Stx2 Induces Differential Gene Expression And Disturbs Circadian Rhythm Genes in The Proximal Tubule Ⅱ

3. Kev sib tham

3.1. Tus Proximal Tubular Pathology hauv STEC Cov Neeg Mob thiab Cov Qauv Tsiaj

Oliguria yog ib qho cim ntawmmob tubular necrosis(ATN). Vim yog ATN, sloughed epithelial hlwb cuam tshuam lub lumen ntawm tubules kom txo cov kua dej uas zoo li oliguria. Hauv cov neeg mob STEC, oliguria thiab anuria feem ntau yog qhov kev tshawb pom uas qhia ATN. Kev txo qis hauv cov tubules zeem muag thaum lub sijhawm ua ntej ntawm STEC kis kab mob tau pom tias nce hauv cov zis 2MG thiab NAG [3,33]. B2MG yog ib qho 12 kDa me me protein uas yog lim los ntawm glomeruli thiab absorbed tag nrho los ntawm cov tubules nyob rau hauv lub xeev noj qab haus huv. Txawm li cas los xij, ib zaug cov tubules ze ze raug puas tsuaj, tus nqi ntawm 2MG hauv cov zis nce ntxiv yog li ua haujlwm biomarker rau kev ua haujlwm ntawm tubular. NAG yog ib qho loj lysosomal enzyme ntawm 130 kDa thiab cov glucosidase loj nyob rau hauv lub tubule proximal. Vim qhov loj me, lub glomerulus tsis tuaj yeem lim NAG qhov uas cov tubules raug puas tsuaj tso NAG hauv cov zis. Yog li, qhov nce hauv cov zis NAG yog lwm biomarker rau kev puas tsuaj rau tubular ze. Glomerular histopathology xws li fibrin-obstructed capillaries yog ib qho kev tshawb pom ntawm cov neeg mob STEC; Txawm li cas los xij, cov ntaub ntawv tso zis saum toj no qhia txog kev puas tsuaj rau tubular hauv cov kab mob ua ntej. Tsis tas li ntawd, cov zis los ntawm cov neeg mob STEC muaj cov casts uas muaj cov tubular epithelium uas yog tshwm sim los ntawm sloughing ntawm cov kab mob proximal tubular puas lawm [1]. Hauv cov qauv tsiaj, sloughing ntawm proximal tubular hlwb feem ntau pom ob qho tib si hauv STEC kab mob thiab Stx txhaj qauv [25–27,34]. Nyob rau hauv txoj kev tshawb no tam sim no, peb pom sloughing ntawm lub proximal tubular hlwb nyob rau hauv Stx2- txhaj murine raum qhia Stx2-induced proximal tubular alteration (Daim duab 1). Tsis tas li ntawd, peb tau kuaj pom Stx2 receptor Gb3 nyob rau hauv ob qho tib si nas thiabtib neeg lub raum proximal tubulesnrog luminal sab qhia uas qhia txog kev sib cuam tshuam ncaj qha ntawm Stx2 nrog cov tubules ze ze (Figures 2 thiab 3). PDK4 phosphorylates pyruvate dehydrogenase inhibit nws cov haujlwm ua rau txo qis hauv kev siv cov piam thaj thiab nce cov roj metabolism hauv cov lus teb rau kev yoo mov thiab tshaib plab. Nws tiv thaiv detachment epithelial hlwb tiv thaiv detachment-induced cell tuag (anoikis) [35]. PDK4 pib muaj lub ncov ntawm 4 h, ces lwm lub ncov ntawm 8 h (Daim duab 5A). Nws tau txo qis ntawm 12 teev, tab sis los ntawm qhov ntawd nws tau maj mam nce mus txog 72 teev nrog lub cav2-fold hloov thiab xaus nrog 3 (12-fold hloov) (Daim duab 5A). Lub detachment ntawm epithelial hlwb tshwm sim nyob rau hauv Stx2- txhaj nas proximal tubular hlwb (sloughing) nrog cov tsis zoo morphology (Daim duab 1), qhov no tej zaum yuav muaj kev cuam tshuam ntawm PDK4 upregulation.

CISTANCHE TUBULOSA EXTRACT POWDER rau lub raum

Raws li NHERF1 (SLC9A3R1) pab txhawb rau lub cev ib txwm ua raws li cov cellular matrix [36], actin cytoskeleton lub koom haum, thiab tswj cov cell morphology, txo qis ntawm cov noob no yuav muaj qhov tshwm sim xws li sloughing ntawm cov hlwb (Daim duab 5K).

Cov nyhuv ntawm Stx2 ntawm cov tubules ze ze zoo li qhov tseem ceeb rau STEC kis kab mob; Txawm li cas los xij, kev hloov pauv ntawm cov noob qhia tsom rau hauv vivo proximal tubular cells tsis muaj ua ntej. Keepers et al. [37] pom cov noob sib txawv (DEGs) nyob rau hauv ob lub raum ntawm Stx2+LPS-txo nas xws li tias feem ntau ntawm cov noob ua ntej thiab hloov pauv loj yog los ntawm LPS cawv thiab cov noob uas tau hloov kho lig yog los ntawm cov nyhuv Stx2. . Lawv cov ntaub ntawv tau pom muaj txiaj ntsig zoo rau qhov sib txawv ntawm cov noob ntawm lub raum tab sis tsis cais cov hom cell uas yog lub luag haujlwm rau qhov kev hloov ntawd. Hauv txoj kev tshawb fawb tam sim no, peb tau sim nrhiav DEGs uas cuam tshuam nrog cov kab mob sib kis los ntawm kev sib piv cov ntaub ntawv microarray los ntawm nas.ob lub raum thiab tib neeg thawj proximal tubularcov hlwb uas tau kho nrog Stx2.

3.2. Proximal Tubular Factors Koom nrog Stx2-Induced Pathology nyob rau hauv Relation to Known Stx2 Activities in Gb3 Expressing Cells

3.2.1. Kev Ua Haujlwm (I), Kev Hloov Pauv Hloov Los ntawm Stxs Binding rau Gb3

B subunits ntawm Stxs induce non-receptor src tyrosine kinase Yes phosphorylation [4]. Phosphorylated Yes activates downstream signaling molecules los ntawm nws cov kev ua kinase thiab ua rau cytoskeleton remodeling [5]. Yog-associated protein (YAP) phosphorylation nyob rau hauv Y357 los ntawm Yes yog paub los ntxias YAP nuclear translocation qhov twg nws ua raws li ib tug transcriptional co-activator los ntxias cov hom phiaj xws li CTGF [38]. Matricellular proteins CYR61 (CCN1) thiab CTGF (CCN2) yog zais cia rau hauv extracellular matrix (ECM) thiab pab txhawb kev tswj cell-ECM adhesion los ntawm kev khi receptor integrins thiab cell nto-txuas nrog heparan sulfate proteoglycans (HSPGs) [39,40]. Cov molecule tseem ceeb ntawm CCN1 thiab 2 transcription yog YAP, thiab nws khaws cia tsis muaj zog (phosphorylated ntawm serine residue 127) nyob rau hauv ib txwm lub xeev nyob rau hauv lub Hippo signaling pathway. Thaum kuaj pom tias tsis muaj cell-cell adhesion, YAP (S127) tsis muaj phosphorylated tso cai rau nws nkag mus rau hauv lub nucleus, thiab kev sau ntawm CCN1 thiab 2 nce [41–43]. CCN1 thiab 2 ua raws li qhov mob-kho cov molecules los kho cov ntaub so ntswg ploj [38,44]. Hauv peb cov ntaub ntawv, CCN1 thiab 2 muaj qhov siab me me ntawm lub sijhawm ua ntej (6 h). Qhov no tej zaum yuav xav txog Stx2 binding-induced Yes activation. Cov sij hawm tom qab, sloughing yuav ua rau YAP ua kom ntxiv los ntawm kev hloov ntawm Hippo signaling txoj hauv kev los ua kom muaj kev qhia ntau dua ntawm CCN1 thiab 2 ntawm 12 teev lossis tom qab (Daim duab 5C).

3.2.2. Kev Ua Haujlwm (II), Inducing ER-Stress / UPR

Stxs ncav cuag ER, qhov twg cleaved A1 fragments thiab B subunits mimic unfolded proteins [9,10,45,46]. Glucose-regulated protein 78 kDa (Grp78, binding immunoglobulin protein (Bip)) yog anchoring peb UPR tseem ceeb proteins Inositol-yuav tsum tau enzyme-1 (Ire-1), protein kinase R-zoo li endoplasmic reticulum kinase (PERK ) thiab activating transcription factor 6 (ATF6) hauv ER membrane thaum lub xeev ib txwm muaj. Txawm li cas los xij, vim tias muaj kev sib raug zoo siab dua ntawm Grp78 rau cov proteins uas tsis tau nthuav tawm, nws tso cov proteins uas tau tso tseg. ATF6 tau qhib tom qab tso tawm thiab nce CHOP (DDIT3) thiab X-box binding protein 1 (XBP-1) mR NAs, whereas Ire-1 splices XBP-1 mRNA los tsim XBP{{ 22}} protein, uas ua rau CHOP (DDIT3) transcription. PERK activation ua rau ATF4 ua kom nce CHOP (DDIT3) mRNA. Yog li ntawd, qhov nce hauv CHOP (DDIT3) mRNA yog qhov cim ntawm UPR [47,48]. Stxs tau tshaj tawm los txhawb UPR thiab CHOP (DDIT3) yog tswj hwm hauv peb cov ntaub ntawv (Daim duab 5D–G).

GDF15 belongs rau tsev neeg transforming growth factor beta (TGF). Tsis ntev los no, GDF15 tau pom los kho mob ntshav qab zib cov tshuaj metformin vim qhov hnyav poob [49]. Hauv cov tshuaj metformin hauv qhov ncauj, cov hnyuv thiab lub raum GDF15 mRNA tau nce ntxiv ua rau nce qib hauv cov ntshav GDF15. Circulating GDF15 ua haujlwm ntawm lub paj hlwb-txhim kho receptor los txo cov khoom noj kom tsawg thiab txo lub cev hnyav [50]. Hauv cov nas uas siv metformin, nce lub raum GDF15 tsawg kawg yog ib feem ntawm kev tswj hwm ntawm CHOP (DDIT3). Hauv peb cov ntaub ntawv ntawm Stx2- txhaj raum nas, CHOP (DDIT3) tau nce thawj zaug ntawm 4 h nrog lub ncov me me thiab tom qab ntawd pib muaj kev nce ntxiv mus rau 48 h uas nws tswj tau qib ntawm lub cav{{16 }}fold hloov nyob ib ncig ntawm 1.3 (Daim duab 5E). Raws li STRING pawg tsom xam, GDF15 tau txais cov tswv yim nquag los ntawm cov ntsiab lus hloov pauv CHOP (DDIT3) thiab ATF3 (Daim duab 5E) [51,52]. Lub Stx2- txhaj tshuaj murine hnyav pib txo lossis sib txawv ntawm kev tswj cov ntsev tom qab 24 teev (Daim duab S3B). Kev nce hauv GDF15 mRNA hauv lub raum ntawm 12 teev tuaj yeem cuam tshuam rau lub hlwb kom txo tau cov khoom noj, uas tau tshwm sim los ntawm kev poob phaus (Figures 5E thiab S3B). Raws li GDF15 qib qhia tau txuas ntxiv siab tom qab 24 teev, nas txuas ntxiv poob phaus (Daim duab 5E thiab S3B).

Raws li ER-kev ntxhov siab, PERK raug tso tawm los ntawm Grp78 (Bip) thiab oligomerizes rau phosphorylate thiab inhibit eIF2 ua rau muaj kev cuam tshuam kev txhais lus thoob ntiaj teb, tshwj tsis yog qee qhov UPR teb cov proteins xws li ATF4, CHOP, thiab Grp78. GADD34 (protein phosphatase 1 regulatory subunit 15A (PPP1R15A)) yog ib qho ntawm cov protein ntau ntxiv nyob rau hauv phosphorylated eIF2 (eIF2 (P))-associated inhibition thiab upregulated hauv peb dataset (Daim duab 5D, G). GADD34 yog ib qho protein phosphatase -1 uas dephosphorylates eIF2 (P) kom tsis zoo tswj UPR-induced protein synthesis inhibition [53]. GADD34 (PPP1R15A) qhia ua cav2-fold hloov loj dua 1 tom qab 12 h ntawm Stx2 hauv peb cov ntaub ntawv thiab txuas ntxiv mus txog 24 teev thaum nws toj siab (log2-fold hloov sib npaug 2.1) thiab tswj qhov no theem kom txog thaum kawg (Daim duab 5D,G). Qhov no qhia tias Stx2-induced UPR muaj cov lus tsis zoo tom qab 12 teev. Kev hloov pauv thiab kev txhais lus ntau ntxiv ntawm CHOP (DDIT3, GADD153) yog qhov cim ntawm ER-kev ntxhov siab thiab cov qauv qhia txawv txawv zoo li GADD34 tshwj tsis yog log2 loj dua 1 tshwm sim tom qab 24 teev thiab maj mam toj siab tom qab ntawd (log2 sib npaug 1.3) (Daim duab 5D, G). Tsis tas li ntawd, CEBPB, qhov tseem ceeb dimerizing tus khub ntawm CHOP, yog upregulated nyob rau hauv peb dataset (Daim duab 5D, G). Cov txiaj ntsig no qhia tias thaum Stx2- ntxias ER kev ntxhov siab pib txij thaum ntxov hauv lub sijhawm kawm, nws ua rau muaj txiaj ntsig mus txog 24 teev thiab tswj hwm qib ntawm ER kev ntxhov siab mus txog thaum kawg thaum qhov kev tawm tswv yim tsis zoo sab caj npab kuj sib npaug nws tus kheej ib txhij.

3.2.3. Kev Ua Haujlwm (III), Ribosome Inactivating Protein (RIP) Kev Ua Haujlwm

Los ntawm cleaving adenine los ntawm rRNA, Stxs inhibit de novo protein synthesis. Depurinated rRNA cuam tshuam rau kev hloov pauv ntawm ribosomal morphology, uas ua rau cov teeb liab hloov pauv [11] hu ua ribotoxic stress response (RSR), ua rau cov yam ntxwv ua kom muaj zog (saib kab lus tom ntej).

3.2.4. Kev Ua Haujlwm (IV), Ribotoxic Stress Response (RSR) Activity

ZAK (a MAPKKK) yog lub npe hu ua kinase hauv RSR thiab qhib cov dej ntws qis JUN N terminal kinase (JNK) thiab p38 MAPK txoj hauv kev [12,15]. Stx1 induces JUN thiab FOS mRNA qhia nyob rau hauv tib neeg txoj hnyuv epithelial hlwb thiab qhov kev tshwm sim yuav tsum tau RIP kev ua-zoo Stx1 uas nws yog xav nyob rau hauv RSR tswj [15]. Lwm RSR downstream molecule, p38 MAPK, paub tias yuav ua rau cov noob teb thaum ntxov xws li JUN, FOS, EGR1, MAFF, DDIT3 thiab ATF3 [54], tag nrho cov no tau qhia txawv hauv peb cov ntaub ntawv. JNK thiab p38 MAPK txoj hauv kev kuj tseem paub tias yuav qhib cov xwm txheej hauv qab ntawm Ire-1 hauv qab UPR (Figures 4B,D thiab 5B,E,F) [47]. Vim li no, Stxs tuaj yeem qhib ob txoj hauv kev los ntawm RSR thiab / lossis UPR. Txoj kev tshaj tawm xov xwm tshaj tawm-txheej txheem kinase (ERK) 1/2 txoj hauv kev kuj tau pom tias yog qis qis ntawm RSR [55] thiab DUSP1 (MKP1) noob paub tias raug ntxias los ntawm ERK1/2 txoj hauv kev [56]. Stx1 thiab anisomycin (lwm cov protein synthesis inhibitor) muaj peev xwm inducing DUSP1 qhia hauv Caco{28}} hlwb [57]. Hauv peb cov ntaub ntawv, DUSP1 kev qhia tau raug ntxias thiab qhov no yuav qhia tau tias ua kom ERK1/2 (Daim duab 4B, D).

3.2.5. Yam Ntxim Saib Ntxim Ua (V), Cov Gene Transcription ua rau Cytokine

Secretion IL-8 secretion in Stxs-treated cells, human intestinal cells in particular, has been reported extensively as a downstream factor of RSR [12,58]. Cytokines and chemokines in animal models revealed CXCL1 and CXCL10 expression within Stx2-treated mouse kidneys [37,59]. IL-8 (CXCL8) and CXCL1 are both neutrophil chemoattractants that utilize CXCR2 as their receptor. IL-8 is expressed in humans but not in mice. In our dataset, human RPTEC showed an increase in IL-8 expression by log2 equals 4.6-fold maximum (log2 values were 4.6, 3.52 and 4.48 at 6, 13, and 19 h after Stx2, respectively). As we extracted common DEGs from mouse kidney and human proximal tubular cells IL-8 was not included in the final dataset. Instead, the molecule with similar function as IL-8, CXCL1 showed a significant increase as a common DEG (Figure 5L). A downstream pathway of UPR is the NF-κB signaling pathway. NFKBIA (IκBα) is an inhibitory factor of the classical (canonical) NF-κB pathway that binds with NF-κB subunits RelA (p65) and p50. Upon phosphorylation by an upstream kinase, NF-kappa B kinase (IKK), NFKBIA (IκBα) is degraded, thus NF-κB can translocate to the nucleus and induce transcription of inflammatory factors. RelB proto-oncogene nuclear factor-kappa B subunit (RelB) is involved in the alternative (non-canonical) NF-κB pathway and induces inflammatory factor expression. RelB-p50 or -p52 dimers act as a transcription factor (NF-κB) when phosphorylation of p100 of RelB-p50-p100 or RelB-p100 complexes is done by IKKα homodimers and this step does not involve IκBα. In our dataset, both NFKBIA and RelB were differentially expressed reaching to log2-fold change >1 tom qab 24 thiab 48 h, ntsig txog (Daim duab 5F). Qhov no qhia tias lub kaus mom noob nyob rau hauv ob qho tib si NF-κB txoj hauv kev, IκB (classical) thiab RelB (lwm txoj kev), raug ntxias. Raws li CHOP (DDIT3) qhov sib txawv ntawm qhov sib txawv tshaj log2 loj dua 1 tom qab 24 teev, nas lub raum nkag mus rau UPR, yog li qhib PERK phosphorylates eukaryotic translation pib qhov tseem ceeb 2 subunit alpha (eIF2) los cuam tshuam kev txhais lus ntawm NFKBIA [60]. Vim li no, kev nce hauv NFKBIA mRNA yuav tsis cuam tshuam ntau ntawm NF-κB txoj hauv kev, yog li kev tso cai ntawm cov kab mob inflammatory (Daim duab 5L).

Sib et al. [61] tau pom tias ob qho tib si classical (RelA-p50) thiab lwm txoj hauv kev (RelB-p52) NF-κB txoj hauv kev tau ua haujlwm hauv Stx-txhaj nas kuaj pom kev txhim kho ntawm RelA-target cytokines CCL20, CXCL1 thiab CXCL10 uas kuj tau qhia txawv hauv peb. dataset (Daim duab 5L).

3.2.6. Kev Ua Haujlwm (VI), Stxs-Induced Apoptosis

Cleaved caspase 3 tau pom nyob rau hauv Stxs-txuas cell tuag [13,15,62]. Nws tau pom tias caspase-3 ua kom lossis induction ntawm apoptosis los ntawm Stxs tshwm sim hauv cov ntsiab lus ntawm RSR thiab UPR. Hauv Stxs-induced RSR, ob qho tib si p38 MAPK thiab JNK txoj hauv kev tau koom nrog [15] nrog rau kev ua kom ERK tau tshaj tawm [55]. UPR ntawm qhov tod tes yog paub tias yuav ntxias NF-κB, JNK thiab p38 MAPK txoj hauv kev. CHOP (DDIT3) nyob rau hauv UPR tau pom tias muaj kev koom tes hauv apoptosis [63] thiab nws kuj tau pom nyob rau hauv Stxs-associated apoptosis [13]. JNK kev koom tes hauv UPR-induced apoptosis tau raug tshaj tawm ib yam nkaus [64]. Hauv peb cov ntaub ntawv, ntau DEGs xws li CHOP (DDIT3), NFKBIA, FOS, JUN, thiab JUNB koom nrog RSR thiab UPR (Daim duab 5B, F).

3.3. Circadian Dysregulation los ntawm Stx2

Kev nce hauv serum endothelin-1 (ET-1, EDN1) tshwm sim hauv cov neeg mob STEC-HUS [65]. Tsis tas li ntawd, kev sim, Stx2 induces EDN1 qhia los ntawm activating teeb liab transduction cascades uas muaj MAPKs [66], thaum UPR paub los induce EDN1 transcription [67]. Hauv peb cov ntaub ntawv, Stx2 kev kho mob-txhim kho EDN1 qhia hauv ob lub raum nas thiab tib neeg lub cev tubular (Daim duab 5G). Raws li EDN1 gene qhia yog ib qho ntawm cov kab mob ntawm nas raum thiab tib neeg cov tubular epithelial hlwb, nws qhia tau hais tias EDN1 tshwm sim, tsawg kawg yog ib feem, nyob rau hauv lub proximal tubular epithelial hlwb nyob rau hauv vivo ntxiv rau endothelial hlwb. Tsis tas li ntawd, secreted ET-1 induces EGR1 qhia ntawm MAPK ua kom [68]. Peb cov ntaub ntawv lees paub qhov kev tswj hwm ntawm EGR1 nyob rau lub sijhawm thaum EDN1 raug kho (Daim duab 5H). EGR1 induces circadian atherosclerosis qhov tseem ceeb ntawm PER1 qhia [69], yog li, kho qhov amplitude ntawm lwm yam circadian yam (Daim duab 5H).

Hauv kev cai circadian, lub heterodimer ntawm CLOCK-BAML1 (ARNTL) upregulates PER1 thiab CRY1. Hauv kev xa rov qab, PER1-CRY1 heterodimer khi rau CLOCK-BAML1 (ARNTL) txhawm rau txhawm rau tshem tawm lawv tus kheej ntxiv, yog li ua rau lub voj tsis zoo ntawm lub voj voog circadian. Yog li, kev qhia txog cov noob caj noob ces zoo (CLOCK thiab BAML1 (ARNTL)) thiab cov noob tswj tsis zoo (PER1 thiab CRY1) tshwm sim nyob rau hauv ib lub sijhawm sib txawv uas thaum CLOCK thiab BAML1 (ARNTL) muaj kev nthuav qhia ntxiv, PER1 thiab CRY1 raug txo, thiab tus qauv hloov hauv cov teev tom qab. Thaum peb ntxiv cov cav2-fold hloov qhov tseem ceeb ntawm CLOCK thiab BAML1 (ARNTL) rau daim duab (Daim duab 5J), thaum lub sijhawm ua ntej cov ntsiab lus, PER1 thiab BAML1 (ARNTL) tau pom qhov sib txawv ntawm qhov sib txawv nrog cov ntse / nqaim peaks, Txawm li cas los xij, tom qab 24 teev lub ncov pib ua npub thiab nce ntxiv tsis tu ncua. Qhov no txhais tau tias Stx2 cuam tshuam rau lub raum circadian atherosclerosis nrog rau hauv cov tubules ze ze. Txawm hais tias yuav tsum muaj kev sim ua kom pom tseeb ntxiv, peb cov ntaub ntawv ua ke nrog cov ntaub ntawv luam tawm uas piav qhia txog PER1 cuam tshuam rau lub raum circadian kev tswj hwm raws li stimuli [70,71], qhia tias muaj peev xwm Stx2 kev koom tes hauv circadian atherosclerosis cuam tshuam thiab cuam tshuam rau kev ua haujlwm ntawm tubular los ntawm kev txo lub raum moos genes- tswj hwm yam xws li SGLT1 (SLC5A1) (Daim duab 5K). Tseeb tiag, peb tau soj ntsuam glucosuria hauv Stx2- txhaj cov nas uas rov tsim dua hauv lwm txoj kev tshawb fawb nrog Stx1- txhaj cov nas [21] qhia tias SGLT1 (SLC5A1) tsis ua haujlwm hauv cov tubules ze ze (Table 3). Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, qhov no yog thawj daim ntawv qhia qhia txog Stx2 kev koom tes hauv lub raum circadian atherosclerosis.

4. Cov lus xaus

Peb tau txiav txim siab cov noob tseem ceeb hais txog cov kab mob sib txuas ntawm tubule uas tau hloov los ntawm Stx2. Kev qhia txawv ntawm cov noob no yog suav tias yog qhov tshwm sim ntawm Stx2 cov haujlwm xws li UPR induction, rRNA depurination-associated RSR, thiab teeb liab transduction induced los ntawm plasma membrane receptor Gb3 khi. Cov noob uas tau hais ntau tshaj plaws, GDF15, tuaj yeem cuam tshuam qhov poob phaus los ntawm Stx2 los ntawm lub hlwb-stem-txhim kho receptors los ntawm kev txo cov khoom noj. Extracellular matrix thiab cell adhesion tej zaum yuav cuam tshuam los ntawm PDK4, NHERF1 (SLC9A3R1), CYR61, thiab CTGF uas ua rau Stx2- tshwj xeeb histopathology, thiab sloughing. Tsis tas li ntawd, kev cuam tshuam hauv lub raum circadian atherosclerosis los ntawm Stx2 tuaj yeem ua rau cov tubular ua haujlwm tsis zoo xws li qabzib reabsorption.

5. Cov ntaub ntawv thiab cov txheej txheem

5.1. Tsiaj nas

(C57BL/6, txiv neej, 19–22 g, tsis muaj kab mob tshwj xeeb) tau yuav los ntawm Charles River Laboratories Japan, Inc. (Yokohama, Nyiv). Khoom noj thiab dej tau muab rau libitum. Cov nas tau tswj hwm nrog tus qauv 12 h: 12 h lub voj voog-teeb tsaus ntuj uas lub teeb nyob ntawm 7 teev sawv ntxov thiab tawm thaum 7 teev tsaus ntuj Tsiaj hauv chav sov tau khaws cia nyob ib puag ncig 24 ◦C. Tag nrho cov txheej txheem tau pom zoo los ntawm University Animal Care Committee.

5.2. Tib neeg lub raum ntaub so ntswg

Cadaver cov ntaub so ntswg yam tsis muaj tus kheej tus kheej tau siv rau hauv txoj kev tshawb no, thiab nws raug suav tias yog kev zam los ntawm tib neeg kev tshawb nrhiav cov txheej txheem ntawm University.

5.3. Cell Culture

Tib neeg lub raum proximal tubular epithelial cell (RPTEC) thawj kab lis kev cai tau yuav los ntawm Clonetics (Walkersville, MD, USA) thiab khaws cia ntawm 37 ◦C, 5% CO2 cua. Cells tau coj mus rau hauv lub raum epithelial cell loj hlob nruab nrab ntxiv nrog tib neeg epidermal loj hlob yam, hydrocortisone, epinephrine, insulin, tri-iodothyronine, transferrin, GA-1000, thiab FBS.

5.4. Kev huv ntawm LPS-Free Stx2 LPS tshem tawm ntawm Stx2 tau ua raws li tau piav qhia yav dhau los [72]. Luv luv, cov anti-Stx2 11E10 antibody-conjugated kem tau siv kom tau Stx2 feem, thiab cov feem tau tom qab dhau los ntawm Detoxi-gel (Thermo Fisher Scientific, Rockford, IL, USA) tshem LPS. Stx2 feem yog lim nrog 0.2 µm lim thiab LPS qib tau sim nrog Limulus amebocyte lysate assay (Pyrotell, Associates of Cape Cod Incorporated, East Falmouth, MA, USA). Cov feem Stx2 tau txiav txim siab tias muaj tsawg dua 0.03 endotoxin unit (EU) / mL. Stx2 variant hom yog Stx2a.

5.5. Stx2 Administration rau Mice A 2LD50 koob tshuaj (5 ng Stx2/20 g lub cev hnyav), uas tua cov nas nyob rau hauv plaub hnub (Daim duab S3A), tau txhaj rau cov nas intraperitoneally hauv ntim ntawm 0.1 mL hauv LPS-dawb saline (Otsuka kws tshuaj, Nyiv). Kev txhaj tshuaj lom tau ua tiav thaum 7 thiab 9 teev sawv ntxov, qhov ntev ntawm cov nas tau txais Stx2 thaum 7 teev sawv ntxov thiab ua raws li 24, 48 thiab 72 teev kuaj ntawm 7 teev sawv ntxov.

5.6. Tso zis thiab ntsuas ntshav qabzib tom qab 2, 6, 8, 12, 24, 36, 48, 60, thiab 84 h ntawm kev txhaj tshuaj Stx2, tso zis thiab 10 µL tau muab tso rau hauv Fuji qhuav chem swb GLU- P III (FUJIFILM, Kanagawa, Nyiv) thiab cov piam thaj tso zis tau ntsuas nrog Fuji Dry Chem 7000 V (FUJIFILM) ntawm lub chaw tswj hwm los ntawm. Cov zis los ntawm kev txhaj tshuaj ua ntej Stx2 tau siv los ua 0 h qauv. Kev sau zis tau ua nrog ob tus nas

5.7. Kev ua cov ntaub so ntswg tom qab 2, 4, 6, 8, 12, 24, 48, thiab 72 h ntawm kev txhaj tshuaj Stx2, peb nas ib lub sij hawm taw tes tau euthanized los ntawm CO2, thiab lub raum tau sau. Ib nrab ntawm ib lub raum tau kho nrog 4% paraformaldehyde / phosphate-buffered saline rau 7 hnub ntawm 4 ◦C thiab ua tiav rau ntu paraffin. Lwm ib nrab ntawm lub raum tau siv los rho RNA rau kev tsom xam microarray. Peb cov nas uas tsis muaj kev kho mob tau siv li qub (naïve lossis 0 h) tswj. Peb cov nas tau txhaj nrog PBS (tsheb) thiab muab txi rau ntawm 72 teev thiab tau txais kev tswj hwm lub tsheb los qhia tias muaj kev hloov pauv ntawm cov noob caj noob ces piv rau kev tswj ib txwm.

5.8. Periodic Acid-Schiff (PAS) Stain Paraffin seem tau hydrated thiab stained nrog 0.5% periodic acid tov, tom qab ntawd yaug nrog dej distilled. Cov seem tau pauv mus rau Schiff reagent. Tom qab cov seem tau ntxuav nrog 0.6% sodium bisulfite tov kom huv si, lawv tau counterstained nrog hematoxylin.

5.9. Stx2 Kev Kho Mob hauv Tib Neeg RPTEC RPTEC cov hlwb raug kho nrog 10 ng / mL ntawm Stx2 rau 6, 13 thiab 19 h hauv qab 37 ◦C, 5% CO2. RPTEC tsis muaj Stx2 kho tau siv los tswj. Ob qhov kev sim tshuaj lom neeg tau ua rau ib lub sijhawm. Tom qab ntxuav nrog PBS, hlwb tau siv rau RNA rho tawm.

5.10. Free-Floating Immunofluorescence Staining Rau kev ua haujlwm pub dawb-floating seem, tus nas ib txwm raug kho-kho raws li tau piav qhia hauv Obata li al. [69]. Murine ob lub raum tau raug txiav thiab muab tso rau hauv 4% PFA / PBS rau 3 teev ntawm 4 ◦C nrog kev ntxhov siab. Cov ntaub so ntswg tau ntxuav nrog PBS, thiab tiv thaiv cryoprotected nrog 30% sucrose / PBS ntawm 4 ◦C thaum hmo ntuj los yog kom txog thaum lub dab dej mus rau hauv qab. Thick frozen sections (50 µm) raug txiav nrog zawv zawg microtome nrog cov chaw khov. Cov ntu raug thaiv nrog 1:50 tshis los tiv thaiv tus nas IgM tshuaj tiv thaiv hauv PBS (F104UN, American Qualex International, Inc., San Clemente, CA, USA) rau 1 h thiab incubated nrog anti-Gb3 / CD77 antibody 1: 100 hauv kev thaiv tshuaj los yog isotype tswj (rat IgM) nyob rau hauv ib tug matched concentration raws li thawj antibody rau hmo ntuj ntawm 4 ◦C. Tom qab ntxuav, ntu tau muab tshuaj tua kab mob thib ob (anti-rat IgM Alexa Fluor 488, 1:2000 dilution hauv PBS) rau 2 teev ntawm 4 ◦C. Cov seem tau stained rau lwm qhov kev sojntsuam AQP1 (1: 1000 dilution, Sigma-Aldrich Nyiv, Tokyo, Nyiv) thiab CD31 (550274, 1:50 dilution, BD Biosciences, Franklin Lakes, NJ, USA) nrog cov tshuaj tiv thaiv thib ob los tiv thaiv luav IgG Alexa Fluor 546 (1: 2000 dilution hauv PBS) thiab anti-nas IgG Alexa Fluor 647 (1: 2000 dilution hauv PBS), feem. Tom qab ntxuav nrog PBS, 40 ,6-Diamidino-2-phenylindole (DAPI, D21490, Thermo Fisher) stain tau ua tiav rau 15 feeb ntawm 4 ◦C. Cov seem tau muab tso rau hauv cov kua dej hauv qhov dej ntawm 96- lub phaj zoo (U-shaped) nrog cov xim me me blush txhua zaus hloov kho. Cov kauj ruam ntxuav tau ua tiav hauv qhov dej loj dua xws li hauv 12- lossis 6- cov phiaj xwm zoo. Aqueous mounting media tau siv los npog npog. Rau tib neeg cov ntaub so ntswg kev npaj, lub raum postmortem tau kho nyob rau hauv 20% formalin rau 2 lub lis piam thiab ua tiav rau free-floating npaj raws li ua rau nas cov ntaub so ntswg nrog ib tug tshwj xeeb anti-tib neeg CD31 antibody (CBL468, 1:20 dilution, Merck KGaA, Darmstadt. , Lub teb chaws Yelemees) tau siv.

5.11. Microarray Analysis

Ib nrab ntawm lub raum yog muab tso rau hauv 2 mL ntawm RNALater (Ambion, Austin, TX, USA) ntawm 4 ◦C thiab tag nrho RNA tau muab rho tawm nrog cov khoom siv Rneasy Midi (Qiagen, Santa Clarita, CA, USA). Mouse Genome 430A 2.0 arrays (Affymetrix, Santa Clara, CA, USA) tau siv. CEL cov ntaub ntawv tau txais thiab ua haujlwm zoo nrog ntau lub nti nruab nrab (RMA), suav RNA tus lej luam, thiab hloov pauv hloov tawm tsam 0 h tau ua hauv R (v.3.6.0) nrog nws pob affy [73] thiab RankProd 2.0 [74]. Cov ntaub ntawv muaj nyob rau hauv cav2-fold hloov qhov tseem ceeb. Cov ntaub ntawv raug xa mus rau Gene Expression Omnibus (GEO) (Accession naj npawb GSE172465). Los ntawm RPTEC hlwb, tag nrho RNA raug rho tawm nrog RNAgents Tag Nrho RNA Isolation System (Promega, Tokyo, Nyiv). Tag nrho tib neeg genome oligonucleotide microarray (44K oligonucleotide DNA microarray, Agilent Technologies, Tokyo, Nyiv) tau siv rau kev sim microarray. Tag nrho cov qauv RNA tau siv rau kev npaj Cy5- thiab Cy{18}}labeled cDNA sojntsuam. Cov qauv fluorophore-labeled tau hybridized ntawm txhua lub iav swb thiab ntxuav, tom qab ntawd luam tawm nrog DNA microarray scanner (tus qauv G2505A; Agilent Technologies). Lub Feature Extraction thiab Image Analysis software (Agilent Technologies) tau siv los nrhiav thiab piav qhia txhua qhov chaw hauv cov array thiab sib xyaw cov kev siv zog, uas tau lim thiab normalized los ntawm kev siv lub hauv paus hnyav scatterplot smoothing txoj kev. Kev rov tsim dua tshiab ntawm microarray tsom xam tau soj ntsuam los ntawm ob qhov kev rov ua dua ntawm cov xim-swap hauv txhua qhov kev sim. Qhov sib txawv hauv mRNA kev qhia ntawm kev tswj thiab Stx2- kho RPTEC tau sau tseg ntawm 6, 10, lossis 19 teev tau muab piv. TXT hom ntaub ntawv los ntawm GeneSpring software tau siv los txiav txim lub npe noob los ntawm kev sojntsuam ID thiab hloov pauv hloov pauv mus rau lub cav2-fold hloov. Cov ntaub ntawv xa mus rau GEO (Accession naj npawb GSE172466).

5.12. Bioinformatic Analysis of Microarray Data thiab Visualization Nyob rau hauv ob lub raum nas thiab RPTEC microarray cov ntaub ntawv, cov noob uas muaj rau ob qho tib si thiab qhov twg qhov kev hloov ntawm kev qhia ntau tshaj 2-fold txo los yog nce (los yog teev2-fold hloov yog me dua −1 lossis loj dua 1) txhua lub sijhawm tau xaiv los ntawm R. Ntxiv mus, hauv cov ntaub ntawv nas raum microarray. Gene alterations uas haum cubic curves tau xaiv siv R pob maSigPro [https://www.bioconductor.org/packages/release/bioc/html/ maSigPro.html (kawg nkag rau 4 Lub Ib Hlis 2022)] [75]. Lub cubic nkhaus yog tsim kom haum rau cov noob 'nce lossis txo qis thaum lub sijhawm kawm. Qhov ntau dua polynomial degree xws li lub cubic nkhaus tso cai rau xaiv cov noob nrog ntau qhov kev hloov pauv thaum lub sijhawm kawm zoo dua li qhov nkhaus plaub. Cov noob uas coj tus cwj pwm zoo sib xws tau sib sau ua ke thiab lawv qhov kev txo qis / nce ntxiv tau pom los ntawm daim ntawv qhia tshav kub siv R pob gplots [https://CRAN.R-project.org/package=gplots (kawg nkag rau 4 Lub Ib Hlis 2022] [76]. Gene ontology (GO) raug xa mus thiab kev ua haujlwm ntawm GO ntawm cov noob caj noob ces tau txhawb nqa siv Metascape [http://metascape.org (kawg nkag rau 4 Lub Ib Hlis 2022)] [77]. Kev sib txuas / kev sib raug zoo ntawm cov noob caj noob ces tau txheeb xyuas siv STRING [https://string-db.org/ (kawg nkag rau 4 Lub Ib Hlis 2022)] [78]. Kev koom tes ntawm cov noob caj noob ces tau tshawb nrhiav manually nrog rau kev siv cov ntawv mining ua haujlwm ntawm STRING.

Cov ntaub ntawv

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