Kev tshem tawm ntawm Alox15 txhim kho raum tsis ua haujlwm thiab inhibits Fbrosis Los ntawm nce PGD2 hauv lub raum
Mar 12, 2022
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Taw qhia
Polyunsaturated fatty acids (PUFA) thiab lawv cov metabolites yog txuas rau o thiab nws cov kev daws teeb meem hauv ntau lub cev [1]. Oxylipins, uas yog tsim los ntawm oxidation ntawm PUFA, yog ib qho tseem ceeb rau PUFA txoj kev lom neeg ua haujlwm li lipid mediators [1, 2]. Biosynthesis ntawm oxylipins yog kho los ntawm ntau lub enzymes, xws li lipoxygenase (LOX), cyclooxygenase (COX), thiab cytochrome P450 (CYP) [3]. Cov enzymes no tsim ntau yam lipid metabolites, xws li prostaglandins, leukotrienes, thiab lipoxins, uas yog tag nrho cov koom nrog kev tswj ntawm o [1, 4]. Piv txwv li, ALOX15, ib tug loj subtype ntawm LOX, muaj ib tug ob nam ntawm proinflammatory thiab anti-infammatory zog los ntawm nws cov metabolites [5]: ALOX15 yog heev qhia nyob rau hauv eosinophils, bronchoalveolar epithelial hlwb, thiab alveolar macrophages nyob rau hauv nonpathological mob [5, 6. ], thiab txhawb kev mob hawb pob [7], mob ntsws [8], thiab lub plawv tsis ua haujlwm [9], thaum nws tiv thaiv kev mob ntawm caj dab [10] thiab ischemic hlwb [11]. Tsis tas li ntawd, lipid mediators thiab lawv cov enzymes cuam tshuam rau lub cev fibrosis nrog rau kev mob. Cov kws kho mob tshwj xeeb lipid yog koom nrog hauv cov kab mob ntawm lub ntsws [12], siab [13], thiab lub plawv [14] fibrosis. Ib yam li ntawd, ALOX15 tau hais los saum toj no kuj tseem txuas nrog cov kab mob ntawm cov kab mob fibrosis xws li dermal fibrosis [15].
Ib qho ntawm cov kab mob uas tshwm sim yog mob ntev mob raum(CKD), uas cuam tshuam txog kwv yees li 8-16 feem pua ntawm cov pej xeem nyob hauv txhua theem ua ke [16]. Txawm hais tias CKD pathogenesis yog qhov nyuaj thiab sib txawv nyob ntawm tus kab mob hauv qab, tusraumcov ntaub so ntswg feem ntau ua tsis zoo, ua rau lub sijhawm kawgraum tsis ua haujlwmtshwm sim los ntawm mob o thiab mob fibrosis tom qab [17]. Raws li tau hais, lipid mediators tau txais los ntawm PUFAs tau txuas nrog rau qhov mob thiab nws qhov tshwm sim fibrosis. Piv txwv li, lipoxins thiab resolvins inhibit lub raum fibrosis [18, 19], tab sis cov kev cuam tshuam no tseem tsis tau kuaj xyuas hauv CKD qauv nrog kev tsis zoo.lub raum ua haujlwm.Ntxiv mus, kev nthuav dav lipidomic profiles hauv CKDraumcov ntaub so ntswg tseem tsis tau tshaj tawm. Yog li, lub luag haujlwm ntawm lipid metabolic enzymes thiab lawv cov khoom hauv CKD pathogenesis tseem tsis to taub.Qhov kev tshawb fawb no tsom mus rau kev qhia txog kev koom tes ntawm fatty acid metabolizing enzymes thiab lawv cov khoom hauvlub raumKev puas tsuaj ntawm 5/6 nephrectomized (Nx) CKD qauv nas. Txoj kev tshawb no qhia tau hais tias ob qho tib si ntawm kev sau thiab cov protein qhia qib ntawm Alox15 tau nce hauv CKDlub raum, thiab Alox15-/- nas pom tau zoo duaraumKev ua haujlwm tsis zoo thiab fibrosis hauv CKD qauv. Ntxiv mus, PGD2, uas yog nce lipid metabolite hauv CKDlub raumntawm Alox15-/- nas, inhibited lub epithelial-mesenchymal hloov (EMT) nyob rau hauv proximal tubular kab lis kev cai hlwb. Yog li, Alox15 inhibition thiab / lossis PGD2 kev tswj hwm tuaj yeem yog lub hom phiaj kho tshiab ntawm CKD thiab fibrosis.
Ntsiab lus:mob raum mob; Lipoxygenase; ALOX 15; Mediator lipidomics; Polyunsaturated fatty acids; lub raum
CISTANCHE yuav txhim kho lub raum / raum ua haujlwm
Cov ntaub ntawv thiab cov txheej txheem
Tsiaj txhu thiab kev simTxoj kev tshawb no tau siv 8-cov txiv neej hnub nyoog ib lim piam C57BL/6 J nas (CLEA Nyiv), uas tau ua kom zoo rau 1 lub lis piam ua ntej txhua qhov kev sim tau ua. Ntxiv mus, Alox15−/− nas tau tsim nyob rau hauv C57BL/6 J keeb kwm (Jackson Laboratory), thiab 5/6 Nx qauv tau tsim raws li kev tshawb fawb yav dhau los [20]. Peb sau cov ntshav kuaj rau kev soj ntsuam ntawmlub raum ua haujlwmthiabraumcov ntaub so ntswg kuaj rau immunoblotting thiab polymerase saw cov tshuaj tiv thaiv ntawm 8 lub lis piam tom qab 5/6 Nx, thiab rau kev soj ntsuam histological ntawm 40 lub lis piam tom qab 5/6 Nx. Txhua qhov kev sim ua tau raws li cov lus qhia rau kev tshawb fawb tsiaj ntawm TMDU, thiab Pawg Saib Xyuas Tsiaj thiab Siv Tsiaj ntawm TMDU tau pom zoo peb txoj kev kawm (tus lej pom zoo: A2019-117C4).
LC-MS/MS-based mediator lipidomicsPeb tau soj ntsuam LC-MS/MS raws li tau piav ua ntej [21, 22]. Cov ntsiab lus ntawm cov txheej txheem tau piav qhia hauv Cov Txheej Txheem Ntxiv,Lwm yam kev simPeb tau piav qhia txog lwm txoj hauv kev sim hauv Cov Txheej Txheem Ntxiv.
Cov txiaj ntsig
mRNA qib ntawm Alox15 tau nce hauv 5/6 Nx raumKev hloov pauv ntau ntawm oxylipin enzymes hauv CKDraumtau tshawb xyuas los ntawm kev thov C57BL/6 nas rau kev ua haujlwm sham lossis 5/6 Nx raws li tau piav qhia yav dhau los [20]. Txhawm rau txheeb xyuas qhov kev qhia ntawm cov oxylipin enzymes loj hauv lub raum (Alox15, Alox5, Ptgs1 (COX1), Ptgs2 (COX2), Cyp4a12 thiab Cyp2c44) [23, 24], peb muab rho tawm.raumcov qauv. Ntawm cov enzymes, Alox15 hauv 5/6 Nxlub raumtau nce qib mRNA ntau heev (P=0.0004) piv nrog rau hauv kev dag ntxiaslub raum(Daim duab 1). Hloov pauv, qib mRNA ntawm lwm cov enzymes loj tsis tau hloov pauv hauv CKD qauv no.
Cov protein ntau ntawm Alox15 tau nce hauv lub raum proximal tubular cells5/6 Nxlub raum, ALOX15 cov protein ntau kuj tau nce ntxiv (P=0.0078), raws li kev cia siab los ntawm kev nce qib transcriptional (Fig. 2a). Txhawm rau txiav txim siab seb hom cell twg tau nce qib Alox15 protein ntau hauv CKDraum,Peb tau tshuaj xyuas qhov chaw ntawm Alox15 mRNA los ntawm qhov chaw hybridization. Nyob rau hauv situ hybridization rau mRNA qhia tau hais tias lub siab qhia theem ntawm Alox15 tau localized ntawmlub raumtubular hlwb hauv 5/6 Nx qauv, tab sis tsis nyob rau hauv glomeruli (Fig. 2b). Cov yam ntxwv histological qhia tias Alox15 mRNA tau hais tawm hauv cov tubules ze ze.
Alox15-/- nas tau tiv taus rau lub raum puas thiab fibrosis hauv 5/6 Nx qauv5/6 Nx CKDlub raum, cov qib transcriptional thiab protein qhia ntawm ALOX15 tau nce siab. Yog li, peb tau tshawb xyuas seb ALOX15 ua lub luag haujlwm hauv CKD li casraumkev ua haujlwm tsis zoo thiab lub raum fibrosis. Rau lub hom phiaj no, Alox15-/- nas tau siv rau 5/6 Nx CKD qauv. Interestingly, ntshav Cre thiab ntshav urea nitrogen (BUN) qib qis dua hauv Alox15−/− 5/6 Nx nas.
dua li cov nas nyob rau hauv cov tsiaj qus (WT) nas (Daim duab 3a), qhia tias Alox15 depletion pom muaj kev tiv thaiv hauv CKD pathogenesis. Raws li, NGAL (alub raum puasmarker) cov protein qhia tau raug txwv tsis pub nyob hauv Alox15−/− nas piv nrog rau hauv WT nas (Fig. 3b). Ntxiv rau, Alox15-/- naslub raumpom tau tias txo qis Col1a1, Fn thiab Acta2 (SMA) mRNA qib (Fig. 3c), thiab kuj pom tias txo qis fibronectin thiab SMA protein qhia (Fig. 3d). Ntxiv mus, Alox15-/- naslub raumnthuav tawm kom meej meej suppressed interstitial fibrotic kev hloov pauv hauv Masson's trichrome staining (Fig. 3e). Yog li ntawd, Alox15 tshem tawm amelioratesraumKev ua haujlwm tsis zoo thiab fibrosis hauv CKD tus qauv tsiaj.
Cov kws kho mob lipidomics tau qhia txog kev hloov pauv PUFA metabolism hauv Alox15-/- CKD ob lub raumRaws li tau hais hauv Tshooj Lus Qhia ntawm daim ntawv no, cov txiaj ntsig lom neeg ntawm Alox15 muaj feem cuam tshuam rau cov lipid mediators tsim los ntawm Alox15. Txhawm rau elucidate profile ntawm lipid metabolites tsim los ntawm Alox15 hauv CKDraum,peb tshuaj xyuas thiab muab piv rau kev dag thiab 5/6 Nxraumcov qauv los ntawm LC-MS/MS-based mediator lipidomics los txiav txim siab lipid mediator profiles ntawm WT nas thiab Alox15-/- CKD nas (Cov Lus Ntxiv 1). Table 1 qhia cov lipid metabolites uas txawv heev ntawm Alox15 ntxiv / ntxiv thiab Alox15−/− nas nyob rau hauv 5/6 Nx mob. Tsis tas li ntawd, peb tau ua cov duab qhia kev ntawm lipid mediators nrog kev faib tawm los ntawm lawv cov substrates thiab lawv cov metabolizing enzymes (Fig. 4). A series ntawm Alox15- muab PUFA metabolites xws li 14-HDoHE, 17-HDoHE, thiab 15-HEPA [5, 25–27] tau nce ntau hauv CKD qauv uas muaj feem cuam tshuam. zoo rau qib nce ntawm ALOX15 qhia hauv lublub raum (P=0.0018, 0.0008,<0.0001, respectively),="" and="" their="" elevations="" under="" ckd="" conditions="" were="" completely="" suppressed="" in="" alox15−/−="" mice="" (fig. 5).="" besides="" 14-hdohe,="" 17-hdohe,="" and="" 15-hepa,="" the="" levels="" of="" 18-hepa,="" 10-hdohe,="" 11-hdohe,="" 13-hdohe,="" 16-hdohe,="" and="" dgla="" were="" also="" significantly="" decreased="" in="" alox15−/−="" ckd="">lub raumpiv rau cov hauv WT CKD ob lub raum, qhov tsuas yog PGD2 tau nce ntau hauv Alox15-/- CKD ob lub raum (Fig. 6).
PGD2 suppressed EMT hauv kab mob raum hlwbQhov cuam tshuam ntawm lipid metabolites uas txawv heev ntawm Alox15 ntxiv / ntxiv thiab Alox15−/− nas nyob rau hauv 5/6 Nx mob tau raug tshuaj xyuas los ntawm kev tswj cov lipids rau NRK-52E hlwb uas tau qhib los ntawm pro-fibrotic cytokine TGF - 1. Peb tau soj ntsuam mRNA kev qhia ntawm Cola1a thiab Acta2 (SMA) hauv kev teb rau TGF- 1 txhawm rau txiav txim siab qhov cuam tshuam ntawm cov lipids hauv kab lis kev cai (Daim duab 7a). Ntawm cov ntawd, PGD2 tau muab cov tshuaj tua kab mob muaj zog ntawm NRK-52E hlwb teb rau TGF- 1. Cov nyhuv suppressive ntawm PGD2 ntawm COL1A1 thiab SMA kev qhia yog nyob ntawm koob tshuaj nrog EC50 ntawm 7.12 μM thiab 6.48 μM, ntsig txog (Daim duab 7b). Tsis tas li ntawd, peb tau ua tib yam kev sim hauv HK-2 hlwb, uas yog immortalized proximal tubule epithelial hlwb los ntawm ib txwm neeg laus.lub raum,thiab pom qhov tshwm sim zoo sib xws, uas yog, COL1A1 thiab SMA inhibition nyob rau hauv ib koob tshuaj, teb rau kev kho mob nrog PGD2 (Fig. 7c). Yog li, nce qib ntawm PGD2 hauv Alox15-/- CKDlub raumtuaj yeem ua rau cov tshuaj tiv thaiv kab mob hauv CKD.
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15-PGDH, ib qho tseem ceeb PGD2-metabolizing enzyme, tau txo qis hauv Alox15-/- CKD ob lub raumTxhawm rau kom paub meej cov txheej txheem ntawm kev nce hauv PGD2 hauvlub raumntawm Alox15-/- CKD qauv nas, peb ntsuas mRNA qib ntawm PGD2 synthase. PGD2 synthase (PGDS) muaj ob isoforms: lipocalin PGDS (L-PGDS) thiab hematopoietic PGDS (H-PGDS) [28]. Ob leeg L-PGDS thiab H-PGDS qib tau nce ntau dua nyob rau hauv 5/6 Nx cov xwm txheej thaum piv nrog cov nyob rau hauv cov xwm txheej sham hauvlub raumntawm WT nas (Fig. 8a, ob leeg P<0.0001) indicating="" that="" increased="" pgd2="" in="" ckd="" is="" due="" to="" increased="" pgd2="" synthases.="" conversely,="" the="" increase="" in="" l-pgds="" and="" h-pgds="" under="" 5/6="" nx="" conditions="" was="" significantly="" inhibited="" in="" alox15−/−="" mice="" (fig. 8a)="" indicating="" that="" the="" increase="" in="" pgd2="" in="" alox15−/−="" ckd="" model="" mice="" was="" not="" due="" to="" increased="" production="" by="" pgds.="" then,="" we="" examined="" mrna="" levels="" of="" cox-1="" and="" cox-2="" as="" more="" upstream="" synthases="" of="" the="" prostaglandin-producing="" pathway.="" we="" also="" measured="" mrna="" levels="" of="" 15-pgdh="" and="">1C18, uas paub tias yog PGD loj2-metabolizing enzymes [29, 30]. Thaum mRNA qib ntawm COX-1, COX-2, thiab AKR1C18 tsis hloov pauv, mRNA qib ntawm 15-PGDH tau txo qis hauv CKD.lub raumntawm Alox15−/− nas thaum piv nrog cov nas WT (Fig. 8b, P=0.0325), uas yuav ua rau muaj kev nce hauv PGD2 hauv CKDlub raumntawm Alox15-/- nas.
Kev sib tham
Txoj kev tshawb no pom tau tias hauv CKDraumcov qauv, ob qho tib si ntawm kev hloov pauv thiab qib protein ntawm Alox15 tau nce ntxiv, thiabraumKev ua haujlwm tsis zoo thiab fibrosis tau ua kom zoo dua hauv Alox15-/- nas. Tsis tas li ntawd, LC-MS / MS-based mediator lipidomics qhia tias Alox15-/- CKD naslub raumtau nce qib ntawm PGD 2 piv nrog WT nas. PGD2 inhibited EMT ntawm NRK-52E thiab HK-2 hlwb; li no, PGD2 nce tuaj yeem ua rau muaj kev tiv thaiv ntawm Alox15-/- nas raumob raumthiab fibrosis. Kev sib raug zoo ntawmmob raumthiab lipid profile siv lipidomics ntawm tib neeg cov ntshav ntshav lossis ntshav tau raug tshawb xyuas ntau [31–33], tab sis tsis nyob rauraumntaub so ntswg. Ib yam li ntawd, txawm hais tias cov ntaub ntawv qhia txog lipidomics siv CKD tsiaj qauv muaj tsawg [34–36], tag nrho cov kev tshawb fawb no tau ua lipidomics ntawm cov plasma kuaj los ntawm CKD tsiaj qauv; Txawm li cas los xij, tsis muaj kev qhia txog lipidomics rauraumcov ntaub so ntswg los ntawm tus qauv CKD tseem tau tshaj tawm. Direct lipidomics ntawm cov ntaub so ntswg kuj zoo, nrog rau cov qauv ntshav plasma, hauv kev txheeb xyuas cov metabolites ua haujlwm. Ntxiv mus, metabolite hloov pauv hauv cov ntaub so ntswg, nrog rau cov ntshav kuaj, muaj qhov sib txawv ntawm lub cev [20]. Hauv txoj kev tshawb fawb tam sim no, peb tau txheeb xyuas qhov profile ntawm PUFA-derived lipid mediators hauvraumcov ntaub so ntswg ntawm CKD tsiaj qauv.
Txoj kev tshawb no tsom rau ALOX15, uas yog ib qho ntawm cov enzymes loj uas metabolize PUFAs. Hauv txoj kev tshawb no, ob qho tib si protein thiab mRNA qhia qib ntawm ALOX15 tau pom meej meej hauv CKDlub raum. Peb tau hais txog hom xovtooj ntawm lub luag haujlwm rau ALOX15 qhia hauv CKDlub raum,thiab nyob rau hauv situ hybridization, peb pom ib qho kev nthuav qhia ntawm ALOX15 mRNA nyob rau hauvlub raumtubular hlwb hauv 5/6 Nx qauv.
ALOX15 koom nrog cov kab mob ntev, xws li atherosclerosis, thiab nws cov kev tshem tawm hauv cov kab mob tsiaj ua rau cov kab mob no zoo dua [5]. Hais txog lub koom haum ntawm ALOX15 nrogmob raum, proteinuria txo qis hauv Alox15-/- nas nyob rau hauv glomerular raug mob los ntawm streptozotocin-induced mob ntshav qab zib nephropathy qauv [37]. Txawm li cas los xij, tus qauv mob ntshav qab zib mellitus nas no tsis pom muaj qhov poob qislub raum ua haujlwm; Yog li, lub mechanism ntawm yuav ua li cas ALOX15 yog txuam nrog impairedlub raum ua haujlwmhauv CKD qauv tseem tsis tau paub meej. Hauv kev tshawb fawb tam sim no, siv tus qauv 5/6 Nx nas, peb pom tias ALOX15 tshem tawm amelioratedraumkev ua haujlwm tsis zoo thiablub raumfibrosis hauv CKD qauv. Txog rau tam sim no, tsuas yog IL-4 thiab IL-13 hauv monocytes paub tias yog cov cai tswj hwm uas ncaj qha nce kev qhia ntawm ALOX15 [5]. Txawm li cas los xij, nws tseem tsis paub meej tias dab tsi nce ALOX15 hauvlub raumtubular hlwb. Ntau yam cytokines thiab uremic toxins tau paub tias yuav nce hauv cov ntshav thiab ob lub raum hauv CKD [38, 39]. Ntawm lawv, tej zaum yuav muaj cov tswj hwm tshiab ntawm ALOX15. Cov kev tshawb fawb ntxiv yog xav tau los piav qhia qhov tseem ceeb ntawm kev tswj hwm ntawm ALOX15 hauv CKD. Hauv peb cov kev tshawb fawb los txheeb xyuas cov kev cuam tshuam oxylipins tshwj xeeb uas txuas Alox15 tshem tawm rau nws cov nyhuv renoprotective hauv 5/6 Nx qauv, peb pom tias PGD2 tuaj yeem cuam tshuam rau kev tiv thaiv.lub raum puastshwm sim los ntawm ALOX15 deletion. Raws li kev nkag siab dav dav, los tsim PGD2, COX-1 thiab
CCOX-2 tsim PGG2 los ntawm arachidonic acids, uas tom qab ntawd hloov mus rau PGH2. Thaum PGH2 yog metabolized los ntawm PGD2 synthase (PGDS), PGD2 yog tsim [28]. PGDS muaj ob qhov sib txawv ntawm cov isoforms, uas yog, lipocalintype PGDS (L-PGDS) thiab hematopoietic-type PGDS (H-PGDS) [28]. Txawm hais tias PGD2 tau nce hauv Alox15-/- CKD naslub raum, tsis yog L-PGDS lossis H-PGDS tau nce hauv Alox15-/- CKD naslub raum, txawm tias qhov nce loj hauv PGDS hauv WT CKD naslub raum(Daim duab 8a). Tsis tas li ntawd, peb kuj tau qhia tias tsis muaj COX-1 lossis COX-2 tau nce ntxiv hauv Alox15−/− CKD nas ob lub raum (Fig. 8b). Cov txiaj ntsig no qhia tau hais tias qhov nce hauv PGD 2 hauv Alox15-/- CKD nas lub raum tsis yog vim muaj kev nce hauv synthases tab sis vim muaj cov substrate ntau ntxiv lossis inhibition ntawm degradation, thiab qhov tseeb, peb tau nthuav tawm qhov txo qis hauv {{ 7}}PGDH, ib qho ntawm PGD2 metabolizing enzymes loj [29], hauv Alox15−/− CKD nas ob lub raum (Fig. 8b). Kev tshawb nrhiav ntxiv yog xav tau los piav qhia qhov laj thawj vim li cas qhov kev tshem tawm ntawm Alox15 ua rau txo qis hauv 15-PGDH.
Raws li tau hais los saum no, PGD2 tuaj yeem koom nrog hauv kev tiv thaivmob raumtshwm sim los ntawm ALOX15 poob. Hauv txoj kev tshawb no, PGD2 inhibited EMT los ntawm TGF- 1 hauv NRK-52E thiab HK-2 hlwb, sawv cev rau cov cell tubular. PGD2 khi rau ob qhov sib txawv G protein-coupled receptors, uas yog, DP1 thiab DP2, uas nws lub luag haujlwm sib txawv [28]. Txawm li cas los xij, hauv peb qhov kev sim hauv vitro, PGD2 tau txais txiaj ntsig zoo ntawm qhov ntau ntawm 4 txog 32 μM, uas yog qhov siab, xav tias Ki qhov tseem ceeb ntawm DP1 thiab DP2 yog 1.7 nM thiab 2.4 nM, feem [28]. Cov txiaj ntsig no qhia tau hais tias PGD2 tuaj yeem siv tshuaj tua kab mob tsis yog los ntawm G protein-coupled receptors tab sis los ntawm lwm txoj hauv kev xws li PPAR , los ntawm qhov uas 15-d-PGJ2, ib qho qis PGD2 metabolite, paub tias yuav ua rau nws cuam tshuam [28]. Hauv txoj kev tshawb no, uas tsom mus rau lipid metabolic enzymes thiab lawv cov metabolites, ALOX15 inhibition thiab / lossis PGD2 kev tswj hwm tuaj yeem yog lub hom phiaj kho mob zoo rau CKD. Hmoov tsis zoo, tsis muaj ALOX15 tshwj xeeb inhibitor uas tuaj yeem siv rau hauv kev kho mob tau tsim [40, 41]. Kev kho lub hom phiaj ntawm downstream functional metabolites, xws li PGD2, es tsis yog inhibition ntawm fatty acid metabolizing enzymes, uas cuam tshuam rau ntau yam metabolites, yuav yog ib tug tshiab zoo tagnrho txoj kev kho CKD.
CISTANCHE yuav txhim kho lub raum / raum mob ntshav qab zib