Ntu 2: Anti-fibrotic Effect Ntawm 6-Bromo-indirubin-3 ′ -oxime (6-BIO) Ntawm Txoj Cai Ntawm Activator Protein-1 (AP-1) Thiab Specificity Protein-1 (SP-1) Transcription Factors hauv raum Cells

Rau Part 1, nyemntawm no.

3.3. 6-BIO attenuates fibrosis markers thiab fibrosis-cuam tshuam kev taw qhia txoj hauv kev nyob rau hauv proximal tubule (HK-2) thiab interstitial fibroblast (NRK49F) hlwb

Tom ntej no, peb tau lees paub qhov cuam tshuam ntawm 6-BIO ntawm cov kab mob sib txuas ntawm cov tubular thiab interstitial fibroblast cells. Peb tau soj ntsuam qhov concentration- thiab lub sij hawm-dependent ntawm GSK3 mRNA qhia los ntawm 6-BIO hauv HK-2 thiab NRK49F hlwb (Fig. S2A thiab B), thiab kev qhia ntawm ECM thiab EMT cov proteins uas cuam tshuam nrog hauvraumcellfibrosiskuj tau txo (Fig. S2E). Ntxiv mus, txhim kho kev qhia ntawm GSK3, raws li TGF raug ntawm cov hlwb, kuj txo qis los ntawm 6-BIO kev kho mob (Fig. S2C thiab D). Fig. 3 qhia cov protein qhia ntawmraumfibrosiscov cim hauv HK-2 thiab NRK49F hlwb kho nrog TGF . Raws li pom nyob rau hauv daim duab 3A, qhov kev qhia ntawm PAI-1 thiab CTGF cov proteins tau nce ntau hauv cov lus teb rau kev kho mob nrog 5 thiab 10 ng/mL TGF hauv HK-2 thiab NRK49F hlwb, raws li. Txawm li cas los xij, lawv cov lus qhia tau txo qis heev tom qab kho nrog 6-BIO (Fig. 3B).SMADthiab MAPK txoj kev taw qhia tau raug kho los ntawm kev kho TGF, uas tau thim rov qab los ntawm 6-BIO kev kho mob (Fig. 4). Tom ntej no, peb tau tshawb xyuas qhov kev qhia ntawm AP-1 thiab SP-1 cov ntsiab lus hloov pauv uas cuam tshuam nrog TGF -mediated signaling hauv HK-2 thiab NRK49F hlwb. Daim duab 4D qhia tau hais tias TGF −mediated nce nyob rau hauv PC-JUN thiab PC-FOS raug txo nyob rau hauv teb rau kev kho mob nrog 6-BIO thiab JNK inhibitor SP600125 thiab raws li dominant-negative C-JUN transfection. Fig. 4E qhia tau hais tias qhov kev hloov pauv ntawm cov protein ntau txheeb ze rau kev tswj. Ntxiv mus, TGF -mediated nce nyob rau hauv SP{18}} qib tau txo qis hauv kev teb rau kev kho nrog 6-BIO thiab SP-1 siRNA (Fig. 4F). Fig. 4G qhia txog kev hloov pauv ntawm cov protein ntau txheeb ze rau kev tswj. Cov txiaj ntsig no qhia tias TGF /SMADthiab MAPK signaling nrog rau cov kev hloov pauv, AP-1, thiab SP-1, yog tswj hwm los ntawm 6-BIO.

3.4. 6-BIO attenuates nuclear qhia ntawm PC-JUN, PC-FOS, thiab SP-1 cov proteins nyob rau hauv proximal tubular thiab interstitial fibroblasts hlwb

Fig. 5A thiab B qhia qhov chaw ntawm PAI-1 thiab CTGF nrog PC-JUN, PC-FOS, thiab SP-1. Liab staining ntawm PC-JUN, PC-FOS, thiab SP-1 qhia tias lawv overexpression nyob rau hauv lub nucleus tom qab TGF kho; Ib yam li ntawd, ntsuab staining ntawm PAI-1 thiab CTGF qhia lawv cov lus tshaj tawm hauv cytosol. Txawm li cas los xij, cov theem qhia ntawm PC-JUN, PC-FOS, thiab SP-1 raug txo los ntawm 6-BIO kev kho mob. Cov txiaj ntsig no tau qhia tias 6-BIO cuam tshuam ncaj qha rau AP-1 thiab SP-1, cov ntsiab lus hloov pauv tswj PAI-1 thiab CTGF kev qhia.

3.5. 6-BIO attenuates PAI-1, CTGF, AP-1, thiab SP-1 txhawb kev ua haujlwm hauv cov kab mob sib luag thiab cov kab mob sib kis

Tom ntej no, peb tshawb xyuas seb 6-BIO cuam tshuam cov kev txhawb nqa ntawm PAI-1, CTGF, AP-1, thiab SP-1 noob. Kev txhawb nqa kev ua haujlwm ntawm PAI-1 thiab CTGF tau nce ntxiv hauv kev teb rau TGF tab sis raug txo los ntawm 6-BIO kev kho mob (Fig. 6A thiab B). Ntxiv mus, qhov txo qis yog qhov tseem ceeb thaum cov hlwb tau kho nrog 6-BIO ib leeg dua li muaj TGF . AP-1 thiab SP-1 khi rau cov neeg txhawb nqa ntawm PAI-1 thiab CTGF. Daim duab 6C thiab 6D qhia txog kev txhawb nqa ntawm AP-1 thiab SP-1. AP-1 thiab SP-1 luciferase kev ua haujlwm tau nce los ntawm kev kho TGF thiab txo qis los ntawm 6-BIO raws li koob tshuaj. Daim duab 6E qhia tau hais tias qhov kev qhia ntawm qhov transcription factor nyob rau hauv nuclear protein extracts. Yog li, peb tau lees paub tias qhov kev hais tawm nuclear ntawm PC-JUN, PC-FOS, thiab SP-1 tau nce ntxiv hauv kev teb rau TGF kev kho mob thiab raug txwv los ntawm 6-BIO kev kho mob. Fig. 6F qhia tau hais tias qhov kev hloov pauv ntawm cov protein ntau txheeb ze rau kev tswj. Tsis tas li ntawd, peb cov txiaj ntsig tau qhia tias 6-BIO ua haujlwm ntawm qhov chaw sib txuas lus sib txuas ntawm tus neeg txhawb nqa kom tsis muaj zog rau kev khi ntawm AP-1 thiab SP-1. Peb tau ua nyob rau hauv vitro txhawb kev sib khi kev soj ntsuam nyob rau hauv lub xub ntiag ntawm 6-BIO kom paub meej tias tib yam. Kev khi ntawm cov proteins nuclear thiab PC-JUN, PC-FOS, thiab SP-1 tsis muaj zog los ntawm kev sib ntxiv ntawm 6-BIO nyob rau hauv ib qho concentration-dependent yam (saib ntxiv Fig. 3). Cov txiaj ntsig no qhia tau tias 6-BIO inhibits PAI-1 thiab CTGF txhawb nqa kev ua ub no nrog rau AP-1 thiab SP-1 ua haujlwm; yog li, nws tuaj yeem muaj peev xwm kho tau hauvraumfibrosis.

4. Kev sib tham

Siv tus qauv nas ntawm UUO thiab TGF -khoraumproximal tubules muab tau los thiab interstitial fibroblast cell kab, peb tau pom tias 6-BIO kev kho mob tuaj yeem txo qisraumkev puas tsuaj. Tus qauv UUO yog tus qauv tsiaj sawv cev ntawm obstructive nephropathy uas yog tus cwj pwm los ntawm kev loj hlob tubulointerstitialfibrosis[43]. Histopathological tsom xam ntawmraumcov ntaub so ntswg tau txais los ntawm tus qauv UUO tau pom tias ECM tsub zuj zuj thiab tubular atrophy nyob rau hauv qhov chaw interstitial tau rov qab los ntawm 6-BIO kev kho mob. Tsis tas li ntawd, siv TGF - khoraumproximal tubule-derived thiab interstitial fibroblast hlwb, peb tau pom tias 6-BIO kev kho mob tuaj yeem txo qisraumkev puas tsuaj. Hauv UUO qauv thiab TGF -kho HK-2 thiab NRK49F hlwb, 6-BIO inhibited qhov kev qhia ntawmraumfibrosiscov cim raws li MAPK thiabSMADqhia txoj kev. Tsis tas li ntawd, 6-BIO kev kho mob txo qis kev qhia ntawm PC-JUN, PC-FOS, thiab SP-1 cov proteins uas raug ntxias los ntawm UUO thiab TGF, nrog rau cov lus qhia nuclear thiab kev txhawb nqa ntawm AP{{6 }} thiab SP-1 hauv TGF- - kho HK-2 thiab NRK49F hlwb. Inhibition ntawm AP-1 thiab SP-1 cov ntsiab lus hloov pauv los ntawm 6-BIO kev kho mob tau cuam tshuam nrog inhibition ntawm ECM tsub zuj zuj uas ua rau exacerbation ntawmraumfibrosis. Qhov no yog ua tiav los ntawm suppressing MAPK thiabSMADkev taw qhia txoj hauv kev uas ua rau txo qis ntawm PAI-1 thiab CTGF. Peb cov txiaj ntsig tau qhia tias 6-BIO tuaj yeem yog tus kws kho mob muaj peev xwm tiv thaivraumkab mob, nrog rau kev tiv thaivraumkev puas tsuaj los ntawm UUO thiab TGF.

Nyem rau organic Cistanche rau mob raum

Lub semisynthetic cell-permeable indirubin derivative 6-BIO belongs rau bis-indole tsev neeg thiab ib txwm tshwm sim nyob rau hauv edible gastropod mollusks thiab nroj tsuag [12]. Indirubin muaj ib daim ntawv cog lus intramolecular hydrogen ntawm 2-C ob daim ntawv cog lus O thiab N1-H pab pawg thiab lwm qhov sib xyaw ua ke hydrogen ntawm cov molecules. 6-BIO muaj cov qauv tshuaj hloov kho nyob rau hauv uas 6- txoj hauj lwm yog hloov nrog Br kom kov yeej qhov tsis zoo ntawm cov tshuaj pharmacokinetic tsis zoo vim cov dej solubility tsawg ntawm indirubin [44]. Ntau indirubins, vim muaj kev sib tw khi nrog ATP, ua raws li ob qhov inhibitors ntawm cyclin-dependent kinase (CDK) thiab glycogen synthase kinase{{10}} (GSK-3 ), nthuav tawm ntau dua {{ 12}}fold selectivity dhau CDK5 [12]. Tsis tas li ntawd, 6-BIO nthuav tawm qhov kev xaiv tshwj xeeb ntawm GSK{16}} nrog tus nqi IC50 ntawm 0.005 μM [12]. Raws li kev tshaj tawm los ntawm ntau pawg, 6-BIO tag nrho inhibits MCF-7 cell proliferation thiab thaiv kev tsiv teb tsaws los ntawm kwv yees li 75 feem pua ​​​​ntawm qhov concentration ntawm 10 μM, thiab inhibits phosphorylation ntawm PDK1. Qhov no tau qhia tias 6-BIO tau muab tso rau hauv lub hnab ntawv khi ntawm PDK1 los ua peb daim ntawv cog lus hydrogen nrog cov seem hauv thaj tsam pob khawm, thiab lub hnab ntawv khi tau yooj yim haum rau Br atoms ntxiv hauv 6-BIO [45]. Nrog rau cov yam ntxwv no, 6-BIO tab tom kawm los ua cov tshuaj tshiab los tswj cov txheej txheem ntawm tes xws li kev laus-txog o, oxidative kev nyuaj siab, cell ciaj sia taus, proliferation, thiab apoptosis hauv mob qog noj ntshav, ntshav qab zib, thiab cov kab mob degenerative [46,47 ]. Txawm li cas los xij, kev tshawb fawb txog kev tswj cov noob thiab cov txheej txheem molecular cuam tshuam nrograumcellfibrosismuaj tsawg. Yog li ntawd, peb nrhiav kev tshawb xyuas seb 6-BIO txo qisraumfibrosislos ntawm inhibiting PAI-1 thiab CTGF gene regulation nyob rau hauvraumcell, yog li txo ECM tsub zuj zuj.

Ntau qhov kev tshawb fawb tau siv tus qauv UUO los qhia txog kev sib raug zoo ntawm ECM tsub zuj zuj thiab ntev.raumkab mob tshwm sim los ntawm tubular interstitialfibrosis[43]. Ntxiv mus, overexpressed PAI-1 thiab CTGF tau tshaj tawm tias muaj feem cuam tshuam nrog ECM ntau ntxiv thaum lub sijhawm muaj kev mob ntev.raumkab mob. Yog li, kev txheeb xyuas cov tshuaj molecule uas inhibits qhov taw qhia thiab kev hloov pauv ntawm cov noob cuam tshuam nrog txhim kho ECM tsub zuj zuj yog qhov tseem ceeb rau kev ua haujlwm zoo.raumfibrosis. Yog li, hauv txoj kev tshawb no, peb tau soj ntsuam 6-BIO, ib qho ntawm cov inhibitors ntawm glycogen synthase kinase 3 (GSK-3 ), hauvraumfibrosis.

Peb tau kawm txog cov txiaj ntsig ntawm PAI-1 thiab CTGF cov proteins ntawmraumcellfibrosisthiab tau lees paub tias lawv cov lus hais tau tswj hwm los ntawm cov xwm txheej hloov pauv, AP-1, thiab SP-1. Tus lej hloov pauv, AP-1, yog heterodimer uas muaj cov proteins uas muaj nyob hauv JUN (C-JUN, JUNB, thiab JUND), FOS (C-FOS, FOSB, Fra-1, thiab Fra{ {6}}), ATF (ua kom cov ntaub ntawv hloov pauv), thiab JDP (JUN dimerization protein) tsev neeg [48]. Nws tswj ntau cov txheej txheem ntawm tes xws li cell proliferation, tuag,

sib txawv, thiab vascularization. Ib txoj kev tshawb fawb dhau los tau tshaj tawm tias AP-1 kev ua haujlwm tau nce hauv daim siab thiab ntsws los ntawm kev ua kom hom VI collagen thiab fibroblasts [49,50]. Ob lub ntsiab lus tseem ceeb ntawm AP-1, C-JUN, thiab C-FOS teb rau ntau yam stimuli, xws li cytokines, kev loj hlob, thiab kev ntxhov siab [51]. Peb tau soj ntsuam phosphorylation ntawm C-JUN thiab C-FOS nyob rau hauv UUO nas qauv thiab TGF -kho HK-2 thiab NRK49F hlwb (Figs. 2D thiab 4D). C-JUN thiab C-FOS daim ntawv heterodimers thiab khi rau AP-1 khi qhov chaw hauv DNA, yog li ua raws li kev hloov pauv los tswj kev qhia ntawm lwm cov noob. Tsis tas li ntawd, phosphorylation ntawm ERK1/2 thiab JNK activates C-FOS thiab C-JUN, feem. Hauv peb txoj kev tshawb fawb, phosphorylation ntawm ERK1/2 thiab JNK, nrog rau C-JUN thiab C-FOS nyob rau hauv lub nucleus tau nce zoo heev hauv kev teb rau kev kho mob nrog TGF. Txawm li cas los xij, 6-BIO kev kho mob tau thim rov qab cov teebmeem no. Peb txoj kev tshawb fawb tau pom tias 6-BIO ua tau zoo inhibited phosphorylation ntawm ERK1/2 thiab JNK thiab txo cov kev ua ntawm transcription factor AP-1, yog li inhibiting phosphorylation ntawm C-JUN thiab C-FOS uas yog nws downstream. cov nyhuv. SP-1 inhibition nyob rau hauv hepatic astrocytes exerts ibanti-fibrotic tshuajeffect [52]. Lwm txoj kev tshawb fawb pom tias SP-1 tau ua lub luag haujlwm tseem ceeb hauvfibrosis[52,53]. Yog li, peb tau soj ntsuam seb puas yog 6-BIO tuaj yeem cuam tshuam qhov kev hloov pauv SP-1. Peb tau pom tias qhov kev qhia ntawm SP-1 cov protein tau nce ntxiv hauv kev teb rau TGF kev kho mob nrog rau hauv UUO qauv, thaum nws txo qis tom qab 6-BIO kev kho mob. Ntxiv mus, cov lus qhia nuclear ntawm SP-1 ntxias los ntawm TGF raug txwv los ntawm 6-BIO kev kho mob. Yog li ntawd, kev txhawb nqa kev ua ub no ntawm PAI-1 thiab CTGF, uas muaj AP-1 thiab SP{10}} khi qhov chaw, kuj raug txo los ntawm kev kho nrog 6-BIO (Fig. 6 ). Yog li, cov kev tshawb pom no qhia tias qhov txo qis hauv kev txhawb nqa kev ua haujlwm ntawm PAI-1 thiab CTGF los ntawm 6-BIO kev kho mob txo qis PAI-1 thiab CTGF cov protein qhia, thiab txo ECM cov khoom sib xyaw, yog li txo qis.raumcellfibrosis.

5. Cov lus xaus

6-BIO kev kho mob txo PAI-1 thiab CTGF qhia hauv UUO qauv thiab TGF khoraumhlwb. Ntxiv rau, 6-BIO inhibited TGF -induced activation ntawmSMADthiab MAPK signaling, thaum tseem inhibiting kev ua ub no ntawm AP-1 thiab SP-1, ob ntawm ntau yam transcription binding rau cov txhawb nqa ntawm PAI-1 thiab CTGF noob. Inhibition ntawm AP-1 thiab SP-1 zoo nkaus li yog ib qho ntawm cov txheej txheem tseem ceeb uas 6-BIO inhibitsraumfibrosis. Yog li, peb cov txiaj ntsig tau qhia tias 6-BIO tuaj yeem yog tus kws kho mob muaj peev xwm tiv thaivraumkab mob.

CREDiT daim ntawv qhia txog kev sau ntawv

Jung Sun Park: Conceptualization, Methodology, Data curation, Writing - original draft preparation, Writing - review & editing, Visualization, Kev nrhiav nyiaj txiag. Hauv Ae Jung: Cov ntaub ntawv curation, Sau - tshuaj xyuas & kho. Hong Sang Choi: Cov ntaub ntawv curation, Sau - tshuaj xyuas & kho. Dong-Hyun Kim: Methodology, Data curation. Hoon In Choi: Methodology, Data curation. Eun Hui Bae: Sau - tshuaj xyuas & kho, Saib Xyuas. Seong Kwon Ma: Sau - tshuaj xyuas & kho,

6. Cov ntaub ntawv

Qhov chaw yog los ntawm Jung Sun Park et, al ntawm Biomedicine & Pharmacotherapy 145 (2022) 112402