PARTⅠ: Novel Protective Effects Of Cistanche Tubulosa Extract Tawm tsam Low-Luminance Blue Light-Induced Degenerative Retinopathy

Hu rau: Audrey Huaudrey.hu@wecistanche.com

Man-Ru Wu Cheng-Hui Lin Jau-Der Hob George Hsiao, Yu-Wen Cheng

Tsev Kawm Ntawv Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Department of Ophthalmology, Taipei Medical University, Taipei, Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Kawm Tiav Lub Tsev Kawm Ntawv Kho Mob, Tsev Kawm Qib Siab Tshuaj, Taipei Medical University, Taipei, Ph.D. Program hauv Biotechnology Research thiab Development, College of Pharmacy, Taipei Medical University, Taipei, Taiwan

Abstract

Keeb kwm/Lub hom phiaj:Blue light-emitting diode light (BLL)-induced phototoxicity plays lub luag hauj lwm tseem ceeb hauv cov kab mob ntawm lub qhov muag thiab ua rau retinal degeneration thiab apoptosis nyob rau hauv tib neeg retinal pigment epithelial (RPE) hlwb.Cistanche tubulosa Extract (CTE)yog cov tshuaj suav tshuaj suav nrog ntau yam kev tiv thaiv zoo; Txawm li cas los xij, ob peb txoj kev tshawb fawb tau tshuaj xyuas lub luag haujlwm tiv thaiv qhov muag ntawm CTE. Hauv txoj kev tshawb no, peb tau tshawb xyuas cov txheej txheem hauv qab ntawm CTE ntawm BLL-induced apoptosis hauv vitro thiab hauv vivo. Txoj kev: RPE hlwb tau siv nyob rau hauv tam sim no nyob rau hauv vitro txoj kev tshawb no thiab cell viability yog txiav txim los ntawm ib tug 3-(4,5-dimethylthiazol- 2-yl)-2, {{6} Kev ntsuam xyuas diphenyltetrazolium bromide. Apoptosis-hais txog protein qhia tau txiav txim siab los ntawm western blot tsom thiab immunofluorescence staining. Brown Norway nas tau siv los tshuaj xyuas qhov raug rau kev lag luam muaj BLL hauv vivo. Hematoxylin thiab eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), thiab western blot assays tau siv los tshuaj xyuas cov retinal morphological deformation. Cov txiaj ntsig: CTE cuam tshuam rau hydrogen peroxide-, tertbutyl hydroperoxide-, sodium azide-, thiab BLL-induced RPE puas. Tsis tas li ntawd, CTE txo cov kev qhia ntawm apoptotic cim xws li cleaved caspase-3 thiab TUNEL staining tom qab BLL raug los ntawm inactivating apoptotic txoj kev, raws li qhia los ntawm immunofluorescent staining. Tsis tas li ntawd, CTE inhibited BLL-induced phosphorylation ntawm c-Jun N-terminal kinase, ntxiv teeb liab ntsig txog kinases 1/2, thiab p38 hauv RPE hlwb. Hauv vivo, kev tswj hwm qhov ncauj ntawm CTE tau cawm 60-hnub ib ntus BLL raug cuam tshuam los ntawm qhov txo qis hauv retinal tuab thiab txo tus naj npawb ntawm TUNEL-zoo hlwb hauv cov qauv nas xim av Norway. Xaus: CTE yog ib tug muaj peev xwm prophylactic tus neeg sawv cev tiv thaiv BLL-induced phototoxicity

Cistanche tubulosa

Taw qhia

Kev tshawb fawb txog hnub nyoog ntsig txog macular degeneration (AMD) tau tshaj tawm tias 8.7 feem pua ​​​​ntawm cov neeg hauv ntiaj teb raug mob los ntawm tus kab mob no [1]. AMD yog tus kab mob neurodegenerative uas ua rau photoreceptor deformation, retinal pigment epithelial (RPE) atrophy, ganglion cell apoptosis, thiab lub hauv paus tsis pom kev hauv cov neeg laus, thiab tau dhau los ua lub nra hnyav thoob ntiaj teb. AMD tau muab faib ua ib daim ntawv qhuav lossis ntub dej [2, 3]. Kev txhim kho hauv thev naus laus zis tau ua rau tib neeg siv sijhawm ntau dua los siv cov khoom siv digital niaj hnub no, thiab yog li ntawd, tib neeg raug rau lub teeb xiav (BL) emissions nyob rau lub sijhawm ntev. Txawm li cas los xij, qhov teeb meem ntawm BL-induced phototoxicity nyob rau hauv lub retina tsis tau tshawb xyuas zoo. Overexposure rau luv luv wavelength BL (450-495 nm) ua rau cov tiam ntawm reactive oxygen hom (ROS), xws li superoxide thiab hydroxyl radicals, uas ua rau kom cov pa oxygen noj thiab induce mitochondrial DNA puas. Kev sib sau ntawm ROS ua rau muaj kev ntxhov siab oxidative thiab nthuav dav ntawm retinoid adducts, uas ua rau lub retina ntxiv [4]. Kev tshawb fawb tau pom tias oxidative kev nyuaj siab yog ib qho tseem ceeb hauv cov kab mob ntawm AMD qhuav [5]. Tsis tas li ntawd, BL irradiation txo qhov kev qhia ntawm retinal antioxidant enzymes, xws li superoxide dismutase thiab catalase, los ntawm kev sib kho Bax/Bcl -2 protein sib cuam tshuam thiab txhawb kev tsim cov endogenous ROS. Raws li xws li, RPE hlwb thiab sab nraud mitochondria ntawm photoreceptors raug kev txom nyem los ntawm BL irradiation-induced raug mob [6, 7]. Ntxiv rau ROS tiam, BL irradiation activates mitogen-activated protein kinases (MAPKs), uas ua lub luag haujlwm tseem ceeb hauv cell ciaj sia thiab apoptosis. mitochondria-dependent apoptosis-related pro-apoptotic proteins, xws li Bax thiab caspase-3, yog qhib los ntawm BL-emitting diode teeb (BLL) irradiation ntawm c-Jun N-terminal kinase (JNK) thiab p38 txoj hauv kev. RPE thiab retinal ganglion hlwb [8]. Ntxiv mus, phosphatidylinositol 3-kinase (PI3K)/Akt thiab nuclear factor erythroid 2-txog yam 2 (Nrf2)-dependent anti-oxidative pathways yog cov khoom ntawm kev tiv thaiv kab mob ntawm RPE hlwb [{{34} }].

Cistanche tubulosa Extract (CTE)muaj phenylethanoid glycoside derivatives thiab tau siv los ua tshuaj suav tshuaj rau ntau xyoo lawm. Echinacoside, acteoside, thiab isoacteoside yog peb lub ntsiab lus tseem ceeb ntawm CTE uas tau pom tias muaj cov tshuaj tiv thaiv neuroprotective, nco-txhim kho, tiv thaiv kab mob, thiab tiv thaiv daim siab fibrosis zog [13, 14]. Echinacoside yog ROS scavenger thiab tiv thaiv 1-methyl-4-phenylpyridinium ion (MPP plus )-induced apoptosis hauv tsiaj qauv ntawm Parkinson tus kab mob [15, 16]. Acteoside tiv thaiv amyloid{10}}induced neurotoxicity thiab txo lipopolysaccharide-stimulated o, tshwm sim los ntawm translocation ntawm Nrf2 los ntawm cytoplasm mus rau lub nucleus thiab khi rau antioxidant teb cov ntsiab lus. Tam sim no muaj kev kho mob rau cov ntub dej AMD, xws li pegaptanib, ranibizumab, bevacizumab, thiab aflibercept, yog siv los tiv thaiv kev mob angiogenesis ntau dhau [17, 18]. Txawm li cas los xij, tsis muaj kev kho mob zoo rau AMD qhuav, vim muaj ntau yam teeb meem ntawm tus kab mob no.

Hauv qhov kev tshawb fawb no, peb qhia txog kev tiv thaiv los ntawm CTE hauv RPE hlwb thiab Brown Norway (BN) nas. Tsis tas li ntawd, peb siv lub sij hawm ntev uas tsis tshua muaj luminance BLL raug rau ob qho tib si hauv vitro thiab hauv vivo qauv los ntsuas qhov kev tiv thaiv ntawm CTE. Peb pom tias CTE txo qhov kev qhia ntawm apoptosis-txog cov proteins tom qab raug rau BLL. Ntxiv mus, CTE txo tus naj npawb ntawm cov davhlau ya nyob twg deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) - cov hlwb zoo hauv cov retina ntawm BN nas tom qab lub sij hawm ntev raug rau cov luminance tsawg BLL. Txoj kev tshawb no muab kev nkag siab ntxiv rau qhov tshiab los tiv thaiv apoptotic cuam tshuam ntawm CTE thiab cov ntaub ntawv hais txog lub tswv yim tshiab rau kev kho cov retinal degeneration.

Cistanche tubulosa extract (CTE): phenylethanoid glycoside

Cov ntaub ntawv thiab cov txheej txheem

Tshuaj thiab cov khoom

CTE tau dav siv los ntawm Sinphar Pharmaceutical Company (Yilan, Taiwan). Hauv qhov chaw kuaj mob cell tuag (Cat. No. 11-684-817-910) tau yuav los ntawm Roche (Mannheim, Lub teb chaws Yelemees).

RPE cell culture

RPE hlwb (ARPE{{0}}} thiab ATCC® CRL-2302™) tau yuav los ntawm American Type Culture Collection (Manassas, VA) thiab cultured hauv Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) ntxiv nrog 10 feem pua ​​​​fetal bovine serum nyob rau hauv ib qho chaw humidified ntawm 5 feem pua ​​CO2 ntawm 37 degree . ARPE-19 hlwb ntawm nqe 19–30 tau khaws cia rau ntawm 10-cm kab lis kev cai tais diav (Orange Scientific, Braine-l'Alleud, Belgium). Thaum dhau nrog 0.05 feem pua ​​​​ntawm trypsin-ethylenediamine tetraacetic acid (Gibco), cov hlwb raug hloov ntawm qhov piv ntawm 1: 4. Ua ntej kev sim, ARPE-19 hlwb tau muab cog rau hauv 48- cov phiaj zoo (Orange Scientific) rau kev soj ntsuam ntawm tes ntawm qhov ntom ntawm 1.0 × 105 hlwb / mL thiab mus rau 6-cm cov tais diav (Orange Scientific ) rau sab hnub poob blotting tsom xam ntawm qhov ntom ntawm 2.0 × 105 hlwb / mL rau 24 teev. Tom qab ARPE-19 cov hlwb mus txog 80 feem pua ​​​​ntawm qhov tsis sib xws, cov kev sim hauv qab no tau ua.

Kev kho tshuaj thiab tshuaj xyuas cell viability

RPE hlwb ntawm nqe 19–30 tau muab noob rau hauv 48- cov phiaj zoo thiab sib koom ua ke rau 24 teev nrog ntau qhov sib txawv ntawm CTE, hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BHP), thiab sodium azide ( NAS 3). Tom qab ntawd cov hlwb raug coj los ua ke nrog 3-(4, 5-dimethylthiazol-2- yl)-2, 5-diphenyltetrazolium bromide (MTT) ntawm qhov kawg ntawm 1 mg. / mL thiab incubated rau 30 min. Tom qab ntawd dimethyl sulfoxide (200 µL) tau ntxiv rau txhua qhov dej kom yaj cov hlwb. Kev nqus tau ntsuas ntawm 570 nm siv Sunrise™ spectrophotometer enzyme-linked immunosorbent assay reader (MRX-TC; Dynex Technologies, Chantilly, VA). Cov txiaj ntsig tau raug kho rau keeb kwm yav dhau los los ntawm kev rho tawm cov blanks tsim nyog. Cov ntaub ntawv yog los ntawm tsib qhov kev soj ntsuam ywj pheej.

Hauv vitro BL exposure raws tu qauv

Lub teeb ci thiab cov phiaj ntawm tes tau muab tso rau 25 cm sib nrug, raws li tau piav qhia hauv peb txoj kev tshawb fawb dhau los [19]. Luminance tau ntsuas nrog lub teeb ntsuas (LM-81LX; Lutron Electronic Enterprise, Taipei, Taiwan). BLL peaked ntawm 460 nm (60 lux) rau lub sijhawm qhia (0–48 h), nyob ntawm qhov kev sim tsim.

Western blotting

Tom qab 0–48-h kev kho mob nrog CTE thiab BLL raug, cov hlwb raug ntxuav ob zaug nrog cov dej khov-txias phosphate-buffered saline (PBS) thiab rov ua haujlwm hauv radioimmunoprecipitation assay lysis buffer (1 feem pua ​​Nonidet P-40, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 1 mM ethylene glycol-bis (-aminothylether)-N, N, N′, N′-tetraacetic acid, 1 mM NaF , thiab protease / phosphatase inhibitor cocktail). Lub cell lysate tau txais los ntawm centrifugation ntawm 12, 000 × g rau 35 min, thiab cov supernatant tau sau los txiav txim siab cov protein ntau ntawm Bradford reagent (Bio-Rad Laboratories, Hercules, CA). Qhov sib npaug ntawm cov qauv protein tau sib cais los ntawm 10-12 feem pua ​​​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis thiab tom qab ntawd xa mus rau polyvinylidene difluoride membrane. Tom qab daim nyias nyias tau thaiv nrog 5 feem pua ​​​​ntawm cov mis nyuj tsis muaj rog thiab ua rau hauv Tris-buffered saline nrog Tween 20 (TBST) rau 1 h, immunoblotting tau ua nrog thawj cov tshuaj tiv thaiv tshwj xeeb rau cleaved caspase -3, phospho-JNK, JNK , phospho-p38, p38, phospho-extracellular signal-related kinase (ERK), ERK, Bcl-2, Bax, Fas-associated protein with death domain (FADD), caspase-8 (GeneTex, Irvine , CA), Fas-ligand (Abcam, Cambridge, UK), thiab b-actin (Sigma- Aldrich, St. Louis, MO). Tom qab ntawd cov blots tau tsim nrog nees radish peroxidase-conjugated thib ob cov tshuaj tiv thaiv kab mob (1: 6000 hauv TBST; Cayman Chemical, Ann Arbor, MI) rau 1 teev ntawm chav tsev kub. Siv cov khoom siv kho mob zoo tshaj plaws (Millipore, Billerica, MA), qhov kev siv ntawm cov bands ntawm txhua blot raug tshuaj xyuas nrog Image J software tom qab normalizing b-actin.

Immunofluorescence

RPE hlwb raug kho nrog 4 feem pua ​​paraformaldehyde hauv PBS rau 15 min. Triton X-100 hauv PBS (0.2 feem pua ​​) tom qab ntawd ntxiv rau cov hlwb rau 15 feeb, tom qab ntawd 5 feem pua ​​​​fetal bovine serum hauv PBS rau 30 min; cov hlwb ces incubated nrog anti-cleaved caspase -3 antibody ntawm 1:250 piv rau 24 h (Cell Signaling Technology, Danvers, MA). Ib tug tshis los tiv thaiv luav IgG thib ob antibody (Dylight 594) tau siv ntawm qhov piv ntawm 1:200 (Vector Laboratories, Burlingame, CA). Cell nuclei raug stained nrog 4′-6-diamidino-2-phenylindole (DAPI; AAT Bioquest, Sunnyvale, CA). Kev hloov pauv hauv fluorescence tau pom nrog lub laser CS SP5 confocal spectral microscope imaging system (Leica, Teban Gardens, Singapore).

TUNEL staining

DNA tawg, uas tuaj yeem kuaj pom los ntawm TUNEL kev soj ntsuam, yog qhov tseem ceeb ntawm apoptosis. Peb siv Cov Khoom Siv Hauv Situ Cell Detection (Cat. No. 11-684-817-910}; Roche) los ntsuas apoptotic programmed RPE cell tuag raws li cov chaw tsim khoom raws tu qauv. Luv luv, peb kho RPE cov hlwb nrog 4 feem pua ​​​​ntawm paraformaldehyde thiab sau npe DNA strand so hauv cov tshuaj tiv thaiv enzymatic los ntes TUNEL-zoo hlwb, uas nthuav tawm nrog cov xim ntsuab-fluorescent. Cov hlwb stained tau raug tshuaj xyuas thiab tshuaj xyuas nrog Tissue FAXS-plus Imaging System (TissueGnostics, Vienna, Austria). Kaum txhiab txheej xwm raug suav nyob rau hauv cheeb tsam RPE gated, thiab peb tau txheeb xyuas cov duab rau tus naj npawb ntawm TUNEL-zoo hlwb nrog TissueQuest/HistoQuest software (TissueGnostics).

Tsiaj sim

Txiv neej BN nas (300-350 g lub cev hnyav) tau yuav los ntawm National Laboratory Animal Center (Taipei, Taiwan) thiab khaws cia rau 2-3 lub hlis. Cov nas tau khaws cia rau hauv 12-h / 12-h lub teeb / tsaus voj voog ntawm 26 ± 1 degree, 35-47 feem pua ​​​​ntawm cov av noo piv, thiab ad libitum nkag mus rau dej thiab zaub mov. Cov nas uas tsis kho tau raug khaws cia hauv qhov tsaus ntuj kom ua haujlwm tswj hwm. Cov txheej txheem kev tshawb fawb tau pom zoo los ntawm Pawg Saib Xyuas Kev Saib Xyuas Tsiaj Tsiaj ntawm Taipei Medical University (tus lej pom zoo: LAC-2016-0442). Tag nrho cov txheej txheem kev sim nrog rau kev siv tsiaj txhu ua raws li Lub Koom Haum rau Kev Tshawb Fawb Txog Kev Pom Zoo thiab Kev Tshawb Fawb Txog Kev Pom Zoo rau kev siv tsiaj txhu hauv kev tshawb nrhiav qhov muag pom kev thiab pom kev.

BLL-induced nas qauv ntawm retinal degeneration

Peb tau siv ib lub sij hawm luv luv-luminance ntev BLL raug tus qauv ntawm retinal degeneration, raws li tau piav qhia hauv peb txoj kev tshawb fawb dhau los [19]. Luv luv, BN nas tau muab faib ua peb pawg: pawg tswj hwm, uas tau khaws cia hauv qhov tsaus ntuj; ib pawg BLL-exposed, uas tau nthuav tawm rau lub sijhawm BLL nyob rau hauv qhov tsaus ntuj yam tsis muaj cov tub ntxhais kawm dilation (460 nm, 150 lux) rau 3 teev ib hnub rau 60 hnub; thiab ib pawg CTE-kho ntxiv rau BLL-exposed pab pawg, uas tau kho ua ntej nrog CTE (100 mg / kg lub cev qhov hnyav) rau 14 hnub thiab tom qab ntawd sib koom nrog BLL raug rau 3 teev hauv ib hnub rau 60 hnub. Tag nrho cov nas raug xa rov qab mus rau cov tsiaj txhu thiab lub voj voog lub teeb / tsaus ntuj (250 lux, 12 h / 12 h) tau rov pib dua thaum kawg ntawm lub sijhawm sim.

Kev txheeb cais

Qhov sib txawv tseem ceeb tau suav nrog siv ib txoj kev tsom xam ntawm qhov sib txawv raws li Tukey's post hoc test thiab ntau qhov kev sib piv. Statistical tseem ceeb tau txiav txim los ntawm p-tus nqi ntawm<>

Cistanche tubulosa khoom

Cov txiaj ntsig

CTE tiv thaiv RPE hlwb los ntawm oxidative kev nyuaj siab puas

Txhawm rau ntsuas cytotoxicity ntawm CTE ntawm MTT kev soj ntsuam, ntau qhov ntau ntawm CTE ({{{{20}}}}}-500 ug/mL) tau siv rau RPE hlwb. Kev kho CTE tsis muaj qhov pom tseeb los ntawm RPE cell viability ntawm ib qho ntawm cov kev soj ntsuam (Fig. 1A). Yog li, 50 thiab 100 ug / mL CTE tau siv rau hauv cov kev sim tom ntej. Txhawm rau tshawb xyuas cov teebmeem tiv thaiv apoptotic ntawm CTE, sib txawv oxidative inducers (H2O2, t-BHP, thiab NaN3) tau koom nrog CTE rau RPE hlwb rau 24 teev. H2O2, NaN3, thiab t-BHP txo qis cell viability thiab puas RPE hlwb nyob rau hauv ib koob tshuaj (slashed tuav, Fig. 1B, 1C, thiab 1D); cell viability tau qis dua hauv cov hlwb kho nrog 0.03 mM H2O2, 0.3 mM NaN3, thiab 0.3 mM t-BHP dua li hauv pawg tswj hwm.

Txawm li cas los xij, kev kho nrog 50 thiab 100 ug / mL CTE txo qis H2O2-, NaN3-, thiab t-BHP- induced RPE cell puas thiab thim rov qab qhov txo qis hauv cell viability (dub bars, Fig. . 1B, 1C, and 1D). Cov ntaub ntawv saum toj no qhia tias CTE cawm RPE hlwb los ntawm oxidative kev nyuaj siab puas tsuaj.

CTE inhibits BLL-induced RPE cell tuag

Hauv peb txoj kev tshawb fawb dhau los, peb pom tias BLL yog cytotoxic rau RPE hlwb, uas yog vim muaj kev hloov pauv ntawm Bcl -2 thiab Bax. Hauv kev tshawb fawb tam sim no, peb tau tshuaj xyuas ntxiv txog kev tiv thaiv los ntawm CTE ntawm BLL-induced RPE cell puas. Peb muab ob daim hlau hluav taws xob xiav LED tso rau hauv lub cell culture incubator. Qhov kev ncua deb ntawm lub teeb ci thiab cell daim hlau yog 25 cm, thiab BLL siv yog 60 lux, raws li ntsuas los ntawm lub teeb meter (Fig. 2A). Qhov kev ntsuam xyuas MTT tau pom tias BLL raug rau lub sijhawm sib txawv (6–48 h) ua rau RPE cell tuag nyob rau lub sijhawm (Fig. 2B). BLL-vim txo qis hauv cell viability tom qab 24 thiab 48 teev tau txo qis thaum cov hlwb raug kho nrog CTE (100 ug / mL) (Daim duab 2C). Cov ntaub ntawv no qhia tias CTE tiv thaiv RPE hlwb los ntawm BLL-induced phototoxicity.

CTE inhibits BLL-induced apoptosis hauv RPE hlwb los ntawm kev tswj Bcl-2/Bax thiab Fas/FasL txoj hauv kev

Raws li qhov muaj peev xwm tiv thaiv kev cuam tshuam ntawm CTE hauv RPE hlwb, peb tau tshawb xyuas ntxiv txog qhov muaj feem cuam tshuam ntawm CTE hauv cov txheej txheem no. Ob txoj kev taw qhia apoptotic tau kuaj xyuas, piv txwv li, Bax/Bcl{{0}} thiab Fas/Fas ligand (FasL) txoj kev, thiab cov proteins uas muaj feem xyuam (xws li, pro-caspase-8 thiab pro-caspase -3; Fig. 3A and 3B). BILL exposure (6–48 h) tau pom los txhim kho cov protein qhia ntawm Bax/Bcl-2, FasL, thiab FADD nyob rau lub sijhawm (Daim duab 3A). Ntxiv mus, BLL tau txo qis cov protein qhia ntawm procaspase-3 thiab procaspase-8, uas ua rau muaj ntau cov ntaub ntawv cleavage ntawm caspase-3 thiab caspase-8 tom qab BLL raug, uas zoo ib yam nrog peb. kev kawm yav dhau los [19]. Txhawm rau ntsuas qhov kev tiv thaiv ntawm CTE ntxiv, qhov sib txawv ntawm CTE tau siv rau BLL-exposed RPE hlwb (Fig. 3B). Tom qab 48-h BLL raug, CTE (50 µg/mL) txo Bax/Bcl{21}} piv. Tsis tas li ntawd, CTE (100 µg/mL) txo qis cov protein qhia ntawm FasL thiab FADD, tab sis nce qib ntawm procaspase -8 thiab procaspase -3 (Fig. 3C thiab 3D). Cov kev tshawb fawb tau pom tias caspase-3 ua kom muaj zog yog qhov cim ntawm apoptosis hauv RPE hlwb [20]. Txhawm rau kom paub meej txog qhov kev tiv thaiv ntawm CTE ntxiv, peb tau ua cov tshuaj tiv thaiv kab mob tiv thaiv kab mob ntawm cleaved caspase-3. BILL raug rau 48 h tau pom tias ua rau caspase -3 cleavage thiab translocation rau hauv lub nucleus, ua rau RPE cell apoptosis (daj xub, Fig. 4A). Txawm li cas los xij, thaum BLL-exposed hlwb raug kho nrog CTE (100 µg / mL), BLL-induced qhia ntawm cleaved caspase -3 yog inhibited. Txhawm rau nkag siab txog kev tiv thaiv apoptotic ntawm CTE zoo dua, peb siv TUNEL staining txhawm rau txheeb xyuas DNA tawg hauv RPE hlwb. Tom qab 48-h BLL raug, TUNEL-zoo hlwb tau nce los ntawm 1.78 ± 0.36 feem pua ​​​​rau 23.82 ± 0.33 feem pua ​​; Cov nyhuv no tau txo qis los ntawm kev kho nrog CTE (23.82 ± 0.33 feem pua ​​​​rau 2.22 ± 0.11 feem pua; Fig. 4B, 4C, thiab 4D). Cov txiaj ntsig no qhia tau tias kev kho CTE tiv thaiv RPE cov hlwb los ntawm BLL-induced apoptosis los ntawm kev kho cov Bax/Bcl-2 thiab FasL/FADD txoj hauv kev.

CTE inhibits BLL-induced phosphorylation ntawm kev ntxhov siab cov proteins hauv RPE hlwb

Cov kev tshawb fawb yav dhau los tau pom tias MAPK txoj hauv kev, xws li ERK, JNK, thiab p38 MAP, tau qhib rau hauv RPE hlwb thiab hauv retina tom qab raug ultraviolet lub teeb lossis BL [21, 22]. Yog li, txhawm rau tshawb xyuas cov kev tswj hwm ntawm CTE ntawm MAPK kev ntxhov siab teb txoj hauv kev tom qab BLL raug, 100 µg / mL CTE tau siv rau RPE hlwb nyob rau hauv lub sijhawm sib txawv BLL (Fig. 5A). Western blot tsom xam qhia tias phosphorylated ERK, JNK, thiab p38 tau qhib thaum BLL raug, tab sis cuam tshuam loj heev thaum kho nrog CTE hauv RPE hlwb (Fig. 5B). Cov txiaj ntsig saum toj no qhia tias CTE inhibits BLL-induced RPE cell puas, uas tej zaum yuav cuam tshuam nrog inhibition ntawm MAPK hauv RPE hlwb.

CTE cawm BLL-induced retinal puas tsuaj tom qab lub sij hawm ntev raug rau hauv tus qauv nas

Txhawm rau ntsuas qhov kev tiv thaiv los ntawm CTE, peb tau nthuav tawm BN nas mus rau qhov ntev BLL raug rau 60 hnub thiab ntsuas qhov cuam tshuam ntawm CTE. Raws li pom nyob rau hauv daim duab 6A, BN ​​nas tau muab faib ua peb pawg: pawg tswj, pawg BLL-exposed, thiab CTE ntxiv rau BLL pawg (Fig. 6A). Hauv pawg CTE ntxiv rau BLL, BN nas tau txuas ntxiv nrog CTE (100 mg / kg lub cev hnyav) los ntawm qhov ncauj gavage ib hnub rau 14 hnub ua ntej BLL raug. Hnub 0, BN nas tau raug kho nrog CTE (100 mg / kg lub cev hnyav) thiab raug rau BLL rau 3 teev ib hnub rau 60 hnub. Tom qab 60 hnub ntawm BLL raug, cov tsiaj tau muab txi rau sab hnub poob blot tsom thiab immunohistochemistry staining. Hauv pawg CTE ntxiv rau BLL, qhov kev qhia ntawm procaspase-3 protein nyob rau hauv nas qhov muag homogenates tau siab dua tom qab kev kho CTE dua li hauv pawg BLL (Fig. 6B thiab 6C). Cov ntaub ntawv no qhia tau hais tias CTE tiv thaiv lub sij hawm ntev uas tsis tshua muaj luminance BLL-induced retinal phototoxicity thiab apoptosis los ntawm inhibiting lub activation ntawm cleaved caspase -3. Immunohistochemistry staining muab pov thawj ntxiv hais txog kev tiv thaiv kev tiv thaiv ntawm CTE ntawm cov qauv thiab txoj hauv kev physiological ntawm retina (Fig. 7). Tsis tas li ntawd, tom qab BLL raug peb pom tias cov txheej txheem neuron sab hauv tau ploj zuj zus thiab pom morphological deformations ntawm txheej txheej neuron sab nrauv (ONL), suav nrog ntu sab hauv / sab nrauv nyob rau hauv nruab nrab thiab sab hauv retina, uas zoo ib yam nrog peb txoj kev kawm dhau los ( Fig. 7B). Qhov tseem ceeb, CTE tiv thaiv RPE cell deformation, neuronal cell puas, thiab tag nrho cov thinning ntawm retina nyob rau hauv pawg CTE ntxiv rau BLL (Fig. 7C). Ntxiv mus, peb tsis tau soj ntsuam TUNEL-zoo hlwb nyob rau hauv nruab nrab thiab peripheral retina ntawm ib txwm nas (Fig. 8Ab thiab 8Bb). Hloov pauv, tom qab BLL raug rau 60 hnub, TUNEL-zoo hlwb tau kuaj pom ntau zaus hauv ONL ntawm lub hauv nruab nrab thiab peripheral retina (Fig. 8Ae thiab 8Be). DAPI staining ntawm ONL ntawm lub hauv paus retina tau poob qis hauv pawg BLL (Fig. 8Ad). Txawm li cas los xij, lub xub ntiag ntawm TUNEL-zoo hlwb tau txo qis thiab DAPI staining ntawm nruab nrab thiab peripheral retina ONL tau nce hauv pawg CTE ntxiv rau BLL (Fig. 8Ai thiab 8Bi).

Cistanche tubulosa khoom

Ntu tom ntej Ⅱ yuav tsum tau txuas ntxiv