Phenylethanoid Glycosides Ntawm Cistanche Deserticola: Mechanism Ntawm Cov nyhuv Neuroprotective

Maiquan Li, et al

Abstract:Nuclear factor erythroid 2-related factor 2 (Nrf2) yog qhov tseem ceeb ntawm kev hloov pauv ntawm oxidative stress thiab neurodegenerative disorders.Phenylethanoid glycosides(PhGs; salidroside,acteoside, isoacteoside, thiabechinacoside cov tshuaj) muaj antioxidant thiabneuroprotectivebioactivities. Qhov kev tshawb fawb no tau ua los tshawb nrhiav qhov tseebneuroprotectivecov nyhuv thiab molecular mechanism ntawmPhGs. PhGspretreatment ho suppressed H2O2-induced cytotoxicity nyob rau hauv PC12 hlwb los ntawm triggering lub nuclear translocation ntawm Nrf2 thiab reversing lub downregulated protein qhia ntawm heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1 ), glutamate-cysteine ​​ligase-catalytic subunit (GCLC), thiab glutamate-cysteine ​​ligase modifier subunit (GCLM). Nrf2 siRNA los yog HO-1 inhibitor zinc protoporphyrin (ZnPP) txo covneuroprotectivenyhuv. PhGs tau pom muaj peev xwm sib cuam tshuam nrog Nrf2 qhov chaw khi hauv Kelch-zoo li ECH-kev koom tes protein 1 (Keap1). Qhov txiaj ntsig no tuaj yeem txhawb nqa qhov kev xav tias PhGs yog cov neeg ua haujlwm ntawm Nrf2. Peb tau pom tias muaj peev xwm khi ntawm PhGs thiab Keapl-activated Nrf2/ARE txoj hauv kev, thiab tias PhGs nrog ntau glycosides tau txhim kho cov teebmeem.

Ntsiab lus:Keap1; Nrf2;Neuroprotective; PC12 hlwb;PhGs(NQO1), and others [5-7]. Cov txheej txheem no sawv cev rau cov kauj ruam tseem ceeb hauv kev tiv thaiv cov hlwb los ntawm oxidative kev nyuaj siab thiab tau tshwm sim los ua lub hom phiaj kho mob zoo rau neuroprotection [8]. Gaia tau tshaj tawm tias Nrf2 txo qis LRRK2- thiab a-synuclein-induced neurodegeneration los ntawm kev txhawb nqa neuronal protein homeostasis hauv cell-autonomous thiab nyob ntawm lub sijhawm [9].

Phenylethanoid glycosides (PhGs) yog tus cwj pwm los ntawm cinnamic acid thiab hydroxyl phenyl ethyl moieties txuas rau ap -glucopyranose los ntawm ester thiab glycosidic linkages, feem. Cov molecules no yog cov tswv cuab ntawm ib pawg ntawm cov dej-soluble natural khoom dav faib nyob rau hauv lub nceeg vaj cog [10]. Hauv vitro thiab hauv vivo kev tshawb fawb tau pom tias cov tshuaj no muaj antioxidant [11],neuroprotective[12], tshuaj tua kab mob,

Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Cistanche muaj cov khoom tiv thaiv neuroprotective, nyem qhov no kom paub ntau ntxiv

1. Taw qhia

Oxidative kev nyuaj siab, uas yog qhov tsis txaus ntawm cov tshuaj tiv thaiv antioxidant homeostasis, induces lipid peroxidation, raug mob rau cov protein thiab DNA, cell aging, thiab cell tuag. Cov txheej txheem no yuav ua rau muaj ntau yam kab mob neurodegenerative, xws li Alzheimer's disease (AD), Parkinson's disease (PD), thiab ischemia / reperfusion [1]. Hydrogen peroxide (H2O2), uas yog ib qho tseem ceeb ntawm cov pa oxygen reactive (ROS), paub tias ua rau lipid peroxidation thiab DNA puas [2]. Ntxiv mus, H2O2 yog ib qho chaw endogenous ntawm hydroxyl dawb radicals uas ua rau cov theem tom ntej ntawm cellular oxidative stress [3,4]. Yog li, cov tswv yim kho mob rau kev tiv thaiv oxidative kev ntxhov siab vim apoptosis tuaj yeem muaj peev xwm hauv kev kho mob neurodegenerative.

Nuclear factor erythroid 2-related factor 2 (Nrf2), yog ib qho kev hloov pauv uas cuam tshuam nrog kev ntxhov siab oxidative. Kev ua kom Nrf2 ua rau muaj kev hloov pauv ntawm ntau cov tshuaj tiv thaiv kab mob antioxidant thiab detoxification noob, suav nrog heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1

anti-inflammatory [13], thiab immunomodulatory [14] bioactivities. Osmanthusfragrans yog ib qho khoom muaj nyob hauv ntau cov zaub mov Esxias thiab tau siv ntev. Peb yav dhau los tau pom tias O.fragrans paj rho tawm txhim kho kev kawm spatial thiab nco, inhibited oxidative puas tsuaj, thiab nthuav tawm cov kev tiv thaiv neuroprotective hauv d-galactose-induced aging hauv ICR nas qauv [15]. Salidroside, acteoside, thiab isoacteoside yog cov lus teb PhGs loj rau cov haujlwm antioxidant ntawm O.fragrans paj rho tawm [16].

Kev tshawb fawb txog cov nyhuv neuroprotective ntawm PhGs tau txais txiaj ntsig zoo. Salidroside txo qis cell apoptosis ntawm PC12 hlwb uas tau nthuav tawm hauv MPP ntxiv [17,18]. Acteoside kuj txo MPP ntxiv rau -induced apoptosis thiab oxidative stress nyob rau hauv PC12 hlwb [19] thiab A p25-35-induced SH-SY5Y cell raug mob [20].Echinacosidetau tshawb xyuas ntawm qog necrosis factor-a (TNFa)-induced apoptosis hauv SH-SY5Y hlwb [21], MPTP-induced dopaminergic toxicity hauv nas [22], glutamate-injured thawj kab lis kev cai ntawm nas cortical hlwb [23], thiab {{ 9}}OHDA-vim kev puas tsuaj hauv PC12 hlwb [24]. Cov txiaj ntsig tau qhia tias PhGs tau nthuav tawm cov txiaj ntsig cytoprotective thiab yog cov neeg ua haujlwm muaj peev xwm los kho cov kab mob neurodegenerative. Cov kev tshawb fawb tau pom cov khoom antioxidant ntawm PhGs hauv qab ntau lwm yam bioactivities rau cov tebchaw no [25]. Txawm li cas los xij, qee qhov kev tshawb fawb tau tshawb xyuas cov txheej txheem molecular ntawm PhGs tiv thaiv oxidative toxicity.

Hauv peb txoj kev tshawb fawb, peb xaiv plaub yam PhGs raws li hauv qab no: salidroside (phenylethanoid monosaccharides), acteoside (phenylethanoid disaccharides), isoacteoside (phenylethanoid disaccharides), thiabechinacoside cov tshuaj(phenylethanoid trisaccharides). Peb siv tus qauv ntawm kev tuag neuronal siv cov PC12 sib txawv [26] los tshawb xyuas cov txiaj ntsig tiv thaiv thiab cov txheej txheem molecular ntawm PhGs ntawm H2O2-induced PC12 cell model. Peb tau pom tias PhGs tau qhib txoj hauv kev Nrf2/ARE los ntawm kev khi rau Kelch-zoo li ECH-koom nrog protein 1 (Keap1). Cov txheej txheem no txhawb nqa cov enzymes antioxidant thiab ua rau muaj zog ntawm PC12 hlwb rau oxidative kev nyuaj siab.

2. Cov txiaj ntsig

2.1. PhGs Txhaum H2O2-Induced Cytotoxicity hauv PC12 Cells

Cytotoxic teebmeem ntawm H2O2 thiab PhGs (0.1,1,5, thiab 10 卩g/mL) ntawm PC12 hlwb raug kuaj. Cov txiaj ntsig tau pom tias H2O2 ua rau poob ntawm PC12 cell viability nyob rau hauv concentration-dependent thiab lub sij hawm-dependent yam ntxwv (Daim duab 1A). Kev nthuav tawm ntawm PC12 hlwb rau 200 H2O2 rau 2 h ua rau lub cev muaj zog ntawm 57.4 feem pua. Kev kho cov hlwb nrog PhGs ntawm 0.1,1, 5, thiab 10 -/mL tsis muaj kev cuam tshuam rau cell viability (Daim duab 1B) thiab muaj kev tiv thaiv PC12 hlwb los ntawm H2O2-induced kev puas tsuaj los ntawm kev txhim kho cell viability raws li 9. . Txawm li cas los xij, salidroside (0.1 ^ g / mL), isoacteoside (0.1, 1, 5, thiab 10 ^ g / mL),echinacoside cov tshuaj(0.1,1, thiab 5 ^g/mL) pretreatment tsis pom qhov txawv ntawm HzOz-induced cell raug mob. Pretreatment ntawm hlwb nrog PhGs kuj ameliorated morphological yam ntxwv induced los ntawm H2O2 (Daim duab 1D).

Daim duab 1. PhGs suppressed H2O2-induced cytotoxicity hauv PC12 hlwb. Cell viability tau kuaj pom los ntawm MTT kev soj ntsuam. Cytotoxic nyhuv ntawm H2O2 (A) thiab PhGs (B) ntawm qhov sib txawv ntawm PC12 hlwb. (C) PhGs attenuated H2O2-ua rau txo qis hauv cell viability. PC12 hlwb raug incubated nrog PhGs ({{10}}.1, thiab 10 wg/mL) rau 24 teev, thiab ces incubated nrog 200 pM H2O2 rau lwm 2 teev tom qab PhGs raug tshem tawm. (D) Kev soj ntsuam morphological. Cells tom qab kev kho mob tau pom los ntawm lub sij hawm-contrast microscope (x100), CK: ib txwm pab pawg neeg, H2O2: H2O2 kho pab pawg, HL: salidroside tsawg npaum li cas kho pab pawg, HH: salidroside siab ntau npaum kho pab pawg, ML: acteoside tsawg npaum li cas kho pab pawg, MH: acteoside high dosage treated group, IL: isoacteoside low dosage treated group, IH: isoacteoside high dosage treated group, SL: echinacoside low dosage treated group, SH: echinacoside siab npaum li cas kho pab pawg. ** p <0.01 piv="" rau="" pab="" pawg="" tsis="" kho;="" #="" p="">< 0.05,="" piv="" rau="" h2o2="" kho="" pab="" pawg;="" ##="" p=""><0.01, piv="" rau="" h2o2="" pab="" pawg="">

2.2. PhGs Ncua tseg H2O2-Induced Intracellular Accumulation ntawm ROS, Lipid Peroxidation (MDA), thiab nce Superoxide Dismutase (SOD) Kev Ua Haujlwm hauv PC12 Cells

Kev tawm ntawm PC12 hlwb rau 200 pM H2O2 rau 2 h nce ROS qib, MDA cov ntsiab lus, thiab txo qis kev ua SOD (Daim duab 2). PhGs pretreatment attenuated ROS theem, salidroside, thiab acteoside, thiab siab npaum li cas ntawm isoacteoside thiab echinacoside pretreatment ho attenuated ROS theem (p <0.01). salidroside="" pretreatment="" tsis="" cuam="" tshuam="" rau="" mda="" cov="" ntsiab="" lus,="" tab="">acteosidepretreatment ho attenuated MDA cov ntsiab lus (p < 0.05).="" isoacteoside="" thiab="" echinacoside="" pretreatment="" txo="" qis="" mda="" cov="" ntsiab="" lus="" rau="" ntau="">

Daim duab 2. PhGs thaiv ROS thiab MDA tsub zuj zuj thiab nce cov dej num ntawm SOD hauv PC12 hlwb. PC12 hlwb raug incubated nrog PhGs (0.1, thiab 10 4g/mL) rau 24 teev, thiab ces incubated nrog 200 |^M H2O2 rau lwm 2 teev tom qab PhGs raug tshem tawm. (A) PhGs thaiv ROS thiab MDA tsub zuj zuj. (B) PhGs thaiv MDA tsub zuj zuj. (C) PhGs tau nce cov haujlwm ntawm SOD. CK: pawg ib txwm, Qauv: H2O2 kho pab pawg, Salidroside: salidroside kho pab pawg, Acteoside: acteoside kho pawg, Isoacteoside: isoacteoside kho pawg, Echinacoside: echinacoside kho pawg. ** p < 0.01="" tiv="" thaiv="" pab="" pawg="" tsis="" kho;="" #="" p="">< 0.05,="" piv="" rau="" h2o2="" kho="" pab="" pawg,="" ##="" p="">< 0.01,="" piv="" rau="" h2o2="" kho="">

2.3.PhGs Reversed H2O2-Induced Apoptosis hauv PC12 Cells

Kev kho H2O2 (200 |1M) rau 2 h nce apoptosis hauv PC12 hlwb, nrog rau tag nrho cov apoptotic tus nqi txog li 16.02 feem pua ​​(Daim duab 3). Txawm li cas los xij, pretreatment nrog PhGs (0.1 thiab 10 卩g / mL) rau 24 h txo tus nqi apoptosis nyob rau hauv ib tug concentration-dependent yam (p <0.01).>acteoside, isoacteoside, thiabechinacoside cov tshuajtxo qis feem pua ​​​​ntawm cell apoptosis los ntawm 4.750-6.627 feem pua ​​, 4.413-5.800 feem pua ​​, 6.593-10.047 feem pua ​​, thiab 1.530-7. 510 feem pua.

Daim duab 3. PhGs thim rov qab H2O2-induced apoptosis hauv PC12 hlwb. PC12 hlwb raug incubated nrog PhGs (0.1, thiab 10 卩g/mL) rau 24 teev, thiab ces incubated nrog 200 pM H2O2 rau lwm 2 teev tom qab cov PhGs raug tshem tawm. Tom qab ntawd, apoptosis tau ntsuas los ntawm qhov ntws cytometry siv PI / FITC fluorescent sojntsuam.

CK: pab pawg ib txwm, Qauv: H2O2 kho pab pawg, Salidroside: salidroside kho pab pawg, Acteoside: acteoside kho pawg, Isoacteoside: isoacteoside kho pab pawg,Echinacoside: echinacoside cov tshuajpab pawg kho mob. ** p < {{0}}.01="" tiv="" thaiv="" pab="" pawg="" tsis="" kho;="" ##="" p=""><0.01, piv="" rau="" h2o2="" pab="" pawg="">

2.4. PhGs Alleviated H2O2-Induced Dysregulation of the Nrf2-Yog Txoj Kev hauv PC12 Cells

Nrf{{0}}YOG txoj hauv kev, yog ib txoj hauv kev tseem ceeb antioxidant hauv ntau hom cell, yog qhov tseem ceeb hauv kev tswj kev loj hlob ntawm tes thiab kev tuag ntawm tes. Yog li, peb tau tshawb xyuas seb Nrf2-Txoj kev puas muaj feem cuam tshuam rau H2O2-induced apoptosis los ntawm immunofluorescence thiab Western blot tsom siv cov tshuaj tiv thaiv tshwj xeeb. Qhov kev ntsuam xyuas immunofluorescence tau pom tias PhGs (0.1 thiab 10 pg / mL) ua rau muaj kev hloov pauv ntawm Nrf2 (Daim duab 4). Tom qab kev kho mob ntawm PhGs, isoacteoside, thiabechinacoside cov tshuajua rau ib nrab rov qab los ntawm lub cell li qub morphology. Cov txiaj ntsig los ntawm Western blotting thiab grey density tsom xam pom tias PhGs ({{0}}}.1 thiab 10 pg/mL) tsis muaj kev cuam tshuam loj rau kev qhia ntawm Nrf2 hauv cytoplasm (Daim duab 5A, B) (p > {{20}}.05) tab sis upregulated Nrf2 qhia nyob rau hauv lub nucleus (Daim duab 5C,D) (p <0.01). tom="" qab="" ntawd,="" peb="" kawm="" txog="" lub="" luag="" haujlwm="" ntawm="" nrf2="" hauv="" h2o2-induced="" apoptosis="" los="" ntawm="" importing="" nrf2="" sirna.="" cov="" protein="" thiab="" mrna="" qhia="" ntawm="" nrf2="" tau="" txo="" qis="" hauv="" txhua="" pawg="" nrf2="" sirna-kho="" (daim="" duab="" 5e-h).="" phgs="" (0.1="" thiab="" 10="" pg/ml)="" tiv="" thaiv="" h2o2-induced="" cytotoxicity="" nyob="" rau="" hauv="" pab="" pawg="" uas="" tsis="" muaj="" nrf2="" sirna="" kev="" kho="" mob="" (p=""><0.01), tab="" sis="" xws="" li="" kev="" tiv="" thaiv="" tau="" thim="" rov="" qab="" los="" ntawm="" nrf2="" sirna.="" tom="" qab="" kev="" kho="" nrf2="" sirna,="" lub="" cell="" viability="" txo="" qis="" txawm="" tias="" nrog="" phgs="" kho="" (daim="" duab="">

Daim duab 5. Kev tiv thaiv ntawm PhGs ntawm Nrf2 hauv H2O2- kho PC12 hlwb. (A) Qhov kev qhia ntawm Nrf2 protein nyob rau hauv cytoplasm tau kuaj pom los ntawm immunoblotting siv cov tshuaj tiv thaiv tshwj xeeb. p-Actin tau siv los tswj kev thauj khoom. (B) Kev soj ntsuam ntau qhov densitometric ntawm Nrf2 protein nyob rau hauv cytoplasm. (C) Qhov kev qhia ntawm Nrf2 protein nyob rau hauv lub nucleus tau kuaj pom los ntawm immunoblotting siv cov tshuaj tiv thaiv tshwj xeeb. Histones H3 tau siv los tswj kev thauj khoom. (D) Kev soj ntsuam ntau qhov densitometric ntawm Nrf2 protein nyob rau hauv lub nucleus. (E, F) PC12 hlwb raug preincubated yam tsis muaj (E) lossis nrog (F) Nrf2 siRNA rau 24 teev, ces incubated nrog los yog tsis muaj PhGs (0.1, thiab 10 昭/mL) rau 24 teev, thiab incubated nrog H2O2 rau lwm 2 teev tom qab cov PhGs raug tshem tawm. Tag nrho Nrf2 protein qhia tau kuaj pom los ntawm immunoblotting siv cov tshuaj tiv thaiv tshwj xeeb, thiab p-actin tau siv los tswj kev thauj khoom. (G) Kev ntsuas densitometric ntawm tag nrho Nrf2 protein. (H) Kev soj ntsuam ntau ntawm Nrf2 mRNA. (I) PC12 hlwb raug preincubated nrog los yog tsis muaj siRNA rau 24 teev, ces incubated nrog los yog tsis PhGs (0.1 thiab 10 -/mL) rau 24 teev, thiab incubated nrog H2O2 rau lwm 2 teev tom qab cov PhGs raug tshem tawm. Tom qab kev kho mob, cov hlwb ciaj sia tau txiav txim siab los ntawm MTT kev soj ntsuam. CK: pawg ib txwm, Qauv: H2O2 kho pab pawg, Tswj Si: tswj siRNA kho pawg, SiRNA Tsis zoo: tsis muaj siRNA kho pawg, SiRNA Zoo: SiRNA kho pab pawg, Salidroside: salidroside kho pawg, Acteoside: acteoside kho pawg, Isoacteoside: isoacteoside kho pab pawg,Echinacoside: echinacoside kho pab pawg. ** p < {{0}}="" 01="" pab="" pawg="" tsis="" kho;="" #="" p="">< 0.05,="" piv="" rau="" h2o2="" kho="" pab="" pawg="" (tsis="" muaj="" nrf2="" sirna="" kho),="" ##="" p="">< 0.01,="" piv="" rau="" h2o2="" kho="" pawg="" (tsis="" muaj="" nrf2="" sirna="" kho);="" &="" p=""><0.05 piv="" rau="" h2o2="" kho="" pab="" pawg="" (nrog="" nrf2="" sirna="" kho),="" thiab="" p=""><0.01, piv="" rau="" h2o2="" kho="" pawg="" (nrog="" nrf2="" sirna="">

2.4. PhGs Reversed H2O2-Induced Downregulation of Protein Expression of HO-1, NQO1, GCLC, and GCLM

HO{{0}}, NQO1, thiab glutamate-cysteine ​​ligase (GCL) yog ib qho tseem ceeb cellular antioxidant enzymes, thiab HO-1, NQO1, thiab catalytic los yog hloov subunits ntawm GCL (GCLC los yog GCLM) yog Nrf2- tswj cov noob caj noob ces [27]. Protein expression of HO-1, NQO1, GCLC, thiab GCLM tau soj ntsuam tom qab kho. Qhov sib txawv pom tseeb tau pom ntawm cov protein qhia ntawm HO-1 thiab NQO1 nrog lossis tsis muaj H2O2 (Daim duab 6A-C) (p < 0.01).="" phgs="" (0.1="" thiab="" 10="" p^g/ml)="" thim="" rov="" qab="" h2o2-induced="" downregulation="" ntawm="" protein="" qhia="" ntawm="" ho-1="" (tshwj="" tsis="" yog="" salidroside="" ntawm="" 0="" }.1="" pg/ml),="" nqo1="" (tshwj="" tsis="" yog="" acteoside="" ntawm="" 0.1="" pg/ml)="" (p=""><0.01). h2o2="" kuj="" txo="" qis="" gclc="" thiab="" gclm="" protein="" qhia="" (p=""><0.05) (daim="" duab="" 6a,="" d,="" e).="" phgs="" (0.1="" thiab="" 10="" pg/ml)="" thim="" rov="" qab="" h2o2-induced="" downregulation="" ntawm="" protein="" qhia="" ntawm="" gclc="" (tshwj="" tsis="">echinacoside cov tshuajntawm {{0}}.1 pg/mL) (p < 0.01)="" thiab="" gclm="" (tshwj="" tsis="" yog="" salidroside="" ntawm="" 0.1="" pg/ml)="" (="" p="">< 0="" 01).="" tom="" qab="" ntawd,="" cov="" tshuaj="" inhibitors="" rau="" ho-1="" tau="" siv="" los="" ntsuas="" ntxiv="" lub="" luag="" haujlwm="" ntawm="" cov="" enzymes="" antioxidant="" hauv="" kev="" tswj="" hwm="" kev="" tiv="" thaiv="" phgs="" tiv="" thaiv="" h2o2-induced="" cytotoxicity.="" phgs="" (0.1="" thiab="" 10="" pg/ml)="" tiv="" thaiv="" h2o2-induced="" cytotoxicity,="" tab="" sis="" cov="" nyhuv="" tiv="" thaiv="" tau="" thim="" rov="" qab="" los="" ntawm="" ho-1="" inhibitor="" znpp="" (p="">< 0.01)="" ntawm="" 20="" pm="" (daim="" duab="" 6f,="" p="">< 0.01)="">

2.5. Keap1 Kev Qhia thiab Molecular Docking Analysis

Raws li lub cev muaj zog, Keap1 ua raws li cov protein repressor ntawm Nrf2 los ntawm kev khi rau Neh2 lub npe ntawm Nrf2 thiab tsom Nrf2 rau Cul3-raws li E3 ubiquitin ligase rau ubiquitination thiab tom qab degradation los ntawm 26S proteasome [28]. Kev khi lub peev xwm rau Keap1 ntawm PhGs tau soj ntsuam los ntawm kev soj ntsuam molecular docking los tshawb xyuas cov txheej txheem raws li lawv cov nyhuv antioxidant.

3. Kev sib tham

Peb tau tshawb xyuas cov neuroprotection ntawm PhGs ntawm H2O2 induced-cytotoxicity hauv PC12 hlwb. Cov txiaj ntsig tau pom tias PhGs pretreatment cuam tshuam HzOz-induced cytotoxicity, attenuated intracellular ROS theem, txhim kho qib ntawm intracellular antioxidant enzymes, thiab thaum kawg thim rov qab H2O2-induced cytotoxicity hauv PC12 hlwb. Ntxiv mus, PhGs tau nce qhov kev hloov pauv ntawm Nrf2, thim rov qab HzOz-induced downregulation ntawm cov protein qhia ntawm HO-1, NQO1, GCLC, thiab GCLM. Tsis tas li ntawd, PhGs tau pom tias muaj peev xwm cuam tshuam nrog Nrf2 khi qhov chaw hauv Keap1 protein.

Hauv H2O2-induced PC12 cell raug mob, lipid peroxidation, uas yog hais txog oxidative degradation ntawm lipid, nce permeability ntawm daim nyias nyias, ua rau cell puas [29]. MDA tsim yog dav siv raws li qhov ntsuas ntawm lipid peroxidation [30]. H2O2 txhim kho ROS ntau lawm thiab tso tawm cov tshuaj tiv thaiv antioxidant, xws li SOD, catalase, thiab GPx. Cov txheej txheem no ua rau muaj kev ntxhov siab oxidative [31], uas ua lub luag haujlwm tseem ceeb hauv kev ua rau thiab kev loj hlob ntawm feem ntau ntawm cov kab mob neurodegenerative. Raws li cov kev tshawb fawb yav dhau los, peb tau pom qhov nce qib ntawm ROS, txo cov enzymes intracellular antioxidant, thiab txhim kho apoptosis ntawm PC12 hlwb tom qab kho H2O2. PhGs pretreatment ho txo ​​qis HzOz-induced nce hauv intracellular ROS, txhim kho intracellular antioxidant enzymes, thiab thaum kawg thim rov qab HzOz-induced cytotoxicity hauv PC12 hlwb.

Kuang et al. [32] qhia tiasechinacoside cov tshuajpom tau tias muaj txiaj ntsig zoo rau cov neuroprotective ntawm H2O2-induced cytotoxicity hauv PC12 hlwb los ntawm txoj kev mitochondrial apoptotic. Hauv txoj kev tshawb no, peb pom tias echinacoside, salidroside, acteoside, thiab isoacteoside tau pom cov teebmeem neuroprotective los ntawm kev txhim kho cov tshuaj tiv thaiv antioxidant ntawm PC12 hlwb vim tias lawv tau nce cov kev ua haujlwm ntawm Nrf2 thiab txhawb nqa cov ntsiab lus qis qis ntawm HO-1, NQO1, GCLC, thiab GCLM. Ntau cov kev tshawb fawb tau pom tseeb tias kev ua kom Nrf2 lub hom phiaj, tshwj xeeb yog HO-1, hauv astrocytes thiab neurons tiv thaiv kev mob, oxidative puas tsuaj, thiab kev tuag ntawm tes. HO-1 system tau raug tshaj tawm tias muaj zog heev hauv lub hauv nruab nrab lub paj hlwb, thiab nws qhov kev hloov kho pom tau ua lub luag haujlwm tseem ceeb hauv cov kab mob ntawm cov kab mob neurodegenerative [33]. Cov kev tshawb fawb tsis ntev los no kuj tau qhia meej txog lub luag haujlwm ntawm Nrf2 hauv kev nce qib thiab kev pheej hmoo ntawm PD [9] thiab Keap1 raws li lub hom phiaj zoo rau kev rov ua haujlwm ntawm Nrf2 hauv AD [34]. Cov txiaj ntsig txhawb nqa cov pov thawj tshiab rau Nrf2 raws li lub hom phiaj kho mob hauv cov kab mob neurodegenerative.

Molecular docking tsom xam pom tias PhGs tuaj yeem khi rau Keap1, nrog rau cov peev txheej hauv qab no:echinacoside cov tshuaj>isoacteoside > acteoside > salidroside. Raws li cov txiaj ntsig no, PhGs pretreatment coj mus rau Nrf2 nuclear translocation, nrog rau Nrf2 qhia hauv qab no:echinacoside cov tshuaj>isoacteoside * acteoside> salidroside. Peb xav tias tus naj npawb ntawm glycosides cuam tshuam rau txoj kev sib khi ua tau ntawm PhGs thiab Keap1, thiab hom kev khi txuas ntxiv ua rau kev tso tawm Nrf2 los ntawm Keap1. Cov txheej txheem no ua rau muaj kev ua kom Nrf2 thiab cov noob caj noob ces thiab thaum kawg tiv thaiv PC12 hlwb los ntawm H2 O2 -induced oxidative stress.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Chemical Compounds thiab Reagents

Salidroside (CAS No. 10338-51-9), acteoside (CAS No. 61276-17-3), isoacteoside (CAS No. 61303-13-7), thiabechinacoside cov tshuaj(CAS No. {{0}}) tau yuav los ntawm YYuanye Biotechnology Company (Shanghai, Suav). Cov PhGs tau yaj hauv PBS los tsim cov tshuaj 10 mg / mL, uas tau khaws cia ntawm -20 degree . H2O2 tau yuav los ntawm Aladdin® (Shanghai, Suav). RPMI-1640 nruab nrab thiab fetal bovine serum tau yuav los ntawm Hyclone (Logan, UT, USA), thiab 0.5 feem pua ​​trypsin EDTA, penicillin, thiab streptomycin tau yuav los ntawm Keyi (Hangzhou, Suav). MDA, SOD cov khoom siv kuaj mob, MTT, thiab DCFH-DA tau yuav los ntawm Beyotime Institute of Biotechnology (Nanjing, Jiangsu, Suav). Annexin V-FITC / PI ob chav staining khoom tau yuav los ntawm Solarbio Life Sciences (Beijing, Suav). Cov tshuaj tiv thaiv rau Nrf2, Histone H3, Keap1, HO-1, NQO1, GCLC, GCLM, thiab p-actin, anti-nas-horseradish peroxide (HRP) IgG, thiab tshuaj tiv thaiv luav-HRP-IgG tau yuav los ntawm Abcam (London, UK). Inhibitors ntawm HO-1 thiab ZnPP tau yuav los ntawm Sigma Chemical Co. (St. Louis, MO, USA). RNAiso Plus, PrimeScript™ RT reagent Kit nrog gDNA Eraser, thiab SYBR® Premix Ex Taq™ II tau yuav los ntawm Takara (Shiga, Nyiv). Lipofectamine® RNAiMAX Transfection Reagent tau yuav los ntawm Thermo Fisher Scientific (Waltham, UK). Nrf2 siRNA sequences tau raws li nram no: pem hauv ntej, CCGAAUUAACUGUCUUAA; and reverse, UAAGACACUGUAAUUGGG. Meanwhile, tswj siRNA sequences raws li nram no: rau pem hauv ntej, UUCUCCGAACGUGUCACGU; thiab thim rov qab, ACGUGACACGUUCGGAGAA.

4.2. Cell Culture

Nas adrenal pheochromocytoma kab (PC12 hlwb) tau txais los ntawm Lub Tsev Haujlwm ntawm Biochemistry thiab Cell Biology, SIBS, (CAS, Shanghai, Suav). Cov hlwb tau khaws cia hauv RPMI-1640 (Hyclone) uas muaj 10 feem pua ​​​​fetal bovine serum (Hyclone), 100 U/mL penicillin, thiab 0.1 mg/mL streptomycin ntawm 37 degree nrog 5% CO2. Qhov nruab nrab tau hloov txhua hnub.

4.3. Cell ViabilityAssay

PC12 hlwb tau cog rau hauv 96-cov phiaj zoo ntawm 2 x 104 cell/well. Tom qab txuas, cov hlwb raug preincubated nrog los yog tsis muaj inhibitor rau 20 min, incubated nrog los yog tsis muaj PhGs rau 24 teev, thiab ces incubated nrog H2O2 rau lwm 2 teev tom qab lub PhGs raug tshem tawm. Tom qab incubation, cov hlwb raug kho nrog 5 mg / mL MTT rau 4 h ntawm 37 degree, thiab cov xov xwm tau ua tib zoo tshem tawm. Cov pob zeb formazan uas tau tsim los ntawm cov hlwb tseem muaj sia nyob tau yaj hauv 150 rL ntawm DMSO los tsim cov xim xiav [35], thiab qhov nqus tau ntsuas ntawm 570 nm ntawm lub phaj nyeem ntawv. Cov tswj tau siv tib qhov concentration ntawm nruab nrab nrog DMSO ib leeg. Cell viability tau normalized raws li feem pua ​​ntawm kev tswj.

Cov concentrations ntawm PhGs (0.1,1,5, thiab 10 Rg/mL) raug xaiv nyob ntawm seb qhov kev tshuaj xyuas cytotoxicity ntawm PhGs thiab cov txiaj ntsig cytoprotective ntawm echinacoside [32]. Raws li tsab ntawv ceeb toom, tsis muaj cytotoxicity tshwm sim hauv qab 10 Rg / mL, thiab echinacoside tau tshaj tawm los qhia cov nyhuv cytoprotective hauv HzOz-mob mob cell qauv.

4.4. Apoptosis Assay

Apoptosis tau kuaj pom nrog Annexin V-FITC / PI ob chav staining Kit (Solarbio). PC12 hlwb tau cog rau hauv 6-zoo daim hlau ntawm 2 x 105 cells/well. Tom qab txuas, hlwb raug kho nrog PhGs (0.1 thiab 10 Rg / mL) rau 24 teev thiab incubated nrog H2O2 rau lwm 2 teev tom qab PhGs raug tshem tawm. Tom qab incubation, lub hlwb raug ntxuav nyob rau hauv txias PBS, centrifuged ob zaug ntawm 1500 rpm rau 10 min, thiab resuspended nyob rau hauv 500 rL ntawm binding tsis. FITC-labeled Annexin V (5 rL) thiab propidium iodide.

4.5. Kev ntsuas ntawm intracellular ROS, MDA Production, thiab SOD

Qib intracellular ROS tau txiav txim siab siv ROS-sensitive fluorescent sojntsuam, uas yog, 2,7-dichlorodihydro fluorescent diacetate (DCFH-DA). PC12 hlwb tau cog rau hauv 6-cov phiaj zoo ntawm 2 x 105 cells/well. Tom qab txuas, cov hlwb raug kho nrog PhGs (0.1 thiab 10 卩g / mL) rau 24 teev thiab incubated nrog H2O2 rau lwm 2 teev tom qab PhGs raug tshem tawm. Tom qab incubation, cov hlwb raug ntxuav nrog PBS peb zaug thiab tom qab incubated nrog 10 卩M DCFH-DA. Tom qab incubation ntawm 37 degree rau 20 min, lub hlwb raug ntxuav nrog PBS thiab sau los ntawm maj mam centrifugation. Intracellular ROS tau ntsuas siv Gallios ™ flow cytometer (Beckman Coulter, Brea, CA, USA).

MDA thiab SOD tau ntsuas los ntawm cov khoom siv tshuaj ntsuam (Beyotime Biotechnology, Nanjing, Jiangsu, Suav). Tag nrho cov txheej txheem ua tiav raws li cov neeg tsim khoom cov lus qhia. Cov ntsiab lus ntawm MDA thiab SOD tau normalized nrog cov ntsiab lus ntawm tag nrho cov protein.

4.6. Nrf2 Nuclear Translocation Immunofluorescence

PC12 hlwb tau cog rau hauv 6-zoo iav slides ntawm qhov ntom ntawm 2 x 105 rau ib qhov dej. Tom qab txuas, cov hlwb raug kho nrog PhGs (0.1 thiab 10 卩g/mL) rau 24 teev thiab incubated nrog H2O2 rau lwm 2 teev tom qab PhGs raug tshem tawm. Tom qab incubation, lub hlwb raug ntxuav nyob rau hauv txias PBS thiab tsau nyob rau hauv 4 feem pua ​​paraformaldehyde rau 15 min ntawm chav tsev kub, ua raws li membrane permeabilization siv 0.5 feem pua ​​Triton X-100 hauv PBS rau 5 min. Cov hlwb tau incubated nrog anti-Nrf2 Luav IgG (Abcam) nrog 5 feem pua ​​FBS thaum hmo ntuj ntawm 4 degree. Tom qab ntawd, cov tshuaj tiv thaiv kab mob thib ob uas sib txuas nrog FITC tau siv rau lub hlwb rau 20 min ntawm chav tsev kub. Lub nuclei tau stained nrog 4z,6-diamidino-2-phenylindole (DAPI) ntawm cov kauj ruam kawg. Cov slides tau yaug luv luv nrog PBS, cua qhuav, thiab mounted nyob rau hauv ib qho anti-fluorescence nyob rau hauv fading nruab nrab. Cov slides tau pom nyob rau hauv lub laser confocal microscope (LMS780, Zeiss, Lub teb chaws Yelemees).

4.7. Protein Extraction

Cov PC12 hlwb tau muab noob rau hauv 100 hli lauj kaub tais diav ntawm 1 x 107 hlwb/phaj. Tom qab txuas, cov hlwb raug kho nrog PhGs (0.1 thiab 10 卩g / mL) rau 24 teev thiab tom qab ntawd incubated nrog H2O2 rau lwm 2 teev nrog PhGs tshem tawm. Tom qab incubation, lub hlwb raug ntxuav ob zaug nrog txias PBS thiab scraped los ntawm cov tais diav nrog 1000 卩L 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. Cov protein ntau ntawm cov qauv tau kuaj pom los ntawm Beyotime BCA protein kuaj cov khoom siv, thiab tag nrho cov qauv raug khaws cia ntawm -80 degree rau Western blot tsom xam.

4.8. Western BlotAnalysis

Western blot tsom xam tau ua los ntawm cov txheej txheem txheem. Luv luv, cov qauv protein (20 lossis 30 卩g) tau muab cais los ntawm SDS-PAGE thiab xa mus rau PVDF daim nyias nyias. Membranes raug thaiv hauv 5 feem pua ​​​​cov mis nyuj-TBST thiab incubated hmo ntuj ntawm 4 degree hauv thawj cov tshuaj tiv thaiv. Antibodies siv suav nrog Nrf2 (Abcam), Keap1 (Abcam), HO-1 (Abcam), NQO-1 (Abcam), GCLC (Abcam), GCLM (Abcam), Histone H3 (Abcam), thiab p-actin (Abcam). Peroxidase-conjugated anti-nas IgG (Abcam) los yog tshuaj tiv thaiv luav IgG (Abcam) tau siv los ua cov tshuaj tiv thaiv thib ob. Cov kab mob protein tau pom pom siv ChemiScope series (Clinx Science Instruments, Shanghai, Suav). Tus nqi grey ntawm cov protein bands tau suav nrog siv ImageJ (National Institutes of Health, Bethesda, MD, USA).

4.9. Quantitative Real-Time PCR

Tag Nrho RNA raug rho tawm siv RNAiso Plus (Takara, Shiga, Nyiv), ua raws li cov chaw tsim khoom cov lus qhia. Tag nrho cov qauv RNA tau thim rov qab nrog PrimeScript™ RT reagent Kit nrog gDNA Eraser (Takara, Shiga, Nyiv) raws li cov chaw tsim khoom qhia. Quantitative real-time PCR tau ua tiav siv SYBR® Premix Ex Taq™ II (Takara, Shiga, Nyiv) hauv Applied Biosystems ViiA™ 7 Real-Time PCR System. Kev txheeb ze ntawm lub hom phiaj noob tau ua rau p-Actin thiab tshuaj xyuas los ntawm 2-AACt txoj kev. Primer sequences rau Nrf2 yog raws li nram no: rau pem hauv ntej, ACAGGTGCTCCTATGCGGTGAA thiab thim rov qab, TCTGGGCGGCGACTTTAT. Primer sequences rau p-Actin yog raws li nram no: rau pem hauv ntej, GCTGTCCCTGTATGCCTCT; thiab thim rov qab, TTGATGTCACGCACGATTT.

4.10. SiRNA Transfection

PC12 hlwb raug coj los rau hauv 6-zoo iav slides ntawm qhov ntom ntawm 2 x 105 ib qhov dej (rau Western blot tsom) los yog hauv 96-zoo iav slides ntawm qhov ntom ntawm 2 x 104 ib qhov dej (rau cell kev soj ntsuam viability). Tswj siRNA thiab Nrf2 siRNA tau hloov mus rau hauv hlwb siv Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) raws li cov chaw tsim khoom cov lus qhia. Tom qab 24 teev ntawm incubation, cov hlwb raug kho nrog lossis tsis muaj PhGs thiab H2O2 rau lub sijhawm qhia thiab tom qab ntawd siv hauv Western blot tsom, qRT-PCR, lossis kev txheeb xyuas ntawm tes raws li tau piav qhia saum toj no.

4.11. Molecular Docking Analysis

Molecular docking analysis was performed to investigate the possible binding mode of PhGs to Keap1. The 3D structure of ligands was obtained from NCBI. The crystallized structure of Keap1 (PDB Code: 4L7B) was prepared using correcting structure issues (such as break bond and miss loop) using SYBYL-X 2.0. In this software simulation, crash represents the inadequate penetration between the protein and the ligand. Polar represents the hydrogen bonding and electrostatic interactions between the protein and the ligand. G-score [36] represents the hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand) energies between the protein and the ligand. PMF-score [37] represents the Helmholtz free energy between the protein and the ligand. D-score [38] represents the charge and van der Waals interactions between the protein and the ligand. Chem-score [39] represents the hydrogen bonding, metal-ligand interaction, lipophilic contact, and rotational entropy, along with an intercept term between the protein and the ligand. C score represents the consensus score between the protein and the ligand, comprehensively reflecting the G, PMF, D, and Chem-scores. Tota-score reflects the binding capacity of the ligand to the protein. The best modes were generated and evaluated using the Total-score (>6) and C-score (>4).

4.12. Kev txheeb cais

Cov ntaub ntawv tau txheeb xyuas siv ib-txoj kev ANOVA nrog LSD tshuaj xyuas siv SPSS. Tag nrho cov txiaj ntsig tau lees paub los ntawm peb qhov kev sim ywj pheej. Cov ntaub ntawv tau qhia raws li txhais tau tias 土 SD. Kev txheeb cais tseem ceeb sib txawv tau txiav txim siab ntawm p <>

Kev lees paub: Txoj kev tshawb fawb no tau txais kev txhawb nqa los ntawm National Major R & D Program ntawm Tuam Tshoj (No. 2017YFD0400200), Zhejiang Provincial Natural Science Foundation ntawm Tuam Tshoj (No. GJFP2018015), Ministry of Agriculture thiab Rural Affairs ntawm Tuam Tshoj.

Tus sau kev koom tes:Baiyi Lu thiab Maiquan Li tsim txoj kev kawm. Maiquan Li tau ua haujlwm hauv chav kuaj, tshuaj xyuas cov ntaub ntawv thiab sau ntawv. Maiquan Li, Tao Xu, Fei Zhou, Mengmeng Wang, Huaxin Song, Xing Xiao, thiab Baiyi Lu tau kho cov ntawv sau.

1 National Engineering Laboratory ntawm Intelligent Food Technology thiab khoom siv,

Lub Tsev Haujlwm Tseem Ceeb rau Agro-Products Postharvest Handling of Ministry of Agriculture thiab Rural Affairs, Lub Chaw Haujlwm Tseem Ceeb rau Agro-Products Nutritional Evaluation of Ministry of Agriculture thiab Rural Affairs, Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science, College of Biosystems Engineering thiab Food Science, Zhejiang University, Hangzhou 310058, Tuam Tshoj;

2 Lub Tsev Kawm Ntawv Qib Siab ntawm Thawj Kev Kho Mob, Guangzhou University of Suav Tshuaj,

Cov ntaub ntawv

1. Sun, AY; Chen, YM Oxidative stress thiab neurodegenerative disorders. J. Biomed. Sci. Xyoo 1998, 5, 401-414.

2. Maheshwari, A.; Misro, MM; Aggarwal, UA; Sharma, RK; Nandan, D. Pathways koom nrog hauv cov kab mob qog nqaij hlav ntawm tes apoptosis tshwm sim los ntawm H2O2 hauv vitro. FEBS J. 2009,276, 870-881.

3. Halliwell, B. Reactive oxygen hom thiab lub hauv paus paj hlwb. J. Neeb. Xyoo 1992, 59, 1609-1623.

4. Richardson, JS; Subbarao, KV; Ang, LC Ntawm lub luag haujlwm ua tau ntawm hlau-vim dawb radical peroxidation hauv neural degeneration hauv Alzheimer's kab mob. Ann. NY Acad. Sci. Xyoo 1992, 648, 326-327.

5. Zhang, DD Mechanistic kev tshawb fawb ntawm Nrf2-Keap1 txoj kev taw qhia. Drug Metab. Rev. 2006, 38, 769-789.

6. Ib., K.; Tong, KI; Yamamoto, M. Molecular mechanism activating Nrf2-Keap1 txoj kev nyob rau hauv cov kev cai ntawm adaptive teb rau electrophiles. Dawb Radic. Biol. Med. 2004, 36, 1208-1213.

7. Kong, AN; Aws, E.; Yus, R.; Hebbar, V.; Chen, C.; Hu, R.; Mandlekar, S. Induction ntawm xenobiotic enzymes los ntawm daim ntawv qhia kinase txoj kev thiab cov antioxidant los yog electrophile teb lub caij (ARE/EpRE). Drug Metab. Rev. 2001, 33, 255-271.

8. Buendia, ib.; Michalska, P.; Navarro, E.; Gameiro, ib.; Eeg, J.; Leon, R. Nrf2-YOG txoj kev: Ib lub hom phiaj tawm tsam oxidative stress thiab neuroinflamation hauv cov kab mob neurodegenerative. Pharmacol. Ther. Xyoo 2016, 157, 84-104. [CrossRef] [PubMed]

9. Skibinski, G.; Hwang, V; Ando, ​​DM; Daub, A.; Li, AK; Ravisankar, UA; Modan, S.; Finucane, MM; Shabby, BA; Finkbeiner, S. Nrf2 mitigates LRRK2- thiab a-synuclein-induced neurodegeneration los ntawm modulating proteostasis. Proc. Natl. Acad. Sci. USA 2016, 114, 1165-1170.

10. Jimenez, C.; Riguera, R. Phenylethanoid glycosides hauv cov nroj tsuag: Cov qauv thiab kev ua haujlwm lom neeg. Nat. Prod. Rep. 1994, 11, 591-606.

11. Georgiev, M.; Alipieva, K.; Orhan, ib.; Abrashev, R.; Denev, P.; Angelova, M. Antioxidant thiab cholinesterases inhibitory kev ua ub no ntawm Verbascum xanthophoeniceum Griseb. thiab nws cov phenylethanoid glycosides. Khoom noj khoom haus Chem. Xyoo 2011, 128, 100-105.

12. Li, N.; Wang, J.; Ma, J.; Gu, Z.; Jiang, C.; Yus, L.; Fu, X. Neuroprotective Effects of Cistanches Herba Therapy on Patients with Moderate Alzheimer/s Disease. Evid.-Based Complement. Hloov. Med. Xyoo 2015, 2015, 103985.

13. Liu, YL; He, WJ; Mo, L.; Shi, MF; Zhu, YY; Pan, S.; Li, XR; Xu, QM; Yang, SL Antimicrobial, tiv thaiv kev ua ub no thiab toxicology ntawm phenylethanoid glycosides los ntawm Monochasma savatieri Franch. ua Maxim. J. Ethnopharmacol. Xyoo 2013, 149, 431-437.

14. Dong, Q.; Yaj, J.; Fang, JN; Ding, K. Cov yam ntxwv ntawm tus yam ntxwv thiab kev tiv thaiv kab mob ntawm ob lub dej txias-dej extractable polysaccharides los ntawm Cistanche deserticola YC Ma. Carbohydrate. Res. Xyoo 2007, 342, 1343-1349.

15. Xiong, L.; Mao, S.; Lu, B.; Yaj, J.; Zhou, F.; Hu, Y.; Jiang, Y.; Shen, C.; Zhao, Y. Osmanthusfragrans Paj Extract thiab Acteoside tiv thaiv d-Galactose-Induced Aging hauv ICR Mouse Model. J. Med. Zaub mov 2016, 19, 54-61.

16. Jiang, Y.; Mao, S.; Huang, W. Lu, B.; Cai, Z.; Zhou, F.; Li, M.; Lou, T.; Zhao, Y. Phenylethanoid Glycoside Profiles thiab Antioxidant Activities ntawm Osmanthusfragrans Lour. Paj los ntawm UPLC/PDA/MS thiab Simulated Digestion Model. J. Agric. Khoom noj khoom haus Chem. Xyoo 2016, 64, 2459-2466.

17. Li, X.; Yog, X.; Li, X.; Sun, X.; Liang, Q.; Tau, L.; Kang, X.; Chen, J. Salidroside tiv thaiv MPP ntxiv -induced apoptosis hauv PC12 hlwb los ntawm inhibiting NO txoj kev. Lub hlwb Res. 2011, 1382, 9-18.

18. Zhou, L.; Ding, W.; Sun, H.; Zhou, Q. Huang, J.; Li, X.; Xie, Y.; Chen, J. Salidroside tiv thaiv PC12 hlwb los ntawm MPP ntxiv rau -induced apoptosis los ntawm kev ua kom txoj hauv kev PI3K/Akt. Khoom noj khoom haus Chem. Toxicol. Xyoo 2012, 50, 2591-2597.

19. Sheng, GQ; Zhang, JR; pua, XP; Ma, J.; Li, CL Tiv thaiv cov nyhuv ntawm verbascoside ntawm 1-methyl-4- phenylpyridinium ion-induced neurotoxicity hauv PC12 hlwb. Eur. J. Pharmacol. 2002, 451, 119-124.

20. Wang, H.; Xu, Y.; Yog, J.; Zhao, X.; Sun, X.; Zhang, YJ; Guo, J.; Zhu, C. Acteoside tiv thaiv tib neeg neuroblastoma SH-SY5Y hlwb tiv thaiv beta-amyloid-induced cell raug mob. Lub hlwb Res. Xyoo 2009, 1283, 139-147.