Oligosaccharides Ameliorate mob raum mob los ntawm Alleviating Cluster Of Differentiation 44-Mediated Immune Responses in Renal Tubular Cells
Jun 05, 2024
Abstract:Mob raum mob (AKI)yog ib ntus tam sim ntawdraum puasuas feem ntau tshwm sim hauv cov neeg mob mus rau hauv tsev kho mob. Txog niaj hnub no, tsis muaj kev kho mob zoo tshaj plaws tau tsim los txo qisAKI qhov hnyav. Oligo-fucoidan(FC) cuam tshuam rau lub raum tubular cell nto protein pawg ntawm qhov sib txawv 44 (CD44) los tiv thaiv lub raum interstitial fibrosis; Txawm li cas los xij, qhov cuam tshuam ntawm oligosaccharides ntawm AKI tseem tsis paub. Hauv txoj kev tshawb no, FC, galacto-oligosaccharide (GOS), thiab fructo-oligosaccharide (FOS) raug xaiv los tshawb xyuas qhov cuam tshuam ntawm oligosaccharides ntawm AKI. Tag nrho peb oligosaccharides tau raug pov thawj tias yog ib feem ntawm txoj hnyuv. Peb pom tias oligosaccharides koob tshuaj-dependently txo CD44 antigenicity thiab suppressed hypoxia-induced qhia ntawm CD44, phospho-JNK, MCP-1, IL-1 , thiab TNF- hauv NRK-52E lub raum tubular cells. Lub caij no, CD44 siRNA transfection thiab JNK inhibitor SP600125 txo qis cov hypoxia-induced qhia ntawm phospho-JNK thiab cytokines. Cov ligand ntawm CD44, hyaluronan, tiv thaiv kev cuam tshuam ntawm oligosaccharides ntawm CD44 thiab phospho-JNK. Thaum 2 hnub tom qab kev phais mob rau ischemia-reperfusion raug mob, oligosaccharides txo cov mob raum, ntshav creatine, MCP-1, IL-1 , thiab TNF- hauv AKI nas. Thaum 7 hnub tom qab kev phais, lub raum rov qab tau nce. Cov txiaj ntsig no qhia tau tias FC, GOS, thiab FOS inhibit qhov hypoxia-induced CD44 / JNK cascade thiab cytokines nyob rau hauv lub raum tubular hlwb, yog li ameliorating AKI thiab raum mob hauv AKI nas. Yog li ntawd,oligosaccharide supplementationyog ib lub tswv yim kho mob muaj peev xwm rau cov neeg mob AKI.
Ntsiab lus: mob raum raug mob; pawg ntawm kev sib txawv 44; fructo-oligosaccharide; galactooligosaccharide; oligo-fucoidan; mob
1. Taw qhia
Mob raum raug mob(AKI) yog tus cwj pwm los ntawm kev txo qis hauv glomerular pom tus nqi thiab qhov ua rau nce hauv cov ntshav creatinine. Qhov kev puas tsuaj no tshwm sim hauv cov neeg mob mus rau hauv tsev kho mob thiab cov chaw saib xyuas hnyav, tshwj xeeb tshaj yog cov neeg laus. Ischemia, sepsis, thiab nephrotoxicity yog lub ntsiab ua rau AKI uas feem ntau ua ke thiab, yog li, nyuaj rau kev kuaj mob thiab kev kho mob [1]. Qhov hnyav AKI tso tawm cov neeg mob uas muaj kev pheej hmoo ntawm cov xwm txheej tsis zoo, xws li mob raum, mob plawv, mob qog noj ntshav, thiab kis kab mob [2]. AKI muaj kev txhim kho sai, thiab tsis muaj kev kho mob zoo kom zam lossis txo nws qhov hnyav. Kev noj cov zaub mov tshwj xeeb tuaj yeem txo cov mob raum tubular raug mob hauv AKI [3]. Yog li ntawd, kawm txog kev cuam tshuam ntawm kev noj zaub mov tshwj xeeb ntawm AKI yuav pab txhawm rau txhawm rau AKI thiab nrog rau cov kev pheej hmoo uas tau ntsib los ntawm cov neeg mob tso tawm.
Kev kho mob thiab kev kho mob ntawm leukocytesyog cov kws kho mob tseem ceeb ntawm AKI. Lub raum, tshwj xeeb tshaj yog rau lub raum tubular hlwb, tsiminflammatory cytokinesthaum AKI, xws li IL-1, IL-6, monocyte chemoattractant protein-1 (MCP-1), thiab qog necrosis factor- (TNF- ) [4,5] . Cov cytokines txuas ntxiv ua rau cov kab mob hauv lub cev thiab leukocyte recruitment. Txawm hais tias ob peb leukocyte subsets tiv thaiv kev tiv thaiv ntawm AKI (xws li T-txheej txheem hlwb), feem ntau neutrophils, monocytes, thiab dendritic hlwb muaj teeb meem rau AKI rov qab [6]. Cov kab mob hauv lub cev kuj tseem cuam tshuam rau kev rov zoo los ntawm AKI vim tias qib cytokine siab [7]. Ntawm cov cytokines secreted los ntawm lub raum, MCP-1 feem ntau yog lub luag hauj lwm rau leukocyte recruitment, teb rau ntau yam cytokines, xws li IL-1 thiab TNF- [8,9], thiab plays ib tug tseem ceeb luag hauj lwm nyob rau hauv lub pathogenesis. kev raug mob raum sib txawv [10] Hauv human proximal tubular cells HK-2, hypoxia-induced MCP{19}} kev qhia yog kho los ntawm c-Jun NH2- terminal kinase (JNK) txoj kev [11]. Txoj kev p38 tseem koom nrog hauv albumin-induced MCP-1 qhia hauv lub raum tubular epithelial hlwb [12]. Yog li, txoj cai ntawm JNK thiab p38 txoj hauv kev tuaj yeem cuam tshuam qhov mob thiab leukocyte nrhiav neeg ua haujlwm hauv AKI.
Oligosaccharides yog saccharide polymers uas muaj 3-10 monosaccharides uas yog indigestible thiab ua ib feem tseem ceeb ntawm tag nrho cov muaj prebiotics raws li kev noj haus additives [13]. Feem ntau cov fructo-oligosaccharides (FOSs) noj hauv tib neeg lub cev yog fermented los ntawm cov kab mob colonic, thiab ib feem me me tau rov qab los hauv cov zis [14]. Galacto-oligosaccharides (GOSs) tuaj yeem nqus tau hauv cov ntshav thiab tom qab ntawd tawm hauv cov zis [15]. Tib qhov xwm txheej no siv rau fucoidan hauv xim av marine algae [16]. Absorbed oligosaccharides tuaj yeem cuam tshuam cov tshuaj biochemical thiab kev paub ntawm tes [17]. Peb txoj kev tshawb fawb yav dhau los qhia tias oligo-fucoidan (FC) cuam tshuam nrog kev cuam tshuam ntawm lub raum tubular cell nto protein CD44 thiab nws cov ligands extracellular, yog li tiv thaiv lub raum interstitial fibrosis [18]. Kev sib cuam tshuam ntawm CD44 thiab nws cov ligands extracellular txhawb JNK signaling txoj kev [19]. Yog li ntawd, kev noj txhua hnub ntawm oligosaccharides tuaj yeem txo cov mob raum hauv cov neeg mob AKI. Txawm li cas los xij, qhov cuam tshuam ntawm cov polymers ntawm AKI tseem tsis paub. Txoj kev tshawb no tshawb xyuas cov txiaj ntsig ntawm FC, FOS, thiab GOS ntawm hypoxia-kho lub raum tubular hlwb thiab AKI nas nrog ischemia-reperfusion raug mob (IRI). Cov txiaj ntsig tau pom tias peb lub oligosaccharides suppress CD44- kho lub raum tiv thaiv kab mob thiab txo qis tom qab AKI qhov hnyav. Yog li ntawd, kev siv cov polymers no ua cov khoom noj khoom haus ntxiv nthuav qhia AKI tshiab txoj kev kho mob
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Khoom siv
FOS (nruab nrab degree ntawm polymerization <10), GOS (nruab nrab degree ntawm polymerization <8), thiab FC (nruab nrab degree ntawm polymerization <5) tau los ntawm Sigma-Aldrich (St. Louis, MO, USA, Cat. No. F8052), Carbosynth (Berkshire, UK, Cat. No. OG32134), thiab Hi-Q Marine Biotech International (Taipei, Taiwan), feem. Oligosaccharides tau yaj nyob rau hauv ob-distilled H2O thiab lim siv 0.22 µm sterile lim (Merck Millipore, Darmstadt, Lub teb chaws Yelemees). Hyaluronan (HA) thiab tag nrho lwm yam tshuaj ntawm reagent qib tau txais los ntawm Sigma. Antibodies tiv thaiv JNK (sc-7345), phospho-JNK (sc-6254), MCP-1 (sc-52701), IL-1 (sc{{17}) }}), thiab TNF- (sc-52746) tau yuav los ntawm Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies tiv thaiv p38 (9212) thiab phospho-p38 (9211) tau txais los ntawm Cell Signaling Technology (Danvers, MA, USA). Antibodies tiv thaiv Ly6G (ab25377) thiab F4/80 (ab100790) tau yuav los ntawm Abcam (Cambridge, UK). Antibodies tiv thaiv HIF-1 (NB100-105) tau yuav los ntawm Novus Biologicals (Centennial, CO, USA).
2.2. Cell Culture
Rat proximal lub raum tubular hlwb (NRK-52E) thiab nas macrophage kab RAW264.7 tau yuav los ntawm Food Industry Research and Development Institute (Hsinchu, Taiwan); kab lis kev cai hauv DMEM ntxiv nrog tshuaj tua kab mob / tshuaj tua kab mob thiab 10% fetal calf serum; thiab khaws cia ntawm 37 ◦C thiab 95% av noo hauv CO2 incubator. Tom qab ncav cuag 70% confluence, cov hlwb tau coj mus rau hauv qhov nruab nrab tsis muaj ntshav qab zib thiab incubated ib hmos ua ntej qhov kev sim. Rau qhov kev sim hypoxia, NRK-52E hlwb thiab Anaero Pob (Mitsubishi Gas Chemical America, New York, NY, USA) tau muab tso rau hauv lub thawv duab plaub (Mitsubishi Gas Chemical America), thiab tag nrho cov teeb tsa tau muab tso rau hauv 37. ◦ C incubator rau 4 h. Cov hlwb raug coj mus rau ib hmos nyob rau hauv ib txwm muaj mob.
2.3. Western Blot Analysis
Hauv luv luv, 15 µg ntawm NRK-52E protein lysate tau siv rau txhua txoj kab, thiab cov txiaj ntsig tau txheeb xyuas los ntawm Western blot. Cov txheeb ze cov qib protein ntau tau ntsuas los ntawm tsib qhov kev sim ywj pheej los ntawm Quantiscan software los ntawm Biosoft (Cambridge, UK).
2.4. Kev cuam tshuam luv luv (si)RNA Hloov ntawm CD44
Rat CD44 siRNA (4390771) tau yuav los ntawm Thermo Scientific (Waltham, MA, USA). Tom qab loj hlob mus rau 70% confluence, cov hlwb tau kis nrog siRNA siv Lipofectamine 2000 transfection reagent (Thermo Scientific) los ntawm manu facturer cov lus qhia.
2.5. Cell Proliferation Assay
Tom qab hypoxic NRK-52E hlwb raug coj mus kuaj rau 24 teev, tib qhov nruab nrab tau siv rau kab lis kev cai RAW264.7 hlwb hauv 96-zoo phaj (2000 hlwb/zoo). Tom qab 24 teev ntawm incubation, lub proliferation ntawm RAW264.7 hlwb raug soj ntsuam los ntawm kev siv SRB Assay Kit (Abcam, Cat. No. ab235935) raws li cov chaw tsim tshuaj paus cov lus qhia.
2.6. CD44 Antigenicity Tests
Recombinant nas CD44 tau txais los ntawm R&D Systems (Minneapolis, MN, USA, Cat. No. 6577-CD) thiab ua hauj lwm los tsim cov tshuaj tiv thaiv nrog HA los ntawm cov chaw tsim khoom cov lus qhia. Hauv luv luv, 20 ng / mL recombinant nas CD44 tau sib xyaw nrog ntau qhov sib txawv ntawm FC, FOS, los yog GOS rau 10 feeb los tshawb xyuas qhov cuam tshuam ntawm oligosaccharides ntawm CD44 antigenicity. Cov khoom sib tov tau raug tshuaj xyuas siv CD44 ELISA cov khoom siv (Abcam, Cat. No. ab213928) raws li cov chaw tsim khoom cov lus qhia.
2.7. Kev sim tsiaj txhu thiab lub raum IRI
Yim lub lis piam txiv neej 129S1 / SvImJ nas tau yuav los ntawm Lasco Technology (Taipei, Taiwan). Tag nrho cov kev sim tsiaj tau pom zoo los ntawm Taipei Medical University Pawg Neeg Saib Xyuas Kev Saib Xyuas Tsiaj thiab Siv (kev pom zoo No. LAC-2019-0511) thiab tsim los ntawm NIH Phau Ntawv Qhia rau Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Txhaj Tshuaj. Rau IRI induction, nas (qhov hnyav 21-23 g) tau raug rau ob sab raum pedicle clamping rau 30 min los ntawm kev ua ib qho kev txiav tawm [20]. Cov nas raug tshuaj loog los ntawm kev tso tshuaj loog nrog isoflurane ua ntej ua haujlwm. Sham-ua haujlwm nas tau ua haujlwm zoo ib yam, tshwj tsis yog rau lub raum pedicles. Txhua tus nas tau noj nrog oligosaccharides los ntawm qhov ncauj gavage los ntawm 1 hnub ua ntej mus rau 2 hnub tom qab IRI phais. Cov nas sim tau muab faib ua tsib pawg hauv qab no nrog n=8 rau cov txiaj ntsig ntawm hnub 3 thiab hnub 7: (1) pawg sham; (2) AKI pab pawg; (3) AKI pab pawg noj nrog FC (10 mg / kg / hnub); (4) AKI pab pawg noj nrog FOS (10 mg / kg / hnub); thiab (5) AKI pab pawg noj nrog GOS (10 mg / kg / hnub). Cov nas tau muab txi rau 3 lossis 7 hnub tom qab kev phais, thiab cov ntshav tau sau thiab tshuaj xyuas siv cov khoom siv biochemical analyzer thiab cov khoom siv ELISA. Cov ntshav creatinine qib ntawm txhua tus nas tau txheeb xyuas los ntsuas kev txhim kho ntawm AKI. Lub raum morphology tau tshuaj xyuas los ntawm ntu ntu ntawm cov kua qaub-Schiff (PAS) staining thiab immunohistochemistry staining (IHC) hauv formalin-taw 2 µm paraffin seem.
2.8. Immunohistochemistry Staining
Lub raum cov ntaub so ntswg tau kho nrog 4% buffered paraformaldehyde, embedded nyob rau hauv paraffin, thiab txiav mus rau hauv 2 µm seem rau staining nrog UltraVision Quanto Detection System HRP DAB cov khoom siv (Thermo Scientific, Fremont, CA, USA) thiab hybridizing nrog cov thawj antibody cov lus qhia los ntawm lub chaw tsim tshuaj paus. .
2.9. PAS Staining
Cov ntu hauv lub raum tau raug stained nrog PAS Stain Kit (Abcam, Cat. No. ab150680) raws li cov chaw tsim khoom cov lus qhia.
2.10. ELISA Kev Tshawb Fawb
Cov kab lis kev cai nruab nrab ntawm NRK-52E hlwb thiab cov qauv kuaj ntshav ntawm cov nas sim tau sau thiab tshuaj xyuas siv MCP-1 ELISA cov khoom siv (Abcam, Cat. No. ab208979) thiab IL-1 ELISA cov khoom siv (Abcam, Cat. No. ab197742). Cov qauv kuaj ntshav kuj tau txheeb xyuas siv cov khoom siv TNF- ELISA (R&D Systems, Cat. No. MTA00B) thiab neutrophil gelatinase-associated lipocalin (NGAL) ELISA kit (R&D Systems, Cat. No. MLCN20) los ntawm cov neeg tsim khoom cov lus qhia.
2.11. Kev Xeem Xeem
Qhov sib txawv tseem ceeb ntawm ob pawg tau txiav txim siab siv Tub Ntxhais Kawm T-test. Qhov sib txawv tau suav tias yog qhov tseem ceeb rau p tus nqi<0.05
3. Cov txiaj ntsig
3.1. Oligosaccharides inhibit qhov nce hauv CD44 thiab Cytokines hauv Hypoxic NRK-52E Cells
CD44 qhia hauv NRK-52E cov hlwb kho nrog FC, FOS, thiab GOS tau raug tshuaj xyuas thawj zaug los ntsuas qhov cuam tshuam ntawm oligosaccharides ntawm lub raum tubular hlwb. Hypoxia-induced CD44 qhia tau inhibited los ntawm tag nrho peb oligosaccharides nyob rau hauv ntau ntawm 0.05–{9}}.5 mg/mL (Daim duab 1A). Yog li, oligosaccharide ntau npaum li cas ntawm 0.1 mg / mL tau siv hauv cov kev sim hauv qab no ntawm tes. Hauv cov txheej txheem npaj, kev kho hypoxia zoo kawg nkaus ua rau muaj kev qhia ntawm hypoxia marker HIF-1 hauv NRK-52E hlwb, qhia tias cov hlwb nyob hauv lub xeev hypoxia (Daim duab 1B). Hypoxia upregulated qib ntawm phosphorylated JNK, MCP-1, IL-1 , thiab TNF- tab sis tsis nyob rau hauv cov hlwb kho nrog oligosaccharides (Daim duab 1B, C). JNK qhia tau nce hauv cov hlwb hypoxic tab sis tsis nyob hauv FC-kho lossis FOS-kho hlwb. GOS tsis cuam tshuam rau JNK kev qhia hauv cov hlwb hypoxic tab sis suppressed upregulation ntawm phosphorylated JNK. Yog li, oligosaccharides tuaj yeem tiv thaiv hypoxia-induced JNK signaling transduction thiab cytokine qhia hauv lub raum tubular hlwb. Txawm hais tias FC thiab GOS upregulated phos phorylated p38 hauv cov hlwb hypoxic, qhov kev qhia ntawm cov protein no tsis nce hauv cov hlwb hypoxic. Qhov kev tshawb pom no qhia tias p38 tsis ua lub luag haujlwm tseem ceeb hauv cov lus teb hypoxia-induced hauv NRK-52E hlwb.
3.2. CD44 thiab JNK Ua lub luag haujlwm tseem ceeb hauv Cytokine nce hauv Hypoxic NRK-52E Cells
Kev qhia ntawm CD44, phosphorylated JNK, thiab cytokines hauv lub raum tubular hlwb tau cuam tshuam tag nrho los ntawm oligosaccharides, tab sis lawv cov koom haum tseem yuav tau piav qhia. Txhawm rau tshawb xyuas lub luag haujlwm ntawm CD44 hauv JNK signaling transduction thiab cytokine qhia hauv cov hlwb hypoxic, NRK-52E hlwb tau kis nrog ntau koob tshuaj CD44 siRNA. CD44 siRNA ntawm 50 thiab 100 PM ua tau zoo thaiv cov kab mob hypoxia-nce CD44 thiab inhibited hypoxia-induced nce qib ntawm phosphorylated JNK, MCP- 1, IL-1 , thiab TNF- (Daim duab 2A). Kev cuam tshuam ntawm CD44 thaiv ntawm JNK qhia tau cuam tshuam nrog siRNA koob tshuaj. JNK qhia nyob rau hauv cov hlwb hypoxic yog induced los ntawm CD44 siRNA ntawm 50 pM tab sis inhibited ntawm 100 pM. Ob koob tshuaj suppressed upregulation ntawm phosphorylated JNK. Cov xaiv inhibitor rau JNK, SP600125, txo qhov kev qhia ntawm MCP-1, IL-1 , thiab TNF- hauv hypoxic NRK-52E hlwb (Daim duab 2B). Cov txiaj ntsig no qhia tias cov nyhuv inhibitory ntawm CD44 siRNA hloov pauv ntawm hypoxia-induced cytokines yog vim qhov txo qis hauv phosphorylated JNK.
Daim duab 1. Inhibitory effect of oligosaccharides ntawm CD44 signal transduction in hypoxic NRK-52E cells. Cov hlwb raug pretreated nrog FC, FOS, lossis GOS rau 30 min thiab tom qab ntawd raug rau cov kab lis kev cai hypoxic. Protein qhia tau txheeb xyuas los ntawm Western blotting. (A) Dose-dependent teebmeem ntawm oligosaccharides ntawm CD44 qhia. (B) Cov nyhuv ntawm oligosaccharides ntawm CD44-txog cov cim. Oligosaccharide concentration yog teem rau 0.1 mg / mL. Cov txheeb ze nce hauv cov protein ntau kuj tau nthuav tawm hauv daim ntawv qhia bar (C). Cov txiaj ntsig tau qhia tias txhais tau tias ± SD (n=4).
Daim duab 2. Lub luag haujlwm ntawm CD44 thiab JNK nyob rau hauv hypoxia-induced inflammatory teb nyob rau hauv NRK-52E hlwb. Protein qhia tau txheeb xyuas los ntawm Western blot. (A) Inhibitory nyhuv ntawm CD44 siRNA transfection ntawm phosphorylated JNK thiab cytokines. (B) Inhibitory nyhuv ntawm SP600125 ntawm cytokines. Cov hlwb raug pretreated nrog 20 µM SP600125 rau 30 min pbeforehypoxia kho. Cov txheeb ze nce nyob rau hauv protein bands kuj muaj nyob rau hauv daim ntawv qhia bar. Cov txiaj ntsig tau qhia tias txhais tau tias ± SD (n=4).
3.3. Hypoxia-Induced Cytokine Secretion nyob rau hauv lub raum Tubular Cells txhawb Macrophage Proliferation
Inflammation yog tus neeg nruab nrab ntawm lub raum raug mob hauv AKI. Yog li ntawd, kev cuam tshuam ntawm oligosaccha caij ntawm leukocytes tau kuaj xyuas. Daim duab 3A qhia tau hais tias FC, FOS, thiab GOS tsis cuam tshuam rau kev loj hlob ntawm macrophage RAW264.7 hlwb. Kev loj hlob ntawm RAW264.7 hlwb tau txhawb nqa los ntawm 24-teev kab lis kev cai nruab nrab rau hypoxic NRK-52E hlwb (Daim duab 3B) tab sis tsis yog los ntawm qhov nruab nrab rau hypoxic NRK-52E hlwb kho nrog Cov tshuaj oligosaccharides. Tus qub tau siab dua MCP-1 thiab IL{10}} ntau dua li tom kawg (Daim duab 3C, D). Tag nrho peb oligosaccharides txo qis MCP-1 thiab IL-1 hauv kab lis kev cai nruab nrab ntawm hypoxic NRK-52E hlwb. Yog li ntawd, FC, FOS, thiab GOS tuaj yeem txo qhov kev qhia thiab tso tawm ntawm cytokines hauv hypoxic lub raum tubular hlwb thiab, yog li ntawd, txo qis kev mob tshwm sim los ntawm lub raum tubular hlwb raug mob.
