Cistanche los kho o thiab kab mob raum: Dab tsi yog Qhov Ua rau Tus Kab Mob Lub Luag Haujlwm Ntawm Qhov Mob hauv Raum Kev Koom Tes hauv Cystinopathy?
Mar 13, 2022
Hu rau:joanna.jia@wecistanche.com
Kev sib cuam tshuam ntawm galectin -3 thiab cystinosin nthuav tawm lub luag haujlwm ntawm kev mob ntawm lub raum kev koom tes ntawm cystinosis
Tatiana Lobry1,2 ua al
Kev mobNws muaj feem xyuam rau lub pathogenesis ntawm ntau yam kab mob. Txawm li cas los xij, lub hauv paus mechanisms feem ntau tsis paub. Ntawm no, peb sim seb puas yogcystinosis, cov protein koom nrogcystinosis, yog ib qho tseem ceeb regulator ntawm galectin-3, ib tug tswv cuab ntawm b-galactosidase binding protein tsev neeg, thaum lub sij hawmmob. CystosisNws yog ib qho lysosomal cia teeb meem thiab, txawm hais tias ubiquitous qhia ntawm cystinosis, tusraumyog lub cev tseem ceeb cuam tshuam los ntawm tus kab mob.Cystosistau pom los txhim kho lysosomal localization thiab degradation ntawm galectin -3. Hauv Ctns–/–nas, tus qauv nascystinosis, galectin -3 yog overexpressed nyob rau hauv lubraum. Qhov tsis muaj galectin -3 hauv cov nas cystinotic ameliorates pathologic lub raum kev ua haujlwm thiab cov qauv thiab txo qis macrophage / monocyte infiltration hauv lub raum ntawm Ctns–/–Gal 3–/–nas piv rau Ctns–/– nas. Cov ntaub ntawv no qhia tau hais tias galectin -3 mediatesmobkoom nrograumKev loj hlob ntawm tus kab mob hauv cystinosis. Tsis tas li ntawd, galectin-3 tau pom muaj kev cuam tshuam nrog cov pro-inflammatory cytokine MonocyteChemoattractant Protein-1, uas txhawb kev nrhiav neeg ua haujlwm ntawm monocytes / macrophages, thiab ua pov thawj tias muaj kev nce ntxiv hauv cov ntshav ntawm Ctns–/– nas. thiab cov neeg mob nrogcystinosis. Yog li, peb qhov kev tshawb pom tau qhia txog lub luag haujlwm tshiab rau cystinosis thiab galectin -3 kev cuam tshuam hauv kev mob thiab muab cov lus piav qhia ntxiv rau covraumkab mob ntawm cystinosis. Qhov no tuaj yeem ua rau kev txheeb xyuas cov hom phiaj tshuaj tshiab kom ncua sijhawmcystinosiskev vam meej.
KEYWORDS:mob raum mob; cystinosis; galectin -3;mob; monocyte chemoattractant protein-1
cistanche tuaj yeem kho mob raum mob thiab cystinosis
Lub hauv paus ua rau mob yog tias nitric oxide tuaj yeem ua rau tsim cov hlwb inflammatory.Cistancheyog ib qho tseem ceeb suav tshuaj ntsuab. Nws cov khoom xyaw nquag tuaj yeem inhibit qhov tso tawm ntawm nitric oxide thiab ua lub luag haujlwm hauv kev tiv thaiv thiab kho mob thiab kab mob raum.
Lus Txhais Lus
Txawm hais tias kev kho mob, cov neeg mob tseem nce mus rau theem kawg ntawm lub raum tsis ua haujlwm. Hauv qhov kev tshawb fawb no, peb tau pom tias qhov tsis muajcystinosistshwm sim hauv impaired galectin-3 (Gal-3) lysosomal degradation, ua raumobthiab kev vam meej ntawmmob raum mobhauvcystinosis. Cov kev tshawb pom no qhib qhov kev xav tshiab ntawm cov hom phiaj kho mob uas tuaj yeem txwv lossis ncua lub raum degeneration hauv cov neeg mob uascystinosis. Xws li, tshuaj tiv thaiv kab mob thiab inhibitors ntawm Gal-3 xws li cov tshuaj uas tsis yog tshuaj steroidal los yog indomethacin tuaj yeem txhim kho lub raum pathogenesis hauv cystinosis.
Kev mobyog ib qho mob hnyav teb ntawm lub cev rau kev raug mob los yog kis kab mob,1tab sis mobmobyog txuam nrog cov ntaub so ntswg puas. Nyob rau hauv cov ntaub ntawv ntawm cov kab mob ntev, xws li ntshav qab zib, cov ntaub so ntswg puas ua rau lub cev tsis muaj zog, ua rau cov lus teb tsis tu ncua thiab, thaum kawg, cov ntaub so ntswg degeneration.2 Cytokines yog qhov tseem ceeb modulators ntawmmobthiab muaj peev xwm ua kom tau lossis daws qhov teeb meemmobprocess.3 Hauv cov neeg mob nrogmob raum mob(CKD), nce ntshav plasma concentration ntawm cytokines (interleukin [IL]-6 thiab qog necrosis factor-a) thiabmobCov cim (C-reactive protein, IL-6, hyaluronan, thiab neopterin) yog txuam nrog kev loj hlob mus rau theem kawg ntawm lub raum kab mob.4 Kev nkag siab txog cov kws kho mob tshwj xeeb uas ua rau cov kab mob o tuaj yeem pab nrhiav pom cov tshuaj tsim nyog los tiv thaiv kev puas tsuaj. .
CystosisNws yog ib qho kab mob autosomal-recessive metabolic uas yog nyob rau hauv lysosomal storage disorder tsev neeg thiab yog tus cwj pwm los ntawm kev tsub zuj zuj ntawm cystine hauv txhua lub cev.5 Lub gene defect nyob rau hauvcystinosis, CTNS, encodes lysosomal cystine / proton co-transporter, cystinosis.6,7 Txawm hais tias CTNS tau nthuav tawm ubiquitously, lubraumyog thawj lub cev cuam tshuam rau cov neeg uas muajcystinosis. Cov neeg mob feem ntau tshwm sim hauv lawv thawj xyoo ntawm lub neej nrog Fanconi syndrome, tus cwj pwm los ntawm cov kua dej hnyav thiab electrolyte cuam tshuam, kev loj hlob tsis zoo, thiab rickets.8CKD tom qab ntawd txhim kho, uas nce mus rau theem kawg ntawm lub raum kab mob thiab xav tauraumhloov pauv. Cystine tsub zuj zuj nyob rau hauv tag nrho cov ntaub so ntswg nws thiaj li ua rau multiorgan tsis ua hauj lwm; Cov neeg mob muaj photophobia thiab dig muag, hypothyroidism, hypogonadism, ntshav qab zib, myopathy, thiab hauv nruab nrab paj hlwb deterioration.9 Hauv keeb kwm yav dhau C57BL / 6, tus qauv nas knockout (Ctns–/–) 10 replicates lub raum phenotype ntawm cov neeg mob cystinotic,11 nrog rau kev tso tawm ntawm cystine crystals nyob rau hauv lub cornea12thiab thyroid tsis ua haujlwm.13
Hauv kev tshawb fawb tam sim no, peb nthuav tawm lub luag haujlwm tshiab raucystinosishauvmoblos ntawm nws txoj kev cuam tshuam nrog Gal -3. Gal-3 yog ib tug tswv cuab ntawm lectin thiab b-galactoside-binding protein tsev neeg 21 thiab koom nrog ntau yam kev ua haujlwm biologic, suav nrogmob.22Hauv cov ntsiab lus ntawmraumCov kab mob, Gal-3 inhibition tau pom tias txo qis cov cim qhia txog kev mob plab thiab lub raum fibrosis thiab tiv thaiv qhov poob ntawm glomerular pom tus nqi hauv hyperaldosteronism, kub siab, lossis rog rog.23–26Ntawm no peb muab pov thawj tias, hauvcystinosis, qhov tsis muajcystinosisua rau Gal-3 overexpression nyob rau hauvlub raum, txhim kho macrophage infiltration thiab CKD kev loj hlob. Tsis tas li ntawd, peb txheeb xyuas cov monocyte chemoattractant protein-1 (MCP-1) ua tus neeg nruab nrab muaj peev xwm, nthuav tawm cov hom phiaj tshiab rau kev kho tshuaj.RESULTS Gal-3 cuam tshuam nrog cystinosis los ntawm nws cov carbohydrate lees paub sau npe txhawm rau txheeb xyuas cov kev cuam tshuam tshwj xeeb Cov neeg koom tes siv ntau qhov loj spectrometry, Madin-Darby canineraum(MDCK) cov hlwb tau hloov pauv nrog cov kab mob lentiviral kom ruaj khov qhia cov cystinosis-ntsuab FL fluorescent protein (GFP) fusion protein. Ntxiv rau 9 subunits ntawm vacuolar-type Hþ adenosine triphosphatase luam tawm yav dhau los,19Gal -3 tau txheeb xyuas tias yog ib qho ntawm cov proteins cuam tshuam nrogcystinosis(Daim duab 1a). Qhov kev cuam tshuam no tau lees paub ntxiv los ntawm kev sib koom ua ke tiv thaiv kab mob ua raws li Western blotting (Daim duab 1b thiab c). Ntxiv rau, peb tau pom tias kev sib cuam tshuam ntawm Gal -3 thiabcystinosistau kho los ntawm Gal -3 carbohydrate recognition domain (CRD) nyob rau hauv C-terminal tail ntawm cov protein. Kev sib cuam tshuam tau cuam tshuam los ntawm thiodigalactoside, muaj zog inhibitor ntawm galectin-carbohydrate kev sib cuam tshuam (Daim duab 1d). Zoo li tag nrho cov galectins, Gal -3 muaj kev sib raug zoo rau glycosylated proteins, 21 yog li nws yuav muaj kev cuam tshuam nrog Gal -3 tshwm sim los ntawm cystinosis glycosylated moiety nyob rau hauv intralysosomal N-terminal tail ntawm cov protein.27
Cystinosin txhim kho Gal -3 lysosomal localization thiab degradation
Txhawm rau txheeb xyuas qhov muaj peev xwm lysosomal localization ntawm Gal -3 thaum cuam tshuam nrogcystinosis, peb tau pom tias Gal -3, zoo li cathepsin D (ib qho intralysosomal protease), tau tiv thaiv kev zom zaub mov los ntawm proteinase K, thaum ob leeg cov proteins tau zom thaum lysosomes permeabilized nrog Triton X- 100 (Daim duab 2a thiab Ntxiv. Daim duab S1). Cov ntaub ntawv zoo sib xws tau txais hauv lysosomes cais los ntawm nas lub siab, lees paub lysosomal localization ntawm Gal-3 hauv vivo (Daim duab 2b thiab c). Vim tias tsis muaj cov tshuaj tiv thaiv kab mob txhim khu kev qha los kuaj pomcystinosis, immunostaining tau ua raucystinosis-GFP-qhia MDCK kab ntawm tes nrog cov tshuaj tiv thaiv-Gal{1}} tshuaj tiv thaiv. Nws paub tseeb tias muaj Gal-3 nyob rau hauv lub lumen ntawm lub lysosomes, whereas cystinosis-GFP thiab Teeb-2 (ib qho lysosomal transmembrane protein) tau pom tsuas yog nyob rau ntawm daim nyias nyias ntawm lub vesicles (Daim duab 2d).
Tshawb nrhiav yog tiascystinosintau koom nrog Gal-3 kev lag luam, peb tau txheeb xyuas qhov muaj zog ntawm ob qho tib sicystinosisthiab Gal-3-cov hlwv zoo siv dual-xim nyob-cell tag nrho sab hauv kev xav FL fluorescence microscopy hauv nas embryonic fibroblasts (MEFs) los ntawm cov tsiaj qus (WT) thiab Ctns–/–nas qhia CTNS-DsRed thiab Gal-3–GFP. Peb qhov kev soj ntsuam tau lees paub tiascystinosisthiab Gal-3 tau txais qhov tseeb colocalization nyob rau hauv Ctns–/– MEFs raws li validated los ntawm qhov zoo sib xws spatiotemporal faib ntawm cov molecules nyob rau hauv tag nrho cov sab hauv xav txog FL fluorescence microscopy cheeb tsam (Daim duab 2e). Cov ntaub ntawv hauv WT MEFs zoo sib xws (cov ntaub ntawv tsis qhia). Ntxiv mus, thaum MEFs kis tau tsuas yog nrog Gal - 3-GFP raug tshuaj xyuas, Gal-3-GFP tau pom nyob rau hauv cytoplasm thiab tsis muaj cov qauv vesicular txawv, tsis sib xws nrog kev sib hloov nrog CTNS-DsRed ( cov ntaub ntawv tsis qhia).
Cov ntaub ntawv no tau lees paub nyob rau hauv 293T hlwb, nyob rau hauv uas muaj zog GFP teeb liab nyob rau hauv lub cytoplasm tau pom nyob rau hauv lub hlwb qhia tsuas yog Gal-3-GFP, whereas nyob rau hauv hlwb qhia ob Gal-3-GFP thiabcystinosin-DsRed, Gal-3–GFP feem ntau yog nyob hauv cystinosis-DsRed–qhia lysosomes (Daim duab 3a). Ntxiv mus, qhov ntau ntawm Gal-3 qhia tawm protein nyob rau hauv cov hlwb kis nrog Gal-3-GFP ib leeg lossis Gal- 3-GFP thiab CTNS-DsRed, thiab Gal-3-GFP thiab DsRed raws li kev tswj hwm, pom tsawg dua Gal-3-GFP cov proteins hauv cov hlwb sib txuas nrog CTNS-DsRed piv nrog cov hlwb hloov nrog Gal-3-GFP ib leeg lossis koom nrog DsRed (Daim duab 3b). Ua ke, cov txiaj ntsig no qhia tiascystinosintxhim kho lysosomal localization thiab degradation ntawm Gal-3. Cystinosin tau pom tias muaj kev koom tes hauv CMA.28 Thaum tshav kub kub poob siab 70 kDa protein (Hsc70) koom nrog CMA los ntawm kev pab lub unfolding thiab translocation ntawm substrate proteins hla daim nyias nyias mus rau hauv lub lysosomal lumen nyob rau hauv ib tug LAMP2A-dependent yam.29,30 Vim Gal{ {8}} tau thov kom degraded los ntawm CMA,31 peb tau tshawb xyuas qhov chaw ntawm Gal{10}} txheeb ze rau Hsc70 hauv cystinotic MEFs. Confocal microscopy tsom xam lees paub qhov colocalization ntawm Gal-3–GFP ntawm Hsc{13}} cov qauv zoo hauv WT (cov ntaub ntawv tsis qhia) thiab Ctns–/–cov hlwb (Daim duab 3c), txhawb nqa kev thauj mus los ntawm Gal-3 nrog Hsc70 thiab qhia tias lysosomal internalization tab sis tsis yog Gal-3 kev lees paub los ntawm tus kws kho mob tsis zoo hauvcystinosis.
Gal -3 koom nrog rau mob raum hauv Ctns–/–nas
Kawm txog lub luag haujlwm ntawm Gal -3 hauvcystinosisHauv vivo, peb tau tsim ob lub khob khob nas qauv tsis muaj nyob hauv ob qho tib sicystinosinthiab Gal-3 (Ctns–/–Gal -3–/–nas). WT, Ctns–/–, thiab Gal -3–/–nas tau siv los tswj cov ntsiab lus. Hauv C57BL / 6 Ctns–/–nas, lub raum anomalies tshwm nyob ib ncig ntawm 10 lub hlis ntawm lub hnub nyoog.11 Yog li ntawd, kev tshawb fawb tau ua nyob rau ntawm 8 mus rau 9 lub hlis, thaumraumCov kab mob tsis zoo pib pom, thiab ntawm 12 mus rau 15 lub hlis thaum lub raum anomalies tau nce. Vim tias cov ntsiab lus cystine tau pom tias txawv ntawm txiv neej thiab poj niam Ctns–/– lub raum, lawv tau txheeb xyuas nyias. Yog tias cov ntsiab lus cystine nce ntxiv nrog lub hnub nyoog hauv ob lub genotypes, tsis muaj qhov sib txawv tseem ceeb hauv cov txiv neej thiab poj niam ob lub raum ntawm Ctns–/– Gal-3–/– nas thiab Ctns–/– nas thaum muaj hnub nyoog 8 txog 9 hli thiab hnub nyoog 12 txog 15 lub hlis (Daim duab 4a).
Lub raum ua haujlwm tau raug soj ntsuam hauv cov ntshav thiab zis thiab tsis muaj qhov sib txawv tseem ceeb ntawm WT thiab Ctns–/–nas ntawm 8 mus rau 9 lub hlis (cov ntaub ntawv tsis qhia), tab sis ntawm 12 txog 15 lub hlis, Ctns–/–Cov nas tau pom ntau dua cov ntshav creatinine thiab urea ntau dua piv nrog WT nas. Interestingly, Ctns–/–Gal -3–/–Cov nas tau nthuav tawm cov kev ua haujlwm rau lub raum zoo dua piv nrog Ctns–/–nas ntawm 12 mus rau 15 lub hlis, nrog qis creatinine (P < 0 05) thiab urea (P < 0.05; Table 1).
Histologic kev tshawb fawb ntawm 8- mus rau 9-hli-hnub thiab 12- rau 15-mos-lausraumcov seem qhia pom muaj qhov tsis txaus ntseeg loj hauv Ctns–/– lub raum, suav nrog tubular atrophy, retracted glomeruli, thiab mononuclear infiltrates. Kev tsom xam qhov muag tsis pom ntawm lub raum thaj tsam thaj tsam ntawm kev puas tsuaj cortical los ntawm 1 (cov ntaub so ntswg khaws cia) mus rau 6. Qhov qhab nia nruab nrab rau Ctns–/–nas yog 2.64 - 0.36 thaum muaj hnub nyoog 9 hli (n ¼ 7) thiab 4.12 0.37 thaum muaj hnub nyoog 12 txog 15 hli (n ¼ 16). Hauv lub raum ntawm Ctns–/–Gal -3–/–cov nas nyob rau tib lub hnub nyoog, cov kev tsis txaus ntseeg no tsis tshua muaj qhov dav: 1.62 - 0.11 ntawm 9 lub hlis (n ¼ 21; P < 0.05,="" mann-whitney="" t-="" test)="" thiab="" 3.="" ntxiv="" mus,="" feem="" pua="" ntawm="" tubular="" atrophy="" tau="" muab="" rau="" txhua="" cov="" ntaub="" so="" ntswg,="" thiab="" qhov="" nruab="" nrab="" rau="">–/–nas thiab Ctns–/–Gal -3–/–nas yog 66 feem pua thiab 42 feem pua, feem, ntawm 12 mus rau 15 lub hlis (P ¼ 0.01). Tsis tas li ntawd, qhov sib txawv ntawm Ctns–/–thiab Ctns–/– Gal -3–/– raumcov seem yog muaj ob peb mononuclear infiltrates hauv Ctns–/–Gal -3–/–ntu, qhov hnyav infiltrates tau pom zoo nyob rau hauv Ctns–/– raum(Daim duab 4b).
Peb cim cov mononuclear infiltrates pom los ntawm kev kuaj histologic hauv 8- rau 9- hli-laus Ctns–/– lub raumraws li macrophages/monocytes los ntawm co-immunostaining nrog CD68 thiab CD45 thiab nrog CD68 thiab loj histocompatibility chav kawm II (Cov duab ntxiv S3), tab sis tsis nrog CD68 thiab CD163, tawm tswv yim tias cov macrophages muaj M1- zoo li proinflammatory profile.32 ,33 Kev txheeb xyuas ntawm CD{10}} cov hlwb zoo tau pom tsawg dua macrophages / monocytes hauv WT, Gal-3–/–, thiab Ctns–/–Gal -3–/– lub raumpiv nrog Ctns–/– lub raum(Daim duab 4c), lees paub cov ntaub ntawv histological (Daim duab 4b).
Ua ke, cov kev tshawb pom no qhia tias Gal -3 koom nrog hauv kev nrhiav cov macrophages/monocytes hauv cystinoticlub raumthiab tias nws qhov tsis tuaj yeem kho cov kab mob raum hauv Ctns–/–nas. Li no nws qhia tiasmobyog lub luag haujlwm, tsawg kawg hauv ib feem, rau lub raum tsis zoo hauv Ctns–/– nas.
Ctns-tsis muaj lub raum pom Gal-3 overexpression
Siv Western blot tsom xam, peb pom tias Gal -3 kev qhia tau nce ntxiv hauvlub raumntawm 12- hli-laus Ctns–/–nas piv nrog WT nas (Daim duab 5a thiab b). Txhawm rau tshawb xyuas seb qhov kev nthuav qhia ntau ntxiv ntawm Gal -3 yog tshwj xeeb rau Ctns–/– lub raum, peb suav Gal-3 qhia ntawm cov ntawv sau thiab cov protein ntau hauvlub raumlos ntawm cov nas nrog CKD induced los ntawm tus qauv subtotal 2- kauj ruam nephrectomy ua haujlwm thiab los ntawm cov tsiaj uas tau ua haujlwm sham. WT thiab Ctns–/–nas tau siv los tswj cov ntsiab lus. Gal -3 cov ntawv sau tseg tau nce ntau hauv Ctns–/– thiab sham ob lub raum tab sis nce siab hauv CKDlub raumpiv nrog WT nas (Daim duab 5c). Hauv qhov sib piv, ntawm qib protein, Gal-3 tau kuaj pom hauv Ctns nkaus xwb–/–lub raum, xav hais tias tsuas yog Ctns–/–ob lub raum tsis tuaj yeem degrade Gal{0}} protein (Daim duab 5d). Immunofluorescent staining ntawm Gal -3 hauv murineraumntawm 8 mus rau 9 lub hlis kuj qhia overexpression ntawm Gal-3 hauv Ctns–/– lub raumpiv nrog WTlub raum(Daim duab 5e). Ntxiv mus, Gal -3 tau qhia los ntawm CD68þ macrophages, nrog rau lub raum hlwb hauv Ctns–/– lub raumntawm 8 mus rau 9 lub hlis (Cov duab ntxiv S4). Ua ke, cov ntaub ntawv no zoo ib yam nrog txo Gal-3 degradation thaum tsis muajcystinosisraws li pom nyob rau hauv vitro (Daim duab 3a thiab b), ua rau tsub zuj zuj ntawm Gal{1}} protein nyob rau hauv Ctns–/– lub raum.
Qhov tsis muaj cystinosin ua rau muaj ntshav ntau ntxiv MCP-1 ntawm Gal-3 upregulation
Gal -3 tswjmoblos ntawm kev sib txawv mechanisms.34 Yog li, kom tau txais kev nkag siab ntxiv rau hauv lub tshuab hauvcystinosis, peb tau tshawb xyuas qhov kev qhia ntawm qhov sib txawv pro-inflammatory lossis anti-inflammatory cytokines nyob rau hauv cov ntshav ntawm 12- mus rau 15- hli-laus WT, Ctns–/–, Gal -3–/–, thiab Ctns–/–Gal - 3–/–nas. Rau qib cytokine tau ntsuas los ntawm nas cytometric hlaws array, MCP-1, interferon-g, qog necrosis factor, thiab IL-10, IL-6, thiab IL-12p70. Tsuas yog MCP-1 qhia tau nce ntau hauv Ctns–/– nas cov ntshav piv nrog WT thiab Gal-3–/– nas thiab rau Ctns–/–Gal -3–/–cov nas, uas nws MCP-1 qib ntshav tau muab piv nrog WT nas (Daim duab 6a). MCP-1 yog tsim los ntawm ntau hom ntawm tes, nrog rau qhov loj ntawm MCP-1 yog macrophages thiab monocytes, 35 thiab ua raws li cov tshuaj tua kab mob rau monocytes thiab macrophages tswj lawv txoj kev tsiv teb tsaws thiab nkag mus. Hauv qhov sib piv, tib lub hnub nyoog, Gal-3 qhia tau pom tias zoo ib yam hauv cov ntshav ntawm Ctns–/– nas (44.33 9.81 ng/ml; n ¼ 5) thiab WT nas (40.{12} }.41 ng/ml; n ¼ 7).
Kev sib raug zoo ntawm Gal-3 thiab MCP-1 tau tshawb xyuas ntxiv los ntawm kev sib koom ua ke immunoprecipitation siv Gal-3–GFP thiab MCP-1–DsRed– lossis Gal-3– GFP thiab CTNS-DsRed- (raws li kev tswj zoo) qhia 293T hlwb. Kev sib cuam tshuam ntawm Gal-3 thiab MCP-1 tau pom (Daim duab 6b), tawm tswv yim ncaj qha ntawm MCP-1 los ntawm Gal-3. Incubation nrog N-Acetyl-D-lactosamine (LacNAc), ib qho inhibitor ntawm Gal -3 CRD, qhia tias, piv raucystinosinkev sib cuam tshuam, CRD tsis koom nrog kev sib cuam tshuam ntawm Gal-3 thiab MCP-1 (Daim duab 6c).
Txhawm rau ntsuas seb qhov kev tshawb pom no puas cuam tshuam rau tib neeg, peb ntsuas Gal{0}} thiab MCP-1 qib hauv cov neeg mobcystinosisleej twg tsis tau txais kev kho mob immunosuppressive lossis noj tshuaj tiv thaiv kab mob. Txawm hais tias Gal-3 qib tau pom tias nce me ntsis hauv cov ntshav ntawm cov neeg mob cystinosis piv nrog cov neeg noj qab haus huv, qhov sib txawv tsis tseem ceeb (Daim duab 6e), lees paub peb cov txiaj ntsig tau txais hauv Ctns–/–nas. Tsuas yog 2 tus neeg mob tau siab Gal{1}} qib (25.34 thiab 39.54 ng / ml); lawv tau raug suav hais tias yog outliers thiab yog li tsis suav nrog hauv daim duab 6e. Hauv qhov sib piv, siv cov enzyme-linked immunosorbent assay qhia tawm tsam tib neeg MCP-1 (Daim duab 6d), ntshav MCP-1 qib tau pom tias muaj ntau ntxiv hauv cov neeg mob uascystinosis(252. 0; hnub nyoog thaj tsam 8–63 xyoo) (P <0.001). tsis="" muaj="" kev="" sib="" raug="" zoo="" ntawm="" lub="" hnub="" nyoog="" thiab="" mcp-="" 1="" qib="" hauv="" ob="" tus="" neeg="" mob="">cystinosisthiab noj qab nyob zoo pub dawb (cov ntaub ntawv tsis qhia).
Cistanchemuajanti-inflammatorykhoom
Kev sib tham
Txawm tias muaj tseeb hais tias cystine accumulates nyob rau hauv tag nrho cov ntaub so ntswg nyob rau hauv cov neeg mob cuam tshuam nrogcystinosis, ob lub raum yog cov kabmob uas muaj feem cuam tshuam rau kev puas tsuaj. Yog li peb ntseeg tias cystine tsub zuj zuj tsis yog lub luag haujlwm rau lub raum degeneration. Ntawm no, peb piav qhia lub luag haujlwm tshiab raucystinosinnyob rau hauv txoj cai ntawmmobkoom nrog hauv kev loj hlob ntawm cov kab mob raum tsis zoo nyob rau hauvcystinosis. Txoj kev tshawb no yuav piav qhia tias yog vim li cas cov neeg mob hloov mus rau theem kawg ntawm lub raum tsis ua haujlwm txawm tias tau txais kev kho cysteamine.
Txoj kev tshawb fawb ntawm cystinosis molecular cov neeg koom tes tau qhia txog kev cuam tshuam ncaj qha nrog Gal -3, uas tuaj yeem cuam tshuam los ntawm inhibitors ntawm Gal -3 CRD. Cov kev tshawb fawb tsis ntev los no tau pom tias Gal -3 tuaj yeem cuam tshuam nrog glycans nyob hauv lysosomal lumen tom qab lysosomal puas xws li siv lead ua buildup.36 Hauv peb txoj kev tshawb fawb, kev cuam tshuam ntawm Gal-3 thiabcystinosintau kawm nyob rau hauv cov hlwb noj qab haus huv qhiacystinosinthiab, yog li ntawd, tsis accumulating cysteine los yog cystine crystals. Tsis tas li ntawd, overexpression ntawm ob Gal-3 thiab cystinosin nyob rau hauv lub hlwb noj qab haus huv hloov lub subcellular localization ntawm Gal-3, uas yog ces localized rau lub lysosomes, piv nrog overexpression ntawm Gal -3 xwb, uas tau nyob hauv cytoplasm. Cov txiaj ntsig no tau pom zoo tias kev sib cuam tshuam ntawm Gal -3 thiab cystinosin tshwm sim ntawm nws tus kheej ntawm kev puas tsuaj lysosomal thiab cystinosin yog lub luag haujlwm rau Gal-3 lysosomal localization. Yav dhau los Gal-3 tau pom tias tau degraded los ntawm lysosomal-dependent proteolysis thaum tsis muaj Abl thiab Arg, 2 tyrosine kinases.31
Ntxiv rau, peb tau qhia tiascystinosisthiab Gal -3 co-localize ntawm dynamic vesicular cov qauv thiab tau ua ke ua ke nyob rau hauv ib tug spatiotemporal yam.Cystinosinyav dhau los tau cuam tshuam nrog kev lag luam mechanisms ntawm lysosomal LAMP2A, tsuas yog paub CMA receptor, uas yog mislocalized hauvcystinosis.28Gal -3 degradation yav dhau los tau pom tias tau kho los ntawm CMA.31Confocal microscopy tsom xam tau lees paub qhov colocalization ntawm Gal-3 ntawm Hsc70- cov qauv zoo hauv ob qho tib si Ctns–/– thiab WT hlwb, txhawb nqa kev thauj mus los ntawm Gal-3 nrog Hsc70 rau kev nthuav qhia ntawm lysosome thiab degradation los ntawm CMA raws li Hsc70 pab txoj kev hloov pauv ntawm cov protein substrate hla daim nyias nyias mus rau hauv lysosomal lumen.29,30Kev txhais tau ntawm peb cov txiaj ntsig yog qhov ntawdcystinosintswj kev lag luam thiab xa khoom ntawm Gal -3 mus rau CMA-active lysosomes rau degradation.
Txoj kev tshiab no ntawmcystinosin-dependent Gal-3 lysosomal localization thiab degradation yog txaus siab nyob rau hauv vivo asCtns-deficient nas qhia overexpression ntawm Gal-3 nyob rau hauv lubraum. Ntxiv mus, qhov nce Gal-3 mRNA raug txwv hauv Ctns–/–ob lub raum piv nrog lwm tus qauv ntawm CKD, ib qho loj ntawm Gal{0}} protein tau kuaj pom tsuas yog inCtns–/– lub raum. Cov ntaub ntawv no txhawb nqa qhov xaus tiascystinosinyog koom nrog rau kev degradation ntawm Gal{0}} protein, uas tuaj yeem tswj tau qhov overexpression ntawm Gal-3 mRNA hauv kev ntxhov siab xws li CKD.
Gal -3 muaj ob peb lub luag haujlwm ntawm biologic, nrog rau lub luag haujlwm hauv kev mob hnyav thiab mob ntevmoblos ntawm kev nyiam cov monocytes thiab macrophages.37,38Hauv Ctns–/– nas, peb pom hnyav mononuclear infiltrates feem ntau nyob rau hauvraumraws li tau piav dhau los,11,39uas yog tus cwj pwm li monocytes / macrophages. Nyob rau hauv sib piv, ob lub raum los ntawm ob knockout Ctns–/–Gal -3–/–Cov nas pom muaj tsawg heev monocytes / macrophage infiltrates, qhia tau hais tias Gal -3 koom nrogmobhauv lub raum ntawm Ctns–/–nas. Hauv cov ntsiab lus ntawm cov ntaub so ntswg rov ua dua, Gal -3 tuaj yeem ua rau muaj kev hloov pauv mus ntevmobthiab fibrosis.34Raws li qhov kev tshawb pom no, peb cov ntaub ntawv qhia txog kev koom tes ntawm Gal-3 hauv kev nce qib ntawm CKD hauvcystinosis. Tseeb tiag, ob chav knockout Ctns–/– Gal -3–/–nas pom zoo duaraummuaj nuj nqi thiab qauv piv nrog Ctns–/–nas. Ib yam li ntawd, Gal-3–cov nas uas tsis muaj peev xwm pom tau tias lub raum fibrosis tsawg duaraumpiv nrog WT nas tom qab unilateral ureteric obstruction,40 thiab Gal-3 cov nas raug mob los ntawm ischemia-reperfusion raug mob muaj lub raum ua haujlwm zoo dua piv nrog WT nas raug mob ischemia-reperfusion.41
Txawm hais tias muaj kev sib raug zoo ntawm qib Gal-3 hauv ntshav thiab kev txhim kho ntawm CKD, 42 tsis muaj qhov sib txawv ntawm Gal-3 qhia tau pom nyob rau hauv cov ntshav ntawm cov nas thiab tib neeg cuam tshuam los ntawmcystinosisthiab tswj kev noj qab haus huv. Los ntawm kev ntsuas ntau qib cytokine hauv cov ntshav, peb pom muaj qhov nce ntxiv ntawm MCP-1 hauv Ctns–/–nas, whereas Ctns–/–Gal -3–/–nas muaj qib piv nrog cov nas WT. MCP-1 yog cytokine uas tuaj yeem tso tawm hauv cov ntshav thiab tsim cov gradient kom nyiam cov monocytes thiab macrophages mus rau qhov chaw raug mob.35 Qhov nce ntawm MCP-1 hauv sera ntawm Ctns–/–nas piv nrog Ctns–/–Gal -3–/–nas yog ywj siab ntawm cystine tsub zuj zuj vimraumcystine cov ntsiab lus zoo ib yam nyob rau hauv ob qho tib si genotypes. Tsis tas li ntawd, txawm tias kev kho mob cysteamine uas tso cai rau kev tawm ntawm cystine tawm ntawm lysosomes, MCP-1 tau pom tias muaj kev nce ntxiv hauv cov neeg mob nrogcystinosispiv nrog cov neeg noj qab haus huv. Cov ntaub ntawv no qhia tau hais tias tsis muajcystinosin, dua li cystine tsub zuj zuj, yog lub luag haujlwm rau MCP -1 nce hauv cov ntshav. Ntxiv rau, peb tau qhia txog kev sib cuam tshuam ncaj qha ntawm Gal-3 thiab MCP-1, uas nyuam qhuav tawm tswv yim los ntawm Gordon-Alonso li al.43 tab sis tsis tau kawm ntxiv lawm. Cov txheej txheem ntawm Gal-3-mediated MCP-1 qhib tsis tau kawm ntawm no. Ib qho kev xav yog qhov muaj lub teeb liab peptide nyob rau hauv tib neeg MCP-1, uas tuaj yeem cleaved los txhim kho nws cov secretion.44 Ntxiv mus, plasmin tuaj yeem tshem tawm C-terminus ntawm MCP-1, uas ua rau nws cov chemoattractant nce. potency.45 Tsis tas li ntawd, sib cuam tshuam nrog peb cov ntaub ntawv, inhibition ntawm MCP-1 hauv tus qauv nas ntawm ntshav qab zib nephropathy tiv thaiv lub raum macrophage infiltration thiab txhim kho lub raum ua haujlwm.46,47 Hauvcystinosis, peb cov ntaub ntawv qhia tau hais tias qhov tsis muajcystinosinua rau muaj qhov nce ntawm Gal-3, uas ua rau MCP-1 kev mob ntshav, txhim kho raummobthiab kev vam meej ntawmmob raum mob.
Cov kev tshawb pom no qhib qhov kev xav tshiab ntawm cov hom phiaj kho mob uas tuaj yeem txwv lossis ncua lub raum degeneration hauv cov neeg mob uascystinosis. Tseeb tiag, cov tshuaj nonsteroidal anti-inflammatory xws li tshuaj aspirin thiab indomethacin tau pom tias inhibit Gal-3 qhia los ntawm inhibiting nws transcription.48 Indomethacin yog siv los tswj polyuria thiab polydipsia hauvcystinosis, 49 thiab kev tshawb fawb tsis ntev los no ntawm 307 European cov neeg mob uas muaj cystinosis tau pom tias muaj kev txhim kho lub raum hauv cov neeg mob kho nrog indomethacin.50 Raws li peb txoj kev tshawb fawb, kev siv cov tshuaj indomethacin los yog nonsteroidal anti-inflammatory tshuaj raucystinosiscov neeg mob tuaj yeem rov xav dua.
Txoj kev
Tsiaj sim
Lub C57BL / 6 Ctns–/–cov nas tau muab los ntawm Dr. Antignac (Inserm U1163, Paris, Fabkis). C57BL/6 galectin -3 null (Gal{4}}–/–) nas tau muab los ntawm Dr. Fu-Tong Liu (University of California, Davis) thiab Dr. Jerrold M. Olefsky (University of California, San Diego). Lub tswv yim yug me nyuam los tsim WT, Ctns–/–, Gal -3–/–, Ctns–/– and Gal- 3–/–Cov nas tau piav qhia hauv Cov Txheej Txheem Ntxiv.
Cell kab
Qhov kev ntxhov siab tau tsim los ntawm cov nqaij mos ntawm daim tawv nqaij ntawm WT thiab Ctns–/–nas. MEF, 293T, thiab MDCK hom II cell kab (ATCC, Manassas, VA) tau loj hlob hauv Dulbecco's modified Eagle medium uas muaj 10 feem pua fetal calf serum lossis fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin , thiab 2 mM L-glutamine.
Co-immunoprecipitation thiab huab hwm coj spectrometry tsom xam
Txhawm rau kawm txog kev sib cuam tshuam ntawm cov protein-protein, MDCK hlwb tau hloov pauv siv cov lentiviral pRRRL.SIN.cPPT.PGK/WPRE vector nraub qaum kom ruaj khov.cystinosin-GFP.51Co-immunoprecipitation tau ua raws li tau piav qhia hauv Cov Txheej Txheem Ntxiv. Co-immunoprecipitated proteins tau daws los ntawm sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) thiab soj ntsuam los ntawm huab hwm coj spectrometry, LTQ Orbitrap raws li tau piav qhia.19
293T hlwb qhia Gal-3–GFP thiab MCP-1–DsRed lossis Gal-3- GFP thiab CTNS-DsRed tau siv rau kev sib koom ua ke tiv thaiv kab mob raws li tau piav qhia hauv Cov Txheej Txheem Ntxiv. Co-immunoprecipitated proteins tau daws los ntawm SDS-PAGE, thiab Gal-3-binding MCP-1 tau kuaj pom siv cov tshuaj tiv thaiv-DsRed antibody (Clontech Laboratories, Mountain View, CA).
Subcellular Fractionation
Yim lub siab tau txais los ntawm C57BL / 6 nas thiab npaj raws li tau piav qhia yav dhau los.52 Cov xwm txheej ntawm cov gradient yog qhov tseem ceeb zoo ib yam li tau piav qhia hauv daim ntawv tshaj tawm thawj, 52 tshwj tsis yog tias Nycodenz tau siv los hloov metrizamide.
Gal-3-inhibitor thiab proteinase K kev kho mob
Lysates los ntawmcystinosin-GFP MDCK hlwb thiab 293T qhia Gal-3–GFP thiab CTNS-DsRed los yog Gal-3–GFP thiab MCP-1–DsRed tau incubated nrog lossis tsis muaj 5 mM thiodigalactoside lossis 5 mM ntawm N -Acetyl-D-Lactosamine, feem, 2 inhibitors ntawm Gal -3 CRD. Tom qab 30 feeb ntawm incubation ntawm 4 - C nrog tas li co, lysates tau incubated nrog 50 ml anti-GFP microbeads, thiab immunoprecipitation tau ua raws li tau hais yav dhau los. Precipitated proteins tau daws los ntawm SDS-PAGE ntawm 10 feem pua gel.
Lysates los ntawmcystinosin-GFP MDCK hlwb thiab nas daim siab lysosome-enriched fractions tau incubated nrog los yog tsis muaj 2 feem pua Triton X-100 rau 10 feeb ntawm 4 - C thiab ces incubated 30 feeb nrog los yog tsis muaj 2.5 mg/ml thiab 25 mg. /ml proteinase K, feem. Tom qab enzyme inactivation nrog 1 mM phenylmethylsulfonyl fluoride rau 5 feeb ntawm 4 - C, cov proteins tau daws los ntawm SDS-PAGE ntawm 10 feem pua gel.
Immunofluorescence tsom xam
Tsau MDCK cov hlwb raug incubated nrog los tiv thaiv teeb-2 (OriGeneTechnologies, Rockville, MD) thiab anti-Gal-3 antibody (CedarlaneLaboratories, Burlington, Ontario, Canada) 2 teev ntawm chav tsev kub, ua raws li Alexa Fluor 555 thib ob antibody (Invitrogen, Carlsbad, CA) rau 1 teev ntawm chav tsev kub. Confocal dluab raug coj los siv Zeiss LSM 700 microscope (Carl ZeissMicroscopy, Jena, Lub teb chaws Yelemees).
293T hlwb ua ntu zus qhia Gal-3–GFP ib leeg, Gal-3–GFP, thiab DsRed lossis Gal-3–GFP thiabcystinosin-DsRed tau raug coj los rau ntawm daim npog npog ua ntej muab tso rau ntawm tus swb. Cells tau yees duab siv lub cuab yeej Keyence BZ-X700 (Keyence Corp., Osaka, Nyiv).
Tsauraumcov seem tau muab tso rau ib hmos ntawm 4 - C nrog anti-CD68 (BioLegend, San Diego, CA) los yog anti-Gal-3 antibody (BioLegend), tom qab ntawd los ntawm Alexa Fluor 488 Secondary antibody (Invitrogen), 1 teev ntawm chav tsev kub. Cov duab tau txais los ntawm Keyence BZ-X700 ntsuas. Qhov ntau ntawm CD68 qhia tau piav qhia hauv Cov Txheej Txheem Ntxiv.
MEFs qhia Gal-3–GFP tau stained siv anti-Hsc70 antibody (Enzo Life Sciences, Farmingdale, NY) thiab duab raws li tau piav qhia yav dhau los.53
Tag nrho sab hauv reflection FL fluorescence microscopy
WT thiab Ctns–/–MEF qhia Gal-3–GFP, thiab CTNS-DsRed tau cog rau ntawm 4-chamber 35- hli borosilicate qab phaj (Cellvis, Mountain View, CA). Tom qab 2 hnub hauv kab lis kev cai, MEFs tau txheeb xyuas los ntawm tag nrho cov kev xav sab hauv FL fluorescence microscopy raws li tau piav qhia yav dhau los54thiab hauv Cov Txheej Txheem Ntxiv. Cov duab tau txheeb xyuas siv ImageJ software.
Gal -3 kev tsom xam
293T hlwb qhia Gal-3–GFP, Gal-3–GFP, thiab DsRed, lossis Gal-3–GFP thiab CTNS-DsRed thiab coglub raumtau lysed hauv radioimmunoprecipitation assay tsis muaj proteinase inhibitor cocktail. Proteins raug cais ntawm 4 feem pua rau 15 feem pua gel thiab qhia tawm siv cov tshuaj tiv thaiv-Gal-3 (Abcam, Cambridge, UK) lossis anti-glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA) antibody .
Lub raumtau homogenized hauv RLT tsis muaj b-mercaptoethanol siv Precellys 24 (Bertin Instruments, Montigny-le-Bretonneux, Fabkis). RNA raug cais tawm thiab Gal-3- cov tshuaj tiv thaiv kab mob tshwj xeeb cov tshuaj polymerase saw tau ua raws li tau piav qhia hauv Cov Txheej Txheem Ntxiv. Gal-3 cov lus sau tseg tau qhia raws li qhov piv nrog rau kev tswj hwm endogenous 18S. Ib qho enzyme-linked immunosorbent assay (Abcam) tau siv los kuaj xyuas Gal-3 qib hauv nas thiab tib neeg sera, raws li cov chaw tsim khoom cov lus qhia.
Lub raum ua haujlwm
Cov ntshav thiab cov zis phosphate, ntshav creatinine, thiab urea qib tau kwv yees siv QuantiChrom Phosphate Assay Kit, Quanti Chrom Creatinine Kit, thiab QuantiChrom Urea Assay Kit (BioassaySystems, Hayward, CA). Cov qib Protein hauv cov zis tau ntsuas los ntawm Pierce BCA Protein Assay Kit (Rockford, IL).
Histology
Thaum cov nas raug tua,lub raumtau sau, tsau rau hauv formalin, thiab embedded nyob rau hauv paraffin. Cov seem uas muaj hematoxylin thiab eosin tau raug tshuaj xyuas los ntawm Dr. Marie Claire Gubler raws li tau piav qhia hauv Cov Txheej Txheem Ntxiv.
Kev ntsuas cov ntsiab lus cystine
Kev ntsuas cov ntaub so ntswg cystine tau ua los ntawm pawg spectrometry (LC-ESI-MS / MS) raws li tau piav qhia dhau los.39
Standard nephrectomy thiab sham ua haujlwm
Cov nas CKD raug ntxias los ntawm tus qauv subtotal 2-theem nephrectomy ua haujlwm raws li tau piav qhia yav dhau los.55,56Pab pawg sham ntawm nas tau ua tib yam txheej txheem tab sis tsis muaj kev txiavraumntaub so ntswg.
Qib Cytokine hauv cov ntshav
Txhawm rau ntsuas qib cytokine hauv cov ntshav nas, nasmobcov khoom siv cytometric hlaws array (BD Biosciences, San Jose, CA) tau ua raws li cov chaw tsim khoom cov lus qhia. MCP-1 concentration nyob rau hauv tib neeg cov ntshav tau txiav txim siab siv cov enzyme-txuas immunosorbent assay qhia tawm tsam MCP-1 (Abcam, Cambridge, UK) raws li cov chaw tsim khoom cov lus qhia.
Kev txheeb cais
Cov txiaj ntsig tau qhia tias txhais tau tias - SEM. Qhov tseem ceeb ntawm qhov tshwm sim tau raug soj ntsuam los ntawm unpaired 2-tailed t-test los yog unpaired 2-tailed t-test nrog Welch qhov kho. Kev sib piv pab pawg ntawm 3 cov xwm txheej lossis ntau dua tau ua nrog kev ntsuas parametric ntawm qhov sib txawv, ua raws li Tukey ntau qhov kev sib piv rau kev sib piv sib piv. Cov qhab nia histologic tau muab piv nrog kev xeem Mann-Whitney. Kev soj ntsuam tau ua tiav siv Prism 6 software (GraphPad, San Diego, CA). P-tus nqi tsawg dua 0.05 tau suav tias yog qhov tseem ceeb.
Kev pom zoo kawm
Kev sim nas tau ua raws li InstitutionalAnimal Use Committee raws tu qauv. Kev siv tib neeg cov ntaub so ntswg hauv txoj kev tshawb no tau pom zoo los ntawm University of California, San Diego, HumanResearch Protections Program.
Qhia tawm
SC yog Pawg Thawj Coj Saib Xyuas Kev Tshawb Fawb thiab cov tswv cuab ntawm Pawg Thawj Coj ntawm Pawg Neeg Saib XyuascystinosisKev tshawb fawb Foundation. SC yog ib tug co-founder, shareholder, thiab cov tswv cuab ntawm ob lub scientific board thiab pawg thawj coj ntawm GenStem TherapeuticsInc. Cov nqe lus ntawm qhov kev npaj no tau raug tshuaj xyuas thiab pom zoo los ntawm University of California-San Diego raws li nws txoj cai tsis sib haum xeeb. Tag nrho lwm tus kws sau ntawv tshaj tawm tsis muaj kev sib tw nyiam.
muab Gal -3–/–nas. Peb ua tsaug rau Lou Devanneaux thiab NicholeFlerchinger rau lawv cov kev pab cuam thiab Elizabeth Souter rau kev tshuaj xyuas cov ntawv sau. Peb lees paub Christopher Alfonso los ntawm BDBioscience rau kev muab nasmobcytometric hlaws array thiab rau nws pab nrog kev txhais cov ntsiab lus. Txoj haujlwm no tau txais kev txhawb nqa los ntawm National Institutes of Health pab nyiaj RO1-DK090058and R01-DK110162, theCystosisTshawb fawb Foundation, thiab California Lub Tsev Haujlwm Saib Xyuas Kev Kho Mob (CIRM, CLIN-09230). TL tau txais kev txhawb nqa los ntawm kev kawm tiav kev sib raug zoo los ntawm Vaincre Les MaladiesLysosomales. AB thiab JZ tau txais kev txhawb nqa los ntawm kev sib raug zoo los ntawmcystinosisKev tshawb fawb Foundation. UCSD Neuroscience MicroscopyShared Facility tau txais nyiaj los ntawm National Institute of Neurological Disorders thiab Stroke (NINDS) pub nyiaj P30-NS047101.
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