Phenylethanoid Glycosides Los Ntawm Paraboea Martinii Tiv Thaiv Rat Pheochromocytoma (PC12) Cells Los Ntawm Hydrogen Peroxide-induced Cell Injury
Mar 04, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Xue Gong†a, Yan Xu†b, Kai Renc, Xiaorong Baid
Oxidative stress tau paub tias ua lub luag haujlwm tseem ceeb hauv kev tsim cov kab mob ntawm ntau yam kab mob. Nws txuas nrog rau etiology ntawm Alzheimer's disease (AD), Parkinson's disease (PD), qaug dab peg, thiab lwm yam kab mob [1–3]. Tam sim no, kev kawm txuas ntxiv tau ua tiav hauv txoj kev tshawb fawb ntawm cov kab mob AD, PD, thiab lwm yam kab mob. Txawm li cas los xij, vim tias tsis muaj kev paub txog cov txheej txheem molecular hauv qab AD, PD, thiab lwm yam kab mob, kev tiv thaiv zoo lossis kev kho mob rau cov kab mob no tsis tau paub txog [4]. H2O2 yog ib qho tseem ceeb ntawm cov pa oxygen reactive (ROS). Raws li, H2O2-induced PC12 cell raug mob yog tus qauv siv feem ntau ntawm oxidative kev nyuaj siab thiab yog dav siv nyob rau hauv kev sim kev tshawb fawb ntawm neuroprotective teebmeem [5]. Heme oxygenase 1 (HO-1) thiab glutamate-cysteine ligase-catalytic subunit (GCLC) yog ib qho tseem ceeb ntawm cov cellular antioxidant enzymes, thiab ob lub noob yog tswj hwm downstream ntawm nuclear factor erythroid 2 (Nrf2), uas tau tshwm sim los ua kev cog lus kho mob. Lub hom phiaj rau kev tiv thaiv neuroprotect [6,7]. Phenylethanoid glycosides, muab los ntawm C-6-C-3 chav tsev, muaj ntau pawg phenolic hydroxyl thiab muaj cov tshuaj tiv thaiv antioxidant thiab tiv thaiv kev laus. Ob peb xyoos dhau los, kev tshawb fawb tau qhia tias ntau cov nroj tsuag tshuaj muaj phenylethanoid glycosides uas qhia txog kev ua haujlwm antioxidant tseem ceeb. Piv txwv li, phenylethanoid glycosides muab los ntawmHerba cistanchepaub tias muaj kev tiv thaiv los ntawm kev paub tsis meej hauv AD. Txoj kev tshawb no tau tshawb xyuas cov txheej txheem tiv thaiv cuam tshuam siv AD senescence-accelerated prone nas 8 (SAMP8) qauv. Cov txiaj ntsig tau qhia tias muaj peev xwm ntawm phenylethanoid glycosides rau ameliorate kev paub tsis txaus hauv SAMP8 nas yuav cuam tshuam nrog kev txhawb nqa ntawm synaptic plasticity uas muaj cov txheej txheem antioxidant [8]. Nyob rau hauv txoj kev tshawb no, ob phenylethanoid glycosides tau muab cais thiab huv si los ntawm Tectona grandis (teak) ntoo knots; Cov tebchaw no tau pom cov teebmeem antioxidant uas tau txiav txim siab siv txoj kev amperometric [9]. Tsis tas li ntawd, caffeoyl phenylethanoid glycoside tebchaw los ntawm Lindernia ruellioides siv tshuaj tua kab mob antioxidant thiab superoxide anion dawb radical-scavenging teebmeem hauv vitro [10]. Cov txheej txheem ntawm kev txiav txim ntawm phenylethanoid glycosides muaj feem xyuam rau cov kev cai ntawm Nrf2/ARE signaling pathway, uas cuam tshuam rau cov synthesis ntawm SOD, CAT, GSH-PX, thiab lwm yam antioxidant enzymes [11]. Tsev neeg Gesneriaceae, uas muaj 150 genera thiab kwv yees li 3700 hom, feem ntau yog muab faib rau hauv thaj chaw sov thiab kub ntawm sab hnub tuaj thiab sab hnub tuaj Asia, Oceania, South America, Africa, Southern Europe, thiab Mexico [12].
Gesneriaceae hauv Suav teb. Nws tau tshaj tawm tias kwv yees li 100 hom muaj kev kho mob tau zoo thiab feem ntau ntawm cov no feem ntau yog muab faib rau hauv Guangxi thiab Guizhou xeev [13]. Nyob rau hauv xyoo tas los no, kev tshawb fawb ntawm Gesneriaceae nroj tsuag tau maj mam nce nyob rau hauv ntau qhov chaw ntawm lub ntiaj teb no, thiab ib co tshiab constituents nrog physiological kev ua ub no tau pom [14]. Paraboea martinii (Levl.) Burtt belongs rau Parabola (Gesneriaceae) thiab yog ib tsob nroj herbaceous. Raws li "Cov Ntaub Ntawv Siv Tshuaj Hauv Suav Teb", nws tau sau tseg tias tag nrho cov nroj tsuag siv los ua tshuaj. Tsis tas li ntawd, nws tuaj yeem ua haujlwm ntau yam tshuaj rau cov mob xws li hematemesis, edema, dysentery, raug mob, thiab pob txha [15]. Bai ua pov thawj tias phenylethanoid glycosides yog ib qho ntawm cov tshuaj tseem ceeb ntawm cov nroj tsuag Gesneriaceae [16]. Qee cov kws tshawb fawb kuj tau pom tias phenylethanoid glycosides muaj cov haujlwm tseem ceeb antioxidant [17]. Li qhia tau tias phenylethanoid glycosides ntawmCistanche deserticola have significant antioxidant activity [18]. In addition, Ju systematically studied the neuroprotective activities of phenylethanoid glycosides using PC12 cell models. The results indicated that phenylethanoid glycosides significantly attenuate the damage induced by Aβ1-42 [19]. In our studies, we isolated eight phenylpropanoid glycosides (paraboside A, paraboside B, paraboside I, paraboside II, paraboside III, nuomioside A, caleolarioside B, isonuomioside A) from P. martini [20]. All compounds were isolated from the genus Parabola for the first time, and the compounds paraboside A, paraboside B, p paraboside I, paraboside II, and paraboside III were new compounds [20]. In a continuing search for molecules with antioxidant properties, phenylpropanoid glycosides were examined. However, whether these phenylpropanoid glycosides from P. martini could protect against H2O2-induced PC12 cell injury was previously unclear. In this study, we examined the protective effects of phenylpropanoid glycosides on H2O2-induced damage to PC12 cells. We also examined the effects and preliminary mechanism underlying the activity of phenylpropanoid glycosides with respect to the activation of HO-1 and GCLC in vitro. Materials and methods Materials Paraboea martini (Level.) Burtt was collected from Daxin County, Guangxi Autonomous Region in August 2014 and verified by Dr. Minhui Li (Baotou Medical College). Voucher specimens were deposited at the Laboratory of Pharmacognosy and Phytochemistry. Eight phenylpropanoid glycosides (paraboside A, paraboside B, paraboside I, paraboside II, paraboside III, nuomioside A, caleolarioside B, isonuomioside A) were extracted and separated from P. martinii (Levl.) Burtt by prof. Xiaoqin Wang [20]. The purity (>98 feem pua ) ntawm yim phenylpropanoid glycosides tau ntsuas los ntawm HPLC peak area normalization method. Ua piv txwv, cov kab kos chromatography ntawm paraboside B thiab paraboside II tau qhia hauv daim duab 1, thiab lawv cov ntsiab lus txheeb ze tau pom tias yog 98.23 feem pua thiab 99.20 feem pua. Cov nas tsis sib txawv ntawm cov nas adrenal pheochromocytoma cell kab (PC12) tau yuav los ntawm Cell Bank ntawm Tuam Tshoj Academy of Science (Shanghai, Suav). Dulbecco's modified Eagle's medium (DMEM) and horse serum was purchased from Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS) tau yuav los ntawm Thermo Fisher Scientific (Hyclone, Waltham, MA, USA). H2O2 tau txais los ntawm Tianjin Fu Yu Reagent Co., Ltd. (Tianjin, Suav). Trizol tau yuav los ntawm Invitrogen (Carlsbad, CA, USA). PrimeScriptTM RT reagent Kit (Lub Sijhawm Zoo Tshaj Plaws) tau txais los ntawm TaKaRa Biotechnology Co. (Dalian, Suav). Primers rau HO-1, GCLC, thiab GAPDH tau tsim los ntawm Sangon Biotech (Shanghai, Suav). CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (MTS) tau txais los ntawm Promega Corp. (Madison, USA). Inhibitors ntawm HO-1 zinc protoporphyrin (ZnPP) tau yuav los ntawm Sigma Chemical Co. (St. Louis, MO, USA). Ib Thermo311 CO2 Incubator (Thermo, USA), StepOnePlusTM Real-Time PCR Instrument Thermal Cycling Block (Thermo), Thermo Scientific Multiskan FC (Thermo), thiab xCELLigence RTCA DPlus Analyzer thiab E-Plate 16 (ACEA Biosciences) kuj tau siv, Tebchaws USA. .
Cistanche deserticola
Cell kab lis kev cai thiab kev kho mob
PC12 hlwb tau loj hlob hauv DMEM uas muaj 5 feem pua FBS thiab 5 feem pua nees cov ntshav ntawm 37 degree nyob rau hauv ib tug humidified 5 feem pua CO2 cua. PC12 hlwb tau muab faib ua peb pawg raws li hauv qab no: pawg tswj hwm, pawg qauv, thiab pab pawg kho mob. Yim phenylpropanoid glycosides tau yaj hauv DMSO (< 0.01%)="" and="" then="" diluted="" with="" a="" complete="" culture="" medium.="" to="" study="" the="" effects="" of="" the="" eight="" phenylpropanoid="" glycosides="" on="" h2="" o2-induced="" cell="" injury,="" pc12="" cells="" were="" cultured="" with="" the="" eight="" compounds="" at="" concentrations="" of="" 3.125,="" 6.25,="" 12.5,="" 25,="" and="" 50="" μm="" for="" 12="">
Kev txiav txim siab ntawm H2O2
Peb txiav txim siab qhov tsim nyog concentration ntawm H2 O2 siv real-time cellular tsom xam (RTCA). PC12 hlwb tau cog rau hauv CIM-phaj-16 microplate ntawm qhov ntim ntawm 100 µL thiab tom qab ntawd incubated rau 12 teev ntawm 37 degree hauv qhov chaw ntawm 5 feem pua CO2. PC12 hlwb tau muab faib ua pawg tswj thiab qauv. Tom ntej no, 10 μL H2O2 (50, 100, 200, thiab 400 μM) tau ntxiv rau cov qauv pawg, thiab cov pab pawg tswj tau raug kho nrog 10 μL ua tiav DMEM, teeb tsa hauv peb qhov chaw ua haujlwm. E-Plate 16 tau muab tso rau hauv xCELLigence RTCA, saib xyuas txhua 15 feeb, thiab cov txiaj ntsig tau kaw rau 32 teev. Cov nyhuv ntawm H2O2 ntawm PC12 hlwb tau soj ntsuam siv xCELLigence RTCA cov ntaub ntawv txheeb xyuas software. Lub cell Performance index (CI) tus nqi tau siv los txiav txim siab qhov zoo H2O2 concentration thiab lub sijhawm ua haujlwm.
Compound extraction thiab cais tawm
Cov seem tau muab cais raws li lawv cov polarity siv cov kev piav qhia yav dhau los. Cua qhuav, hmoov P. martini (4 kg) tau ua tiav peb zaug nrog 75 feem pua aqueous ethanol tov [20]. Tom qab ntawd cov extracts tau txais yog concentrated siv lub teb evaporator kom tshem tawm ethanol, thiab cov extracts crude tau txais. Cov qauv no tau yaj hauv dej deionized thiab tom qab ntawd muab rho tawm nrog roj av ether, ethyl acetate, n-butanol, thiab dej. N-butanol extract (400} g) yog fractionated siv AB-8 macroporous resin kem thiab ethanol-H2O2 tov (0, 1{{47} }, 30, 50, 70, thiab 95 feem pua ). Rau feem (feem 1–6) thaum kawg tau. Tshooj 3 thiab 4 tau raug cais los ntawm MCI kem nrog cov kauj ruam-gradient ntawm MeOH-H2O2 (10–100 feem pua). Tom ntej no, cov khoom raug cais tawm siv Sephadex LH-20 nrog 50 feem pua MeOH ua qhov zoo. Cov tshuaj 1 (41.0 mg) thiab 6 (15.6 mg) raug cais tawm ntawm feem 3. Cov tshuaj 2 (62.0 mg), 3 (34.7 mg), 7 (11.0 mg), thiab 8 (26.0 mg) tau los ntawm feem 4 siv Sephadex. LH -20 chromatography nrog 50 feem pua MeOH raws li qhov tseem ceeb. Kev rho tawm zaum kawg tau ua los ntawm ib nrab-prep, thim rov qab-theem HPLC (Merck LiChrosorb, RP-18, RP-18, 250 × 10 mm) nrog 50 feem pua MeOH thiab UV nrhiav tau ntawm 280 nm los ua cov tebchaw 4 (39.0 mg) thiab 5 (9.0 mg) [20].
Cell viability assay
PC12 hlwb tau cog rau hauv 96-cov phiaj zoo ntawm qhov ntom ntawm 2 × 1{{10}}4 hlwb/zoo ntawm qhov kawg ntim ntawm 100 µL . PC12 hlwb raug incubated ntawm 37 degree nrog 5 feem pua CO2 rau 12 h. Tom qab ntawd, cov hlwb raug kho nrog ntau qhov sib txawv ntawm cov khoom siv raw (0.025, 0.05, 0.1, 0.2, thiab 0.4 mg / mL) thiab cov tshuaj 1, 2, 3, 4, 5, 6, 7, thiab 8 ntawm tsib concentrations (3.125, 6.25, 12.5, 25, thiab 50 μM). Tom qab pretreatment rau 24 teev, cell viability tau txiav txim los ntawm MTS kev soj ntsuam. PC12 hlwb raug preincubated nrog los yog tsis muaj HO-1 inhibitor yam tsis muaj ZnPP (15 μM) rau 30 min, ces incubated nrog los yog tsis muaj caleolarioside B, paraboside B, thiab paraboside II (3.125 μM) rau 12 h, thiab thaum kawg incubated nrog 400 μM H2O2 rau lwm 6 h. Tom qab kev kho mob, tus naj npawb ntawm cov ciaj sia taus tau txiav txim siab los ntawm MTS kev soj ntsuam [6].
Kev tiv thaiv los ntawm crude extracts thiab monomeric compounds ntawm H2O2- kho PC12 hlwb
Cov hlwb raug coj mus rau hauv ib qho {{0}}zoo phaj ntawm qhov kawg ntim ntawm 100 µL. Serial dilutions ntawm crude extracts (0.025, 0.05, 0.1, 0.2, thiab 0.4 mg/mL) tau ntxiv rau pawg kho. Tom qab pretreatment rau 12 h, 400 μM ntawm H2O2 tov (qhov zoo tshaj plaws concentration) tau ntxiv rau cov pab pawg kho mob thiab H2O2 pawg ntawm txhua qhov dej rau 6 h. MTS tov tau ntxiv rau txhua lub qhov dej, thiab cov phiajcim tau muab tso rau hauv qhov cub rau 3 teev ntawm 37 degree C. Cov phaj tau oscillated ntawm qis ceev rau 5 min ntawm chav tsev kub kom txog thaum tag nrho cov crystals tau yaj tag. Kev tiv thaiv ntawm tes tau txiav txim siab raws li MTS kev soj ntsuam cov txiaj ntsig raws li cov chaw tsim khoom 2204 X. GONG ET AL. Tau txais los ntawm https://academic.oup.com/bbb/article/83/12/2202/6044145 los ntawm qhua ntawm 27 Lub Yim Hli 2021 cov lus qhia. Qhov ntom ntom ntawm qhov muag tau txiav txim siab ntawm qhov nqus dej ntawm 490 nm. Peb ntxiv cov kev ua ntawm cov tebchaw 1, 2, 3, 4, 5, 6, 7, thiab 8 cais los ntawm feem 3 thiab 4. Serial dilutions ntawm cov tebchaw 1, 2, 3, 4, 5, 6, 7, thiab 8 (3.125, 6.25, 12.5, 25, thiab 50 μM) tau ntxiv rau pawg kho mob los txiav txim siab txog lawv qhov kev tiv thaiv ntawm H2O2-induced PC12 hlwb.
phenylethanoid glycosides los ntawm Cistanche deserticola: Tiv thaiv neurons & tiv thaiv kev laus
Quantitative real-time PCR (qRT-PCR)
PC12 hlwb raug sau thaum 12 teev tom qab kho nrog 3.125 μM caleolarioside B, paraboside B, thiab paraboside II. Tag Nrho RNA raug rho tawm siv TRIzol-reagent, thiab aliquots ntawm tag nrho RNA tau thim rov qab siv Primescript RT Master Kit raws li cov chaw tsim khoom cov lus qhia. Cov primers hauv qab no tau ua haujlwm rau kev sim ua raws li daim ntawv tshaj tawm dhau los [21]. HO-1: Forward 5′ -GC CTGCTAGCCTGGTTCAAG-3′, Rov qab 5′ -AGC GGTGTCTGGGATGAACTA-3′; GCLC: Forward 5′ - GTCCTCAGGTGACATTCCAAGC-3′, Rov qab 5′ -TG TTCTTCAGGGCTCCAGTC-3′; GAPDH: Forward 5′-AAGCTGGTCATCAACGGGAAAC-3′, Reverse 5′- GAAGACGCCAGTAGACTCCACG-3′. Aliquots ntawm cov tau txais cDNAs tau nthuav dav los ntawm PCR. Cov kev mob tshwm sim tau raws li hauv qab no: pib denaturation ntawm 95 degree (30 s), ua raws li 40 lub voj voog ntawm 95 degree (5 s), 60 degree (34 s), thiab 72 degree (35 s). Tag nrho cov primers raug kuaj thiab kuaj pom FL fluorescent teeb liab; Tom qab ntawd, tus nqi △Ct raug xam thiab cov nqi txheeb ze tau muab piv rau cov pawg tswj hwm siv cov kab zauv hauv qab no: △Ct=Ct (piv txwv) − Ct (endogenous tswj), △△Ct {{36} } △Ct (cov qauv) −△Ct (tsis kho), thiab hloov pauv=2−△△Ct. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tau siv los ua kev tswj hwm sab hauv.
Protein extraction
PC12 hlwb tau cog rau ntawm 100- hli lauj kaub tais diav ntawm 1 × 107 hlwb / phaj. Tom qab kev sib txuas, cov hlwb raug kho nrog caleolarioside B, paraboside B, thiab paraboside II (3.125 µM) rau 12 teev. Tom qab ntawd cov glycosides raug tshem tawm, thiab cov hlwb raug tsim nrog H2O2 rau lwm 6 teev. Tom qab incubation, lub hlwb raug ntxuav ob zaug nrog txias PBS thiab scraped los ntawm cov tais diav nrog 1 mL ntawm PBS. Cell homogenates tau centrifuged ntawm 1500 rpm rau 10 min. Tom qab kev kho mob, cellular proteins tau muab rho tawm siv Beyotime RIPA cell lysis buffer nrog 1 mM PMSF raws li cov chaw tsim khoom cov lus qhia. Tsis tas li ntawd, cytoplasmic thiab nuclear proteins raug cais raws li tau piav qhia hauv Beyotime nuclear thiab cytoplasmic extraction kit raws tu qauv. Cov protein ntau ntawm cov qauv tau kuaj pom siv cov khoom siv tshuaj ntsuam xyuas Beyotime BCA protein, thiab tag nrho cov qauv raug khaws cia ntawm −80 degree rau kev tsom xam sab hnub poob [6].
Western blot tsom xam
SDS-PAGE electrophoresis tau ua tom qab rho tawm thiab tom qab denaturation ntawm tag nrho cov protein los ntawm ntau pawg. Tom qab electrophoresis, cov protein tau pauv mus rau PVDF daim nyias nyias, uas tau raug thaiv nrog 5 feem pua ntawm cov mis nyuj hmoov rau 4 teev. Tom qab ntxuav, thawj cov tshuaj tiv thaiv (luv polyclonal antibody GAPDH (1: 10, 000 dilution), luav polyclonal antibody HO-1 (1: 1000 dilution), los yog luav polyclonal antibody GCLC (1: 1000 dilution) tau muab ntxiv rau hauv daim nyias nyias, uas tau muab tso rau ib hmos ntawm 4 degree. Hnub tom qab, PVDF daim nyias nyias tau ntxuav peb zaug hauv TTBS. horseradish peroxidase tau ntxiv rau hauv daim nyias nyias, uas tau muab tso rau hauv chav sov li 2 h. Cov PVDF daim nyias nyias tau ntxuav thiab raug rau ECL chemiluminescence detection reagent rau signaling raug rau X-ray zaj duab xis, uas yog ces tsau thiab scanned [22].
Kev txheeb cais
Cov ntaub ntawv tau raug tshuaj xyuas siv SPSS 19.0, thiab cov txiaj ntsig tau nthuav tawm raws li qhov txhais tau tias nrog tus qauv sib txawv (SD) ntawm peb qhov kev sim ywj pheej. Tus nqi ntawm p < 0.05="" raug="" txiav="" txim="" siab="" los="" qhia="" txog="" qhov="" sib="" txawv="" tseem="">
Cov txiaj ntsig Tsim H2O2 qauv
Raws li pom hauv daim duab 2, H2O2 ntawm 50, 100, thiab 200 μM tsis ua rau cov qib tsim nyog ntawm cell tuag. Txawm li cas los xij, 400 μM H2O2 ua rau PC12 hlwb hauv 2-6 h, thiab oxidative puas hlwb rau cov hlwb zoo li ruaj khov. Yog li ntawd, peb xav tias induction nrog H2O2 ntawm ib tug concentration ntawm 400 μM rau 6 h yog tsim nyog rau mimicking oxidative stress-induced raug mob rau PC12 hlwb.
Kev tiv thaiv los ntawm crude extracts ntawm PC12 hlwb kho nrog H2O2
Piv nrog rau qhov ntawd hauv pawg qauv H2O2 (Daim duab 3), n-butanol extract ntawm qhov ntau ntawm 0.05, 0.1, {{10} }.2, thiab 0.4 mg/mL tseem ceeb txhim kho oxidative puas tsuaj rau PC12 hlwb induced los ntawm H2O2 (p < 0.05)="" nyob="" rau="" hauv="" ib="" tug="" concentration-dependent="" yam.="" cov="" ntaub="" ntawv="" no="" tau="" qhia="" tias="" cov="" ntu="" n-butanol="" muaj="" cov="" kev="" tiv="" thaiv="" muaj="" zog="" dua.="" txawm="" li="" cas="" los="" xij,="" petroleum="" ether="" thiab="" ethyl="" acetate="" extracts="" pom="" muaj="" kev="" tiv="" thaiv="" tsuas="" yog="" nyob="" rau="" hauv="" siab="" concentrations="" (petroleum="" ether:="" 0.1="" thiab="" 0.2="" mg/ml,="" p="">< 0="" 0).="" 5;="" ethyl="" acetate:="" 0.2="" thiab="" 0.4="" mg/ml,="" p=""><0.05). ntxiv="" mus,="" qhov="" sib="" txawv="" ntawm="" qhov="" sib="" txawv="" ntawm="" aqueous="" theem="" rho="" tawm="" tsis="" pom="" muaj="" kev="" tiv="" thaiv="" (p=""> 0.05). Tsis tas li ntawd, peb tau ntxiv cov ntaub ntawv los ntawm kev tshawb fawb siv tau siv SH-SY5Y human neuroblastoma cell kab (SH-SY5Y) hauv Cov Ntaub Ntawv Ntxiv. Kev kho mob nrog 250 μM H2O2 ua rau txo qis cell viability; Txawm li cas los xij, caleolarioside B, paraboside B, thiab paraboside II (3.125, 6.25, 12.5, 25, thiab 50 μM) kev kho mob tseem ceeb txo qis SH-SY5Y cell tuag. Qhov kev sim tau ua pov thawj tias caleolarioside B, paraboside B, thiab paraboside II tiv thaiv SHSY5Y hlwb los ntawm H2O2-induced cell raug mob.
Cov qauv ntawm cov tebchaw cais tawm los ntawm n-butanol feem
Txhawm rau kom ntseeg tau tias cov n-butanol extract muaj cov khoom nquag, peb ntxiv fractionated yim lub tebchaw los ntawm kev rho tawm. Cov qauv ntawm cov khoom sib txuas 1, 2, 3, 4, 5, 6, 7, thiab 8 tau txheeb xyuas los ntawm cov ntaub ntawv sib txawv (1 H-NMR, 13C-NMR, thiab ESI-MS) raws li kev sib piv rau cov ntaub ntawv luam tawm rau paraboside A , paraboside B, paraboside I, paraboside II, paraboside III, nuomioside A, caleolarioside B, thiab isnuomioside A, raws li [20]. Piv txwv li, cov ntaub ntawv rau paraboside B yog raws li nram no: cov pos hniav xim av; ½ 20 D −55.7 (c 0.05, CH3OH) [20]; UVMeOH max nm (logε): 220 (3.61), 291 (3.54), 330 (3.66) [20]; IRKBr max cm-1 : 3392, 2943, 2885, 1699, 1683, 1606, 1521, 1361, 1282, 1163, 1114, 1039, 995, 817 cm; rau 1 H thiab 13C NMR spectroscopic ncauj lus kom ntxaws cov ntaub ntawv qhia nyob rau hauv siv 20; HR-ESI-MS (negative) m/z 741.2231 [MH]− (calcd. for C33H41O19, 741.2242) [20]. Cov ntaub ntawv rau paraboside II yog raws li nram no: dawb amor phous hmoov; ½ 20 D −80.5 (c 0.05, CH3OH) [20]; UVMeOH max nm (logε): 229 (3.74), 322 (3.73) [20]; IRKBr max cm-1 : 3419, 1699, 1625, 1596, 1515, 1456, 1419, 1263, 1159, 1139, 1068, 1022, 808 cm-1 [20]; rau 1 H thiab 13C NMR spectroscopic ncauj lus kom ntxaws cov ntaub ntawv qhia nyob rau hauv siv 20; ESI-MS (tsis zoo) m/z 637
Kev tiv thaiv ntawm cov tshuaj sib cais tawm tsam H2 O2-induced toxicity hauv PC12 hlwb
MTS kev soj ntsuam tau siv los tshawb xyuas cov txiaj ntsig ntawm cov tshuaj sib cais ntawm qhov muaj peev xwm ntawm PC12 hlwb raug rau H2O2-induced toxicity. Raws li pom nyob rau hauv daim duab 5, piv rau cov tswj pawg, PC12 hlwb pretreated nrog tsib txawv concentrations ntawm caleolarioside B, paraboside B, paraboside II, los yog paraboside A (3.125, 6.25, 12.5, 25, thiab 50 μM) rau 24 h tsis pom qhov sib txawv tseem ceeb hauv cell viability (p> 0.05). Yog li, pretreatment ntawm PC12 hlwb nrog caleolarioside B, paraboside B, thiab paraboside II ua rau tsis muaj cytotoxicity. Raws li pom nyob rau hauv daim duab 6, 400 µM H2O2 txo qis cell viability piv nrog rau hauv pawg tswj. Ntxiv mus, lub cell viability ntawm H2O2- kho PC12 hlwb tau nce ntxiv tom qab pretreatment ntawm PC12 hlwb nrog ntau qhov sib txawv ntawm caleolarioside B, paraboside B, thiab paraboside II; Ntxiv mus, cell viability yog qhov siab tshaj tom qab kev kho mob nrog 50 µM caleolarioside B, 50 µM paraboside B, thiab 12.5 µM paraboside II. Lwm tsib lub tebchaw tsis pom muaj kev tiv thaiv tseem ceeb ntawm H2O2- kho PC12 hlwb. Txhawm rau piav qhia qhov kev tshawb pom no, peb tham txog ib qho ntawm cov piv txwv no, qhov twg lwm tus tsis tau piav qhia hauv phau ntawv sau.
Kev hloov pauv qib ntawm HO-1 thiab GCLC
Peb tom ntej no tau sim cov qib transcriptional ntawm HO{{0}} thiab GCLC hauv PC12 hlwb kho nrog caleolarioside B, paraboside B, thiab paraboside II. Tag nrho mRNA tau sau los ntawm kev kho thiab tsis kho PC12 hlwb rau 12 teev thiab tom qab ntawd raug rau kev tshuaj xyuas los ntawm qRT-PCR. Raws li pom nyob rau hauv daim duab 7(a,c,e), cov qib transcriptional ntawm HO-1 tau nce ntau tom qab PC12 hlwb raug pretreated nrog qhov qhia tau hais tias concentrations ntawm caleolarioside B, paraboside B, thiab paraboside II rau 12 h (p < 0="" 05).="" raws="" li="" pom="" nyob="" rau="" hauv="" daim="" duab="" 7(b),="" cov="" transcriptional="" theem="" ntawm="" gclc="" kuj="" nce="" ho="" nce="" nyob="" rau="" hauv="" pc12="" hlwb="" pretreated="" nrog="" cov="" qhia="" concentrations="" ntawm="" caleolarioside="" b="" rau="" 12="" h.="" txawm="" li="" cas="" los="" xij,="" theem="" ntawm="" gclc="" hauv="" pc12="" hlwb="" ua="" ntej="" kho="" nrog="" paraboside="" b="" rau="" 12="" h="" poob="" qis="" dua="" piv="" rau="" cov="" hauv="" pawg="" qauv="" (p=""><0.05; daim="" duab="" 7(d)).="" tsis="" tas="" li="" ntawd,="" nrog="" kev="" kho="" paraboside="" ii,="" qib="" ntawm="" gclc="" hauv="" pc12="" hlwb="" tsis="" sib="" txawv="" heev="" piv="" rau="" cov="" hauv="" pawg="" qauv="" (p=""> 0.05; Daim duab 7(f)).
Kev nthuav qhia ntawm HO-1 thiab GCLC
HO{{0}} thiab GCLC yog cov cellular antioxidant enzymes tseem ceeb, thiab ob qho tib si yog Nrf2- tswj cov noob caj noob ces. Cov protein qhia ntawm HO-1 thiab GCLC kuj tau pom tom qab kho [6]. Daim duab 8 qhia txog qib protein ntawm HO-1 thiab GCLC hauv PC12 hlwb tom qab kho nrog 400 μM H2O2; Cov txiaj ntsig tau qhia tias FL fluorescent intensities ntawm HO-1 hauv caleolarioside B thiab paraboside B pawg tau ntau dua li cov hauv H2O2- pab pawg kho (Daim duab 8(a,c)). Txawm li cas los xij, qhov kev qhia ntawm GCLC tsis tau hloov pauv los ntawm cov tebchaw, tshwj tsis yog caleolarioside B (Daim duab 8(b)). Tom ntej no, cov tshuaj inhibitors ntawm HO-1 tau siv los ntsuas ntxiv lub luag haujlwm ntawm cov enzymes antioxidant hauv kev tswj cov kev tiv thaiv los ntawm caleolarioside B, paraboside B, lossis paraboside II tiv thaiv H2O2-induced cytotoxicity. Caleolarioside B, paraboside B, thiab paraboside II (3.125 µM) tiv thaiv H2O2-induced cytotoxicity, tab sis cov kev tiv thaiv zoo li no tau thim rov qab los ntawm HO-1 inhibitor ZnPP (p < 0.01)="" ntawm="" 15="" µm="" (daim="" duab="" 8.="">
Cov txiaj ntsig ntawm Cistanche: Ua kom txiv neej yug me nyuam
Kev sib tham
Kev laus ntawm pej xeem yog ib qho teeb meem loj thoob ntiaj teb. Ntau yam kab mob muaj feem xyuam rau kev laus, xws li AD thiab PD, uas yog kev hem thawj rau pej xeem noj qab haus huv. AD thiab PD tau tshwm sim los ua tus thib peb cov kab mob tuag coob tshaj plaws hauv ntiaj teb, tom qab kab mob plawv thiab mob qog noj ntshav. Yog li ntawd, neurodegeneration tau dhau los ua cov ncauj lus tshawb fawb ceev thoob ntiaj teb. Txawm li cas los xij, pathogenesis ntawm AD thiab PD, cov kab mob neurodegenerative ntawm lub hauv paus paj hlwb, tseem tsis tau paub meej txog hnub no. Tsis tas li ntawd, tsuas muaj ob peb yam tshuaj uas tuaj yeem kho AD lossis PD zoo vim lawv cov txheej txheem pathogenic nyuaj [23]. Cov kev tshawb fawb yav dhau los tau qhia tias ROS, xws li superoxide anion (O2−) thiab H2O2, yog qhov tseem ceeb ua rau cov kab mob neurodegenerative. Pathological hloov thaum lub sij hawm PD suav nrog neuronal degeneration thiab tuag ntawm substantia nigra thiab striatal dopamine [24,25]. Yog li ntawd, nws yog ib qho tseem ceeb los tsim cov tshuaj antioxidant zoo los kho PD, thiab kev tshawb pom cov tshuaj no tau dhau los ua ib qho kev tshawb fawb tseem ceeb [26]. Tam sim no, Nrf2/ARE qhia txoj hauv kev zoo nkaus li yog qhov tseem ceeb tshaj plaws endogenous antioxidant txoj kev ntxhov siab [27]. Ares nyob rau hauv 5′- ua kom tiav thaj tsam ntawm cov noob, xws li HO-1 thiab GCLC [28,29]. HO-1 thiab GCLC yog Nrf2- cov noob muaj feem cuam tshuam uas tau tswj hwm qis ntawm cov protein no [6]. AREs tuaj yeem cuam tshuam rau qib ntawm cov tshuaj tua kab mob antioxidant thiab ua kom cov dej ntws qis ntawm HO-1 thiab lwm yam tshuaj tiv thaiv, uas tuaj yeem txhim kho kev tiv thaiv ntawm tes [30,31]. Tsis tas li ntawd, kev tshawb pom pom tau hais tias qhov kev tiv thaiv ntawm resveratrol tiv thaiv A {{2{{5{54}}}}}} induced toxicity hauv PC12 hlwb tshwm sim los ntawm upregulation ntawm HO-1 qhia los ntawm kev ua kom Nrf2 intracellular. signaling pathway [32] .PC12 hlwb yog cov qauv lees paub ntawm cov hlwb neural thiab tau siv dav hauv cov kev tshawb fawb neuroprotective effect [33]. H2O2 tuaj yeem yooj yim dhau los ntawm cov cell membrane thiab ua ke nrog hlau los tsim cov dawb radicals uas ua rau muaj kev ntxhov siab oxidative thiab cell raug mob [34]. H2O2 yog ib qho tseem ceeb ROS, thiab cov dawb radicals tau yooj yim tsim thiab ruaj khov, yog li, lawv feem ntau yog siv los tsim cov qauv kev ntxhov siab oxidative siv PC12 hlwb [5]. Nyob rau hauv txoj kev tshawb no, pretreatment ntawm PC12 hlwb nrog nontoxic concentrations ntawm cov nroj tsuag extract tiv thaiv hlwb los ntawm H2O2-induced cytotoxicity los ntawm txo cov tiam ntawm ROS. Phenylethanoid glycosides muaj ntau phenolic hydroxyl pawg uas muaj cov tshuaj tiv thaiv antioxidant thiab tiv thaiv kev laus. Hauv txoj kev tshawb no, peb pom tias n-butanol extraction tsim kev tiv thaiv zoo dua li lwm cov polarity feem (Daim duab 3). Yog li, peb tau soj ntsuam ntxiv cov kev tiv thaiv ntawm yim lub tebchaw uas cais tawm ntawm n-butanol feem. Cov txiaj ntsig tau pom thawj zaug uas daim duab 8. Kev soj ntsuam ntawm HO-1 thiab GCLC protein ntau ntawm PC12 hlwb. (a) Western blotting tsom xam ntawm kev qhia ntawm HO-1 thiab GCLC thiab GAPDH tau siv los ua kev tswj xyuas kev thauj khoom. (b) Western blot tsom xam GCLC protein qhia. (c) Western blot tsom xam HO-1 protein qhia. (d) HO-1 inhibitor ZnPP txo cov nyhuv neuroprotective. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± SD, n=3. ***p < 0.001="" pawg="" tswj="" hwm;="" #p=""><0.05 piv="" rau="" h2o2="" pab="" pawg="" (tsis="" muaj="" znpp="" kho),="" ##p=""><0.01 piv="" rau="" h2o2="" pawg="" (tsis="" muaj="" znpp="" kho),="" ###p=""><0.001 tiv="" thaiv="" h2o2="" pawg="" (tsis="" muaj="" znpp="" kho);="" thiab="" p=""><0.01, piv="" rau="" h2o2="" pawg="" (nrog="" znpp="" kho),="" thiab="" &="" p=""><0.001, piv="" rau="" h2o2="" pawg="" (nrog="" znpp="" kho).="" (control="" pawg;="" pawg="" qauv:="" h2o2;="" znpp="" pawg="" tsis="" zoo:="" caleolarioside="" b="" ntxiv="" rau="" h2o2,="" paraboside="" b="" ntxiv="" rau="" h2o2,="" paraboside="" ii="" ntxiv="" rau="" h2o2;="" ntxiv="" rau="" h2o2).="" 2210="" x.="" gong="" et="" al.="" downloaded="" los="" ntawm="" https://academic.oup.com/bbb/article/83/12/2202/6044145="" los="" ntawm="" qhua="" ntawm="" 27="" lub="" yim="" hli="" 2021caleolarioside="" b,="" paraboside="" b,="" thiab="" paraboside="" ii="" muaj="" kev="" tiv="" thaiv="" tseem="" ceeb="" tiv="" thaiv="" h2o2-="" induced="" oxidative="" kev="" puas="" tsuaj="" hauv="" pc12="" hlwb.="" cov="" txiaj="" ntsig="" kuj="" tau="" qhia="" tias="" kev="" kho="" h2o2="" ua="" rau="" pc12="" raug="" mob="" tseem="" ceeb="" thiab="" txo="" qis="" ntawm="" cell="" viability,="" thaum="" caleolarioside="" b,="" paraboside="" b,="" thiab="" paraboside="" ii="" kho="" tau="" txo="" qis="" cov="" teebmeem="" no="" (daim="" duab="" 6).="" ib="" txoj="" kev="" tshawb="" fawb="" yav="" dhau="" los="" tau="" tshaj="" tawm="" tias="" txoj="" hauv="" kev="" nrf2/are="" yog="" ib="" txoj="" hauv="" kev="" tseem="" ceeb="" tshaj="" plaws="" endogenous="" antioxidant="" txoj="" hauv="" kev,="" thiab="" tuaj="" yeem="" txhim="" kho="" qhov="" kev="" qhia="" ntawm="" cov="" cim="" hauv="" qab,="" xws="" li="" ho-1,="" los="" tiv="" thaiv="" cov="" hlwb="" [7].="" cov="" txiaj="" ntsig="" ntawm="" qrt-pcr="" thiab="" western="" blotting="" tsom="" xam="" pom="" tias="" caleolarioside="" b,="" paraboside="" b,="" thiab="" paraboside="" ii="" tau="" nce="" qib="" ntawm="" ho{102}}.="" txawm="" li="" cas="" los="" xij,="" qib="" transcriptional="" ntawm="" gclc="" tau="" nce="" ntau="" hauv="" pc12="" hlwb="" pretreated="" nrog="" caleolarioside="" b="" tab="" sis="" txo="" qis="" hauv="" cov="" pretreated="" nrog="" paraboside="" b="" piv="" rau="" cov="" hauv="" pawg="" qauv.="" tsis="" tas="" li="" ntawd,="" paraboside="" ii="" tsis="" tau="" hloov="" qhov="" kev="" qhia="" ntawm="" tus="" cim="" no,="" piv="" rau="" qhov="" hauv="" pawg="" qauv="" (figures="" 7="" thiab="" 8(a–c)).="" tsis="" tas="" li="" ntawd,="" kev="" tiv="" thaiv="" los="" ntawm="" caleolarioside="" b,="" paraboside="" b,="" thiab="" paraboside="" ii="" tiv="" thaiv="" h2o2-induced="" pc12="" cell="" raug="" mob="" raug="" thim="" rov="" qab="" los="" ntawm="" ho-1="" inhibitor="" znpp="" (daim="" duab="" 8(d)).="" hauv="" kev="" xaus,="" phenylethanoid="" glycosides="" caleolarioside="" b,="" paraboside="" b,="" thiab="" paraboside="" ii="" zoo="" tiv="" thaiv="" pc12="" hlwb="" los="" ntawm="" h2o2-induced="" oxidative="" puas.="" yog="" li,="" cov="" tshuaj="" no,="" cais="" tawm="" los="" ntawm="" p.="" martini,="" tuaj="" yeem="" sawv="" cev="" rau="" cov="" neeg="" siv="" tshuaj="" muaj="" peev="" xwm="" los="" kho="" cov="" kab="" mob="">
Qhov txiaj ntsig ntawm Cistanche:kho cov kab mob neurodegenerative
Cov ntaub ntawv
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