PART 1 Echinacoside Inhibits Glutamate Release By Suppressing Voltage-Dependent Ca2 plus Entry And Protein Kinase C in Rat Cerebrocortical Nerve Terminals

Cheng Wei Lu 1,2, Tzu Yu Lin 1,2, Shu Kuei Huang 1 thiab Su Jane Wang 3,*

1 Department of Anesthesiology, Far-Eastern Memorial Tsev Kho Mob, Pan-Chiao District, New Taipei City 22060, Taiwan; drluchengwei@gmail.com (CWL); drlin1971@gmail.com (TYL);nskh9450n@yahoo.com.tw (SKH)

2 Department of Mechanical Engineering, Yuan Ze University, Taoyuan 32003, Taiwan

3 Tsev Kawm Ntawv Tshuaj, Fu Jen Catholic University, No. 510, Zhongzheng Rd., Xinzhuang Dist., New Taipei 24205, Taiwan.

Abstract:Lub glutamatergic system tuaj yeem koom nrog cov teebmeem ntawm kev kho mob neuroprotectant. Echinacoside, phenylethanoid glycoside muab rho tawm los ntawm cov tshuaj suav tshuaj ntsuabHerba Cistanche,muaj cov teebmeem neuroprotective. Txoj kev tshawb no tshawb xyuas qhov cuam tshuam ntawm echinacoside ntawm 4-aminopyridine-evoked glutamate tso tawm hauv nas cerebrocortical paj hlwb terminals (synaptosomes). Echinacoside inhibited Ca^{2 plus}-dependent, tab sis tsis yog Ca^{2 plus}-independent, 4-aminopyridine-evoked glutamate tso tawm nyob rau hauv ib tug concentration-dependent yam. Echinacoside kuj txo qhov 4-aminopyridine-evoked nce hauv cytoplasmic dawb Ca^{2 plus}concentration tab sis tsis hloov lub synaptosomal membrane peev xwm. Cov nyhuv inhibitory ntawm echinacoside ntawm 4-aminopyridine-evoked glutamate tso tawm tau tiv thaiv los ntawm cu-conotoxin MVIIC, qhov dav-spectrum blocker ntawm Cav2.2 (N-type) thiab Cav2.1 (P / Q-type) channels , tab sis yog insensitive rau intracellular Ca^{2 plus}tso tawm-inhibitors dantrolene thiab 7-chloro-5-(2-chlorophenyl)-1, 5-dihydro-4, 1-benzodiazepine{{ 8}}(3H)-ib (CGP37157). Tsis tas li ntawd, echinacoside txo qis 4-aminopyridine-induced phosphorylation ntawm protein kinase C, thiab protein kinase C inhibitors tshem tawm cov nyhuv ntawm echinacoside ntawm glutamate tso tawm. Raws li cov txiaj ntsig no, peb xav tias qhov cuam tshuam ntawm echinacoside ntawm evoked glutamate tso tawm yog cuam tshuam nrog txo qis voltage-dependent Ca.^{2 plus}nkag mus thiab tom qab tsub zuj zuj ntawm cov protein kinase C kev ua.

Ntsiab lus:echinacoside; glutamate tso tawm; cerebrocortical paj hlwb terminals; voltage-dependent Ca²ntxiv cov channel; protein kinase C ntawm Parkinson's disease, Alzheimer's disease, thiab nruab nrab cerebral artery occlusion[9-12]. Txawm li cas los xij, cov txheej txheem los ntawm echinacoside induces neuroprotection tsis nkag siab tag nrho.

Yog xav paub ntxiv thov hu rau:Joanna.jia@wecistanche.com

Echinacosidehauv cistanche muaj ntau txoj haujlwm

1. Taw qhia

Echinacosideyog ib qho tseem ceeb phenylethanoid glycoside tam sim no hauvHerba Cistanche, ib lub npe nrov suav tshuaj siv los kho qhov tsis nco qab, tsis muaj zog, thiab cem quav [1].Echinacosidemuaj ntau yam bioactivities xws li tshuaj tiv thaiv oxidation, tiv thaiv kab mob, tshuaj tiv thaiv kab mob, hepatoprotection, thiab kev tiv thaiv kab mob [2-4]. Qhov tseem ceeb,echinacoside cov tshuajmuaj cov teebmeem neuroprotective; Piv txwv li, nws muaj peev xwm tiv thaiv oxidative kev nyuaj siab-los yog neurotoxin-induced neurotoxicity nyob rau hauv thawj nas cortical neurons, tib neeg neuroblastoma SH-SY5Y hlwb, thiab pheochromocytoma (PC12) hlwb [5–8]. Tsis tas li ntawd,echinacoside cov tshuajattenuates hlwb puas thiab txhim kho kev txawj ntse muaj nuj nqi nyob rau hauv cov tsiaj qauv ntawm Parkinson tus kab mob, Alzheimer's kab mob, thiab nruab nrab cerebral hlab ntsha occlusion [9-12]. Txawm li cas los, lub mechanism ntawm uasechinacoside cov tshuajinduces neuroprotection tsis to taub tag nrho.

Neuroprotection yog cov txheej txheem nyuaj ntawm kev khaws cov qauv neuronal thiab ua haujlwm raws li kev thuam thuam. Glutamate excitotoxicity txo yog suav tias yog lub peev xwm txheej txheem koom nrog hauv hlwb neuroprotection. Glutamate, ib qho excitatory amino acid neurotransmitter, muaj lub luag haujlwm tseem ceeb hauv ntau lub hlwb ua haujlwm [13]. Txawm li cas los xij, overactivation ntawm glutamate receptors nyob rau hauv siab glutamate concentrations ua rau intracellular Ca2 ntxiv rau overload, mitochondrial dysfunction, dawb radical ntau lawm, thiab neuronal tuag [14,15]. Cov txheej txheem pathological no cuam tshuam rau ntau lub hlwb mob xws li cerebral ischemia, raug mob lub hlwb, mob vwm, thiab kab mob neurodegenerative [16,17]. Li no, inhibitors thaiv kab mob pathophysiological glutamatergic kis tau tus mob yog suav hais tias muaj peev xwm neuroprotective tshuaj. Cov piv txwv tseem ceeb ntawm cov no yog glutamate receptor antagonists [18,19]; Txawm li cas los xij, kev sim tshuaj rau cov tshuaj no tau ua tsis tiav vim tias tsis muaj txiaj ntsig thiab tsis xav tau, lossis txawm tias muaj kev mob tshwm sim cytotoxic [20,21]. Ntxiv rau qhov ncaj qha glutamate receptor blockade, glutamate tso tawm inhibition tuaj yeem yog lub tswv yim zoo rau kev tiv thaiv kab mob. Ntau cov neuroprotectants (xws li, memantine thiab riluzole) tuaj yeem txo glutamate tso tawm hauv nas hlwb [22–24].

Xav txog lub luag haujlwm ntawm glutamate hauv excitotoxicity thiab neuroprotective profile ntawmechinacoside cov tshuajKev tshawb fawb tam sim no tau siv cov paj hlwb cais tawm (synaptosomes) purified los ntawm nas cerebral cortex los tshawb xyuas cov txiaj ntsig ntawmechinacoside cov tshuajntawm glutamate tso tawm thiab tshawb nrhiav ntxiv muaj peev xwm mechanisms. Kev npaj cov paj hlwb cais tawm yog ib qho qauv tsim zoo rau kev kawm cov kev cai presynaptic ntawm neurotransmitter tso tawm los ntawm cov tshuaj thaum tsis muaj cov teebmeem postsynaptic [25]. Los ntawm kev siv cov qauv no, peb tau soj ntsuam cov nyhuv ntawmechinacoside cov tshuajntawm glutamate tso tawm, membrane muaj peev xwm, presynaptic Ca2 ntxiv rau influx, thiab protein kinase C kev ua. Raws li peb qhov kev tshuaj xyuas ntawm cov ntaub ntawv, qhov no yog thawj daim ntawv tshaj tawm uas sau cov txheej txheem los ntawm qhov ntawdechinacoside cov tshuajinhibits endogenous glutamate tso tawm ntawm qib presynaptic.

Echinacoside tuaj yeem tiv thaiv oxidation

2. Cov txiaj ntsig

2.1. EchinacosideInhibits 4-Aminopyridine-Evoked Glutamate tso tawm los ntawm nas Cerebrocortical Nerve Terminals los ntawm Txo Cov Vesicular Exocytosis

Daim duab 1 qhia txog qhov concentration-dependent nyhuv ntawmechinacoside cov tshuajntawm {{0}}aminopyridine-evoked glutamate tso tawm los ntawm purified nas cerebrocortical synaptosomes. Hauv synaptosomes incubated nrog 1 mM CaCl2, 1 mM 4-aminopyridine evoked glutamate tso tawm ntawm 7.4 ˘ 0.1 nmol / mg / 5 min, uas tau txo los ntawm 1, 5, 10, 30, thiab 50 µMechinacoside cov tshuajrau 6.5 ˘ 0.2, 5.8 ˘ 0.3, 4.8 ˘ {{10}}.2, 4.1 ˘ 0.1, lossis 2.3 ˘ 0.4 nmol/mg/5 min, ntsig txog (F(5,24)=67.1, p=0.000). Tus nqi IC50 rauechinacoside cov tshuaj-mediated inhibition ntawm 4-aminopyridine-evoked glutamate tso tawm, muab los ntawm cov koob tshuaj tiv thaiv kab mob, yog 24 µM. Ntxiv mus, glutamate tso tawm evoked los ntawm 1 mM 4-aminopyridine nyob rau hauv ib qho extracellular Ca2 ntxiv -free tov uas muaj 300 µM ethylene glycol bis (-aminoethyl ether)-N, N, N1, N {{ 13}}tetraacetic acid (EGTA) yog 2.1 ˘ 0.2 nmol/mg/5 min (F(2,12)=310.65, p=0000), thiab qhov no Ca2 ntxiv -independent tivthaiv ntawm 4-aminopyridine-evoked glutamate tso tawm tsis cuam tshuam los ntawm 20 µMechinacoside cov tshuaj(1.8 ˘ 0.2 nmol/mg/5 min; p=0.58; Daim duab 1). Hauv synaptosomes kho nrog 0.1 µM bafifilomycin A1, vesicular transporter inhibitor [26], 4-aminopyridine-evoked glutamate tso tawm tau txo qis (2.2 ˘ 0.2 nmol/mg/ 5 feeb; F (2,12)=249.518, p=0.000). Nyob rau hauv lub xub ntiag ntawm bafifilomycin A1, 20 µM echinacoside ua tsis tau tejyam kom tsis txhob muaj qhov tso tawm ntawm glutamate (2.1 ˘ 0.2 nmol/mg/5 min; p=0.94; Daim duab 1). Los ntawm qhov sib piv, 10 µM DL-threo-beta-benzyl-oxyaspartate (DL-TBOA, glutamate reuptake inhibitor) [27], nce 4-aminopyridine-evoked glutamate tso tawm rau 11.8 ˘ 0.4 nmol / mg / 5 min ( t(8)=´11.31, p=0 000). Txawm nyob rau hauv lub xub ntiag ntawm DL-TBOA, 20 µMechinacoside cov tshuajinhibited {{0}}aminopyridine-evoked glutamate tso tawm qhov tseem ceeb (7.7 ˘ 0.2 nmol/mg/5 min; F(2,12)=87.23, p=0.{{ 12}}; Daim duab 1)

2.2. EchinacosideTxo Cytosolic Ca²ntxivConcentration tab sis Tsis Hloov Synaptosomal

Mem brane Potential

Synaptosome depolarization tshwm sim los ntawm 1 mM 4-aminopyridine nce Ca²ntxiv concentration(p= 0000; Table 1). Daim ntawv thov ntawm 20 |vMechinacoside cov tshuajtsis cuam tshuam rau baral Ca²ntxiv concentration (t(8) = 0.06, p= 0.95) tab sis txo qis ntawm 4-aminopyridine-induced nce iia Ca²ntxiv concentration (t(10) = 6.16, p =0 000). Tsis tas li ntawd, 1 mM 4-aminopyridine nce hauv 3',3',3'-dipropylthiadicarbocyanine iodide [DiSC₃(5)] fluorescence (p= 0 000). Qhov ntxiv o⁽20 fdM echinacoside tsis tau hloov lub so ntawm daim nyias nyias muaj peev xwm (t(8)=0.976,p= 0.36) lossis hloov pauv qhov 4-aminopyridine-mediated nce hauv DiSC₃(5) fluorescence (t(8) = —0.014, p= 0.99; Table 1).

Cistancheechinacoside cov tshuajtuaj yeem kho mob raum

2.3. Txo - CO2 plusInflux los ntawm Cav2.2 (N-Type) thiab Cav2.1 (P/Q-Type) Raws Tej zaum yuav koom nrog Inhibition ntawm 4-Aminopyridine-Evoked Glutamate tso tawm los ntawmEchinacoside

Daim duab 2 qhia tau tias 2 cu-conotoxin MVIIC, ib N-thiab P / Q-type Ca²ntxiv rau channel blocker,

txo {{0}}aminopyridine-evoked glutamate tso tawm los ntawm 7.4 土 0.2 rau 2.0 土 0.1 nmol/mg/5 min (t(9) = 25.35, p{{0}}000). Nyob rau hauv lub xub ntiag ntawm cu-conotoxin MVIIC, cov nyhuv ntawm 20 pM echinacoside ntawm 4-aminopyridine-evoked glutamate tso tawm tsis tseem ceeb (1.8 土 0.2 nmol / mg / 5 min;t(8) = 1.06, p= 0.32). Dantrolene (10 ^ M), ib qho inhibitor ntawm intracellular Ca²ntxiv rau tso tawm Crom lub endoplasmic reticulum [28], txo 4-aminopyridine-evoked glutamate tso tawm (5.6 土 0.3 nmol/mg/5 min; F(2,14)=104. 95,p= 0 000). Txawm li cas los xij, nyob rau hauv lub xub ntiag ntawm dantrolene, 20 pJMechinacoside cov tshuajtseem tuaj yeem inhibit glutamate tso tawm ntau heev (3.3 土 0.2 nmol/mg/5 min;p= 0 000). Cov txiaj ntsig zoo sib xws tau pom siv 100 pM 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin -2(3H)-ib (CGP37157), ib daim nyias nyias-permeaWe blocker ntawm mitochondrial Na^ntxiv/Ca²plus pauv. Hauv tsib qhov tshuaj xyuas synaptosomal npaj, 20 pMechinacoside cov tshuajua ke nrog 100 pM CGP37157 txo 4-aminopyridine-evoked glutamate tso tawm los ntawm 48.3 feem pua ​​​​土 5.2 feem pua ​​(F(2,13) ​​= 136.79,p= 7000), zoo ib yam li inhibition los ntawm echinacoside ib leeg (46.2 feem pua ​​​​土 2.3 feem pua ​​;p = 0.89;

2.4. EchinacosideInhibcts 4-Aminopyridine-Evoked (Glutamate tso tawm los ntawm Kev Tshaj Tawm los ntawm Protein Kinase C

Raws li pom hauv daim duab 3, 10 degree jlM 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X), gen er al protein kinase C inhibitor [29], reduc ed 4-aminopyridine-evoked glutamate tso (F(2,13) ​​= 19.46, p=0 000). Hauv GF109203X-kho synaptosomes, 20 pM echinacoside txo 4-aminopyridine-evoked glutamate tso tawm tsuas yog 5.5 feem pua ​​​​土 1.8 feem pua ​​(p=0.89), tsawg dua li ntawm echinacoside ib leeg (42.42 feem pua ​​​​土 . feem pua ​​; p=f.000). Cov txiaj ntsig zoo sib xws tau txais nrog 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3 ,4-c]carbazole-12-propanenitrile (Go6976), ib qho kev xaiv rau Ca2 ntxiv -dependent protein kinase C isoforms (a, |3I, |3II, y) [29]. Nyob rau hauv lub xub ntiag ntawm 3 pM Go6976, 20 p.Mechinacoside cov tshuajtxo glutamate tso tawm los ntawm 11.9 feem pua ​​​​土 2.9 feem pua ​​(p=0.59), qhia txog kev txo qis piv nrog qhov ntawd los ntawm echinacoside ib leeg (42.4 feem pua ​​土 2.3 feem pua; p=0.000; Daim duab 3). Los ntawm qhov sib txawv, 3 pM rottlerin, ib qho Ca2 ntxiv -independent protein kinase C6 inhibitor [30], tsis tau hloov pauv 4-aminopyridine (1 mM)-evoked glutamate tso tawm (p=0.45). Txawm li cas los xij, nyob rau hauv lub xub ntiag ntawm rottlerin, 20 pM echinacoside ua tau zoo ua rau qhov nruab nrab inhibition ntawm 37.1 feem pua ​​​​土 5.6 feem pua ​​​​ntawm kev tso tawm (F(2,13) ​​= 19.72, p=0.{{33 } }), zoo ib yam li ntawm echinacoside ib leeg (p=0.41; Daim duab 3). Tsis tas li ntawd, mitogen-activated protein kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-ib) (PD98059 ) (50 pM) thiab cov protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) (100 pM) txo 4-aminopyridine-evoked glutamate tso tawm (p=0 000). Txawm nyob rau hauv lub xub ntiag ntawm PD98059 los yog H89, 20 pMechinacoside cov tshuajtxo qhov tso tawm tau zoo (F(2,13) ​​= 52.3, p=0.000; Daim duab 3).

Daim duab 4 qhia tha11 mM 4-aminopyridine nce phosphorylation ntawm protein kinase C hauv synaptosomes (t(4) = —6.871, p= 0.002). Thaum synaptosomes tau pretreated nrog 20 pM echinacoside rau 10 min ua ntej ntxiv ntawm 4-aminopyridine, 4-aminopyridine induced phosphorylation ntawm protein kinase C txo qis (F(2)_a6) = 29.202, p = 0.00 1).

Echinacosidehauvcistanchetuaj yeem txhawb nqativ thaiv kab mob

2.5. Echinacoside-Mediated Inhibition ntawm Glutamate Release Tsis Koom Nrog Gamma-Aminobutyric Acid Hom A (GAB³A_A) Receptor

Hauv daim duab 5, cov nyhuv ntawm echinacoside ntawm 4-aminopyridine-evoked glutamate tso tawm thaum tsis muaj lossis muaj SR95531 (tus antagonist ntawm GABA_Areceptor) tau muab piv. Tsis tas li ntawd, 100 pM SR95531 tsis tau hloov pauv 4-aminopyridine (1 mM)-evoked glutamate tso tawm. Hauv

SR95531-kho synaptosomes, daim ntawv thov ntawm 20 pM echinacoside ua rau 43 feem pua ​​inhibition ntawm 4-aminopyridine-evoked glutamate tso tawm (F(2,12)=42.63,p= 0.000), uas tsis txawv ntawm qhov inhibition tsim los ntawm echinacoside ib leeg (40 feem pua ​​;p= 0 000). Ib qho txiaj ntsig zoo sib xws tau txais nrog lwm tus GABAa receptor antagonist, bicuculline (50 pM). Kev tso tawm ntsuas nyob rau hauv lub xub ntiag ntawm bicuculline thiab