淫羊藿苷对H2O2诱导的PC12细胞内质网应激的保护作用

Vol. 42 No. 3
Jun.2019
第42卷第3期2019年6月
遵义医学院学报
Journal of Zunyi Medical University 淫羊）+对HO ?诱导的PC12细胞内质网应激的保护作用
张洋洋，李祖高，刘义伟，陈霞，李菲
（遵义医科大学药理学教研室暨基础药理教育部重点实验室暨特色民族药教育部国际合作联合实验室，贵州遵义563099）
［摘 要］目的观察淫羊LN (ICA )对H 2O 2诱导的PC12细胞内质网应激的作用。

方法采用H 2O 2制备PC12细胞应激 损伤模型，观察ICA 在6. 25& 12. 5&25 "M 不同浓度下的作用。

采用MTT 和LDH 测定法检测细胞活力和细胞死亡率%
Western blot 检测GRP78、PERK 、eIF2a 、ATF4和CHOP 的蛋白表达，以及PERK 和eIF2a 的磷酸化水平%实时荧光定量PCR
检测GRP78、PERK 、ATF4和CHOP 的mRNA 水平%结果结果显示H 2O 2可诱导PC12细胞损伤和内质网应激，而ICA 预处 理可增加PC12细胞活力、减轻细胞损伤,并抑制HO ：诱导的GRP78、ATF4和CHOP 的mRNA 高表达，同时降低了 PERK 、 eIF2a 磷酸化水平以及GRP78、ATF4和CHOP 的蛋白表达％结论本研究表明ICA 可减轻H 2O 2诱导的PC12细胞内质网应
激损伤，其作用主要与抑制PERK 信号通路的激活有关％
［关键词］淫羊#%;H 2O 2 ；内质网应激;蛋白激酶R 样内质网激酶;嗜｛细胞瘤［中图法分类号］R964
［文献标志码］A ［文章编号］1000-2715(2019)03-0254X)6
Protection of icariic agaicsi H 2 O 2 induced erdoplasmic reticulum stress it PC 12 cells
Zhang Yangyang ，Li Zugao ，Liu Yiwei ，Chen Xia ，Li Fei
(Key Laboratory of Basic Pharmacology of Ministry of Education ，Joine International Research Laboratory of Eth ­nomedicine of Ministry of Education ，Zunyi Medical University ，Zunyi Guizhou 563099，China)
［Abstraci ］ Objective To investigaie the effect of icariin (ICA) on H2O2 induced endoplasmic reticulum (ER) irest in PC 12 celUt. Methods HO ? wat used at an inducea of ER ires t ，and PC12 celUt were pry - treated with vvriout concentratione of ICA (6. 25，12. 5 and 25 "M ) . Celt proliferation and death were detected using MTT and LDH usayt. The protein expressions of GRP78，PERK ，eIC2#，ATF4 and CHOP ，at weli at the phosphoryla ­tion levvit of PERK and eIC2# were tested by Western blog The mRNA level of GRP78，PERK ，ATF4 and CHOP were measured by reai - time qPCR analysit. Results The date demonstrated that R z Ozinduced PC12 alls I oss and ER stress. Howevee ，pre - treatmeni with ICA enhanced PC12 celt viability and repressed cells loss ； de ­creased the lsSs of PERK ，eIF2# phosphorylation and the expressions of GRP78，ATF4，and CHOP protein ; as wel as reduced GRP78，ATF4 and CHOP mRNA Isis in HO 2 - injuried PC12 ti l s. Conclusion The present study revealed that ICA exerts protective effect on H2O2 - induced ER stress in PC12 cens ，mainOy related te sup ­pression of the PERK signaling pathway activetion.
［Key words ］ H 2O 2 ; endoplasmic reticulum stress ; protein kinase R - like endoplasmic reticulum kinase ; phee- 6heomo6yeoma
Eukaryotic ceUs execute verious functions in sub- cellulay compartmenIs oo oryanUles. Endoplasmic re-
eiuaum ( ER ) paaysamauoeeoaein peoeeoseasisby ehe6o e e6eeoading ， modieiaeion and quaaiey6oneeoaoe appeoeimaeeayoneehied oea a se6eeeoeyand membeane
peoeeins (1］ .Rea6eieeoeygen spe6ies ( ROS ) hasbeen shown eomoduaaeeER eun6eion.ROS ， ER seee s ， and
peoeein aggeegaeion aeea s o6iaeed wieh eheeeioaogyoe diseases such as type 2 diabetes ，canceo ，and Alzhei- mey + s disuse ( AD ) . AD ，a aneU neurodeaenerative
［基金项目］国家自然科学基金地区资助项目（NO ：81560594）；贵州省国际科技合作计划项目（NO ：黔科合外G 字（2014］
7011）%
［通信作者］李菲，女，博士，畐教授，研究方向：神经药理学，E-m — ： lifei@ zmu. edu. cn %・254
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3张洋洋等•淫羊L昔对@。

2诱导的PC12细胞内质网应激的保护作用
disease and no remission in its pmvmssion,now af-aicts more than50million people worldwigv(2).The disaoveeyoedisease-modieyingdeugsisnow oethe neueophaemaaooogiaaoeeseaeah peioeities.
Icamin(ICA),a Chinese traditional herbal med-iaineeiteaated eeom Epimedium,eihibitsawideeange oephaemaaoogiaa=eunationsina=udinganti-ineam-mation,antioxigant ations⑶.It alse has a protection neueon,again,toiygen-gouao,edepeivation and oii-dative stmss⑷.ICA R wiOely dRtributed in the body and can pas through the blood-brain b*mer(5).We haveaa e ied outthat CCA aan peoteatneueonsagainst ER stress by inducing Hrd1expression(6).However, thee e atoeCCA on ER stee s isnotaoeaeoy,theee-eoee,thispeesentstudyisaimed toevaouatethee e at oeCCAon ERstee s in PC12ae s induaed byH2020
1Materials and methods
1.1Mgsmts Ica/i n(purity#98%by HPLC)was pueahased eeomNanjingWeoangMediaaoTeahnooogyCoe-poeation Ltd(Nanjing,China),and di s ooved in dimeth-yosuoeoiide(DMS0)and eioteeed.0theeeeagentsweee eeagent-geaded and aommeeaia y avaioaboe.
1.2Cel l culture and treatment Highly diRemntia-ted eatpheoaheomoaytoma PC12ae s(Roakvi o e, MD,USA)were cultured in DMEM supplemented with100U/ml penicillin,100U/ml streptomycin,and 10%FBS at37°C in a humidified atmosphere of95% aieand5%C0
2.Themediumwas ahanged eveeyoth-er day.First,we tested the ifect of ICA alone on PC12cells by treating cells with concentra­tions of ICA(
3.125,6.25,12.5,25,50and100 "M)for48h.Subsequently,the cells injury induced by@02(100,200,400,800"M)was evvluated.To inve,tigatethepeoteativee e atoeCCA on H202-in-duced PC12cells injury,cells were pretreated with ICA at ermus concentrations(6.25,12.5and25 "M)eoe2h,and Ghen ao-inaubaed wih400"M HO z ir another48h.
1.3Cell viability assay Cell viability was meas­ured by the MTT assay(Beijing Solarbio Scienc一a& Technology,China).Cel l s were seeded onto96wel l s plate at1X105cells/pel l density in100"l culture medium for the indicated time points Another while cells were incubated with MTT for4h at37°C till the end of experimenO The dark blue cmsths in cils weee,ooubioieed in DMS0.Theoptiaaoden,ity(0D) of each wel t was measured at570nm with a Micm-poaeReadee.Ce o viabioiywasaaoauoaed aspeeaen-aga of celt viability(OD treated^OD control)X100.
1.4LDH leakaga assay The PC12celts(2X104 ae s peewe o)weeepee-teeated with oewithoutCCA asdesaeibed aboveand thesupeenatantwasanaoyeed byaLDH deteation kit(NanjingjianahengBioengi-neemng Institute,China),according to the manufac­turer s pmtocol.The makaga of LDH was determined at490nm waveoength usingaMiaeopoateReadee.
1.5Real-Oma quantitative PCR Total RNA from PC12ae s wasisooated with Teieooeeagent.Then1"g mRNA waseeveese teansaeibed to aDNA with the qScript cDNA synthesis kit.After30s pm-denature at95C,40cycles wil t ba perfomned: denature at95C for15s,annealing and extension at60C for30s. Di s oaiation au vewaspe7to7med atte7tinishing40 ayaoestove7itythequaoityotp ime7sand ampoitiaa-tion.Quantitative eao-timePCR(qPCR)waspe-formed with SYBR®Gun Supmio(BioRad).Ri-ativeeipee s ion wasnoemaoieed to0-aatin0Speaitia peimeesused in thisstudyweeeoistin taboe10
Gene GeneCD Foewaed peimee Reveesepeimee TabSe1Sepicific qPCR primers used in this stedy
GRP78NM_013083.2CTACGAAGGTGAACGACCCC GCAGGAGGGATTCCAGTCAG PERK NM_001313918.1GCGGCAGGTCCTTGGTAATC GGCACAGCTGTAGGTTGGTT
ATF4NM_024403.2TTAAGCCATGGCGCTCTTCA GACATTAAGTCCCCCGCCAA CHOP XM_006241445.3CCTAGCTTGGCTGACAGAGG CTGCTCCTTCTCCTTCATGC
0-actin NM031144.3CGCGAGTACAACCTTCTTGC CGTCATCCATGGCGAACTGG
1.6Western Blot Westeen bootanaoysis wasused to weeehomogenieed with RCPA oysisbu t ee,whiah aon-
detemnina the level of PERK,iF2#,ATF4,CHOP, phospho-PERK(Thr982),phospho-iF2# (The51),0-aatin and Tubuoin(A inity,USA).Ce s tained thepeoteaseinhibitoeand phosphataseinhibitoe aoaktaios.Totaopeoteinsweeesepaeated on10%SDS-PAGEgeosand teanse e ed toPVDFmembeanes,whiah
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weeebaoaked wieh non -eaemiak eoe2 h aeeoom eem- peraturo , and then incubated with first - antibodies o- vey night at 4 °C with rotation. Aftea washing , mem-
b anesweeinaubaeed wieh app op iaeeHRPaonuuga- ted second - antibodies ( Beyotime , China ) foy 1 h at
room tempsature ; the blots were visualized by Beyo- ECL ( Thsmo , Waltham , MA , USA ) . The image was scanned , and the band intensity was quantibed using Quantity One softwarev 4.52 ( BioRad ).
1. 7 Statistical Analysis Statistical analysis was peeeoemed u,ingSPSS 18. 0. A a da ea we ee e ep ee ,ed as mean 土 SD and were analyzed fgy statistical signif ­
icance using a one - way anUysis of veriance ( ANO- VA ) , followed by Bonferroni multipS comparisons
W s U P <0. 05 was considered statisticaUy significant.
2 Resulhs
2. 1 ICA inhibits H 2O 2 - induced PC12 ceUs injury
The ceU viability was measured using MTT assay in
ehisseudyaeeeeeaeiouseeeaemenes.Asshown in Fig 1A , less than 50 "M ICA did not affect ceU viability and caused no aU toxicity. The resuis indicated that H O 2 reduced aU injury in a concentration - depend- enemannee ,and eheaonaeneeaeion ae400 "M eoe48 h
eimepoinewasaoneiemed eobean appeopeiaeeER stress model w vitro with approximateiy 50% aU via-
bOity inhibition rate [ Fig 1B ]. As shown in Fig 1C , H2 O2 decreased ceU viability , which was partiy re ­
versed by ICA ( 12. 5 and 25 "M ) .In parallel , 400
"M H 2O 2 significantiy increased LDH release. Howev-
ee , peeeeeaemenewieh CCAeeduaed eheamouneoeLDH release [ Fig 1D ]. This protective effect of ICA was also evidenced by morphologic observations of PC 12
al i s. As shown io Fig 2, H 2O 2 induced aUs shOnk-
age and eoatation , pretreated with ICA reduced the in- uuey.
A
B
H2O2 (400 gM) H2O2 (400 yM)
Celi viability was measured by MTT assay followed treatment with indicated concentrations of ICA ( A) and H 2O 2
(B) for 48 h. Cells were pro 一 treated with indicated concentrations of ICA for 2 h followed cc 一 incubation with 400
"M H 2O 2 for 48 h ,celi viability was measured by MTT assay ( C) and LDH release assay ( D) . (7 ± SD , n = 3 inde ­pendent experiments ,every experiment contained 5 samples/group ) * :P <0. 05, * * :P <0.01 vs controi ,#：P <
0.05,##：P<0.01v H 2O 2.
Fig 1 Effect of ICA on H 2 O 2 - inducee PC 12 cells injury
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张洋洋等・淫羊L 苛对@。

2诱导的PC12细胞内质网应激的保护作用
Fig 2
ICA (6.25 jiM)
ICA (12.5 jiM) ICA(25 卩M)
The effect of ICA on H 2 O 2 - induced morphologicaS alternations in PC 12 cells (Arrows : injured cells )
2. 2 ICA represses H 2O 2 induced - ER stress in PC12 celts In order to obsemv the eiect of ICA on ER stress , we tested the level of marker of ER stress : glucose - reaulated protein 78 ( GRP78 ) , and the re ­sults showed that H2O2 increased high expression of
GRP78 peoein , in eeesingoy , peeeeaed wih CCA sig ­nificantly decreased GRP78 level [ Fig 3 A and D ]. Wetuetheeeipooeed whetheethepeotein kinaseRNA
-oikeERkinase ( PERK ) pathwayisinvooved in the
ifect of ICA on H 2O 2 - induced - ER stress. The phosphorylation level of PERK and # - subunit of eu-
koyotic translation initiation factor - 2 ( iF2# ) , as we o astheietotaopeoteinsoeveosweeetested byWest-
een boot.Theeesuotsshowed in Fig3 thatPERK and
iF2# phosphorylmion levels were induced higher by @2O2than conhot group , however , petreated with ICA
eeduaed theiephosphoeyation eves.Neit , we ei- pored the downstream factors of PERK - iF2# sig-
naoing pathway : aativating teansaeiption aatoe- 4
(ATF4) and C/PBP homologous protein ( CHOP ), and ound thattheiepeotein oeveoseeduaed in CCA
gmup compared with alone
group.
p-EERK Tubulin
G H
A : Representative bands ot GRP78 and p - PERK ；
B ：p - iF2# and iF2#;
C : ATF4 and CHOP;
D - H :respectivelu ,the
relative vvlue normalized with that ot tubulin , 0 - actin , or total protein. ( 7 土 SD , n = 4 independent experiments ) * ： P <
0. 05 , ! * :P <0. 01 5 control ,#：P <0. 05 ,##：P < 0. 01 vs H 2O 2.
Fig 3 Effect of ICA on ER stress - induced by H 2 O 2 in PC 12 cells
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This study also tested the mRNA levei of markery of ER stress,as shown in Fig4,HO z domUial i y in­creased the mRNA levei of GRP78,PERK,ATF4and CHOP in PC12ccl i.Pretreated with ICA could ds crease the mRNA level of GRP78,ATF4and CHOP, but not affect PERK mRNA level.
Control H2O26・2512・52S
ICA Q1M)
The mRNA expression of abovementioned genes were normalized to!-actin.(7±SD,n=4independ­ent experiments)*:P<0.05,**:P<0.01vs control,#：P<0.05,##：P<0.01vs H2O2.
Fig4Effect of ICA on the mRNA level of GRP78,ATF4,and CHOP in也O2-induced PC12cell
3Discussion
As an impoiant oryanUle in eukaryotio cell,ER is closely related te the synthesis of proteins,the pro­duction of lipids and the storage of calcium ions(7"8). It has been reported that a vvyeta of causes,such as ischemia,low oxygen,drug or poison,oxidative stress and other pathological physiology s/muit could induce ER stress Orther te induce the cell self-adjustment and even induce apoptosis(9).It also has been repea­ted that ER stress widely presents in the brain of AD patients,deteyorates neurons loss and neurological function(10),and could be one of the main causes of AD(11-12).We have been demonstrated that ICA could ameliorate the cognOive function of AD and neuron in­jury in multiple models^13"14).This study grther sup­plied the conclusion that ICA protected neuron w让h evidences of the increased cell viability and decreased LDH release using H2O2-induced cell injuees model W vitro.
There are thre e main sensors,PERK,activetiny transengtion fectoy-6(ATF6)andinos/oi-requt-yng protein-1(IRE1)located in ER membrane,ncy-ma i y combin9d wieh GRP78which cios9seh9iesig-nais.Und9eERsee s condieion,GRP78peoein di s o-ciates Oom the sensors and binds to non-folded and folded proteins,which is an impoOant marker to in­duce and reUect the stress level of ER(15).Then the sensor proteins:PERK,ATF6and IRE1wil l be acti­vated and migyes their downstream signaling pathway, respectively.ER stress reaction usually includes three mechanisms:the instantaneous adaptive response coues9sin eh99aeiyphas9swieh e9ducingpeoein syn-eh9siseh9ebyeh9numb9eoopeoeinseeansoeineoeh9 ER has been decreased#the second phase cel l s trig-yes the long-term adaptive response,reyulate the co e spondingg9n9eeansceipeion eo9nhanc9eh9abiiiey of ER treated with non—folding protein#if the Orst two mechanisms do not reestablish cell homeostasis, then the apoptosis reaction will be toggered to protect oryanisms Oom damaged cells(16"17).H2O2at the con­centration of400"M incubated PC12cells foy48h induced cells loss and severe ER stress evidenced by eh9ince9as9d ooie9iGRP78,PERK and iesdown-see9am9CF2#,asw9i asa i eiaed eh9phosphoeyia-eion ooPERKand9CF2#.P e9ee9a ed wieh CCA ooe2h reduced the expression of GRP78protein and mRNA in H2O2-induced PC12cells#suppressed the activi­ty of PERK and eIF2#through decreasing their phos­phorylation level,but not altered the mRNA level
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3张洋洋等•淫羊L昔对@。

2诱导的PC12细胞内质网应激的保护作用
PERK and the total protein of iF2#This vvidence maybeindiaatesthatCCA eeguoatesthetunation ot PERK and iF2#via inhibiting their activity,not their eipee s ion.
Aatieated PERK phospho ey oates and aatieatesits downstream iF2#at sine51site.p-iF2#sup-pee s estheteansoation initiation aompoeieCF2-GTP -Mi-tRNAi Mi,and then reduces the mostly pro­teins synthesis load in the ER(18),but increases ATF4 peotein,ynthe,i.ATF4enaode,aaAMPeeaation eoe-mentthataan peomoteae o,ueeieaotheough induaing gene,eipee,ion in aminoaaid metabooim,eedoiee-aation,,tee,ee,pon,eand peotein,eaeetion eea ation, o ep eomote apopto,i th eough induaingCHOPeipee,-sion(19-20).The level of ATF4and CHOP has been in­creased in PC12cits after incubation with H2O2-ICA p eteatmentto72h aouod7ep e s thep7otein and mRNAoeeeootATF4and CHOP,theetoeimp7oeed theae s su7eieao.
Cn aonaousion,thisstudysuppoted thehypothe-sisthatCCA po s e s es the p oteation againstER ste s,espeaia o y supp7e s ingtheaatieation otPERKP eIF2#signaling pathway.
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