PART 1: Cistanche Extracts Ameliorates Lub Neurotoxicity Induced Los ntawm Hydrogen Peroxide nyob rau hauv Tshiab Mutant DJ-1-transfected Neuroblastoma Cellular Models

Hu rau:joanna.jia@wecistanche.com

1. Kev Taw Qhia

Cov kab mob neurodegenerative tseem ceeb suav nrogAlzheimer tus kab mobkab mob, Parkinson's disease (PD), Huntington's disease, thiab lwm yam. Ntawm cov kab mob no, qhov tshwm sim ntawm PD (Tus kab mob Parkinson) yog thib ob hauv ntiaj teb thiab ua tiav 1 feem pua ​​-2 feem pua ​​​​ntawm cov neeg muaj hnub nyoog tshaj 60 xyoo (Burke & O'Malley, 2013; Mullard, 2017; Shen & Ji, 2013). Raws li tus yam ntxwv tseem ceeb ntawm PD, dopaminergic neurons nyob rau hauv substantia nigra pars compacta yog cov kev loj hlob degenerative (Glizer & MacDonald, 2016), uas yog nrog los ntawm cov tsos ntawm -synuclein inclusions hu ua Lewy lub cev (Zhang, An, Zhang, & Pu , 2010). Nyob rau hauv lub pathogenesis ntawm PD, so tremors, rigidity, bradykinesia, thiab postural abnormalities raug kuaj raws li cov tsos mob tseem ceeb ntawm PD nyob rau hauv lub tsev kho mob. Txawm hais tias qhov tseeb ntawm cov txheej txheem degenerative ntawm dopaminergic neurons tsis tuaj yeem nkag siab, ib puag ncig ua rau suav nrog cov tshuaj tua kab, cov kab mob neurotoxic, thiab cov hlau hnyav, lossis cov caj ces xws li kev hloov pauv ntawm Parkin (Kitada li al., 1998), -synuclein (Polymeropoulos thiab. al., 1997), thiab DJ-1 (Biosa et al., 2017; Bonifati li al., 2003), tau ntseeg tias yuav ua rau muaj qhov tshwm sim ntawm PD. Txog rau tam sim no, tus naj npawb ntawm kev hloov pauv tsis zoo, hloov pauv hloov pauv, thiab tawg tawg loj tshwm sim los ntawm DJ-1 noob hloov pauv ntau dua 10 (Andres-Mateos li al., 2007; Bonifati li al., 2003; Hague et al., 2003). Tsis pub dhau DJ-1 protein, kev hloov pauv ntawm Leucine166Proline (L166P) yog qhov tseem ceeb. Tsis tas li ntawd, nws kuj tau pom tias oxidative kev nyuaj siab tshwm sim hauv PD (Dexter et al., 1989, 1994; Nikam, Nikam, Ahaley, & Sontakke, 2009) thiab koom nrog hauv nws cov kab mob (Hague et al., 2003).

DJ-1 noob muaj yim exons faib tshaj 24 kb, nrhiav tau ntawm chromosome 1p36 nyob rau hauv tib neeg (Bonifati li al., 2003), thiab encodes ib tug heev conserved protein uas muaj 189 amino acids. Cov protein muaj nyob rau hauv tsev neeg ThiJ / PfpI, muaj nyob rau hauv homodimer daim ntawv. Glial hlwb nyob rau hauv lub cortex thiab substantia nigra thiab striatum yog lub ntsiab pom nyob rau hauv cov cheeb tsam ntawm DJ-1 protein (Olzmann li al., 2007). Hauv cov hlwb no, DJ-1 ua lub luag haujlwm hauv qab no, xws li oncogene (Nagakubo li al., 1997), kev tswj hwm kev hloov pauv (Kim li al., 2005; Niki, Takahashi- Niki, Taira, Iguchi-Ariga, & Agria, 2003. ), antioxidative stress (Taira li al., 2004; Zhou & Freed, 2005), chaperone (Shendelman, Jonason, Marlinat, Leete, & Abeliovich, 2004; Zhou, Zhu, Wioson, Petsko, & Fink, 2006), thiab protease (Abou-Sleiman, Healy, Quinn, Lees, & Wood, 2003).

Kev kho mob rau cov kab mob neurodegenerative: cistanche extract

Cistancheyog cov tshuaj suav tshuaj thiab tau siv los kho cov kab mob andrology hauv cov hnub nyoog. Cov khoom xyaw tseem ceeb ntawmCistancheyog phenyl glycosides suav nrog 34 tebchaw, xws li acteoside, echinacoside, thiab lwm yam.Cistanche extractsuav nrog kev txhim kho kev ua haujlwm ntawm kev sib deev, kev tiv thaiv kev laus, nce kev kawm thiab kev nco muaj peev xwm, neuroprotection, immunomodulation, antifatigue, antiischemic, thiab kev tiv thaiv lub siab (Tu et al., 2011). Caffeic acid yog ib qho ntawm cov metabolites loj ntawm Cistanche (Yan, 2018).

Cov qauv siv cellular feem ntau ntawm PD suav nrog 1-methyl-4- phenyl pyridinium ion-induced PC12 hlwb (Abou-Sleiman li al., 2003) thiab hydrogen peroxide (H2O2)-induced neuroblastoma (SH-SY5Y) hlwb (Zhang li al. Ib., 2009). Txawm li cas los xij, cov yam ntxwv pathological ntawm PD tsis muaj nyob hauv cov qauv no. Txhawm rau tsim kom muaj lub cev muaj txiaj ntsig zoo ntawm cov qauv ntawm PD, hauv txoj kev tshawb no peb tsim H2O2 ntxias L166P thiab C106S DJ-1-transfected SH-SY5Y hlwb, thiab tshawb xyuas cov teebmeem ntawmCistanche extractthiab tseem ceeb bioactive compounds, xws li acteoside, echinacoside, caffeic acid, thiabCistanche tag nrho glycosidesntawm ob tus qauv no, thawj zaug.

Cistanche extract anti-neurodegenerative kab mob

2. MATERIAL S THIAB MUAB

2.1|Plasmids, tshuaj, tshuaj, thiab hlwb

FLAG-L166P DJ-1, FLAG-C106S DJ-1 plasmids, thiab anti-DJ-1 poly- clonal antibodies tau ua siab zoo muab los ntawm Dr. Hiroyoshi Ariga, Tsev Kawm Ntawv Qib Siab Pharmaceutical Sciences, Hokkaido University. Lub cev qhuav dej tsawg tsawg qhov nruab nrab (MEM) thiab F-12 nruab nrab tau yuav los ntawm Gibco. Lipofectin yog los ntawm Invitrogen. FastDigest Xho I thiab FastDigest EcoR Kuv yog los ntawm Fermentas. Cov khoom siv endotoxin-dawb plasmid npaj yog los ntawm biotech. SH-SY5Y cell kab yog los ntawm Cell Bank ntawm Tuam Tshoj Academy ntawm Kev Kho Mob Sciences. BCA protein assay reagent cov khoom yog los ntawm Pierce. PVDF daim nyias nyias yog los ntawm Millipore. Anti-FLAG polyclonal antibody yog los ntawm GeneTex.Cistanche extract, suav nrog acteoside, echinacoside, caffeic acid, thiab Cistanche tag nrho glycosides, tau muab los ntawm Department of Natural Medicines, Tsev Kawm Ntawv Pharmaceutical Sciences, Peking University.

2.2|Plasmids amplification, extraction, thiab purification

Tom qab culturing Escherichia coli JM109 hlwb hauv LB kua nruab nrab mus txog OD600=0.5, cov hlwb muaj peev xwm tau npaj siv txoj kev calcium chloride. Cov muaj peev xwm E. coli tau hloov pauv nrog plasmid DNA siv txoj kev ua kom sov, tom qab ntawd cov kev hloov pauv tau raug coj mus rau LB daim hlau uas muaj ampicillin rau 16-24 teev ntawm 37 degree. Tom qab ntawd ib qho kev hloov pauv monoclonal colony tau raug xaiv thiab coj mus rau hauv LB kua nruab nrab uas muaj 50 ug / mL ampicillin rau 12 teev ntxiv ntawm 37 degree. Cov plasmids tau muab rho tawm thiab ua kom huv si siv cov khoom siv endotoxin-dawb plasmid npaj raws li cov chaw tsim khoom cov lus qhia.

2.3|Plasmid ID nkag mus

Extracted plasmids raug rau enzymatic digestion, tom qab ntawd cov plasmids thiab digested fragments raug tshuaj xyuas siv 1 feem pua ​​agarose gel electrophoresis. Cov txiaj ntsig gel tau pleev xim rau hauv cov tshuaj EB rau 20-25 min ntawm chav tsev kub thiab yees duab.

2.4|Plasmid hloov mus rau SH-SY5Y hlwb

Purified plasmid DNA tau hloov mus rau hauv SH-SY5Y hlwb kab lis kev cai hauv MEM / F-12 nruab nrab uas muaj 10 feem pua ​​​​fetal bovine serum. Cov kab mob kis tau raug xaiv nyob rau hauv ib qho nruab nrab uas muaj 400 ug / mL G418, tom qab ntawd 200 ug / mL G418 tau siv los tswj cov kab mob sib kis tau zoo.

2.5|Kev txheeb xyuas cov hlwb hloov pauv los ntawm Western blot

Transfected SH-SY5Y hlwb tau lysed nyob rau hauv RIPA tsis. Tom qab centrifugation ntawm lysate, tag nrho cov protein concentration nyob rau hauv lub supernatant tau txiav txim siab siv BCA protein assay reagent cov khoom. Qhov sib npaug ntawm cov protein extract tau raug xa mus rau 12.5 feem pua ​​SDS-polyacrylamide gel electrophoresis thiab xa mus rau PVDF daim nyias nyias. PVDF daim nyias nyias tau thaiv nrog 5 feem pua ​​​​cov mis tsis muaj roj qhuav hauv cov tshuaj TBST thiab ua tiav rau kev tiv thaiv kab mob. Anti-FLAG thiab anti-DJ-1 tau siv los ua thawj cov tshuaj tiv thaiv, thiab HRP-conjugated IgG yog cov tshuaj tiv thaiv thib ob. Ib qho kev txhim kho chemiluminescence nrhiav tau qhov system tau siv los txheeb xyuas cov hom phiaj protein.

2.6|Immunocytochemical kev txheeb xyuas ntawm cov kab mob sib kis

Transfected SH-SY5Y hlwb raug kho nrog 4 feem pua ​​​​cov tshuaj paraformaldehyde, permeabilized nrog 0.3 feem pua ​​Triton X-100 tov, thiab thaiv nrog 10 feem pua ​​tshis ntshav. Cov hlwb uas thaiv tau raug tsim ua ntej nrog anti-FLAG thiab anti-DJ-1 polyclonal antibody, thiab tom qab ntawd nrog FITC-conjugated tshis antirabbit theem nrab antibody. Lub cell nuclei tau stained nrog Hoechst 33342, thiab cov hlwb tau pom nyob rau hauv ib qho inverted fluorescence microscope (IX-71, Olympus).

2.7|Cell viability assay

Untransfected thiab transfected SH-SY5Y hlwb tau plated rau hauv 96- rho tawm, suav nrog acteoside, echinacoside, caffeic acid, thiab Cistanche tag nrho glycosides (tag nrho 10, 20, thiab 40 ug / mL, raws li), tau ntxiv thiab cov hlwb tau incubated rau 6 hr ua ntej kev kho mob nrog 0.2 mM H2O2 rau ib tug ntxiv 1 hr. Qhov nruab nrab tau muab pov tseg, thiab kev muaj peev xwm ntawm tes tau soj ntsuam siv MTT raws li tau piav qhia saum toj no.

2.8|Cov teebmeem ntawm Cistanche extracts ntawm cell viability

rho tawm, suav nrog acteoside, echinacoside, caffeic acid, thiab Cistanche tag nrho glycosides (tag nrho 10, 20, thiab 40 ug / mL, raws li), tau ntxiv thiab cov hlwb raug incubated rau 6 hr ua ntej kho nrog 0.2 mM H2O2. rau ib qho ntxiv 1 hr. Qhov nruab nrab tau muab pov tseg, thiab kev muaj peev xwm ntawm tes tau soj ntsuam siv MTT raws li tau piav qhia saum toj no.

2.9|Kev txheeb cais

Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± SD ntawm peb qhov kev sim ywj pheej. Qhov sib txawv ntawm pawg tau txheeb xyuas siv ib txoj kev ANOVA thiab LSD txoj kev nrog SPSS 22.0 software. Cov nqi ntawm p < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="">

Cistanchetshuaj ntsuab

3|TSEEM CEEB

3.1|Plasmids tau zoo hloov pauv thiab ua kom huv si. Tom qab cais tawm hauv E. coli JM109, plasmids tau muab rho tawm thiab tshuaj xyuas siv 1 feem pua ​​​​agarose gel electrophoresis tom qab zom nrog cov enzyme txwv (Daim duab 1). Lub 6.2-kb plasmids tau cleaved rau hauv 5.4- thiab 0.8-kb linear DNA fragments, uas tau lees tias cov L166P thiab C106S DJ-1 plasmids tau ob leeg ntse qhia thiab purified.

3.2|Kev txheeb xyuas cov kab mob kis los ntawm Western blotting. Raws li pom nyob rau hauv daim duab 2, peb kuaj FLAG-L166P DJ-1 thiab FLAG-C106S DJ-1 bands ntawm txog 70 kDa. Cov qib siab ntawm FLAG-tagged protein nyob rau hauv cov kis tau tus mob SH-SY5Y hlwb qhia tias L166P thiab C106S DJ-1 mutants tau hais tawm hauv lawv cov kab mob sib kis.

3.3|Immunocytochemical kev txheeb xyuas ntawm cov kab mob sib kis. Nws qhia tau hais tias muaj zog fluorescent signals los ntawm transfected SH-SY5Y hlwb tom qab incubation nrog anti-DJ-1 los yog anti-FLAG antibodies. Qhov no lees paub qhov siab ntawm L166P thiab C106S DJ-1 qhia hauv cov tshuaj tiv thaiv kab mob.

3.4|Cov hlwb hloov pauv tau nkag siab ntau dua rau H2O2 dua li cov hlwb tsis hloov pauv. Tom qab incubation rau 1 hr nyob rau hauv lub xub ntiag ntawm graded concentrations ntawm H2O2 (0.1, 0.2, 0.3, 0.4, {{15} }.5, thiab 1 mM, ntsig txog), qhov viabilities ntawm SH-SY5Y hlwb hloov nrog L166P los yog C106S DJ-1 raug txo koob tshuaj raws li piv rau cov hlwb tsis hloov pauv.

3.5|Cistanche extracts inhibited H2O2-vim txo qis SH-SY5Y cell viability koob tshuaj-nyob ntawm inhibited los ntawm kev kho mob nrogCistanche extract, suav nrog acteoside, echinacoside, caffeic acid, thiab Cistanche tag nrho glycosides (tag nrho 10, 20, thiab 40 ug / mL, feem).

Cistanche