob qhia lub cev mus sij hawm ntev muaj kev cuam tshuam rau TGFB teeb liab uas tuaj yeem tswj tau.raummatrix ntau lawm hauv AD. Kev hloov pauv ntawm cov kev taw qhia no yuav ua rau txo qis raumfibrosis thiab normalize AD clearance. Yog li ntawd, peb lub hom phiaj yog los qhia txog kev sib raug zoo ntawm TGFB-kev taw qhia thiab cov txiaj ntsig zoo ntawm kev cob qhia lub cev ntawmraumfibrosis hauv AD. Hauv peb qhov kev sim, peb nthuav tawm cov pov thawj tias kev cob qhia mus sij hawm ntev muaj kev cuam tshuam ncaj qha rau TGFB signalization hauvlub raumntawm AD nas. Tsis tas li ntawd, peb qhia tias kev tawm dag zog lub cev muaj zog txo qisraumfibrosis ntawm normalizing canonical thiab non-canonical TGFB signalization.
Ntsiab lus:Alzheimer's kab mob, fibrosis, lub cev ua si, Smad, kab mob raum; lub raum
CISTANCHE yuav txhim kho lub raum / raum mob ntshav qab zib
MATERIAL THIAB MUAB
TsiajTxiv neej Alzheimer-transgenic (n=5) (B6C3-Tg (APPswe, PSEN1dE9)85Dbo/J) nas tau siv los ua raws li AD. Cov tsiaj qus muaj peb hlis (WT) (tsis muaj kev hloov pauv hloov pauv thiab tsis muaj kev cob qhia) (n=5), Alzheimer's transgenic nas (AD) (n=5), thiab kawm AD (TAD) (n=5) Cov nas tau khaws cia hauv lub teeb / tsaus ntuj ntawm 12/12 teev nrog zaub mov thiab dej ad libitum. Interval treadmill khiav tau thov rau kev tawm dag zog thiab ua ke pab pawg. Yav dhau los tag nrho cov tsiaj qoj ib ce tau nyob nrog lub cev muaj zog treadmill (Columbus Inst., Columbus Ohio, USA) thiab khiav ceev rau 2 lub lis piam. Kev cob qhia tau ua plaub zaug hauv ib lub lis piam, rau 60 feeb. Txhua qhov kev cob qhia tau siv sijhawm 10 lub voj voog, txhua lub voj voog muaj 4 min ntawm kev siv siab thiab 2 min ntawm kev siv qis. Tsis siv neeg khiav ceev tau ruaj khov thaum lub sij hawm sim txhais tau tias ceev ntawm 10 m / min. Thaum lub siab siv khiav ceev pib ntawm 16 m / min thiab tau nce txhua lub lim tiam thib peb nrog 1 m / min mus txog 20 m / min. Pab pawg tswj hwm thiab noj zaub mov kuj tau nyob nrog lub treadmill thiab nyob ntawd rau 5 min / hnub ntawm lub treadmill sawv [28]. Kev sim tau pib nrog 3-cov tsiaj muaj hnub nyoog hli thiab xaus rau thaum muaj hnub nyoog 10 hli. Txoj kev tshawb no tau ua tiav raws li cov lus qhia txog kev coj ncaj ncees (tus naj npawb tso cai rau kev kawm no: PEI/001/2105-6/2014, Semmelweis University, Hungary). Phire Animal Tissue Direct PCR Kit tau siv rau genotyping cov tsiaj (Thermo Fischer Scientific, Waltham, MA, USA) raws li cov chaw tsim khoom cov lus qhia.
Lub teeb microscopic morphologyTom qab tag nrho cov kev ntsuam xyuas kev txawj ntse tiav lawm, {{0}} cov nas muaj hnub nyoog ib hlis tau tshuaj loog nrog kev txhaj tshuaj intraperitoneal ntawm ketamine (Richter, concentration: 100 mg / ml) / xylazine (Produlab Pharma, concentration: 20 mg / ml) cocktail nyob rau hauv ib koob ntawm 0.1 ml / 10 g lub cev qhov hnyav thiab transcardially perfused nrog heparinized ice txias saline [28] thiablub raumraug tshem tawm. Cov qauv ntaub so ntswg raug ntxuav hauv PBS peb zaug thiab kho hauv 4: 1 sib xyaw ntawm cov ethanol thiab 40 feem pua formaldehyde, ces muab tso rau hauv paraffin. Rau Masson's trichrome staining (Sigma-Aldrich, MO, USA) serial seem tau tsim thiab cov ntaub so ntswg fibrotic tau pom. Cov txheej txheem staining tau ua raws li cov neeg tsim khoom cov lus qhia. DP74 lub koob yees duab (Olympus Corporation, Tokyo, Nyiv) ntawm Olympus Bx53 microscope (Olympus Corporation, Tokyo, Nyiv) tau siv los thaij duab.
Immunohistochemistrytau ua rauraumCov ntaub so ntswg los ntawm WT, AD nas, thiab TAD nas kom pom qhov chaw ntawm Smad2, PCNA (proliferating cell nuclear antigen), thiab collagen hom I (Col. I) [30].Lub raumtau kho nyob rau hauv 4: 1 sib tov ntawm absolute ethanol thiab 40 feem pua formaldehyde thiab ntxuav nyob rau hauv 70 feem pua ethanol. Tom qab embedded serial seem tau ua, deparaffinization tau ua raws li los ntawm kev yaug hauv PBS (pH 7.4). Cov chaw tsis tshwj xeeb khi tau raug thaiv nrog 1 feem pua bovine serum albumin yaj hauv PBS (Amresco LLC, Solon, OH, USA). Cov seem tau tsim nrog polyclonal Smad2 (Sigma-Aldrich, MO, USA) ntawm dilution ntawm 1: 500, PCNA (Cell Signaling, Danvers, MA, USA) ntawm dilution ntawm 1: 800 lossis Col. I (Sigma-Aldrich, MO, USA) cov tshuaj tiv thaiv kab mob ntawm dilution ntawm 1:500 ntawm 4◦C thaum hmo ntuj. Thawj cov tshuaj tiv thaiv tau pom nrog qhov sib ntxiv ntawm cov tshuaj tiv thaiv luav Alexa Fluor 555 cov tshuaj tiv thaiv kab mob thib ob (Life Technologies Corporation, Carlsbad, CA, USA) tau siv los ntawm dilution ntawm 1: 1000. Cov qauv tau muab tso rau hauv Vectashield mounting nruab nrab (Vector Laboratories, Peterborough, England) uas muaj DAPI (4,6-Diamidino-2-phenylindole dihydrochloride) rau nuclear DNA staining. Anti-Lavbit Alexa Fluor 555 tau siv yam tsis muaj thawj cov tshuaj tiv thaiv rau kev tswj tsis zoo. Photomicrographs tau siv lub koob yees duab DP74 (Olympus Corporation, Tokyo, Nyiv) ntawm Olympus Bx53 microscope (Olympus Corporation, Tokyo, Nyiv) rau kev pom ntawm Col. I. Cov duab tau txais los ntawm kev siv cellSense nkag 1.5 software (Olympus, Shinjuku, Tokyo, Nyiv ) nrog kev teeb tsa lub koob yees duab tas li tso cai rau kev sib piv ntawm FL fluorescent teeb liab siv. Rau Smad2 thiab PCNA nrhiav pom, Olympus FV3000 confocal microscope (Olympus Co. Tokyo, Nyiv) tau siv los siv 60 × roj immersion lub hom phiaj (NA: 1.42). Rau excitation, laser kab ntawm 543 nm tau siv. Lub sijhawm nruab nrab pixel yog 4Bs. Z duab series ntawm 1m optical thickness tau kaw nyob rau hauv sequential scan hom. Cov duab ntawm Alexa555 thiab DAPI tau dhau los siv Adobe Photoshop version 10.0 software. Qhov sib piv ntawm cov duab tau sib npaug nce ntxiv yam tsis hloov qhov chaw tas li.
RT-PCR tsom xamLub raumntawm WT (n=5), AD (n=5), thiab TAD (n=5) yog mechanically hauv av thiab tau yaj hauv Tri zol (Applied Biosystems, Foster City, CA, USA ), tom qab 30 min incubation ntawm 4◦C thiab tag nrho RNA raug rho tawm [30]. RNA tau sau rau hauv RNase-dawb dej thiab khaws cia ntawm -70◦C. Cov Khoom Siv Muaj Peev Xwm Muaj Peev Xwm RT tau siv rau kev thim rov qab (Applied Biosystems, Foster City, CA, USA). Rau cov kab ke ntawm cov khoom siv primer thiab cov ntsiab lus ntawm polymerase chain reactions, saib Table 1. Amplifications tau ua nyob rau hauv lub thermal cycler (Labnet MultiGene™ 96-zoo Gradient Thermal Cycler; Labnet International, Edison, NJ, USA) raws li raws li nram no: 95◦C, 2 min, ua raws li 35 cycles (denaturation, 94◦C, 30 s, annealing rau 45 s ntawm optimized kub raws li muab nyob rau hauv Table 1; ncua, 72◦C, 90 s) thiab ces 72◦C ,
7 feeb. Actin tau siv los ua kev tswj hwm sab hauv. PCR cov khoom raug tshuaj xyuas siv 1.2 feem pua agarose gel uas muaj ethidium bromide thiab sau nrog FluorchemE geldocumetary system (ProteinSimple, CA, USA). Optical densities ntawm PCR cov khoom lag luam teeb liab tau txiav txim siab los ntawm kev siv ImageJ 1.40 g freeware. Ua ntej kev ntsuas qhov muag pom qhov ntom ntom, peb tau ua tus kheej calibrations ntawm txhua photomicrograph. Tom qab ntawd peb tau nce qhov kev nthuav dav ntawm cov duab kom ncav cuag tus neeg pixel loj thiab nrog rau txoj kev freehanded txoj kab tau meej meej thiab ntsuas qhov sib xyaw ua ke ntawm thaj chaw. Cov pixels tau ntsuas hauv 5 qhov kev sim ywj pheej los ntawm 3 tus neeg ua haujlwm ywj pheej. Cov teeb liab thawj zaug tau muab piv nrog WT actin rau kev sib piv zoo dua ntawm kev qhia qhov sib txawv hauv AD thiab TAD nas. Cov lej hauv qab ntawm txoj kab qhia kev txheeb xyuas qhov kev sim ntawm qhov kev sim uas txhua qhov kev sim cais (tsawg kawg 5) tau ua rau nws tus kheej actin teeb liab thiab tom qab ntawd suav nrog qhov sib txawv ntawm cov lej.
Western blot tsom xam
Lub raumntawm WT (n {{{0}}), AD (n=5), thiab TAD nas (n=5) tau ntxuav hauv physiological saline N thiab khaws cia ntawm -7{{1 0}}◦C. Cov qauv tau ua hauv av hauv cov kua nitrogen thiab tau sau rau hauv 100mL ntawm homogenization RIPA (Xov tooj cua Immuno Precipitation Assay)-tsis (150 mM sodium chloride; 1.0 feem pua NP40, 0.5 feem pua sodium deoxycholate; 50 mMTris, pH 8.0) inhibitors (proteasein inhibitors) 10 g/mL), 5 mM Benzamidine, Leupeptin (10ug/mL), Trypsine inhibitor (10 g/mL), 1 mM PMSF, 5 mM EDTA, 1 mM EGTA, 8 mM NaFluoride, 1 mM Na-orthovanadate). Pellets tau sonicated los ntawm pulsing tawg rau 30 s ntawm 40 A (ColeParmer, IL, USA). Tag nrho cov cell lysates rau westernblot tsom xam tau npaj [30]. 20 g protein raug cais nyob rau hauv 7.5 feem pua SDS-polyacrylamide gels rau kev kuaj pom ntawm TGF-1, TGF R1, TGF-R2, Smad2, Smad3, ERK1/2, P-ERK1/2, p38, P-p38,
JNK, PP2A, PP2B, p21, PCNA, Cleaved caspase3, MMP9, collagen hom I (Col. I), thiab actin. PhosphoSer/Thr detection kit (Millipore, Billerica, MA, USA) tau siv los txheeb xyuas cov theem phosphorylation ntawm Ser/Thr amino acid sab chains. Proteins raug blotted rau nitrocellulose daim nyias nyias thiab raug rau cov thawj cov tshuaj tiv thaiv thaum hmo ntuj ntawm 4◦C nyob rau hauv lub dilution raws li muab nyob rau hauv Table 2. Incubation yog ua raws li 30 min PBST ntxuav ces daim nyias nyias tau them nrog cov peroxidase-conjugated thib ob antibody antibody IgG nyob rau hauv ib tug 1. :1500 (Bio-Rad Laboratories, CA, USA) los yog anti-nas IgG hauv 1:1500 (Bio-Rad Laboratories, CA, USA) dilution. Rau kev tshawb nrhiav txhim kho chemiluminescence (Advansta Inc., Menlo Park, CA, USA) tau siv raws li cov lus qhia ntawm cov chaw tsim khoom. Actin tau siv los ua kev tswj hwm sab hauv. Cov teeb liab raug ntes nrog lub koob yees duab txias ntawm cov ntaub ntawv gel (Fluorchem E, ProteinSimple, CA, USA). Optical densities ntawm cov teeb liab tau ntsuas los ntawm kev siv ImageJ 1.40g freeware. Ua ntej kev ntsuas qhov muag pom qhov ntom ntom, peb tau ua tus kheej calibrations ntawm txhua photomicrograph. Tom qab ntawd peb tau nce qhov kev nthuav dav ntawm cov duab kom ncav cuag tus neeg pixel loj thiab nrog rau txoj kev freehanded txoj kab tau meej meej thiab ntsuas qhov sib xyaw ua ke ntawm thaj chaw. Cov pixels tau ntsuas hauv 5 qhov kev sim ywj pheej los ntawm 3 tus neeg ua haujlwm ywj pheej. Cov teeb liab thawj zaug tau muab piv nrog WT actin rau kev sib piv zoo dua ntawm kev qhia qhov sib txawv hauv AD thiab TAD. Cov lej hauv qab ntawm txoj kab qhia kev txheeb xyuas cov kev sim uas txhua qhov kev sim cais (tsawg kawg 5) tau ua rau nws tus kheej actin teeb liab thiab tom qab ntawd suav nrog qhov sib txawv ntawm cov lej.
Kev txheeb caisTag nrho cov ntaub ntawv yog tus sawv cev ntawm tsawg kawg tsib qhov kev sim ywj pheej. Rau txhua tus lej, cov qauv ntawm tib WT, AD thiab TAD tsiaj raug xaiv nrog lawv cov kev tswj sab hauv kom muaj kev sib piv zoo dua. Tsuas yog ib daim duab qhia los ntawm tib pawg tsiaj tau siv hauv txhua daim duab. Kev txheeb xyuas txheeb cais tau ua los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA), ua raws li Tukey's HSD tom qab kev xeem. Qhov chaw pib rau qhov sib txawv tseem ceeb raws li piv rau cov qauv tswj tau teeb tsa ntawm ∗p < 0.05="" thiab="" rau="" ad="" cov="" qauv="" #p="">< 0.05.="" ∗="" qhia,="" qhov="" sib="" txawv="" wt="" piv="" nrog="" ad="" thiab="" tad,="" #="" qhia="" qhov="" sib="" txawv="" ad="" piv="" nrog="">
TSEEM CEEB
Kev ua si lub cev ua haujlwm zoo li canonic TGF qhia txoj hauv kev hauv ob lub raum ntawm AD nasKev nthuav qhia ntawm cov ntsiab lus ntawm canonical TGFB signaling txoj kev tau tshawb xyuas kom meej. Protein thiab mRNA qhia ntawm TGFB1 tsis qhia qhov sib txawv tseem ceeb thaumlub raumntawm WT thiab AD nas tau muab piv tab sis qhov nthuav qhia tau pom hauv TAD cov qauv (Fig. 1A, B). Kev nthuav qhia ntawm TGFRI txo qis hauv AD tab sis kev ua lub cev ua haujlwm zoo li qub (Daim duab 1A, B), thaum qhov kev qhia ntawm TGFBRII nce siab hauvlub raumntawm AD nas tab sis poob qis tom qab kev ua si lub cev (Fig. 1A, B). Nyob rau hauv txoj kab nrog cov kev soj ntsuam no, qhov kev qhia ntawm Smad2 tau nce hauv AD cov qauv thiab nws tau normalized tom qab lub cevkev ua si (Fig. 1A, B). Ntawm qhov tsis sib xws, qhov kev qhia ntawm Smad3 tau pom qhov txo qis hauvlub raumntawm AD nas uas tau them rau hauv cov qauv TAD (Fig. 1A, B). Qhov siab ntawm Smad2 cov cim zoo tau pom nyob rau hauv tubular system ntawm lub cortex tab sis tsis nyob rau hauvlub raumcorpuscles hauv AD nas. Qhov siab no tau txo qis hauv tubules ntawm TAD tsiaj (Daim duab 1C).
Cov kev taw qhia tsis yog canonical tau hloov kho hauv lub raum ntawm AD nas tom qab lub cev ua siMAPK-es, xws li ERK, p38, thiab JNK, tsim ib feem tseem ceeb ntawm tsis-canonical TGFsignaling [22]. mRNA kev qhia ntawm ERK tsis tau hloov pauv hauvlub raumntawm AD nas tab sis pom qhov txo qis hauv TAD cov qauv (Fig. 2A). Protein qhia ntawm no kinase tsis nyob rau hauv correlation nrog lub mRNA qhia thiab ib tug muaj zog txo tau pom nyob rau hauvlub raumntawm AD nas nrog ib tug nruab nrab normalization tom qab muaj zog lub cev ua si (Fig. 2B). Daim ntawv nquag, phosphorylated tau nthuav dav hauv cov qauv AD thiab pom tias muaj qhov txo qis hauvlub raumntawm TAD nas (Fig. 2B). mRNA kev qhia ntawm p38 tsis tau hloov pauv loj hauv ib qho qauv (Fig. 2A). Protein nthuav qhia ntawm p38 tau nce ntxiv hauvlub raumntawm AD nas uas tau them ib feem los ntawm kev ua lub cev (Fig. 2B). Interestingly lub active phosphorylated daim ntawv qhia ib tug txo nyob rau hauv AD cov qauv uas
tau normalized hauv TAD tsiaj (Fig. 2B). mRNA qhia ntawm JNK raug txo ob qho tib si hauv AD thiab TAD cov qauv (Fig. 2A). Protein nthuav qhia ntawm 2 isoforms ntawm JNK tau kuaj pom los ntawm kev tsom xam sab hnub poob. Cov haujlwm uas tsis yog rov ua haujlwm rau JNK1 thiab JNK2 tau tshwm sim [34] thiab nws tau kuaj pom qhov txo qis ntawm 57 kDa isoform thiab ntawm 46 kDa isoform hauv AD cov qauv (Daim duab 2B). 57 kDa thiab 46 kDa isoforms pom qhov nce siab tom qab kev ua si hauv lub cevlub raumntawm TAD nas (Fig. 2B).
Kev hloov pauv ntawm Ser / Thr phosphatase thiab hloov pauv protein phosphorylation hauv ADHloov pauv Ser/Thr tshwj xeeb protein phosphatase kev qhia thiab kev ua haujlwm tau tshwm sim hauv AD [35, 36]. Peb tau pom qhov nthuav qhia ntau ntxiv ntawm PP2B hauvlub raumntawm AD nas, tab sis nws raug txo los ntawm kev ua lub cev (Fig. 2A, B). Ntawm qhov tsis sib xws, kev qhia ntawm lwm cov cellular loj Ser / Thr tshwj xeeb phosphatase, PP2A, tshwm sim nrog kev txo qis hauvlub raumntawm AD nas (Fig. 2A, B). Hauv TAD cov qauv, mRNA thiab protein qhia ntawm PP2A catalytic subunit tau nce (Fig. 2A, B). Kev soj ntsuam ntawm cov protein phosphorylation ntawm Ser / Thr residues pom lub teeb liab muaj zog ntawm 55 kDa hauvlub raumntawm AD nas, sawv cev qhov hnyav molecular ntawm tau (Fig. 2C). Lwm qhov siab tau tshwm sim ntawm 10 kDa uas sib haum rau qhov hnyav molecular ntawm cleaved C-terminal ntawm APP (Fig. 2C). Cov phosphorylation ntawm ob qho tib si ntawm cov proteins no tau txo qis thaum qoj ib ce (Daim duab 2C).
TGF signaling cuam tshuam lub voj voog ntawm tes thiab apoptosisRaws li cov cim ntawm cell proliferation p21 thiab PCNA qhia qib tau saib xyuas. Kev ua kom TGFB paub tias yuav ua rau kev qhia ntawm p21 [37]. Tsis tas li ntawd, TGFB1 ua kom ua rau muaj kev hloov pauv ntawm cov cell proliferation [38]. Yog li ntawd, proliferating cell nuclear antigen (PCNA) qhia tau tshawb xyuas. Cov qib protein ntawm p21 tau nce siab hauv AD tab sis tau txo qis hauv TAD cov qauv (Fig. 3A, B). PCNA teb txawv: AD ua rau txo qis ntawm cov protein qhia, thaum PCNA protein ntau ntxiv thaum qoj ib ce (Fig. 3A, B). Immunohistochemistry tau ua los txheeb xyuas PCNA cov hlwb zoo thiab ua rau txo qis kev tiv thaiv kab mob hauv tubular hlwb hauv ob lub raum ntawm AD nas. Interestingly ib tug normalized PCNA zoo tau pom nyob rau hauv tubular hlwb nyob rau hauv TAD nas. Tsis muaj PCNA zoo hlwb raug kuaj pom hauvlub raumcorpuscles (Fig. 3C). Raws li TGF1 signaling tuaj yeem cuam tshuam rau apoptosis [39], daim ntawv nquag ntawm proapoptotic caspase3 tau tshawb xyuas. Txawm hais tias me ntsis mRNA qhia ntawm caspase3 tau kuaj pom hauv WT cov qauv, tsis muaj cleaved, nquag capase3 tau pom. Ntawm qhov tsis sib xws, ib qho muaj zog mRNA thiab cleaved caspase3 tshwm sim hauv AD cov qauv, thaum muaj zog lub cev ua kom txo tau qhov kev qhia ntawm proapoptotic protein (Fig. 3A, B).
Lub raum fibrosis tau downregulated los ntawm lub cev ua siCov txiaj ntsig yav dhau los qhia tau tias muaj fibrosis hauv ob lub raum ntawm AD tsiaj. Ua ntej, MMP9 qhia, uas tuaj yeem tswj hwm ntawm TGFB1 ua kom, tau tshawb xyuas [21]. mRNA ntawm MMP9 tau qhia nyob rau hauv tag nrho cov qauv tab sis pom tau hais tias ib tug tseem ceeb nce nyob rau hauv lub raum ntawm TAD nas. Protein qhia ntawm matrix metalloproteinase yog nyob rau ntawm qhov kev kuaj pom hauv WT cov qauv tab sis pom qhov nce siab hauv AD tsiaj. Kuj ceeb tias, kev tawm dag zog lub cev muaj zog nce MMP9 qhia (Fig. 4A, B). Lwm qhov kev tivthaiv ntawm cov txheej txheem fibrotic yog qhov nce ntxiv ntawm collagen hom I hauv covraum[40]. Hauv WT cov qauv, mRNA qis thiab cov protein qhia ntawm collagen hom kuv tau kuaj pom tab sis muaj zog nce ntxiv hauv AD tsiaj. Active lub cev txav mus normalized ob qho tib si mRNA thiab protein qhia ntawm collagen hom I (Fig. 4A, B). Collagen hom kuv ib txwm nyob ib puag ncig cov tubules thiab yog nyob rau hauv lub interstitium. Nrog niaj hnub pathohistological staining ntawm Masson's trichrome, xim xiav sawv cev rau qhov muaj ntawm txhua hom collagen, uas pom nyob ib ncig ntawm cov tubules,lub raumcorpuscles, thiab ib qho me me hauv interstitium hauv WT ob lub raum. Hauv cov qauv AD, qhov sib txuam muaj zog tau pom nyob rau hauv interstitium thiab hauv qee cov tubules. Tom qab kev ua si lub cev, tus nqi ntawm collagen raug txo, thiab tsis muaj kev txuam nrog hauv interstitium (Fig. 4C). Collagen hom I immunohistochemistry tau ua kom paub meej cov collagen ntau lawm. Hauv WTlub raum, zoo ib yam li Masson cov staining, cov cim tshwm nyob ib ncig ntawm cov tubules,lub raumcorpuscles thiab ib qho tsis muaj zog interstitial teeb liab kuj pom. Hauv cov qauv ntawm AD nas, muaj zog tiv thaiv kab mob tau pom nyob rau hauv interstitium thiab nyob ib ncig ntawm lub tubular system. Kuj ceeb tias, qhov txo qis tau pom nyob rau hauv cov ntaub so ntswg interstitial, tab sis ib qho teeb meem tseem ceeb tshwm sim nyob rau hauv lub apical ib feem ntawm tubule epithelial hlwb hauv TAD tsiaj (Fig. 4D).
CISTANCHE yuav txhim kho lub raum / raum mob
Kev sib tham
Txawm hais tias tus txheej txheem ntawm kev tsim AD tau tham dav dav hauv CNS, kev hloov pauv thiab qhov ua tau ntawm cov kab mob peripheral nrog rau cov kab mob tsis tau tshawb xyuas kom meej. Peb qhov kev xav loj yog qhov ntawdraumfibrosis yog txheej txheem ua tau uas cuam tshuam rau AD manifestation thiab A tsub zuj zuj. Ntawm qhov tod tes, kev cob qhia lub cev mus sij hawm ntev tau muaj txiaj ntsig zoo rau AD uas yuav muaj kev cuam tshuam nrog kev hloov pauv ntawm TGFB txoj hauv kev. Cov kab mob peripheral tau pom tias muaj kev koom tes hauv AD pathogenesis, ib yam li lwm tus, kev hloov pauv metabolic enzyme tau kuaj pom hauv cov neeg mob AD [41] thiab Kev Tshaj Tawm tau pom nyob rau hauv pancreas [42] thiab hauvraum[30]. Cov kab mob no cuam tshuam rau ntau yam kabmob ua rau AD kuaj pom ntxov thiab kho tsis yooj yim. Kev tshem tawm ntawm AD los ntawm CNS yog ib feem tswj hwm los ntawm astrocytes thiab microglia tab sis qee qhov tom qab dhau los ntawm cov hlab ntsha-hlwb barrier tuaj yeem raug tshem tawm hauv thaj tsam [43]. Qhov no peripheral clearance yog suav hais tias yog ib qho ntawm cov tshuaj siv tau los txo AD concentration hauv cov ntshav [44]. Kev tso tawm ntawm cov me me los ntawm cov tubules ze ze ntawm lub raum sawv cev yog qhov tseem ceeb hauv homeostatic muaj nuj nqi thiab tuaj yeem koom nrog hauv kev tso tawm ntawm co toxins lossis proteins rau hauv cov zis [45]. Nws tau raug pom tias AD yog zais cia hauv cov zis thiab tuaj yeem ua qhov kev kuaj mob ntawm dementia [46]; Tsis tas li ntawd, lub raum tsis ua haujlwm hauv cov neeg laus tuaj yeem ua rau muaj kev pheej hmoo ntawm dementia [47], thiab nws tau pom tias cov ntshav AD qib siab dua hauv cov neeg mob CKD [10]. Cov ntaub ntawv no qhia txog qhov ua tau ntawm lubraumnyob rau hauv AD clearance nyob rau hauv periphery. Kev tawm dag zog lub cev tau pom tias muaj txiaj ntsig zoo rau lub raum ua haujlwm raws li nws khaws cia kom zoo glomerular morphology thiab tiv thaiv tubular secretion [48]. Kev ua kom lub cev muaj zog tau pom tias yuav txo qis TGFB signalization hauv lub raum thiab kev tawm dag zog lub cev tuaj yeem yog qhov zoo tshaj plaws antifibrotic [32]. Ua ke, kev ua kom TGF tuaj yeem muaj kev sib txuas ncaj qha rau kev ua lub cev muaj feem cuam tshuam nrog cov teeb liab uas yuav tswj tau qhov kev tshem tawm AD hauv qhov chaw. Yog li ntawd, peb tau tshawb xyuas TGFB kev taw qhia txoj hauv kev hauv WT, AD, thiab cov nas uas tau kawm ntev AD.
Hauv txoj kev tshawb fawb tam sim no, nws tau pom tias TGFFRII qhia tau nce hauv ob lub raum ntawm AD nas thiab qee qhov normalized hauv TAD cov qauv. Qhov siab TGFBRII qhia txog kev ua kom zoo dua ntawm TGFB kev taw qhia txoj hauv AD [49]. Qhov txo qis TGF RI qhia pom tias TGFbinds nrog ntau dua affinity rau TGFBRII thiab homodimer tsim yog yuav nyob rau hauv AD [50]. Zoo ib yam li cov txiaj ntsig no, TGFFRII homodimer tsim tau pom hauv macrophages hauvlub raumfibrosis [51]. Ntawm qhov tod tes, kev cob qhia mus sij hawm ntev sib npaug qhov kev qhia ntawm TGFB receptors qhia txog kev tsim heterodimer receptor complex uas tuaj yeem ruaj khov los ntawm kev qhia TGFB1 siab [50]. Ntxiv mus, Smad2 thiab Smad3 qhia tau hloov pauv tsis sib haum xeeb hauv AD. Smad2 paub tias yuav tsum tau qhib ua classical los ntawm TGF receptors thiab hloov mus rau hauv lub nucleus thaum Smad3 muaj qhov txawv txav [52]. Nws tau tshaj tawm tias Smad3 down-regulation cuam tshuam rau fibrosis [53]. Qhov kev qhia zoo ntawm Smad transcription yam hauv TAD cov qauv qhia
tias kev ua si lub cev rov sib npaug TGFB qhov taw qhia thiab txo nws txoj kev ua kom muaj kab mob. Tsev neeg mitogen-activated protein kinase (MAPK) muaj p38MAPK, JNK, thiab ERK muaj kev cuam tshuam nrog TGF signalization [54]. Kev nthuav qhia siab ntawm p38 tau pom nyob rau hauv tubular epithelial hlwb ua rau lub raum fibrosis thiab nws qhov thaiv tuaj yeem yog lub hom phiaj zoo ntawm kev kho mob fibrosis [55]. Hauv AD nas tau nce p38 protein qhia tau pom tias ntxiv txhawb kev tsim fifibrosis. Ntawm qhov tsis sib xws, p38 phosphorylation raug txo qis hauv AD ob lub raum qhia txog kev ua haujlwm ntawm fibrosis theem ntawm p38 MAPK [56]. Ntawm qhov tod tes, kev ua kom lub cev muaj zog ib feem ua rau ob qho tib si p38 thiab phosphorylated daim ntawv muaj nyob rau hauv lub raum qhia txog kev sib npaug ntawm kev cob qhia hauv p38-kev kho mob fibrosis tsim. Zoo ib yam li p38 kinase, inhibition ntawm JNK tuaj yeem cuam tshuam kev tsim fibrosis [57]. JNK activation muaj ntau yam haujlwm hauv ntau qhov chaw ntawm lub raum, xws li glomerular filtration thiab interstitial fibrotic processes [58]. Cov protein extract hauv peb qhov kev sim tau muab los ntawm tag nrho lub raum lysate qhia tau hais tias thaum lub sij hawm AD txoj kev loj hlob JNK yuav muaj ib tug txawv kev ua hauj lwm nyob rau hauv lub tubular thiab glomerular qhov chaw nrog cell-specific isotype activation nyob rau hauv AD-cuam tshuam raum. TGFB1 induced MMP activation tau tshaj tawm nyob ntawm p38 muaj nuj nqi [59], thaum inhibition ntawm ERK kev ua kom txo qis interstitial fibrosis [60]. Cov ntaub ntawv no txhawb nqa cov haujlwm sib txawv ntawm MAPK tsev neeg hauv cov kab mob ntawm lub raum fibrosis hauv AD. Peb tau kuaj pom ERK qis hauv ob lub raum ntawm AD nas, thaum tus nqi ntawm nws cov phosphorylated, active daim ntawv ho nce. Hauv kev sim pab pawg nas koom nrog kev cob qhia lub cev mus sij hawm ntev, peb tau kuaj pom ERK thiab phospho-ERK qhia thiab muaj txiaj ntsig zoo raulub raum ua haujlwm.Cov kev soj ntsuam no txhawb kev koom tes ntawm ERK hauv kev hloov pauv ntawm lub raum fibrotic hauv AD thiab ua kom pom qhov pom tseeb zoo ntawm kev cob qhia lub cev ntawm lub raum morphology thiab kev ua haujlwm ntawm AD nas. Tsis ntev los no, peb lub chaw soj nstuam tau ua pov thawj kev koom tes ntawm ERK hauv kev tsim kho cov pob txha mos [61].
TGFBRII, Smad, JNK, p38, thiab ERK phosphorylation tshwm sim ntawm Ser residues [62] uas tawm tswv yim ua kom muaj peev xwm ntawm Ser / Thr phosphatases. PP2B dysregulation tau pom nyob rau hauv AD [35, 63]. Ntawm qhov tod tes, PP2B tsis zoo tswj phosphorylation ntawm tau hauv lub hlwb, txawm hais tias nws muaj peev xwm dephosphorylate nws ntawm ntau qhov chaw phosphorylation [35]. Lwm cov cellular Ser / Thr phosphatase, PP2A kuj dephosphorylates proteins ntawm Ser residues thiab nws cov dej num yog txo nyob rau hauv AD thiab nws muaj peev xwm tswj tau phosphorylation nrog ntau affinity tshaj PP2B [64]. Qhov txo qis PP2A, tab sis qhov nce PP2B qhia hauv lub raum ntawm AD nas tau pom, zoo ib yam li CNS. Kev nce phosphorylation ntawm Ser residues, raws li qhov tshwm sim ntawm PP2A qhia txo qis, tuaj yeem ua rau hyperphosphorylation ntawm tau protein lossis APP raws li nws tau kwv yees hauv ob lub raum ntawm AD nas. Dephosphorylation ntawm JNK thiab p38 tuaj yeem tswj tau ncaj qha los ntawm PP2B [65], yog li ntawd, qhov txo qis ntawm cov kinases hauv AD tuaj yeem yog qhov tshwm sim ntawm PP2B qhia. Ntawm qhov tod tes, ERK kinase dephosphorylation tuaj yeem tshwm sim los ntawm PP2A ua kom [66], tom qab txo qis ntawm PP2A tuaj yeem ua rau nce ERK phosphorylation hauv AD. Kev cob qhia mus sij hawm ntev ntawm AD nas ua rau qhov normalization ntawm PP2B thiab PP2A qhia uas zoo li hloov pauv Ser / Thr phosphorylation qib ntawm MAPK enzymes ze rau qhov qub. Lub tshuab ua haujlwm tau ua kom muaj kev sib npaug ntawm cov phosphorylation rov qab los ntawm kev koom tes ntawm PP2B thiab PP2A hauv kev sib txawv ntawm chondrocytes ib yam nkaus [61].
Kev ua kom lub cev tsis yog-canonical TGFB signaling txoj hauv kev tswj cov cell proliferation thiab apoptosis. Zoo ib yam li peb cov txiaj ntsig, p21 cov hlwb zoo tau pom nyob rau hauv tubular system ntawm lub raum [67], thiab peb kuj tau nthuav tawm lawv cov lus qhia ntxiv hauv AD. Qhov kev nce siab no qhia txog kev kaw lub voj voog ntawm tes hauv AD kho los ntawm MAPK ua kom, thaum p21 cuam tshuam nrog PCNA thiab thaiv cov hlwb hauv S-theem ntawm lub voj voog ntawm tes [68]. Peb tau kuaj pom p21 ua kom muaj zog thiab qhov ua rau muaj zog PCNA txo qis hauv tubular system ntawm AD nas. Zoo ib yam li peb qhov kev tshawb pom yav dhau los, kev ua kom lub cev muaj zog ua kom lub cev muaj zog hauv cov hlwb ntawm cov hlab ntsha. Ntawm qhov tod tes, p21 kuj muaj kev sib txuas nrog caspase activation thiab JNK, thaum ERK kuj induces caspase3 cleavage [69, 70]. Kev ua kom Caspase3 tau raug txheeb xyuas los ua lub luag haujlwm tseem ceeb hauv AD pathogenesis ntawm A accumulation thiab caspase-mediated ABPP cleavage-induced synoptical disorders [71]. Hauv ob lub raum ntawm AD nas, peb tau kuaj pom cov kab mob siab cleaved caspase 3 uas qhia tias muaj apoptosis siab thiab tsis ua haujlwm ntawm tubular thiab glomerular cells. Cov txheej txheem no tuaj yeem txhawb nqa qhov sib txuam ntawm AD hauv lub raum interstitium raws li peb tau qhia ua ntej [30].
CISTANCHE yuav txhim kho lub raum / raum kab mob
TGFB mediated signalization tau pom tias yuav nce collagen qhia thiab tsub zuj zuj hauv interstitium [40]. Raws li cov txiaj ntsig tau tshwm sim hauv ob lub raum ntawm AD nas, peb tau ua kom pom qhov nce siab ntawm collagen hom kuv qhia tau sau rau hauv interstitium. Cov ntaub ntawv no qhia tau hais tias hloov pauv canonical thiab non-canonical TGFB signalization hauv AD plays lub luag haujlwm tseem ceeb hauv lub raum fifibrosis [18, 72]. Ntawm qhov tsis sib xws, kev cob qhia mus sij hawm ntev txo qhov interstitial collagen hom I localization tsawg kawg ib nrab los ntawm kev rov ntsuas qhov TGFB signalization. Txo fifibrosis tuaj yeem ua rau AD clearance thiab txo nwslub raumKev sib sau ua ke raws li peb tau pom ua ntej [30] Qhov kev xav no tau txais kev txhawb nqa los ntawm peb qhov kev tshawb pom, raws li kev cob qhia lub cev, ua rau muaj kev sib sau ntawm collagen hom I hauv tubular epithelial hlwb, uas tau pom nyob rau hauv ob lub raum ntawm TAD nas implying cov txheej txheem collagen degradation. MMP9 tuaj yeem qhib tau los ntawm TGFB teeb liab nyob rau hauv lub raum [73] thiab ua rau cov degradation ntawm matrix fibrils. Txawm hais tias MMPs muaj ob qho tib si inhibitory thiab stimulatory luag hauj lwm nyob rau hauv fibrosis, MMP9 yog suav hais tias yog ib tug profibrotic neeg sawv cev kho los ntawm TGFB ua kom [74]. Ntawm qhov tsis sib xws, nws tau pom tias MMP9 tuaj yeem ua lub luag haujlwm tswj hwm hauv collagen hom I degradation hauv lub raum [75]. Hauv ob lub raum ntawm AD nas, peb tau pom ib qho kev qhia siab MMP9 qhia nws txoj haujlwm hauv cov txheej txheem fibrotic tswj los ntawm TGFB signaling cascade. Ntawm qhov tod tes, kev nce ntxiv tau ntsuas tom qab kev cob qhia mus sij hawm ntev uas tuaj yeem yog vim li cas ntawm kev txo qis hauv kev qhia thiab tubular tsos ntawm collagen hom I. Hauv kev txhawb zog txhua yam, peb lub chaw kuaj mob tau pom tias MMP9 qhia tau nce siab hauv cov kab lis kev cai chondrogenic. [76], thiab cov txiaj ntsig zoo sib xws tau luam tawm hauv kev tsim cov caws pliav qhov twg cov tshuab compression tau nce qhov kev qhia ntawm MMP9 [77]. Hauv kev raug mob raum mob, qhov siab ntawm MMP9 muaj kev tiv thaiv apoptotic thiab tiv thaiv S3 ib feem ntawm cov tubules ze ze [78]. Tsis tas li ntawd, shear stress-activated TGFB signaling pathway and shorted fifibrosis formation has also been detected in the ntsws [79]. Ntxiv mus, txhua yam kev ntxhov siab nyob rau hauv lub ntsws epithelial hlwb induced lub remodeling ntawm ECM los ntawm kev ua kom MMP9 thiab hloov collagen qhia [80]. Yog li, peb tuaj yeem txiav txim siab tias kev ua kom lub cev muaj zog ua rau kev hloov kho ntawm matrix ntau lawm ntawm kev hloov pauv TGFB signalization tsis yog hauv zos nkaus xwb tab sis kuj ua haujlwm zoo ib yam. Cov kev txwv ntawm peb txoj haujlwm yog tias cov nas transgenic tuaj yeem muaj kev tsim AD sai dua thiab lub raum ua haujlwm tsis raug tshawb xyuas kom meej. Nws yuav tsum tau soj ntsuam ntxiv tias tsuas yog TGFB induced matrix ntau lawm yog hloov nyob rau hauv fibrotic ob lub raum ntawm AD nas los yog cov kev cai ntawm sodium thiab poov tshuaj tshuav nyiaj li cas thiab AD clearance kuj koom nyob rau hauv kev cob qhia mus sij hawm ntev. Tsis tas li ntawd, kev sib sau ntawm collagen hom I hauv cov zis kuj tuaj yeem ua raws li ntxiv los txhim kho qhov kev cob qhia lub cev ntev hauv AD ua rau txo qis ntawm lub raum fibrosis. Ntawm qhov tod tes, peb tau ua pov thawj hauv vivo tias kev cob qhia lub cev mus sij hawm ntev muaj kev cuam tshuam rau kev ua haujlwm ntawm canonical thiab non-canonical TGFB signaling. Hauv vivo peb tau pom tias kev cob qhia lub cev txo qis kev tsim ntawm lub raum fifibrosis hauv AD uas muaj txiaj ntsig zoo rau kev tshwm sim ntawm tus mob. Hauv kev xaus, peb cov ntaub ntawv qhia tias kev ua kom TGFB teeb liab txoj hauv kev ua lub luag haujlwm tseem ceeb hauv fibrotic transformation ntawm ob lub raum ntawm AD nas. Raws li txoj hauv kev yooj yim heev kom ua tiav qhov kev hloov pauv zoo hauv tus mob no, peb pom zoo kom mus sij hawm ntev kev cob qhia lub cev, raws li nws muaj peev xwm ua kom cov lus qhia thiab phosphorylation ntawm cov tswv cuab ntawm canonical thiab non-canonical TGFB qhia txoj hauv kev thiab ua rau kev txhim kho raum fifibrosis. Tsis tas li ntawd, kev tawm dag zog lub cev inhibiting fifibrosis tuaj yeem rov ua kom lub raum ua haujlwm tau zoo thiab tuaj yeem ua rau muaj kev tshem tawm siab ntawm cov txheej txheem AD hauv AD.
