Metformin tiv thaiv Lens Epithelial Cells tawm tsam Senescence hauv Tus Qauv Nas Laus Lawm

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Taw qhia

Lens epithelial hlwb (LECs) tsim ib lub monolayer ntawm cov hlwb nyob rau hauv lub anterior capsule ntawm lub lens thiab txuas mus rau lub equatorial lens hneev nti [1, 2]. Kev tsim kho ib txwm muaj thiab kev ua haujlwm ntawm LECs yog qhov tseem ceeb rau kev saib xyuas cov pob tshab thiab metabolic homeostasis ntawm tag nrho lub lens [3]. Cov pov thawj ntxiv qhia tau hais tias qhov tsis zoo ntawm LECs tuaj yeem ua rau muaj kev hloov pauv, denaturation, thiab sib sau ua ke ntawm lens proteins nrog rau cov enzymes thiab crystallins, yog li thaum kawg ua rau lub lens opacification lossis txawm tias cataracts [3-5]. Hnub nyoog ntsig txog cataract (ARC) tseem yog qhov ua rau pom kev tsis pom kev thiab qhov muag tsis pom kev, uas cuam tshuam rau lub neej zoo ntawm cov neeg laus [6]. Tam sim no, tsis muaj cov tswv yim zoo rau kev tiv thaiv kev nce qib ntawm ARC. Yog li ntawd, nws yog ib qho tsim nyog los tsim kom muaj kev cuam tshuam cov tshuaj pharmacological uas tuaj yeem txhim kho qhov pom tseeb ntawm lub lens thiab ncua kev nce qib ARC.

Ob peb xyoo dhau los, cov kws tshawb fawb tau ua tiav qhov kev vam meej hauv kev txhim kho kev noj qab haus huv thiab txuas ntxiv lub neej siv ntau yam tshuaj, kev noj haus, thiab tshuaj noj [7]. Metformin (MET) yog ib qho ntawm cov kev tshawb fawb ntau los tiv thaiv kev laus. Cov kev tshawb fawb tseem ceeb qhia tias MET tuaj yeem txo qis kev noj qab haus huv thiab txuas ntxiv kev noj qab haus huv nyob rau hauv ntau yam tsiaj qauv. MET tuaj yeem tsis tsuas yog ncua lub neej ntawm Caenorhabditis elegans tab sis kuj txhim kho nws txoj kev noj qab haus huv thiab kev loj hlob [8]. MET tuaj yeem txo qhov muaj hnub nyoog ntsig txog thiab oxidative kev ntxhov siab cuam tshuam txog DNA puas tsuaj hauv Drosophila midgut qia hlwb kom ntev lawv txoj sia [9]. Cov kev tshawb fawb yav dhau los kuj tseem qhia tau tias kev noj tshuaj tsawg tsawg MET txhim kho kev noj qab haus huv thiab kev ua neej nyob ntawm nas [10]. Ntxiv mus, cov pov thawj tshwm sim qhia tau hais tias thaum cov neeg mob ntshav qab zib tau kho nrog MET, lawv pom cov txiaj ntsig muaj sia nyob, txawm tias piv nrog cov tswj tsis muaj ntshav qab zib [11].

KSL09

Thov nias ntawm no kom paub ntxiv

Ntxiv nrog rau kev txhawb nqa kev ua neej nyob ntawm MET hauv ntau hom kab mob, MET tsom mus rau ntau lub xov tooj ntawm tes-ling txoj hauv kev los kho cov kab mob uas muaj hnub nyoog. MET tau pom tias yuav hloov kho autophagy feem ntau los ntawm adenosine monophosphate (AMP)-activated protein kinase (AMPK) ua kom thiab tiv thaiv neurodegeneration thiab neuroinflammation los ua lub luag haujlwm neuroprotective hauv Parkinson's disease [12]. Ib yam li ntawd, MET ameliorates hnub nyoog txog kev hloov pauv hauv daim siab sinusoidal endothelial cell ntawm AMPK thiab endothelial nitric oxide txoj kev [13].cistanche noj redditTsis tas li ntawd, kev ua haujlwm ntev AMPK los ntawm MET tiv thaiv cardiomyopathy los ntawm kev txhim kho autophagy hauv OVE26 nas nrog ntshav qab zib [14]. Tsis tas li ntawd, MET txo cov hnub nyoog cuam tshuam nrog kev mob plawv los ntawm kev txhim kho autophagy thiab caloric txwv [15]. Tsis tas li ntawd, MET kev kho mob tuaj yeem cuam tshuam cov hnub nyoog uas muaj feem cuam tshuam nrog atherosclerosis hauv ApoE-7- nas thiab luav vim yog kev noj zaub mov muaj roj ntau, tiv thaiv atherosclerotic plaque calcification, txo qis C-reactive protein ntau ntau, thiab txo qis kev ua haujlwm ntawm cov plaques. nuclear factor kappa B(NF-kB) txoj hauv cov hlab ntsha [16-18].

Raws li cov kev soj ntsuam no, txoj kev tshawb fawb tam sim no tau sim tshawb xyuas seb MET puas tuaj yeem ncua ARC kev laus thiab cov txheej txheem hauv qab molecular. Peb qhov kev tshawb pom muaj raws li hauv qab no: (i) kev kho mob qis MET qeeb qeeb ntawm LECs nyob rau hauv cov nas muaj hnub nyoog, yog li inhibiting lens opacity; (i) AMPK txoj hauv kev tsis ua haujlwm, thiab autophagic flux yog impaired hauv senescent LECs;(i) ) Kev kho mob qis MET qeeb qeeb ntawm LECs hauv cov nas uas muaj hnub nyoog los ntawm kev ua kom txoj hauv kev AMPK thiab txhim kho autophagy. Peb qhov kev tshawb pom pom tseeb tias lub luag haujlwm tiv thaiv kev laus ntawm MET thiab muab pov thawj txaus tias MET tuaj yeem ua pov thawj tias qhov kev xaiv kho tau rau ARC.

TSEEM CEEB

Senescence ntawm LECs tau txuam nrog ARC hauv cov nas uas muaj hnub nyoog ib txwm muaj

Ntau lub peev xwm ntawm ARC, suav nrog cov txiaj ntsig ntawm kev laus, tau raug tshawb xyuas ntau.cistanche extract cov txiaj ntsig,Txawm li cas los xij, kev sib koom ua ke ntawm kev tsis sib haum xeeb ntawm LECs thiab ARC hauv vivo tseem tsis paub meej. Peb tau xav tias qhov kev loj hlob ntawm LECs tuaj yeem ua rau ARC hauv vivo. Yog li ntawd, peb tsim tus qauv nas ntawm ARC. Peb cov txiaj ntsig tau qhia tias qhov pom tseeb ntawm lub lens hauv cov nas muaj hnub nyoog (cov nas laus) tau txo qis dua li piv nrog rau hauv pawg tswj hwm (cov nas hluas)(Fig.1a). Raws li pom nyob rau hauv Fig.1b, qhov tshwm sim ntawm cataracts nyob rau hauv ib txwm muaj hnub nyoog pab pawg neeg yog 95.23 feem pua. H&E staining assay tau qhia tias LECs hauv pawg tswj hwm tau hloov pauv hauv cov duab thiab teeb tsa tsis tu ncua, thiab qhov ceev ntawm cov LECs ib txwm muaj qhov siab, qhov LECs ntawm cov nas uas muaj hnub nyoog ib txwm muaj nyob hauv cov duab thiab teeb tsa tsis sib xws, thiab qhov ntom ntawm senescent LECs tau poob qis heev (Fig.1c). Tsis tas li ntawd, SA- -Gal staining tau pom tias qhov piv ntawm senescent LECs tau nce siab hauv cov nas muaj hnub nyoog (daim duab 1d). Tsis tas li ntawd, peb tau soj ntsuam cov protein qhia ntawm cov cim kev sib txuas ntawm cov cim ntawm IHC staining thiab western blot tsom xam. IHC staining tau qhia tias qhov kev qhia ntawm p21 thiab p53 tau nce hauv LECs ntawm cov nas uas muaj hnub nyoog ib txwm muaj piv nrog cov nas me (Fig. 1e, f). Ib yam li ntawd, peb tau tshawb pom ntxiv tias cov protein qhia ntawm p21 thiab p53 yog siab dua nyob rau hauv lub lens capsules ntawm ib txwm muaj hnub nyoog nas tshaj nyob rau hauv lub lens capsules ntawm cov nas me (Fig. 1g). Cov txiaj ntsig no tau qhia tias qhov kev loj hlob ntawm LECs tau txuam nrog ARC hauv cov nas muaj hnub nyoog ib txwm muaj.

KSL10

Cistanche tuaj yeem tiv thaiv kev laus

Kev noj tshuaj tsawg tsawg MET tau txhim kho qhov pom tseeb ntawm lub lens thiab txo qhov tsis zoo ntawm LECs Ua ntej peb kuaj cov nyhuv ntawm metformin ntawm ARC, peb txiav txim siab seb qhov kev tswj hwm qis MET puas muaj kev nyab xeeb rau cov nas. Tom qab MET kev kho mob rau 10 lub hlis, 28 nas (18 nyob rau hauv ib txwm muaj hnub nyoog pab pawg neeg thiab 10 nyob rau hauv pab pawg neeg MET) tuag los ntawm ntuj tsim. Peb pom tias cov nas ciaj sia nyob hauv ob pawg neeg noj qab haus huv, thiab tsis muaj qhov sib txawv ntawm lub cev hnyav thiab qhov hnyav ntawm ob pawg (Table 1). Yog li, cov txiaj ntsig no tau qhia tias ntau npaum li cas ntawm metformin siv hauv txoj kev tshawb fawb muaj kev nyab xeeb rau cov tsiaj thiab tuaj yeem siv rau kev sim tom ntej.

Tom qab ntawd, cov koob tshuaj tsawg tsawg MET tau muab tshuaj xyuas seb MET puas tuaj yeem cuam tshuam lub lens opacity thiab txo qhov muaj hnub nyoog ntsig txog LECs hauv cov nas muaj hnub nyoog. Raws li qhov xav tau, qhov pom tseeb ntawm lub lens hauv pawg MET tau txhim kho zoo heev thaum piv nrog cov pob tshab ntawm lub lens hauv pawg neeg laus (Fig.2a). Tsis tas li ntawd, cov kev soj ntsuam morphological tau lees paub tias lub lens ntawm MET pawg pom tau tias txo qis ntawm opacity. Qhov tshwm sim ntawm cataracts nyob rau hauv ib txwm muaj hnub nyoog pab pawg neeg yog 95.23 feem pua, whereas nyob rau hauv cov pab pawg neeg MET yog 19.00 feem pua (Fig.2b).H&E staining assay qhia tau hais tias qhov ceev ntawm LECs nyob rau hauv pab pawg MET yog tshwj xeeb tshaj yog siab dua. dua li cov nas uas muaj hnub nyoog lawm (Fig. 2c). Ntxiv mus, LECs ntawm pab pawg MET tau zoo ib yam li piv nrog cov neeg ntawm cov hnub nyoog ib txwm muaj (Fig.2c). SA- -Gal staining pom tias qhov piv ntawm senescent LECs tau txo qis hauv pawg MET raws li piv nrog rau hauv pawg neeg laus (Fig.2d). Kev tshuaj xyuas IHC tau qhia tias qhov kev qhia ntawm p21 thiab p53 tau txo qis ntau dua hauv LECs ntawm pawg MET dua li cov neeg laus (Fig.2e, f). Ib yam li ntawd, western blot tsom xam pom tias cov protein qhia ntawm p21 thiab p53 tau txo qis hauv lub lens capsules ntawm MET pawg piv nrog rau cov neeg ntawm ib txwm muaj hnub nyoog (Fig.2g). Cov txiaj ntsig no tau qhia tias kev noj tshuaj tsawg tsawg MET tuaj yeem txhim kho qhov pom tseeb ntawm lub lens thiab txo qhov tsis zoo ntawm LECs.

KSL11

Kev noj tshuaj tsawg tsawg MET ua rau txo qis ntawm LECs hauv cov nas uas muaj hnub nyoog ib txwm muaj los ntawm kev ua kom AMPK Cov ntaub ntawv ntau dhau los txhawb qhov kev txiav txim siab tias kev ua kom AMPK txaus los txuas ntxiv lub neej nyob rau hauv cov qauv kab mob, ua rau nws rov hais dua raws li lub hom phiaj ntev [19,20] . Xav txog qhov kev xav no, peb tau soj ntsuam qhov kev qhia ntawm AMPK txoj hauv kev hauv LECs ntawm nas. Tus txheeb ze FAS mRNA qib tau txo qis hauv cov neeg muaj hnub nyoog ib txwm muaj thiab nce hauv pawg MET (Fig. 3a). Raws li qhia hauv Fig.3b,c, qhov kev qhia ntawm phosphorylated AMPKa (Thr172) thiab phosphorylated ACC (Ser79) tau txo qis hauv LECs ntawm cov nas uas muaj hnub nyoog li qub piv nrog cov nas me. Txawm li cas los xij, qhov kev qhia ntawm phosphorylated AMPKa (Thr172) thiab phosphorylated ACC (Ser79) tau nce siab nyob rau hauv LECs ntawm pawg MET piv nrog rau cov pab pawg neeg laus. Tsis tas li ntawd, western blot tsom xam tau qhia tias cov protein qhia ntawm phosphorylated AMPKa (Thr172) thiab phosphorylated ACC (Ser79) tau txo qis hauv lub lens capsules ntawm ib txwm muaj hnub nyoog, thiab cov protein qhia qib ntawm phosphorylated AMPKa (Thr172) thiab phosphorylated ACC ( Ser79) tau nce nyob rau hauv lub lens capsules ntawm MET Group (Fig.3d). Cov ntaub ntawv no tau qhia tias txoj hauv kev AMPK tsis ua haujlwm hauv LECs ntawm cov nas muaj hnub nyoog ib txwm muaj. Tsis tas li ntawd, kev noj tshuaj tsawg tsawg MET ua rau txo qis ntawm LECs hauv cov nas uas muaj hnub nyoog dhau los ntawm kev ua kom AMPK.

Kev noj tshuaj tsawg tsawg MET cov thawj coj tau rov qab kho cov kab mob autophagic kom txo tau qhov tsis zoo ntawm LECs hauv cov nas uas muaj hnub nyoog dhau los tau qhia tias autophagy tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev tawm tsam qhov tsis zoo ntawm kev laus [21,22]. Txhawm rau txheeb xyuas ntxiv cov kev hloov pauv ntawm cov molecular thiab cellular hauv lub lens tom qab kev kho mob MET, peb txiav txim siab qhov kev qhia ntawm LC3 thiab p62 vim tias lawv tau txais dav raws li biomarkers ntawm qib ntawm autophagy. Raws li tau pom nyob rau hauv daim duab 4a, qhov txheeb ze p62 mRNA qhia theem tau nce siab nyob rau hauv cov neeg laus uas muaj hnub nyoog li piv nrog rau pawg tswj hwm; Txawm li cas los xij, qhov txheeb ze mRNA qhia theem ntawm p62 tau txo qis-tswj hauv pawg MET piv nrog rau hauv pawg neeg laus.cistanche genghis khanTsis tas li ntawd, IHC staining tau qhia tias qib ntawm microtubule-txuas nrog cov protein 1 lub teeb saw 3 (LC3-II/l) thiab p62 tau nce hauv LECs ntawm cov nas muaj hnub nyoog ib txwm muaj. Tsis tas li ntawd, peb cov txiaj ntsig tau qhia tias theem ntawm LC3-II / l thiab p62 tau poob qis heev hauv LECs ntawm MET Group (Fig. 4b, c). Cov qib protein ntau ntawm LC3-ll thiab p62 tau nce ntau ntxiv hauv lub lens capsules ntawm pawg neeg laus (Fig.4d). Ua ke, peb tau kwv yees tias autophagic flux yog qhov tsis zoo nyob rau hauv LECs ntawm cov nas uas muaj hnub nyoog ib txwm muaj thiab tuaj yeem rov qab los ntawm kev siv tshuaj tsawg tsawg MET los txo qhov tsis zoo ntawm LECs hauv cov nas muaj hnub nyoog.

Kev sib tham

Cov kab mob cataracts tsis kho yog qhov ua rau qhov muag tsis pom kev thoob ntiaj teb uas yog tus cwj pwm los ntawm opacification ntawm crystalline lens [23]. Cov kev tshawb fawb txog kab mob kis tau pom tias muaj hnub nyoog yog qhov tseem ceeb tshaj plaws rau cov kab mob cataracts, hu ua ARC, thiab tsuas yog kev kho mob yog phais tshem tawm [24]. Cov kev tshawb fawb yav dhau los tau pom tias qhov tsis zoo ntawm LECs yog lub ntsiab ntawm ARC [25]. Txog rau tam sim no, tsis muaj cov tshuaj kho tau zoo los tiv thaiv qhov cloudiness ntawm lub lens thiab txo qis qhov tsis zoo ntawm LECs yam tsis muaj kev phiv tsis zoo. Lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog txhawm rau txheeb xyuas cov molecular biological hauv paus rau kev laus ntawm LECs thiab cov txheej txheem ntawm kev tiv thaiv ib txwm LECs tawm tsam senescence. Peb cov txiaj ntsig tau tshaj tawm tias kev tsis ua haujlwm ntawm AMPK thiab kev puas tsuaj ntawm autophagy tau cuam tshuam nrog kev loj hlob ntawm LECs thiab ARC. Tsis tas li ntawd, peb pom tias MET tuaj yeem tiv thaiv LECs tiv thaiv kev laus thiab cuam tshuam qhov huab cua ntawm lub lens los ntawm kev ua kom AMPK thiab txhim kho autophagy.

Senescence yog ib qho tseem ceeb ntawm cov txheej txheem lom neeg nrog rau kev poob qis hauv cov ntaub so ntswg ua haujlwm. Raws li lub neej nce ntxiv, cov hnub nyoog cuam tshuam txog kev ua haujlwm tsis zoo xws li ARC ua rau mob hnyav

cov ntsiab lus [26]. Senescence ntawm LECs tau raug tshaj tawm tias koom nrog qhov tshwm sim thiab kev loj hlob ntawm ARC [5]. Peb cov txiaj ntsig tau nthuav tawm tias LECs ntawm cov nas uas muaj hnub nyoog ib txwm muaj nyob hauv cov duab thiab cov txheej txheem tsis sib xws, thiab qhov ceev ntawm LEC poob qis heev (Daim duab 1c, d). Tsis tas li ntawd, kev qhia txog cov noob caj noob ces (p21 thiab p53) tau raug kho nyob rau hauv LECs ntawm cov nas muaj hnub nyoog (Fig. 1e, f, g). Peb cov txiaj ntsig tau qhia tias qhov kev loj hlob ntawm LECs tau cuam tshuam nrog qhov tshwm sim thiab kev loj hlob ntawm ARC. Yog li ntawd, kev nkag siab meej ntawm cov txheej txheem molecular hauv qab ntawm qhov tsis txaus ntseeg ntawm LECs yuav tsum tau muab qhov laj thawj rau cov tswv yim kho tshiab.

MET yog cov tshuaj kho qhov ncauj ntau tshaj plaws rau cov ntshav qab zib hom 2 thoob ntiaj teb. Cov ntaub ntawv pov thawj tshwm sim qhia tias MET muaj txiaj ntsig zoo rau kev noj qab haus huv tshaj qhov cuam tshuam nrog kev ua kom zoo dua hauv glycemia [27]. Nws tau raug lees paub tias cov txheej txheem ntawm MET yog qhov tseem ceeb rau lub hom phiaj tseem ceeb hauv kev laus hauv kev laus, xws li ua kom AMPK, augmenting autophagy, inhibiting o, thiab exerting anti-oxidative teebmeem [28]. Hauv tib neeg, MET tau siv rau hauv kev kho mob rau ntau tshaj 60 xyoo; nws muaj kev nyab xeeb siab profile thiab yog qhov tshwj xeeb nyob rau hauv kev cuam tshuam hauv ntau txoj hauv kev tseem ceeb uas lub luag haujlwm rau kev laus thiab cov kab mob muaj hnub nyoog. Txawm li cas los xij, cov teebmeem ntawm MET ntawm ARC thiab cov txheej txheem tswj hwm ntawm cov teebmeem no yeej tsis tau tshaj tawm. Yog li ntawd, hauv peb txoj kev tshawb fawb, kev noj tshuaj tsawg tsawg MET hauv cov nas muaj hnub nyoog tau txhim kho qhov pom ntawm lub lens (Daim duab 2a) thiab txo qis qhov tsis zoo ntawm LECs hauv vivo (Fig. 2).

KSL12

Nws tau lees paub dav dav tias MET qhib txoj hauv kev AMPK uas yog lub peev xwm ntawm MET kom ua rau muaj kev tiv thaiv kev laus [29]. AMPK, lub cellular zog sensor, plays lub luag haujlwm tseem ceeb hauv kev tswj hwm cellular zog tshuav [30]. Ntau qhov kev tshawb fawb tau piav qhia tias AMPK ua haujlwm ua tus koom ua ke thiab tus neeg nruab nrab ntawm ntau txoj hauv kev thiab cov txheej txheem koom nrog kev muaj zog nrog lub neej ntev. Hauv peb txoj kev tshawb fawb, peb pom tias qhov tsis ua haujlwm ntawm AMPK yog ib qho ntawm senescent LECs hauv cov nas uas muaj hnub nyoog lawm (Fig.3). Tom qab ntawd, MET tau pom los txhawb txoj hauv kev AMPK los tiv thaiv qhov tsis zoo ntawm LECs (Fig.3). Raws li tus tsim AMPK activator, lub luag haujlwm ntawm MET hauv kev tiv thaiv kev laus feem ntau yog los ntawm nws cov teebmeem ntawm kev hloov kho cov kev taw qhia hauv qab, xws li kev rov ua haujlwm ntawm autophagy, ua kom Sirt1 thiab Foxo1 thiab kev tawm tsam ntawm mTOR [31,32]. Peb txoj kev tshawb fawb tau muab cov ntaub ntawv pov thawj tshiab hais txog qhov tseem ceeb ntawm AMPK kev ua haujlwm tau qhib los ntawm MET hauv kev tiv thaiv qhov tshwm sim ntawm ARC.

Raws li tau hais los saum no, AMPK sib txuas lus nrog ntau txoj hauv kev thiab cov proteins kom ua rau muaj kev tiv thaiv kev laus, suav nrog kev txhawb nqa ntawm autophagy. Autophagy, tawm los ua cov txheej txheem tseem ceeb rau kev ruaj ntseg ntev, tau txais kev txaus siab thoob plaws hauv lub tebchaws raws li lub hom phiaj kho mob rau cov kab mob muaj hnub nyoog. Autophagy yog cov kua dej, ntau cov txheej txheem lom neeg. Cov txheej txheem ua tiav ntawm autophagy yog hu ua autophagic flux, uas yog dav siv los cuam tshuam txog qib ntawm autophagy. Raws li qhov taw qhia ntawm autophagy, LC3-Il yog nruj nreem khi rau autophagosomal membrane uas yog degraded los ntawm lysosomal enzymes.cistanche lub neej extensionIb yam li ntawd, p62, ib qho autophagy-specific substrate, feem ntau degraded hauv autophagolysosome, thiab nws cov lus qhia tsis ncaj qha cuam tshuam txog qib ntawm autophagy. Hauv txoj kev tshawb fawb tam sim no, peb qhov kev tshawb pom pom tau hais tias theem ntawm autophagy txawv txav tsis zoo nyob rau hauv senescent LECs (Fig. 4), whereas MET reversed lub hnub nyoog-hais txog kev puas tsuaj ntawm autophagy (Fig.4). Kev tiv thaiv kev laus molecular mechanism ntawm MET yog ntaus nqi rau lub luag haujlwm tseem ceeb ntawm AMPK hauv hlwb vim nws feem ntau txhim kho autophagy ntawm AMPK ua kom [33]. Tsis tas li ntawd, MET tau pom tias yuav txhawb nqa autophagy ncaj qha, yog li pab txhawb kev tiv thaiv kev laus [34]. Txawm li cas los xij, cov kev tshawb fawb ntxaws ntxiv yog xav tau los piav qhia cov txheej txheem hauv qab no, ua pov thawj tias MET augments autophagy ntawm txoj kev ywj pheej lossis txoj hauv kev AMPK.

Nws tau raug pom tias qhov ntau ntawm ARC tsis tau cuam tshuam nrog kev sib deev hauv cov neeg Esxias keeb kwm [35, 36]. Yog li ntawd, peb tsis tau xav txog kev sib deev yog ib yam uas yuav cuam tshuam rau ARC. Peb txoj kev tshawb fawb yuav tsum tau txhais los ntawm cov kev txwv saum toj no.

XAIV

Hauv cov ntsiab lus, peb tau ua pov thawj tias kev tsis ua haujlwm ntawm AMPK txoj hauv kev thiab muaj hnub nyoog cuam tshuam txog kev tsis txaus ntseeg ntawm autophagy yuav ua rau muaj qhov tsis zoo ntawm LECs thiab qhov tshwm sim ntawm ARC. Tsis tas li ntawd, cov ntaub ntawv los ntawm peb qhov kev sim tau qhia tias MET ua haujlwm tau zoo ncua sijhawm ntawm LECs thiab tsim ntawm ARC los ntawm kev ua kom txoj hauv kev AMPK thiab augmentation ntawm autophagy. Peb qhov kev tshawb pom yog ib txoj kab yooj yim rau kev tshawb nrhiav ntxiv ntawm cov txheej txheem molecular uas muaj txiaj ntsig zoo hauv lub luag haujlwm ntawm MET thaum lub sijhawm kev laus ntawm ARC thiab yuav yog cov ntaub ntawv rau kev tshawb fawb ntau ntxiv txog kev tsim cov tswv yim tshiab tawm tsam qhov tshwm sim thiab kev loj hlob ntawm ARC.

KHOOM HAUJ LWM THIAB KEV PAB CUAM

MET tau yuav los ntawm Bristol-Myers Squibb Company (Tuam Tshoj).Antibodies tiv thaiv p21 (ab188224), p53 (ab131442), phosphorylated AMPKa(p-AMPK)(Thr172)(ab133448), AMPKa1(ab32047), -26 (ab82) SQSTM1/p62 ({14}}) tau txais los ntawm Abcam(MA, USA).Antibodies tiv thaiv LC3 (12741) thiab phospho-acetyl CoA carboxylase (p-ACC)(Ser79)(3661) tau muas los ntawm Cell Signaling Technology ( MA, USA).

Tsiaj txhu thiab kev kho mob

Txiv neej C57BL6J nas uas muaj hnub nyoog 3 thiab 8 hli tau yuav los ntawm Beijing Vital River Laboratory Tsiaj Technology Co. Ltd.(Beijing, Suav). Cov nas tau nyob hauv qhov ntsuas kub (kub, 20-25} degree thiab txheeb ze av noo, 55 ± 15 feem pua) cov xwm txheej nrog lub voj voog 12 h lub teeb / tsaus nti, nrog kev nkag mus rau zaub mov thiab dej dawb. Tag nrho cov txheej txheem thiab cov txheej txheem ua rau tsiaj raug pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Harbin Medical University.

Cov nas tau muab faib ua peb pawg hauv qab no: (i) pab pawg tswj hwm muaj cov nas me (hnub nyoog 3 hlis, lub cev hnyav=29.72± 1.66 g, n =40) tau txais cov qauv purified nas thiab dej ad libitum; Thaum cov nas muaj hnub nyoog 10 hli, 120 nas tau muab faib ua ob pawg: pawg neeg laus thiab MET pawg. (i) ib txwm muaj hnub nyoog pab pawg (cov nas laus, 20 lub hlis, lub cev hnyav=32.98±1.93g, n=60) tau txais cov qauv purified nas noj zaub mov thiab dej ad libitum;(ii) MET pawg ( 20 lub hlis, lub cev hnyav =31.21 ± 2.81 g, n=60) tau khaws cia ntawm tus qauv purified nas thiab dej kom txog thaum cov nas muaj hnub nyoog 10 hli; tom qab ntawd, cov kev kho mob tau pib. Cov nas nyob hauv pawg MET tau noj zaub mov ntxiv nrog 0.1 feem pua MET (Bristol Myers Squibb, Tuam Tshoj) thiab dej dawb rau 10 lub hlis.

Kev sib cais ntawm cov lo ntsiab muag thiab lub lens capsules

Cov nas tau euthanized los ntawm lub ncauj tsev menyuam dislocation. Tag nrho lub qhov muag raug excised tam sim ntawd los ntawm tag nrho cov tsiaj sim thiab muab tso rau hauv sterile saline. Lub paj hlwb tau muab tso rau saum thiab kho nrog tweezers. Tom qab ntawd ib qho kev phais tau ua rau ntawm lub paj hlwb uas nkag mus rau lub qhov muag, thiab lub sclera raug rub rov qab los nthuav tawm lub lens.ceg nzTom qab ntawd, peb siv cov tweezers maj mam tshem tawm cov ciliary lub cev fragments txuas rau lub dav hlau equatorial ntawm lub lens. Cov duab ntawm lub lens tshiab tau muab tam sim ntawd, thiab lub lens tau kho sai nrog 4 feem pua paraformaldehyde ntawm chav tsev kub. Tag nrho lub lens capsules tau sai sai tev tawm ntawm lub lens ntxiv, snap-kho hauv cov kua nitrogen, thiab khaws cia ntawm 80 degree C rau kev sim tom ntej.

Hematoxylin thiab eosin (H&E)

Lub lens tshiab tau kho tam sim nrog 4 feem pua ntawm paraformaldehyde ntawm chav tsev kub thiab muab tso rau hauv paraffin. Rau kev kuaj histological, cov ntaub so ntswg raug txiav rau hauv 4-μm slices. Tom qab deparaffinization thiab rehydration, cov khoom npaj paraffin tau stained nrog hematoxylin (H) tov rau 8min, tom qab ntawd 5 dips hauv 1 feem pua cov kua qaub ethanol (1 feem pua HCI hauv 70 feem pua ethanol)

thiab yaug nrog dej distilled. Tom qab ntawd, cov seem tau stained nrog eosin (E) tov rau 3 min, tom qab ntawd lub cev qhuav dej nrog qib cawv thiab tshem tawm nrog xylene. Kev hloov pauv ntawm morphological tau pom nyob rau hauv lub tshuab kuaj kab mob (Nikon, dab noj hnub, Nyiv).

Senescence-associated -galactosidase (SA- -Gal) assay Lub senescence-associated -galactosidase (SA- -Gal) kev ua haujlwm tau ntsuas los ntawm kev siv SA- -Gal staining kit (Sigma, CS{ {12}}030). Raws li cov chaw tsim tshuaj paus cov lus qhia, cov ntaub so ntswg, tom qab ib tug series ntawm kev kho mob, tau ntxuav nrog phosphate-buffered saline (PBS) rau 5 min (thrice) thiab ces incubated nyob rau hauv SA- -gal staining tov (pH 6.0) ntawm 37 degree tsis muaj CO2 rau 24 teev. Cov duab tau raug ntes nyob rau hauv lub teeb pom kev zoo (Nikon, Eclipse, Nyiv). Qhov feem pua ntawm SA- -gal-positive hlwb tau kwv yees raws li tus lej nruab nrab ntawm cov hlwb zoo / tus lej nruab nrab ntawm tag nrho cov hlwb.

Quantitative real-time polymerase chain reaction (gRT-PCR) Tag nrho RNA los ntawm lub lens capsules ntawm nas tau muab rho tawm siv TRlzol reagent. Kev hloov pauv rov qab tau ua nrog ExScript RT Reagent Kit (Invitrogen). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) kev soj ntsuam tau ua tiav siv SYBR Green Supermix

cov khoom siv (Takara, Tokyo, Nyiv), nrog -actin raws li kev tswj hwm endogenous. Cov ntawv sau tau raug tshawb xyuas rau ntau lub hom phiaj, suav nrog FAS (rau pem hauv ntej, 5'-TTGCTGGCACTACAGAATGC-3'thiab thim rov qab,5'-AACAGCC TCAGAGCGACAAT-3'), p62 (forward,5'-GGCGCACTACCGCGATGAGGA{{ 10}} thiab thim rov qab, 5'-TGTTCCCGCCGGCACTCCTT-3') thiab -actin (forward, 5 '-TCGTGGGCCGCCCCTAGGCAC-3'and rov qab,5'-TGGCCTTAGGGTTC AGGGGGG-3'. theem ntawm txhua tus gene qhia tau

txoj kev. normalized rau -actin thiab xam siv cov 2-△△ct r Immunohistochemical (IHC) staining Tag nrho ntawm 5 tus neeg sawv cev seem los ntawm txhua qhov ntawm 10 lo ntsiab muag xaiv rau ib pab pawg tau siv rau kev tshuaj xyuas immunohistochemical (IHC) los soj ntsuam qhov qhia ntawm p21, p53, p-AMPK, p-ACC, LC3, thiab p62. Tom qab cov qauv npaj tau muab tso rau ntawm 60 degree rau 2 h, lawv tau dhau los ntawm xylene thiab graded cawv series rau deparaffinization. Tom qab ntawd, cov qauv raug muab rhaub hauv 0.1 M ntawm citric acid (pH 6.1) rau 30 min thiab txias rau chav tsev kub. Tom qab ntxuav peb zaug nrog PBS, cov qauv tau tsau nrog 0.3 feem pua HO, txhawm rau inhibit endogenous peroxidase kev ua haujlwm. Tom qab ntawd, thawj cov tshuaj tiv thaiv tau ntxiv thiab txias ntawm 4 ° C, thaum hmo ntuj. Cov tshuaj tiv thaiv kab mob thib ob tau muab tso rau hauv 1 teev ntawm 37 degree C. Tom qab ntxuav nrog PBS, cov qauv tau muab tso rau hauv diaminobenzidine (DAB) kom txog rau thaum qhov xav tau stain siv tsim. Cov duab ntawm cov tshuaj tiv thaiv kab mob tau raug ntes siv Nikon Eclipse microscope (Nikon, dab noj hnub, Nyiv).

Western blot tsom xam

Tag nrho cov protein los ntawm lub lens capsules ntawm nas tau muab rho tawm siv xov tooj cua-immunoprecipitation assay (RIPA) tsis nrog protease inhibitor cocktail (Pierce, USA), thiab cov khoom siv bicinchoninic acid (BCA) (Thermo Fisher Scientific, USA) tau siv los ntsuas cov protein ntau. . Tag nrho cov protein (40 ug) tau soj ntsuam siv sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) thiab electrophoretically xa mus rau polyvinylidene difluoride lim (PVDF) daim nyias nyias (Millipore, Lub teb chaws Yelemees). Cov membranes tau incubated ib hmos nrog ib tug thawj antibody ntawm 4 degree C. Hnub tom qab, cov membranes tau incubated nrog coj lwm yam antibodies rau 1 h ntawm 37 degree C. Cov immunoreactive bands pom tau siv txoj kev chemiluminescent substrate nrog ib tug Super Signal West Pico. Cov khoom siv (Pierce Biotechnology, USA). Peb qhov kev sim ywj pheej tau ua.

Kev txheeb cais

Cov ntaub ntawv tau qhia raws li qhov nruab nrab ± tus qauv sib txawv (SD). Kev txheeb xyuas txheeb cais tau ua los ntawm GraphPad Prism 7.0 software thiab Microsoft Excel. Protein tau suav nrog siv Image J software. Cov tub ntxhais kawm qhov kev xeem t-test thiab kev soj ntsuam ntawm qhov sib txawv (ANOVA) tau siv los xam qhov tseem ceeb ntawm cov ntaub ntawv sim. Qhov sib txawv nrog P-tus nqi<0.05 were="" considered="" statistically="" significant.="" all="" experiments="" were="" repeated="" independently="" at="" least="" three="">