Metformin txhim kho stemness ntawm tib neeg adipose-Drived qia hlwb los ntawm downmodulation
Jul 14, 2022
Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv
Abstract:Cov ntaub so ntswg adipose ua lub luag haujlwm tseem ceeb hauv kev tswj hwm cov metabolism hauv homeostasis los ntawm khaws cov roj ntau dhau thiab tiv thaiv lwm yam kabmob ntawm lipotoxicity. Kev laus yog cuam tshuam nrog kev faib cov roj hauv nruab nrab, ua rau txo qis ntawm cov tshuaj insulin-rhiab subcutaneous thiab nce hauv insulin-tiv taus visceral adipose depots. Adipose-derived qia hlwb (ASCs) ua lub luag haujlwm tseem ceeb hauv kev tsim cov ntaub so ntswg adipose. Cov hnub nyoog ASCs qhia txog kev txo qis thiab cov peev xwm rov tsim dua tshiab vim yog qhov sib txuam ntawm oxidative kev nyuaj siab thiab mitochondrial tsis ua haujlwm cuam tshuam txog kev puas tsuaj ntawm tes. Metformin yog ib qho tshuaj tiv thaiv kab mob ntshav qab zib zoo uas tau pom tias muaj kev tiv thaiv kev laus hauv cov kab mob sib txawv thiab cov qauv tsiaj. Hauv txoj kev tshawb no, peb tau txheeb xyuas cov txiaj ntsig ntawm kev kho metformin ntawm lub stemness ntawm tib neeg ASCs hauv kab lis kev cai ntawm tes thiab tag nrho cov qauv kab lis kev cai adipose. Peb cov txiaj ntsig tau pom tias metformin txhim kho lub stemness ntawm ASCs, txo lawv cov kev loj hlob thiab kev sib txawv ntawm adipocytes. Kev tshawb xyuas cov txheej txheem hauv qab no, peb pom qhov txo qis hauv mTOR thiab ERK kev ua haujlwm hauv metformin-kho ASCs. Tsis tas li ntawd, peb tau pom qhov nce hauv kev ua haujlwm autophagy thaum kho metformin. Peb xaus lus tias kev kho metformin txhim kho ASCs stemness los ntawm kev txo mTOR thiab ERKsignaling thiab txhim kho autophagy. Yav tom ntej nyob rau hauv vivo kev ntsuam xyuas hauv cov qauv tsiaj thiab tib neeg yuav ua rau txoj hauv kev rau kev kho kom haum xeeb ntawm cov tshuaj no tau tsim kom muaj kev txhawb nqa lub cev ntawm cov laus qia hlwb.
Thov nias ntawm no kom paub ntxiv
Ntsiab lus:adipose qia hlwb; cov ntaub so ntswg adipose; stemness; kev sib txawv; kev loj hlob; metformin, mTOR; ERK; autophagy
1. Taw qhia
Cov ntaub so ntswg adipose yog lub cev muaj zog uas ua rau muaj lub luag haujlwm tseem ceeb hauv homeostatic hauv kev tswj cov khoom noj muaj txiaj ntsig, kev nkag siab ntawm insulin, thiab kev tiv thaiv kab mob. Cov ntaub so ntswg adipose ua lub luag haujlwm no los ntawm kev khaws cia thiab oxidizing ntau cov roj fatty acids, yog li tiv thaiv steatosis thiab lipotoxicity hauv lwm yam kabmob [1]. Hnub nyoog cuam tshuam nrog insulin tsis kam yog cov kab mob metabolic tshaj plaws. Kwv yees li ntawm 25 feem pua ntawm cov neeg nyob hauv Asmeskas tshaj li 65 xyoo raug mob ntshav qab zib, ua rau cov neeg tuag ntau dua, txo qis kev ua haujlwm, nce kev pheej hmoo ntawm kev mus pw hauv tsev kho mob, thiab lub nra hnyav rau kev noj qab haus huv [2]. Txawm hais tias kev koom tes ntawm ntshav qab zib thiab rog rog tau lees paub ntev, qhov sib txuas ntawm kev laus thiab ntshav qab zib hom 2 tseem tsis tau paub [3]. Thawj qhov kev soj ntsuam hais txog qhov poob qis hauv cov piam thaj rau kev laus tau ua rau xyoo 1921 [4]. Ntau qhov kev tshawb fawb tau pom txij li thaum txo qis insulin rhiab heev ua thawj qhov ua rau muaj hnub nyoog cuam tshuam ntawm cov piam thaj metabolism [5,6]. Raws li anatomical localization, cov ntaub so ntswg adipose tuaj yeem muab faib ua ob pawg: subcutaneous thiab visceral adipose depots. Qhov chaw thiab kev ua haujlwm ntawm cov ntaub so ntswg adipose yog qhov tseem ceeb ntawm cov tshuaj insulin rhiab heev; Piv txwv li, lub visceral depot yog resistant dua whereas subcutaneous depot yog rhiab heev rau insulin [7]. Cov hnub nyoog cuam tshuam txog kev poob qis ntawm subcutaneous adipose cov ntaub so ntswg ntim ua rau txo cov piam thaj thiab lipid uptake, ua rau lipid dhau thiab ectopic deposition ntawm lipids nyob rau hauv cov nqaij thiab daim siab, pab txhawb rau kev loj hlob ntawm insulin tsis kam [8].
Cov ntaub so ntswg adipose yog tsim los ntawm ntau hom cell. Enzymatic digestion ntawm cov ntaub so ntswg adipose nrog collagenase ua ob feem tseem ceeb: adipocytes feem thiab stromal vascular feem (SVF) [9]. SVF suav nrog adipose-derived qia hlwb (ASCs), lymphocytes, endothelial hlwb, pericytes, thiab fibroblasts [10]. ASCs txhais tau tias kev tiv thaiv kab mob-phenotypically li CD34 ntxiv rau CD90 ntxiv rau CD29 ntxiv rau CD45-CD31 hlwb ntawm mesenchymal keeb kwm, nthuav tawm tri-lineage sib txawv peev xwm rau hauv pob txha, pob txha, lossis rog [11,12]. Hauv cov ntaub so ntswg adipose dawb, cov hlwb no feem ntau nyob hauv vascular stroma nyob ib ncig ntawm cov hlab ntsha me thiab tuaj yeem loj hlob thiab sib txawv rau hauv adipocytes [11]. Adipose-derived stem cells (ASCs) ua lub luag haujlwm tseem ceeb hauv kev tsim cov ntaub so ntswg adipose los ntawm kev loj hlob, rov ua dua tshiab, thiab sib txawv. Kev laus yog txuam nrog qhov poob ntawm cov stemness zog hauv ASCs [13,14]. Kev poob qis ntawm lub hnub nyoog ntawm subcutaneous fat depot loj yog ntseeg tau tias yog vim hloov pauv thiab sib txawv ntawm ASCs[15-17]. Findeisen et al. qhia txog oxidative kev nyuaj siab nyob rau hauv cov ntaub so ntswg adipose thaum laus, uas cuam tshuam tsis zoo rau lub peev xwm sib txawv; [15] txawm li cas los xij, cov txheej txheem tseeb hauv qab qhov poob ntawm qia hauv ASCs tsis to taub. Cov kev tshawb fawb tsis ntev los no tau pom tias txo qis autophagy, siab dua lub hom phiaj ntawm rapamycin (mTOR) txoj hauv kev ua haujlwm thiab sib sau ntawm cov pa oxygen reactive (ROS) txuas rau poob ntawm stemness hauv ASCs thiab ntxov ntxov senescence induction hauv ASCs [18-20].
Cistanche tuaj yeem tiv thaiv kev laus
Metformin yog cov tshuaj tiv thaiv kab mob ntshav qab zib uas nquag siv los txo cov ntshav qab zib thiab txhim kho cov tshuaj insulin. Nws tuaj yeem siv los kho ntau yam kev laus cuam tshuam txog kev mob metabolic, xws li kab mob plawv, mob qog noj ntshav, thiab kev paub tsis meej [21,22]. Kev ua kom lub neej ntev-txhim kho cov teebmeem ntawm metformin tau pom nyob rau hauv cov cab, nas, thiab nas los ntawm kev txwv kev noj haus, uas ua rau cov nyhuv ntawm kev txhawb nqa ntawm adenosine monophosphate-activated protein kinase kev ua haujlwm (AMPK) [23]. Ntau yam kev tshawb fawb tau pom tias metformin inhibits hom tsiaj ntawm rapamycin (mTOR) [24,25]. AMPK-mTOR qhia txoj hauv kev tshwj xeeb tswj cov yam ntxwv ntawm cov cellular homeostases, xws li autophagy, proliferation, zog metabolism, thiab protein synthesis [26]. Metformin tau pom tias inhibit kev loj hlob, sib txawv, thiab mob, txo cov oxidative kev nyuaj siab thiab mitochondrial muaj peev xwm thiab txhim kho tag nrho stemness nyob rau hauv ntau hom neeg laus qia hlwb [27,28]. Cov nyhuv ntawm metformin ntawm cov metabolism thiab stemness ntawm ASCs hauv vitro thiab hauv vivo tsis meej.
Hauv txoj kev tshawb no, peb tau ua haujlwm 2D kab lis kev cai ntawm tib neeg ASCs thiab tag nrho tib neeg cov ntaub so ntswg kab lis kev cai los simulate hauv vivo cellular lub koom haum thiab cov ntaub so ntswg microenvironment. Siv cov qauv kev sim no, peb tau kawm txog cov txiaj ntsig ntawm kev kho metformin ntawm kev sib txawv, kev loj hlob, thiab stemness ntawm ASCs thiab tshawb xyuas cov txheej txheem molecular koom nrog hauv cov txheej txheem ntawm tes.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1.Txoj kev qhia tshwj xeeb
Cov ntaub so ntswg adipose tau sau los ntawm tsib tus poj niam uas tsis yog mob ntshav qab zib hauv cov hnub nyoog 39 ± 13 thiab BMI ntau ntawm 27 ± 4 tab tom ua cov txheej txheem phais yas xaiv ntawm University of Pittsburgh Medical Center (UPMC). Cov txheej txheem ntawm kev sau cov ntaub so ntswg tau pom zoo los ntawm University of Pittsburgh Institutional Review Board (IRB No.0511186).
2.2. Kev rho tawm thiab cog qoob loo ntawm tib neeg ASCs
Lub hlwb adipose precursor raug cais raws li hauv qab no[19,29]. Adipose cov ntaub so ntswg biopsies tom qab cov txheej txheem phais raug xa mus rau lub chaw kuaj mob hauv lub thawv ntim tsis muaj menyuam. Cov ntaub so ntswg tau muab yaug nrog Dulbecco's phosphate-buffered saline (PBS; Sigma-Aldrich, St.Louis, MO, USA) ua raws li kev tshem tawm cov khoom siv fiber ntau thiab cov hlab ntsha los ntawm kev txiav tawm. Cov ntaub so ntswg raug txiav rau hauv daim (1-2 mg) thiab zom hauv plab zom mov tsis zoo (HBSS muaj 200 U/mL collagenases (CLS Type II, Worthington Biochemical Corp., Lakewood, NI , USA) thiab 2 feem pua w/o BSA(Sigma-Aldrich, St. Louis, MO, USA)) nyob rau hauv nplawm rau 60 min ntawm 37 degree ; 1 mg adipose ntaub so ntswg / 3 mL digestion tsis. Cov ntaub so ntswg dispersed tau centrifuged rau 10 min ntawm 200 × g ntawm chav tsev kub. Cov floating adipocytes tau aspirated, thiab cov pelleted hlwb ntawm stromal vascular feem (SVF) raug tshem tawm nyob rau hauv erythrocyte lysis tsis (0.155 M NH4CI, 5.7 mM K2HPO4, 0.1 mM EDTA, pH 7.3) thiab incubated ntawm chav tsev kub 1. Txhawm rau tshem tawm cov ntaub so ntswg, qhov kev ncua ntawm tes tau lim los ntawm cov nylon mesh (pore loj 100 um; Thermo Fisher Scientific, Waltham, MA, USA). Tom qab lwm kauj ruam centrifugation (10 min ntawm 200 × g) cov pelleted SVF raug tshem tawm hauv ASC nruab nrab (Dulbecco's modified Eagle's nruab nrab (DMEM) / F-12 nruab nrab (1: 1) nrog HEPES thiab L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA), ntxiv nrog 33 μM biotin (Sigma-Aldrich, St. Louis, MO, USA), 17 μM pantothenate (Sigma-Aldrich, St.Louis, MO, USA), 10ng / mL EGF, 1 ng/mL bFGF, 500 ng/mL insulin, 2.5 feem pua fetal bovine serum, thiab 12.5 μM/mL gentamicin (Sigma-Aldrich, St. Louis, MO, USA) thiab noob ntawm qhov ntom ntawm 50, 000 hlwb /cm2 hauv T175 flasks.
Tom qab ncav cuag 70 feem pua ntawm qhov sib xyaw, cov hlwb raug ntxuav nrog PBS thiab trypsinized siv 0.05 feem pua trypsin-EDTA 1x tov (Sigma-Aldrich, St.Louis, MO, USA). Trypsin yog inactivated los ntawm qhov sib ntxiv ntawm ASC nruab nrab ntxiv rau 10 feem pua FBS thiab tshem tawm los ntawm centrifugation ntawm 300 × g rau 5 min ua ntej lub hlwb tau noob ntawm ib tug ceev ntawm 5000 hlwb / cm². ASCs tau nce ntxiv mus rau 70 feem pua ntawm qhov sib xyaw ua ntej sib cais.ASCs hauv nqe lus 3-4 tau siv hauv txoj kev tshawb no. Cov hlwb raug kho nrog ntau qhov sib txawv ntawm metformin (Sigma-Aldrich, St.Louis, MO, USA), rapamycin (Sigma-Aldrich, St. Louis, MO, USA), lossis U0126 inhibitor raws li kev sim.
2.3. Adipogenic sib txawv
Rau qhov induction ntawm adipogenesis, ASCs tau noob ntawm qhov ntom ntawm 50, 000 hlwb / cm² hauv 6-cov kab lis kev cai zoo ntawm tes. Adipogenesis raug ntxias siv qhov sib txawv nruab nrab, muaj li ntawm 0.2 uM insulin (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM 1-methyl{{9 }}isobutylxanthine (IBMX) (Sigma-Aldrich, St. Louis, MO, USA), 0.25 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), thiab 10 ug/mL transferrin (Sigma-Aldrich, St. Louis, MO, USA) in ASCmedium. Tom qab 3 hnub ntawm kev sib txawv, qhov nruab nrab tau hloov pauv, thiab cov hlwb tau cog rau hauv qhov sib txawv nruab nrab yam tsis muaj IBMX rau 14 hnub.
2.4.Adipose ntaub so ntswg kab lis kev cai
Adipose nqaij kab lis kev cai tau ua nyob rau hauv 6-zoo cell kab lis kev cai daim hlau, nrog ib co kev hloov kho, raws li luam tawm los ntawm Harms et al. [30]. Tag nrho cov ntaub so ntswg adipose raug txiav rau hauv cov me me 3-5 hli qhov loj thiab coj mus rau hauv 5 mL kab lis kev cai xov xwm ntawm tes. Cell strainers nrog lub pore loj ntawm 70 μM tau khaws cia hla cov ntaub so ntswg kom cov ntaub so ntswg submerged hauv xov xwm. Cov ntaub so ntswg tawg tau zom nrog collagenase, raws li tau piav qhia saum toj no rau ASCs kev cais tawm, thiab SVF tau txheeb xyuas rau kev qhia ntawm ASC-txuas nrog cov noob caj noob ces.
2.5. Roj Liab O Staining
Rau kev pom cov lipid droplets, cov hlwb raug kho nrog 4 feem pua ntawm paraformaldehyde hauv PBS rau 1 h thiab stained nrog 0.3 feem pua Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) hauv isopropanol / dej ( 60:40) rau 1h. Kev ntxuav zaum kawg yog ua ob zaug nrog dej distilled. Rau kom muaj nuj nqis ntawm absorbed Roj Liab O, cov stain yog eluted nrog isopropanol (Sigma-Aldrich, St.Louis, MO, USA), thiab optical ntom yog ntsuas ntawm 518 nm.
2.6. Lub cev Staining
Hnub 14 post-induction ntawm adipogenesis, hlwb tau stained nrog Bodipy (Invitrogen, Waltham, MA, USA) thiab Hoechst (Sigma-Aldrich, St. Louis, MO, USA) rau 30 min ntawm 37 degree . Cov duab tau txais los ntawm kev siv lub fluorescent microscope thiab tshuaj xyuas nrog ImageJ software.
2.7. FlouCytometry
ASCs hauv nqe lus peb tau siv rau kev ntsuas cytometric rau qhov kev qhia saum npoo ntawm CD29 (Biolegend, San Diego, CA, USA), CD90 (Biolegend, San Diego, CA, USA), CD45 (BD, Franklin Lakes, NJ, USA) , CD24(Biolegend, San Diego, CA, USA), and CD31(BD, NJ, USA). Cells tau trypsinized, ntxuav nrog PBS, thiab stained nrog cov tshuaj tiv thaiv hauv FACS staining buffer (1 feem pua BSA hauv PBS). Unstained ASCs tau ua haujlwm ua tus tswj. Fluorescent teeb liab raug ntsuas los ntawm kev siv lub cytometer ntws (Fortessa, BD, Franklin Lakes, NJ, USA), thiab cov ntaub ntawv raug tshuaj xyuas siv FlowJo software (BD, Franklin Lakes, NJ, USA).
2.8. Osteogenic sib txawv
ASCs tau loj hlob mus rau qhov sib xyaw ua ke hauv {{0}} cov phiaj zoo, thiab kev sib txawv ntawm osteogenic raug ntxias siv qhov nruab nrab osteogenic (DMEM 10 feem pua FBS, 0.1uM dexamethasone, 10 mM -glycerol phosphate, thiab 50 μM Ascorbic Acid). Hnub 21 tom qab kev sib txawv, cov hlwb raug kho hauv 4 feem pua ntawm paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) thiab stained nrog Alizarin Liab Stain (Sigma-Aldrich, St. Louis, MO, USA). Quantification ntawm staining. tau ua tiav siv Image J software.
2.9.Apoptosis Detection los ntawm Annexin V-APC/PI Staining
Apoptotic cell tuag tau ntsuas siv cov khoom siv Annexin V-APC/PI (Biolegend, San Diego, CA, USA). Tag nrho ntawm 10,000(cells/well) ASCs tau cog rau hauv 6- cov phiaj xwm zoo thiab incubated ntawm 37 degree nrog 5 feem pua CO2. Cov hlwb raug kho nrog metformin (0.25 mM, 0.5 mM, 1 mM, 2.5 mM, thiab 5 mM) (Sigma-Aldrich, St.Louis, MO, USA). Tom qab 72 teev ntawm kev kho mob, cov hlwb tau raug kho. sau los ntawm trypsinization thiab stained nrog 2.5μL AnnexinV-APC thiab 2.5 μL PIin binding buffer, incubated nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub rau 15 mus rau 20 min, thiab soj ntsuam los ntawm flow cytometry (Fortessa, BD, NJ, USA).
2.10.Reactioe Oxygen Species Detection
ROS qib tau txiav txim siab siv fluorescent dye 2',7'-dichlorofluorescein diacetate (DCFH2-DA) sojntsuam (Invitrogen, Waltham, MA, USA). ASCs(10,000 hlwb/zoo) tau coj mus rau hauv ib lub cev 6-zoo phaj thiab incubated nrog metformin (5 mM) ntawm 37 degree , 5 feem pua CO, rau 72h. Tom qab incubation, ASCs tau trypsinized thiab stained nrog 50μM DCFH2-DA dye rau 30 min ntawm 37 degree thiab soj ntsuam siv lub fluorescence microscope.
2.11. TMRM Staining
ASCs(10,{1}} hlwb/zoo) tau coj mus rau hauv ib lub cev 6-zoo phaj, kho nrog ntau koob tshuaj metformin (5 mM) rau 72 h, thiab incubated ntawm 37 degree nrog 100 nM TMRM (Sigma-Aldrich , St. Louis, MO, USA) rau 30 feeb ntawm 37C. Tom qab incubation, hlwb raug soj ntsuam nyob rau hauv ib tug fluorescence microscope. Duab] tau siv los ntsuas qhov fluorescence ntawm cov duab microscopic.
2.12. Quantitative RT-PCR Gene Expression
Tag nrho RNA raug cais nrog RNeasy Micro Kit (Qiagen, Hilden, Lub teb chaws Yelemees), thiab cDNA synthesis tau ua nrog RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene-specific primers tau muas los ntawm Sigma-Aldrich, St. Louis, MO, USA. Quantitative expression analysis tau ua los ntawm Quantstudio 3 (Thermo Fisher Scientific, Waltham, MA, USA). Kev ntsuas mRNA tau ua tiav siv -actin rau normalization. Cov ntaub ntawv rau txhua qhov kev sau cov noob tau raug ua kom zoo los ntawm kev suav qhov sib txawv (ACT) ntawm Ct-tsev tu thiab Ct-Target genes. Cov txheeb ze nce lossis txo qis hauv kev qhia tau suav los ntawm kev sib piv cov qauv siv nrog lub hom phiaj gene (AACT) siv cov qauv rau kev qhia txheeb ze (=24ACt).
2.13. Western Blot
Western blot tsom xam tau ua qhov tseem ceeb raws li tau piav qhia [19]. Txoj kev siv los normalize cov protein ntau yog "Pierce BCA Protein Assay Kit" (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (15 ugs tag nrho cov protein ib txoj kab) tau npaj nyob rau hauv sodium dodecyl sulfate (SDS) qauv tsis, sib cais los ntawm SDS-PAGE, thiab blotted ntawm polyvinylidene difluoride daim nyias nyias. Cov tshuaj tiv thaiv hauv qab no tau siv: nas los tiv thaiv tib neeg -actin (Proteintech, IL, USA), perilipin (Cell Signaling, MA, USA), phosphor-P70S6K (Cell Signaling, MA, USA), tag nrho P70S6K (Cell Signaling, MA, USA), pERK1/2 (Cell Signaling, MA, USA), tag nrho ERK1/2 (Cell Signaling, MA, USA), anti-nas IgG HRP conjugate (Proteintech, IL, USA), thiab luav anti-rat IgG HRP( Proteintech, IL, USA). Duab J software tau siv rau kev ntsuas densitometric. 2.14.Txoj Kev Tshawb Fawb
Kev txheeb xyuas txheeb cais tau ua nyob rau hauv GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA).FlowJo tau siv rau kev tshuaj xyuas FACS. Qhov tseem ceeb ntawm qhov sib txawv ntawm kev txhais tau tias raug soj ntsuam los ntawm Tus Tub Kawm Ntawv t-test los yog kev txheeb xyuas qhov sib txawv. Qhov yuam kev liab qab yog sawv cev raws li qhov nruab nrab ± SEM. Cov nqi tseem ceeb ntawm p tus nqi ntawm<><0.01 and=""><>
3. Cov txiaj ntsig
Metformin yog cov tshuaj tiv thaiv kab mob ntshav qab zib tau tsim tau zoo uas tau pom tias ua lub luag haujlwm tseem ceeb hauv kev ua kom lub neej ntev [31]. Txhawm rau kawm txog cov txiaj ntsig ntawm kev kho metformin ntawm lub stems ntawm adipose-derived qia hlwb, peb cais ASCs los ntawm cov ntaub so ntswg adipose ntawm tib neeg pub dawb los ntawm kev zom cov collagenase. Cov ASCs cais tau cov yas adherent thiab nthuav tawm ib qho fibroblastic lossis spindle-shaped morphology. Flow cytometry tsom xam pom tias ASCs tau zoo rau mesenchymal qia cell markers, xws li CD29, CD90, thiab CD24, tab sis tsis zoo rau hematopoietic lineage marker CD45 thiab endothelial kab cim CD31 (Daim duab 1A). cov ASCs tau pom muaj zog adipogenic thiab osteogenic sib txawv muaj peev xwm, txheeb xyuas los ntawm Bodipy thiab Alizarin Liab S staining, ntsig txog (Daim duab 1B).cistanche แอมเวCov yam ntxwv ASCs tau siv rau kev sim ntxiv hauv peb txoj kev tshawb fawb.
Nws tau raug tshaj tawm nyob rau hauv cov ntaub ntawv hais tias metformin txo cov adipogenic sib txawv ntawm cov qia hlwb. Peb tsom mus tshawb nrhiav cov kev cai ntawm adipocytes sib txawv ntawm ASCs raws li kev kho mob metformin. ASCs tau kho nrog 1,2.5, thiab 5 metformin pib 2 hnub ua ntej adipogenic induction thiab thoob plaws hauv cov txheej txheem sib txawv. Bodipy thiab Roj Liab Ostains tau siv los pom cov roj tee nyob rau hnub 14 tom qab kev sib txawv ntawm qhov sib txawv. Cov txiaj ntsig ntawm Roj Liab O (Daim duab 1C) thiab Bodipy staining (Daim duab 1D) qhia tias kev kho metformin cuam tshuam qhov sib txawv ntawm adipogenic nyob rau hauv koob tshuaj raws li piv rau kev tswj cov hlwb. Qhov inhibition ntawm adipogenic sib txawv tau lees paub ntxiv siv Western blot tsom ntawm adipocyte sib txawv marker protein perilipin. Rau qhov no, cell lysates tau sau los ntawm cov hlwb sib txawv nyob rau hnub 14 ntawm kev sib txawv ntawm adipogenic, thiab perilipin qhia tau txheeb xyuas. Peb cov txiaj ntsig ntawm Western blot qhia pom qhov txo qis ntawm perilipin qhia txog kev kho metformin thaum lub sij hawm sib txawv ntawm adipogenic (Daim duab 1E), uas cuam tshuam nrog kev txo qis hauv Bodipy thiab Roj Liab O stains. Peb txiav txim siab tias kev kho metformin inhibits qhov sib txawv ntawm qhov sib txawv ntawm tib neeg ASCs, nrog rau qhov loj ntawm inhibition zoo sib xws nrog cov tshuaj metformin.
Ntau qhov sib txawv thiab qhov loj hlob tuaj ua rau poob ntawm stemness. Txij li thaum peb tau pom tias metformin txo qis qhov sib txawv ntawm adipogenic, peb xav tshawb xyuas cov txiaj ntsig ntawm kev kho metformin ntawm kev loj hlob ntawm ASCs hauv vitro. ASCs tau kho nrog metformin, thiab cov hlwb raug suav rau hnub 4 tom qab kev coj noj coj ua hauv lub xub ntiag lossis tsis muaj metformin. Los ntawm peb cov txiaj ntsig, peb pom tias metformin txo qis ntawm cov cell proliferation piv rau cov tswj ASCs (Daim duab 2A). Stem cells ua lub luag haujlwm tseem ceeb hauv kev tsim cov ntaub so ntswg thiab kev saib xyuas.npaum li cas cistanche cojYuav kom ua tau zoo ua lawv lub luag hauj lwm regenerative, qia hlwb yuav tsum tswj lawv cov yam ntxwv stemness. Peb tau txheeb xyuas cov txiaj ntsig ntawm kev kho metformin ntawm kev qhia ntawm cov noob caj noob ces hauv ASCs. Txog qhov kawg no, peb tau siv PCR ntau npaum li lub sijhawm los tshuaj xyuas cov noob muaj feem xyuam nrog kev saib xyuas lub cev hauv ASCs. Peb tau pom tias metformin tau tswj hwm qhov kev qhia ntawm adipose qia cell cim, xws li BMP7, DPP4, Sox2, Oct3/4, Wnt2, ICAM1, CD90, thiab DLK1, piv nrog cov tswj ASCs (Daim duab 2B). Peb cov txiaj ntsig tau pom tias kev kho metformin ua tau zoo ua rau txo qis ASCs kev loj hlob, yog li tiv thaiv cov hlwb los ntawm kev ua kom qaug zog los ntawm kev txhim kho qhov kev qhia ntawm stemness kos npe noob.
Daim duab 1. Kev kho Metformin inhibits adipogenic sib txawv ntawm ASCs raws li koob tshuaj. (A) ASCs tau raug sim rau kev qhia ntawm CD29, CD90, CD24, CD31, thiab CD45 los ntawm kev ntws cytometry. Ob feem pua zoo pom nyob rau hauv histograms thiab txhais tau tias fluorescence siv (MFI) qhia nyob rau hauv lub rooj ntawm kev tswj (tsis muaj xim ASCs) thiab stained (ASCs kho nrog cov tshuaj tiv thaiv kab mob) tau nthuav tawm. (B) ASCs tau raug rau adipogenic lossis osteogenic sib txawv siv cov kab mob tshwj xeeb sib txawv cocktails. Kev sib txawv rau adipocyte kab mob tau lees paub los ntawm Bodipy staining, thiab osteogenesis tau lees paub los ntawm Alizarin Liab staining. Qhov feem pua ntawm Alizarin Red stained cheeb tsam tau suav nrog siv Image J. (C) ASCs tau kho nrog adipocytes sib txawv cocktail nyob rau hauv lub xub ntiag los yog tsis muaj ntau koob tshuaj metformin rau 14 hnub. Kev loj hlob ntawm adipocytes nyob rau hnub 14 tom qab induction tau soj ntsuam los ntawm staining lipid droplets siv roj O stain. Absorbed Roj Liab O stain tau muab rho tawm, thiab ntsuas qhov muag pom. Optical density los ntawm kev tswj ASCs raug coj los ua 100 feem pua, thiab cov txheeb ze hloov pauv raws li kev kho mob metformin tau npaj tseg (n =3). (D) Lipid cov ntsiab lus ntawm hnub 14 tau txiav txim siab siv Bodipy stain (Green). Hoechst (Blue) tau siv los stain nuclei.dab tsi yog cistanche(E) Western blot tsom xam ntawm perilipin protein qhia. -Actin tau ua haujlwm ua tus tswj kev thauj khoom. Quantification ntawm perilipin protein band densities normalized rau -Actin siv Image J.Images thiab graphs yog cov neeg sawv cev ntawm peb ywj siab replicates (n=3). *p<0.05,**><0.01,***p><0.001 and="" ***=""><0.0001 as="" compared="" with="">
Txhawm rau kom tsis suav nrog qhov txo qis ntawm kev sib txawv thiab kev loj hlob tsis yog vim muaj kev cuam tshuam cytotoxic ntawm metformin ntawm ASCs, peb tau txheeb xyuas qhov muaj peev xwm ntawm cov cell kho cov tshuaj metformin. Kev soj ntsuam ntawm AnnexinV / PI staining los ntawm kev ntws cytometry pom tau tias tsis muaj kev nce ntxiv hauv cov hlwb apoptotic lossis necrotic thaum kho nrog kev nce siab ntawm metformin mus txog 5 piv rau cov kab lis kev cai nruab nrab ntawm tes (Daim duab 3A). Mitochondrial kev ua ub no thiab tiam ntawm reactive oxygen hom (ROS) yog ib qho tseem ceeb rau kev sib txawv ntawm tes, thiab nce qib ntawm mitochondrial metabolism thiab ROS tiam tau pom thaum lub sij hawm adipogenic sib txawv [32]. Los ntawm peb cov txiaj ntsig, peb tau pom tias kev kho mob metformin txo qis qhov sib txawv ntawm adipogenic ntawm ASCs. Peb txuas ntxiv mus tshawb xyuas cov teebmeem ntawm kev ua haujlwm mitochondrial thiab ROS ntau lawm. ASCs tau kho nrog metformin thiab stained nrog TMRM los ntsuas mitochondrial membrane kev ua thiab nrog DCF2DA rau kwv yees ROS concentration. Fluorescent microscope cov duab thiab qhov ntau ntawm cov teeb liab fluorescent qhia tias kev kho metformin txo qis kev ua haujlwm ntawm mitochondrial (Daim duab 3B, C) thiab ROS ntau lawm (Daim duab 3D, E). Peb txiav txim siab tias metformin-mediated txo qis hauv kev ua haujlwm mitochondrial thiab ROS ntau lawm ua rau muaj kev nce hauv stemness ntawm ASCs.
Txhawm rau nkag siab txog qhov ua tau teeb liab cascade lub luag haujlwm rau kev txo qis adipocytes sib txawv, kev loj hlob, thiab kev txhim kho ntawm cov noob caj noob ces hauv ASCs thaum kho metformin, peb tau tsom mus rau mTOR, autophagy, thiab extracellular signal-regulated kinase (ERK) signaling cascades. Cov kev tshawb fawb yav dhau los tau qhia txog lub luag haujlwm tseem ceeb ntawm txoj hauv kev no hauv kev tswj hwm lub stemness hauv ASCs [19,33]. Ob qho ERKand mTOR signaling plays lub luag haujlwm tseem ceeb hauv kev txhawb nqa cell proliferation thiab sib txawv. Tom qab ASCs tau kho nrog ntau koob tshuaj metformin, cell lysates tau sau thiab Western blotted rau LC3, phosphorylated-p70S6 kinase, tag nrho p70S6 kinase, phosphorylated-ERK42/44, thiab tag nrho ERKp42/44 antibodies. Beta-actin tau ua haujlwm raws li kev tu vaj tse. Western blot tsom xam pom tias piv nrog kev tswj hwm ASCs, kev kho metformin txo qis-tswj cov lus qhia ntawm phosphorylated p70S6K thiab phosphorylated-ERK42/44 kinase (Daim duab 4A, B, D). Ob qho ERK thiab mTOR signaling ua lub luag haujlwm tseem ceeb hauv kev tswj hwm txoj kev autophagy.bioflavonoidsWestern blot cov txiaj ntsig tau qhia tias kev kho mob metformin txhim kho kev ua haujlwm autophagy hauv ASCs (Daim duab 4A, C). Peb tau soj ntsuam ntxiv txog qhov qhia txog cov kab mob autophagy kab mob raws li kev kho mob metformin ntawm qib mRNA los ntawm kev siv PCR ntau npaum li lub sijhawm. Nrog qPCR tsom xam, peb tau pom qhov nce ntxiv hauv cov noob caj noob ces autophagy VMP1, P62, ATG5, thiab ATG7 raws li kev kho mob metformin (Daim duab 4E), uas zoo ib yam nrog kev nce hauv autophagy kev ua hauv peb cov txiaj ntsig Western blot. Ib qho kev nce tsis tseem ceeb hauv kev qhia ntawm ATG 9 kuj tau pom hauv peb qhov kev tshuaj ntsuam. Peb xaus lus tias kev kho mob metformin txo ERK thiab mTOR kev ua haujlwm thiab txhim kho autophagy hauv ASCs raws li lub hauv paus txheej txheem rau txo kev loj hlob thiab sib txawv thiab nce stemness.
Txhawm rau qhia txog lub luag haujlwm ncaj qha ntawm ERK thiab mTOR teeb liab hauv metformin-kev kho mob-kev kho mob inhibition ntawm kev sib txawv thiab nce hauv stemness hauv ASCs, peb cais inhibited ERK signaling thiab mTOR txoj kev siv ERK inhibitor U0126 thiab rapamycin, raws li, thiab soj ntsuam cov teebmeem ntawm ASC. sib txawv thiab stemness [19,33]. Qhov sib ntxiv ntawm ERK inhibitor U0126 thaum lub sij hawm adipocytes sib txawv ntawm ASCs ua rau txo qis qhov sib txawv, raws li qhia los ntawm Bodipy stained dluab thiab fluorescence kom muaj nuj nqis (Daim duab 5A, B).Quantitative real-time PCR cov txiaj ntsig uas qhia txog kev txhim kho ntawm stemness noob raws li inhibitor-mediated tshwj xeeb inhibition. ntawm ERK signaling (Daim duab 5C) ntxiv txhawb nqa peb qhov kev tshawb pom tias ERK txoj kev txo qis los ntawm kev kho mob metformin pab txhawb kom nce stemness.
Tom qab ntawd peb siv rapamycin, tus paub zoo mTOR txoj hauv kev inhibitor uas tuaj yeem ua rau autophagy, thiab tshuaj xyuas cov teebmeem ntawm adipogenesis thiab stemness noob qhia [19]. Cell lysates ntawm ASCs kho nrog rapamycin tau sau thiab soj ntsuam nrog phosphorylated-p70S6 kinase, tag nrho p70S6 kinase, thiab -actin antibodies. Peb pom tias rapamycin txo qis kev qhia ntawm phosphorylated-p70S6 kinase (Daim duab 6A). Txhawm rau txiav txim siab lub luag haujlwm ua haujlwm hauv kev sib txawv ntawm adipogenic, ASCs tau kho nrog rapamycin thiab induced nrog cov nruab nrab adipogenic. Tom qab 14 hnub, cov hlwb tau stained nrog Bodipy stain los soj ntsuam adipogenesis. Peb tau pom tias kev kho mob rapamycin txo qis qhov sib txawv ntawm adipogenic (Daim duab 6B, C). Tom ntej no, peb txheeb xyuas qhov cuam tshuam ntawm kev thaiv mTOR teeb liab hloov ntawm lub stemness ntawm ASCs. Txog qhov kawg no, ASCs tau kho nrog rapamycin, thiab PCR lub sijhawm tiag tiag tau siv los ntsuas qhov kev qhia ntawm cov ntaub ntawv sau tseg ntsig txog stemness. Peb tau pom tias rapamycin ua rau muaj txiaj ntsig zoo rau kev nthuav qhia ntawm cov kab ntsig txog cov cim (Daim duab 6D). Peb xaus lus tias inhibition ntawm ob qho tib si ERK thiab mTOR txoj hauv kev ua rau muaj kev txo qis hauv adipocytes sib txawv thiab nce hauv stemness hauv tib neeg ASCs.
Tom ntej no, peb tau sim seb peb puas tuaj yeem tsim cov stemness-txhim kho cov teebmeem ntawm kev kho metformin ntawm 2Dcultured ASCs hauv qhov nyuaj dua thiab hauv vivo txhais tau tag nrho cov qauv kab lis kev cai adipose. Rau qhov no, peb tau hloov kho cov txheej txheem kab lis kev cai adipose uas luam tawm los ntawm Harms et al.30] siv cell strainers es tsis txhob transwell inserts kom cov ntaub so ntswg adipose submerged hauv xov xwm (Daim duab 7A). Tom qab culturing 3-5 hli fragments ntawm tib neeg adipose cov ntaub so ntswg nrog los yog tsis muaj metformin rau 5 hnub, peb cais cov stromal vascular feem (SVF) los ntawm collagenase zom ntawm adipose cov ntaub so ntswg thiab soj ntsuam cov kev qhia ntawm stemness txog cov noob caj noob ces. Cov ntaub ntawv PCR ntawm lub sijhawm tiag tiag tau pom tias SVF los ntawm cov ntaub so ntswg adipose kho nrog metformin tau pom muaj kev txhim kho tseem ceeb hauv kev qhia cov cim stemness xws li BMP7, DLK1, MYC1, DPP4, OCT3/4, Sox2, Nanog, KLF4, PDGFR, thiab Wnt2 (Daim duab 7B. ).
4. Kev sib tham
Adipose qia hlwb (ASCs) rov tsim cov ntaub so ntswg adipose los ntawm kev rov ua dua nws tus kheej thiab kev sib txawv, yog li tswj cov ntaub so ntswg lub qia cell. Txawm li cas los xij, kev laus thiab kev rog rog yog cuam tshuam nrog kev poob ntawm stemness zog hauv ASCs. Metformin yog ib qho tshuaj tiv thaiv kab mob ntshav qab zib thiab tau pom cov txiaj ntsig tau zoo raws li kev tiv thaiv kev laus. Peb tau tsom mus rau kev tshuaj xyuas cov txiaj ntsig ntawm metformin ntawm lub stemness ntawm ASCs thiab nkag siab txog lub hauv paus txheej txheem, nrog rau lub hom phiaj los tsim cov metformin ua tus neeg sawv cev muaj peev xwm los tswj cov qia hauv ASCs nrog hnub nyoog. Rau kev txhais lus yooj yim ntawm peb cov txiaj ntsig rau tib neeg cov ntsiab lus, peb ua haujlwm 2D cell kab lis kev cai qauv ntawm tib neeg ASCs thiab siv tib neeg cov ntaub so ntswg kab lis kev cai los simulate ntau cov ntaub so ntswg ib puag ncig. Peb siv cov ntaub so ntswg subcutaneous adipose ua cov ntaub so ntswg rau peb cov kev tshawb fawb. Siv cov kev sib txawv ntawm cov hlwb thiab cov ntaub so ntswg kab lis kev cai, peb tau kawm txog cov txiaj ntsig ntawm kev kho metformin ntawm lub stemness ntawm ASCs.
Kev rog dhau ua rau muaj kev hloov pauv hauv ASC sib txawv kinetics, uas txo qis cov qia cell. Peb cov txiaj ntsig tau pom tias, piv nrog ASCs tsis tau kho, kev kho metformin txo qis adipogenesis tus nqi raws li koob tshuaj. Qhov kev soj ntsuam no yog ua raws li cov ntaub ntawv dhau los uas tau pom tias metformin inhibits adipogenesis hauv adipose qia hlwb, pob txha pob txha stromal hlwb, cov kab mob ligament qia hlwb, thiab 3T3 cell kab [34-37]. Kev loj hlob ntawm cov qia hlwb ua lub luag haujlwm tseem ceeb hauv kev tswj cov qia cell pas dej ua ke, stemness, thiab muaj peev xwm rov ua dua nws tus kheej. Peb pom tias metformin inhibited qhov kev loj hlob ntawm ASCs yam tsis muaj kev cuam tshuam cytotoxic ntawm ASCs, uas pom zoo nrog lwm cov kev tshawb fawb luam tawm ntawm nas adipose-derived qia hlwb [28], stromal hlwb, thiab fibroblasts [38,39].muab cistancheIb txoj kev tshawb fawb tsis ntev los no siv cov qia hlwb los ntawm cov nees tau pom tias muaj txiaj ntsig zoo ntawm metformin [27]. Qhov feem ntau yuav piav qhia rau cov kev soj ntsuam tsis sib xws yog qhov sib txawv ntawm cov hom pub dawb thiab qhov chaw anatomical ntawm cell sau, raws li nees ASCs tau sau los ntawm tus Tsov tus tw. Agnieszka Smieszek et al. pom tias metformin inhibits cell proliferation thiab txhawb kev tuag ntawm tes nrog kev nce koob tshuaj (5 thiab 10 mM) hauv nas pob txha hlwb-derived stromal hlwb thiab Balb / 3T3 embryonic fibroblast cell kab [40]. Tsis tas li ntawd, metformin inhibits kev loj hlob ntawm satellite hlwb cais los ntawm cov leeg nas |41. Txhawb nqa peb qhov kev soj ntsuam, Min-lung Park li al. tau tshaj tawm tias metformin txhim kho cov cuab yeej tiv thaiv kab mob ntawm tib neeg ASCs los ntawm kev txo qis kev qhia qib ntawm IL-1, IL-6, thiab TGF- ; Metformin kuj ua rau kom muaj sia nyob ntawm kev hloov pauv ASCs thiab tiv thaiv apoptotic cell tuag [42]. Cov txiaj ntsig no qhia txog qhov sib txawv ntawm kev kho mob metformin ntawm kev muaj peev xwm ntawm cov hlwb tau los ntawm ntau hom thiab tias tib neeg ASCs tiv taus cov teebmeem cytotoxic ntawm kev kho metformin.
Kev sib txawv ntawm ASCs rau hauv adipocytes yog txuam nrog kev xav tau ntawm lub zog ntau dua thiab cov nqi metabolic, ua rau muaj kev ua haujlwm siab dua mitochondrial thiab ROS ntau lawm. ROS ua lub luag haujlwm tseem ceeb hauv kev tswj hwm qhov tsis txaus ntseeg thiab stemness hauv cov neeg laus qia hlwb, raws li qib siab ntawm ROS thawb cov hlwb mus rau qhov sib txawv thiab thaum kawg qia cell senescence thiab cell tuag [43]. Peb tau pom tias metformin ua rau txo qis ntawm mitochondrial membrane muaj peev xwm thiab muaj cov pa oxygen reactive. Raws li peb cov ntaub ntawv, cov kev tshawb fawb dhau los kuj tau tshaj tawm tias metformin txo qis oxidative kev nyuaj siab thiab txhim kho cov tshuaj tiv thaiv antioxidant hauv nas embryonic fibroblasts [44], nas ASCs [38], thiab tib neeg aortic endothelial hlwb [45]. Ntxiv mus, hauv kev pom zoo nrog peb txoj kev tshawb fawb, Chien-Hung Lin li al, tau tshaj tawm tias metformin ua raws li cov tshuaj tiv thaiv neuroprotective los ntawm kev txo qis oxidative kev nyuaj siab hauv tib neeg neural qia hlwb [46]. Ntau qhov kev tshawb fawb pom tseeb tias metformin ua raws li cov tshuaj tiv thaiv antioxidant los ntawm kev txo qis oxidative kev nyuaj siab thiab ua rau cov tshuaj tiv thaiv kab mob antioxidant, xws li superoxide dismutase hauv nas ASCs, murine C2C12 myoblasts, thiab circulating human endothelial progenitor cells [38,47,48]. Ntxiv mus, txoj kev kawm ntawm Alejandro Martin-Montalvo li al. txhawb peb txoj kev soj ntsuam, ua kom pom tias metformin cuam tshuam rau mitochondrial complex, inhibiting mitochondrial respiration nyob rau hauv nas [49] Cov teebmeem ntawm metformin rau txo qis reactive oxygen hom muaj peev xwm kho tau los ntawm kev nce hauv txo glutathione [50] hauv ib lub tswv yim nrog rau redox- rhiab heev transcription factor Nrf2 [51].
Metformin tau pom los txhawb lub xeev quiescence ntawm satellite cell los ntawm kev txo qis cov metabolism uas xav tau rau kev loj hlob thiab kev sib txawv. Ntau cov kev tshawb fawb kuj tau lees paub tias metformin txhawb nqa lub stemness thiab rejuvenation ntawm qia hlwb [23,39,41,49,52,53]. Nws kuj tseem paub tias txhim kho qhov tseem ceeb thiab txhim kho cov theem qhia ntawm cov kab mob qia cell. Txij li thaum peb pom tias metformin inhibits proliferation thiab adipogenesis los ntawm inhibiting oxidative kev nyuaj siab thiab mitochondrial kev ua, peb nrhiav kom paub tseeb tias seb metformin txhim kho lub qia hauv ASCs los yog hauv vitro adipose cov ntaub so ntswg kab lis kev cai qauv. Raws li, peb tau pom tias metformin tau txhim kho qhov cim qhia txog kev cuam tshuam ntawm stemness ntawm DPP4, Wnt2, THP1, DLK1, PDGFR, ICAM1, thiab Annexin3, Oct3/4, Nanog, BMP4, SOX2, thiab Mycl hauv 2D ASCculture thiab adipose ntaub so ntswg kab lis kev cai qauv. . Kev siv tag nrho cov ntaub so ntswg adipose muaj lub sijhawm tshwj xeeb los simulate hauv vivo tib neeg cov ntaub so ntswg microenvironment, qhov twg cov hlwb tau taw qhia raws li hauv lawv cov qauv tsim thiab muaj kev sib tham sib txhawb los ntawm ntau hom cell woven hauv cov ntaub so ntswg matrix. Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, peb txoj kev tshawb fawb pom thawj zaug ib qho kev txhim kho ntawm cov tshuaj metformin rau tib neeg ASCs, ob qho tib si hauv 2D thiab tag nrho cov kab mob adipose. Peb txiav txim siab peb cov txiaj ntsig uas qhia txog kev txhim kho ntawm cov noob caj noob ces kos npe hauv ASCs cais los ntawm metformin-kho cov ntaub so ntswg adipose cov ntaub so ntswg raws li kev tawm tswv yim rau kev tshawb nrhiav ntxiv ntawm kev siv metformin ua kev kho mob kom qeeb cov txheej txheem kev laus thiab rov ua dua cov laus cov ntaub so ntswg.
Lub hom phiaj ntawm lub hom phiaj ntawm rapamycin (mTOR) plays lub luag haujlwm tseem ceeb hauv kev loj hlob thiab kev sib txawv ntawm cov qia hlwb. Kev txo qis ntawm mTOR kev ua haujlwm los ntawm kev tswj cov caj ces, kev cuam tshuam kho mob, lossis kev hloov pauv hauv kev ua neej ua rau nce stemness thiab tev [19,20]. Txij li thaum peb pom tias metformin inhibits adipogenesis, peb tau sim cov nyhuv ntawm metformin ntawm mTOR signaling. Interestingly, cov txiaj ntsig tau pom tias metformin txo qis- tswj cov qib phosphorylation ntawm PS70S6 kinase qhov tseem ceeb, uas yog lub cim tseem ceeb ntawm cov dej ntws ntawm mTOR thiab tus tshaj tawm ntawm mTOR txoj hauv kev. Cov kev tshawb fawb yav dhau los kuj qhia tias metformin inhibits adipogenesis los ntawm inhibiting mTOR signaling txoj hauv kev nas embryonic fibroblasts (MEFs), C3H10T1/2 nas mesenchymal qia hlwb, thiab 3T3-L1 preadipocytes [34]. Lub hom phiaj ntawm lub hom phiaj ntawm rapamycin yog tsim los ntawm ob txoj hauv kev, mTORCl, thiab mTORC2, thiab cov teeb liab ua lub luag haujlwm tseem ceeb hauv adipogenesis. Inhibition ntawm mTORC1 signaling nrog rapamycin yog pom tias ua rau tsis zoo adipogenesis los ntawm kev txo qis hauv phosphorylation ntawm PS6kinase hauv 3T3-L1 preadipocytes. Cov kev tshawb fawb no kuj tau pom tias inhibition ntawm mTOR signaling inhibits adipogenesis los ntawm kev txo qis kev qhia theem ntawm adipocytes kab cim xws li PPARgamma, CEBPI, CEBP1 , thiab adiponectin [54-56]. Ntxiv mus, mTOR signaling yog kho los ntawm phosphorylation ntawm S6 kinase. Lub knockdown ntawm S6 kinase tau pom tias muaj kev tiv thaiv kev rog thiab qhov hnyav nce thaum lub sijhawm noj zaub mov muaj roj ntau vim yog qhov tsis zoo ntawm adipogenesis [57]. Peb tau lees paub peb qhov kev soj ntsuam ntawm metformin-mediated downregulation ntawm mTOR kev ua ub no raws li tus neeg tuaj yeem kho tau ntawm kev nce hauv stemness thiab txo qhov sib txawv ntawm ASCs los ntawm kev siv rapamycin, uas tshwj xeeb thaiv m TOR txoj hauv kev.
Ntxiv rau mTOR, ERK signaling txoj hauv kev ua lub luag haujlwm tseem ceeb hauv kev loj hlob thiab adipogenesis. Xiaomin Ning li al., hauv lawv cov ntawv tshaj tawm, pom zoo tias adipogenesis yog qhib los ntawm ERK signaling [58]. Txij li thaum peb pom tias metformin inhibited adipogenesis thiab kev loj hlob, peb txiav txim siab los tshawb xyuas cov txiaj ntsig ntawm metformin ntawm ERK signaling. Raws li cov ntaub ntawv tshaj tawm ua ntej, peb cov txiaj ntsig kuj tau qhia tias metformin tuaj yeem cuam tshuam adipogenesis los ntawm kev tawm tsam ntawm ERK signaling nrog kev nce hauv stemness-related gene expression.mTOR signaling yog xav tau rau kev loj hlob ntawm tes thiab kev sib txawv thiab yog tus tswj tsis zoo ntawm autophagy [{{2. }}]. Txawm li cas los xij, kev poob ntawm autophagy yog txuas nrog kev poob ntawm qia, txawm hais tias kev kho mob metformin tau txhim kho cov kab mob ntsig txog cov noob qhia los ntawm kev ua kom muaj kev cuam tshuam ntawm autophagy cov cim qhia xws li LC3Il. Yog li, peb cov txiaj ntsig tau qhia tias metformin cuam tshuam adipogenesis los ntawm kev txhim kho stemness hauv tib neeg ASC lossis tib neeg adipose nqaij kab lis kev cai qauv ntawm kev ua kom autophagy thiab inhibiting mTOR signaling pathways. Yav tom ntej hauv vivo tsiaj thiab tib neeg kev tshawb nrhiav kev tshawb fawb tsom mus rau kev soj ntsuam cov teebmeem ntawm metformin ntawm biology ntawm ASCs yuav coj kev hloov pauv ntawm kev siv metformin ua cov qia cell tiv thaiv kev laus.
Kab lus no yog muab rho tawm los ntawm Biomedicines 2021, 9, 1782. https://doi.org/10.3390/biomedicines9121782 https://www.mdpi.com/journal/biomedicines
