Kaempferol Alleviates LD-mitochondrial Kev Puas Tsuaj Los Ntawm Kev Txhawb Autophagy: Kev cuam tshuam hauv Parkinson's Disease Part 1

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Abstract:Cov pov thawj tshwm sim qhia tau hais tias kev npaj txhij txog lipid droplet (LD) deposition thiab peroxidation tuaj yeem ua kom lub cev muaj zog thiab ua lub luag haujlwm tseem ceeb hauv cov kab mob ntawm cov kab mob neurodegenerative (NDDs). , nthuav tawm cov teebmeem neuroprotective rau nas nrog LPS-induced Parkinson's disease (PD). Tsis tas li ntawd, cov kev tshawb fawb yav dhau los tau pom tias autophagy plays lub luag haujlwm tseem ceeb hauv kev tswj hwm ntawm cellular LD deposition. Nyob rau hauv txoj kev tshawb no tam sim no, peb tau pom tias Ka tiv thaiv TH ntxiv rau neuronal poob thiab kev coj tus cwj pwm tsis zoo hauv MPTP / p-induced PD nas, nrog rau kev txo qis lipid oxidative kev nyuaj siab hauv substantia nigra pars compacta (SNpc).In cultured neuronal cells, Ka nthuav tawm ib qho kev nyab xeeb ntawm ntau yam thiab cuam tshuam LD ntau ntxiv thiab cellular apoptosis tshwm sim los ntawm MPP ntxiv. Kev tshawb fawb ntxiv tau qhia tias qhov kev tiv thaiv ntawm Ka yog nyob ntawm autophagy, tshwj xeeb tshaj yog lipophagy. Critically, Ka txhawb autophagy los kho LD degradation nyob rau hauv lysosomes, uas tom qab ntawd alleviated lipid deposition thiab peroxidation thiab ua rau mitochondrial puas tsuaj, thiaj li yuav txo tau neuronal tuag. Tsis tas li ntawd, AAV-shAtg5-mediated Atg5 knockdown tshem tawm cov teebmeem neuroprotective ntawm Ka tiv thaiv lipid oxidation hauv PD nas. Qhov kev ua haujlwm no qhia tau tias Ka tiv thaiv dopaminergic neuronal degeneration hauv PD los ntawm kev cuam tshuam ntawm lipid peroxidation-mediated mitochondrial kev puas tsuaj los ntawm kev txhawb nqa di ncauj-autophagy thiab muab cov tswv yim tshiab kho tshiab rau PD thiab lwm yam NDDs.

Thov nias ntawm no kom paub ntxiv

1. Taw qhia

Parkinson's disease (PD) yog ib hom kab mob neurodegenerative (NDD) uas yog pathologically tus cwj pwm los ntawm kev poob ntawm dopaminergic (DA) neurons nyob rau hauv substantia nigra pars compacta (SNpc)[]. Txawm hais tias qhov tseeb etiology thiab tej yam ntuj tso ntawm qhov tsis yooj yim no tseem tsis tau qhia meej, ntau cov txheej txheem-theem thiab kev ua haujlwm tsis zoo, suav nrog kev ua haujlwm mitochondrial, dopamine homeostasis, neuroinflammation, thiab autophagy, tau cuam tshuam rau hauv pathogenesis ntawm PD [1, 2]. Cov ntawv ceeb toom tsis ntev los no tau qhia tias lipid droplet (LD) ntsig txog lipotoxicity tuaj yeem koom nrog PD pathology [3,4]. LDs yog cov organelles muaj zog heev uas tshwm sim los ntawm endoplasmic reticulum (ER) membrane thiab ib txwm ua haujlwm hauv cov chaw hauv nruab nrab ntawm nruab nrab lipid cia [5]. Tsis ntev los no LDs tau pom tias ua lub luag haujlwm dav dua li kev khaws cia ntawm fatty acid (FA) thiab koom nrog ntau yam kab mob. Piv txwv li, cov hlwb myeloid, suav nrog cov macrophages, leukocytes, thiab eosinophils, tsim LDs hauv kev teb rau kev mob thiab kev ntxhov siab, thiab LDs yog qhov chaw ntawm inflammatory cytokine ntau lawm thiab khaws cia thiab koom nrog hauv kev nthuav qhia antigen thiab pathogen clearance [6]. Qhov tseem ceeb, nyob rau hauv atherosclerosis, LD-nplua nuj ua npuas ncauj hlwb tau pom tias yuav deleterious nyob rau hauv tag nrho cov theem ntawm tus kab mob [7].

Cistanche tuaj yeem pab txhim kho kev tiv thaiv

Txawm li cas los xij, LDs tsis tau kawm ntau yam hauv lub hauv nruab nrab paj hlwb (CNS), nrog ob peb cov ntaub ntawv qhia txog cov keeb kwm ntawm LDs hauv tib neeg lub hlwb [8,9]. Txawm li cas los xij, LDs tuaj yeem muaj lub luag haujlwm tseem ceeb hauv lub hlwb, raws li kev tshawb fawb tsis ntev los no tau lees paub tias cov LDs sib sau ua ke hauv glia ua rau muaj kev cuam tshuam ntawm neurodegeneration hauv Drosophila qauv [10]. Tsis ntev los no tau tshaj tawm tias hauv cov nas, cov roj liab O-positive lipid-laden hlwb, suav nrog cov neurons, glial fibrillary acidic protein (GFAP) ntxiv rau astrocytes thiab ionized calcium-binding adapter molecule-1 (IBA-1} ) microglia, muaj nyob hauv ntau thaj tsam ntawm lub hlwb thiab tau pom tias koom nrog cov txheej txheem muaj hnub nyoog [11]. Peb cov hauj lwm yav dhau los kuj tau pom tias muaj LD ntau ntxiv hauv lub hlwb hauv PD nas qauv [12]. Ua ke, cov kev tshawb fawb no qhia tias LDs tuaj yeem koom nrog hauv cov kab mob ntawm PD, nrog rau lwm yam NDDs.

Feem ntau, intracellular LDs yog degraded nyob rau hauv lysosomes thiab xa FAs rau mitochondria rau lawv noj raws li ib tug lwm lub zog qhov chaw nyob rau hauv lub sij hawm ntawm nutrient depletion [13]. Txawm li cas los xij, neurons muaj lub peev xwm tsawg rau mitochondrial FA noj rau lub zog tsim [14]. Cov yam ntxwv no ua rau cov neurons tshwj xeeb tshaj yog rau LD txuam thiab peroxidation. Tsis tas li ntawd, qhov sib xyaw ntawm LDs txhim kho FA oxidation tus nqi thiab ua rau lub siab tsis tu ncua ntawm mitochondrial electron thauj saw, uas ua rau kom cov pa oxygen reactive (ROS) ntau lawm los ntawm electron thauj saw complexes Iand II [15] kom nthuav tawm oxidative kev nyuaj siab. Lipid overload kuj ua rau ROS ntau lawm los ntawm extramitochondrial qhov chaw xws li nicotinamide adenine dinucleotide phosphate (NADPH) oxidases thiab lwm yam oxidative enzymes. Ua ke nrog kev txo qis ntawm cov enzymes antioxidant thiab lub peev xwm qis rau FA noj nyob rau hauv neurons, LD sacumulation mediates oxidative kev nyuaj siab thiab tuaj yeem ua rau kev puas tsuaj mitochondrial, lysosomal dysfunction, tsis zoo autophagy, thiab ua kom cov inflammatory teb [16,17]. Tshwj tsis yog cov LDs khaws cia tuaj yeem cuam tshuam lossis tshem tawm, cov neurons muaj zog heev tau txais kev kho mob pathophysiology, ua rau nce mus rau neurodegeneration [17]. Cov ntaub ntawv pov thawj qhia tias cov txheej txheem lysosome-mediated catabolic hu ua autophagy plays lub luag haujlwm tseem ceeb hauv kev tswj hwm cellular LD homeostasis hauv ntau cov ntaub so ntswg [17,18]. Hauv kev nthuav dav, LDs tuaj yeem xaiv tau hauv autophagosomes thiab xa mus rau lysosomes rau degradation los ntawm lysosomal acid lipases, txheej txheem tshwj xeeb hu ua lipophagy [19]. Yog li ntawd, tsom LDs thiab lawv txoj kev tshem tawm autophagic tuaj yeem sawv cev rau txoj hauv kev tshiab rau kev tswj cov neurodegeneration.

Txoj kev tshawb fawb ntawm cov khoom ntuj thiab cov khoom noj khoom haus raws li cov peev txheej rau kev kho mob NDD thiab cov tswv yim tau txais kev txaus siab ntau hauv xyoo tas los [20]. Kaempferol (Ka) yog lub ntuj polyphenolic me me molecule uas tuaj yeem pom nyob rau hauv ntau cov tshuaj suav tshuaj thiab cov khoom noj khoom haus [21]. Ka tau raug tshaj tawm kom siv tshuaj tua kab mob, tiv thaiv kev laus, thiab kev tiv thaiv kab mob, nrog rau kev ua haujlwm antioxidant thiab neuroprotective [20,22,23]. Yav dhau los, Ka tau tshaj tawm tias inhibit qhov mob ntawm BV2 microglial hlwb hauv vitro [24] thiab ua rau muaj kev tiv thaiv ntawm DA neurons rau neuroinflammation hauv vivo [25]. Txawm li cas los xij, qhov cuam tshuam ntawm Ka ntawm LDs hauv PD tseem tsis tau tshawb xyuas.

Muab qhov tseem ceeb ntawm LDs thiab kev sib raug zoo ntawm LDs, mitochondria, thiab autophagy [16], uas txhua tus koom nrog PD, peb xav tias Ka inhibits LD peroxidation thiab tiv thaiv DA neurodegeneration hauv PD. Hauv txoj kev tshawb fawb tam sim no, peb tau pom tias Ka tau tiv thaiv kev tiv thaiv neurodegeneration hauv murine PD qauv los ntawm inhibiting LD tsub zuj zuj. Txoj kev tshawb fawb ntxiv tau qhia tias Ka ua rau autophagy thiab txo qhov sib xyaw ntawm oxidized LDs kom txo qis mitochondrial dysfunction thiab mitochondrial ROS (mtROS) ntau lawm, yog li tiv thaiv neuronal apoptotic tuag. Cov kev tshawb pom tau los ntawm txoj kev tshawb fawb no tuaj yeem pab ncaj qha kev txiav txim siab txog kev siv lub ntuj molecule Ka hauv NDDs xws li PD.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Kev sim tsiaj

C57BL/6J nas (txiv neej,4- hli) tau txais los ntawm Nanjing Medical University Animal Core (Nanjing). Cov nas tau khaws cia thiab bred nyob rau hauv Animal Resource Center ntawm Kws Kho Mob, Nanjing Medical University thiab nyob rau hauv cov tsiaj chaw ntawm Nanjing Drum Tower Tsev Kho Mob. Cov nas yog pub dawb nkag mus rau zaub mov thiab dej hauv chav nrog qhov kub thiab txias ntawm 22 ° ± 2 degree thiab lub teeb / tsaus voj voog ntawm 12:12 teev. Txhua cov txheej txheem tsiaj tau ua raws li cov lus qhia ntawm National Institutes of Health Guide for the Care and Use of Laboratory Tsiaj.

2.2. Reagents

MPTP (M0896),3-MA (M9281), penicillin-streptomycin (V900929), sodium pentobarbital, and probenecid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Kaempferol (HPLC>95%)for in vivo treatment was purchased from Nanjing Jingzhu Biotechnology Co., Ltd (Nanjing, China), kaempferol(HPLC>99. BODIPY 493/503 (D3922), BODIPY 581/591 C11 (D3861), DHE (D11347) tau yuav los ntawm Thermo Fisher Scientific. Rau kev sim tsiaj, Ka tau yaj hauv kev daws ntawm 10 feem pua ​​​​DMSO hauv 16 feem pua ​​​​SBE- -CD hauv sterile saline. Rau kev sim ntawm tes, Ka tau npaj hauv DMSO (Sigma-Aldrich) thiab PBS (qhov kawg DMSO concentration yog 0.01 feem pua), pH 7.4.

2.3. Induction thiab kev kho ntawm methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced PD nas qauv

Txhawm rau ntsuas qhov cuam tshuam ntawm Ka hauv PD tus qauv ntawm MPTP/p-toxicity, nas (txiv neej, hnub nyoog 4-5 lub hlis)) tau muab faib rau hauv pawg tshuaj saline, pawg MPTP-kho, MPTP ntxiv rau Ka-kho pab pawg, thiab Ka ib leeg kho pab pawg. Hauv pawg MPTP-kho, nas tau txais kev tswj hwm MPTP ntev nrog cov txheej txheem zoo ib yam li tau piav qhia yav dhau los [26]: MPTP tau yaj hauv saline thiab txhaj tshuaj subcutaneously ntawm 25 mg / kg tom qab 250 mg / kg probenecid (solved hauv dimethyl sulfoxide) intraperitoneally (ip) txhaj tshuaj ntawm 1 teev txhua 3.5 hnub nyob rau lub sijhawm 5 lub lis piam. Cov nas tswj tau txais kev txhaj tshuaj saline thiab probenecid. Hauv pab pawg MPTP ntxiv rau Ka-kho, nas tau txais Ka (50 mg / kg, ib zaug txhaj tshuaj / hnub thaum lub sijhawm sim, Jingzhu Biotechnology, Nanjing, Tuam Tshoj) intraperitoneally (p.) txhua hnub 3d ua ntej kev kho mob nrog MPTP thiab tshaj 5. MPTP txhaj lub lis piam. Ib lub lis piam tom qab txhaj tshuaj MPTP kawg, cov nas tau raug kuaj tus cwj pwm tsis pom kev rau cov pab pawg. Tom qab ntawd, txhua tus nas tau muab tshuaj loog nrog 40 mg / kg sodium pentobarbital thiab txi rau kev kuaj pom lub hlwb thiab kev tiv thaiv kab mob-histochemistry lossis qPCR tsom xam.

2.4. Hauv vivo stereotaxic phais thiab kev sim kho

Stereotaxic phais nyob rau hauv sodium pentobarbital anesthesia (40 mg/kg, ip) tau ua raws li tau piav qhia [25]. Nas Atg5 thiab tswj shRNA tau kis nrog cov khoom ntim khoom (AAV-U6-CMV-shR-NA-EGFP) los tsim AAV. Rau microinjection, cov nas tsuag tshuaj loog tau muab tso rau hauv lub tshuab stereotaxic. Lawv tau txhaj nrog 1μLAAV los ntawm iav electrode aiming ntawm DG(AP:-0.3 hli; ML: ±{15}}.13 hli; DV:-0.45 hli) ntawm tus nqi ntawm 0.25 μL/min. Tom qab ntawd cov koob tau khaws cia rau ntxiv 2-min.2 lub lis piam tom qab tus kab mob microinjection, nas tau raug rau MPTP-induced PD qauv conduction thiab (lossis) Ka kho.

Atg5 shRNA tau txais los ntawm Hanbio Biotechnology (Shanghai, Tuam Tshoj) Co., Ltd. Rau Atg5 silence, cov primers tau qhia nyob rau hauv Ntxiv Table 1.

2.5. Kev txheeb xyuas tus cwj pwm

Ib lub lis piam tom qab qhov kawg txhaj tshuaj ntawm MPTP los yog saline, rotarod xeem thiab ncej xeem tau ua raws li tau piav ua ntej [27]. Rau qhov kev sim rotarod, nas tau acclimatized rau rotarod hauv ob qhov kev sim (6 min nrog ceev ceev ntawm 12-20 rpm) ib hnub rau 2 hnub sib law liag ua ntej pib qhov kev sim. Tom qab ntawd, cov nas tau sim ntawm 20 rpm rau 300 s rau lwm 3 hnub sib law liag. Lub latency rau lub caij nplooj zeeg tau sau tseg siv Rotarod Analysis System (Jiliang, Shanghai, Suav). Rau kev ntsuas tus ncej, nas tau muab tso rau lub taub hau rau saum toj ntawm tus ncej ntoo ntsug (nruj-surfaced, qhov siab 50 cm, txoj kab uas hla 1 cm). Txhua tus nas tau raug siv rau lub cuab yeej 2 hnub ua ntej kuaj thiab tom qab ntawd sim peb zaug hauv peb hnub. Tag nrho lub sij hawm (T-tag nrho, kom txog thaum tus nas mus txog hauv pem teb nrog nws plaub paws) thiab lub sij hawm tig (T-tig, rau nas tig kiag lub taub hau downward) raug kaw. Qhov kev sim tau ua qhov muag tsis pom rau cov pab pawg nas rau txhua qhov kev ntsuas tus cwj pwm thiab qhov kev sim tau ua peb zaug.

2.6. Lub hlwb cov qauv sau

Tom qab qhov kev ntsuam xyuas tus cwj pwm zaum kawg, cov nas tau muab tshuaj loog nrog sodium pentobarbital (40 mg / kg, ip).Rau qPCR, western blotting, thiab high-performance kua chromatography (HPLC) tsom xam, tag nrho lub hlwb tau sai tshem tawm ntawm cov tsiaj ces lub midbrain thiab Cov ntaub so ntswg striatum tau sai sai dissected, ua ntej khov los ntawm kua nitrogen. Tag nrho cov qauv tau muab khaws cia ntawm -80 degree kom txog thaum kev tshuaj ntsuam.

Rau kev soj ntsuam immunohistochemical, nas tau perfused trans-cardially nrog 4 feem pua ​​​​paraformaldehyde (PFA). Lub hlwb raug muab rho tawm, tom qab kho, lub cev qhuav dej, kos rau hauv OCT (Tissue-Tek), thiab cov ntu ntu ntawm lub hlwb tau raug kaw (30 um ib daim) los ntawm txhua qhov stratum thiab midbrain siv lub microtome khov (Leica CM1950, Nussloch, Lub teb chaws Yelemees ). Tag nrho cov seem tau muab sau rau hauv rau qhov sib cais thiab lub paj hlwb tau muab khaws cia rau hauv 50 feem pua ​​​​glycerin thiab khov rau hauv -20 degree kom txog thaum kev tshuaj xyuas.

Rau TEM, nas tau perfused nrog 2.5 feem pua ​​glutaraldehyde thiab 2 feem pua ​​paraformaldehyde raws li tau piav 【12】. Ib feem me me (<1 mm³）="" of="" the="" mesencephalon="" was="" carefully="" sectioned="" and="" incubated="" in="" the="" same="" fixative="" for="" 2="" h="" at="" 4°c.="" specimens="" were="" postfixed="" in="" 1%="" osmium="" tetroxide,="" stained="" in="" aqueous="" uranyl="" acetate,="" and="" then="" dehydrated="" and="" embedded="" in="" epoxy="" resin.="" ultrathin="" sections="" were="" stained="" using="" lead="" citrate="" and="" examined="" with="" a="" transmission="" electron="" microscope(jem-1010,="" tokyo,="">

2.6. Immunohistochemical tsom xam

Lub paj hlwb tau muab yaug hauv PBS ua raws li 3 feem pua ​​​​H2O2 rau 10 min ces incubated nrog 0.3 feem pua ​​Triton X-100 hauv PBS ntxiv nrog 5 feem pua ​​BSA rau 1 h. Tom qab ntawd, cov slides tau incubated nrog cov thawj cov tshuaj tiv thaiv ntawm 4 degree thaum hmo ntuj hauv PBS uas muaj 5 feem pua ​​BSA ntawm 4 degree thaum hmo ntuj, ces ntxuav thiab incubated nyob rau hauv lwm cov tshuaj tiv thaiv rau 1 h nyob rau hauv chav tsev kub, ces incubating nrog diaminobenzidine (DAB) los yog mounting nyob rau hauv DAPI (Lub Neej Technologies, Cat P36931) ua cov tshuaj tiv thaiv kab mob fluorescent rau 5 feeb. Rau Nisslstaining, cov swb tau muab tso rau hauv cresyl violet (CV) kev daws teeb meem (0.1 g cresyl violet, 99 ml H2O thiab 1 feem pua ​​​​acetic acid 1 ml) rau 30 min ntawm chav tsev kub ces lub cev qhuav dej nrog cawv thiab xylene. Cov duab tau pom thiab cov duab tau raug ntes hauv Olympus BX52 microscope (Olympus America Inc., Melville, NY, United States). Tag nrho cov naj npawb ntawm TH-positive thiab Nissl's-positive neurons hauv SNpc tau txais serologically los ntawm kev siv cov txheej txheem kho qhov muag nrog MicroBrightField Stereo-Investigator software (MicroBrightField, Williston, VT, USA).

2.7. High-performance kua chromatography (HPLC) tsom xam

Cov qauv nas striatal tau homogenized nrog {{0}}.1 M perchloric acid (1 mg cov ntaub so ntswg hauv 100 μL perchloric acid) thiab 0.1 mM EDTA, kho los ntawm

2.17. Flow cytometry

Cells tau zom thiab yaug nrog D-hank's ces stained nrog Annexin V-FITC (5 ul diluted hauv 500ul PBS)/PI (0.1 ul diluted hauv 500 ul PBS) apoptosis kit (CA1020, Solarbio, Beijing, Chian) raws li cov chaw tsim khoom raws tu qauv. Tom qab ntawd, cov hlwb apoptotic tau txheeb xyuas siv cov cytometry ntws (guava easyCyteTM 8, Millipore, USA). Mitochondrial ROS thiab mitochondrial membrane muaj peev xwm ntsuas tau ua raws li luam tawm 【12】.SH-SY5Y hlwb tau stained nrog MitoSOX (2.5 μM, Invitrogen, USA) lossis nrog JCl (10 ug / ml, T-3168, Invitrogen, USA ) ntawm 37 ℃ rau 30 min. Tom qab ntxuav nrog PBS ob zaug, cov hlwb tau rov ua dua nyob rau hauv txias PBS uas muaj 1 feem pua ​​​​FBS rau ntws cytometric tsom xam. Cov ntaub ntawv raug tshuaj xyuas nrog FCS Express software (Guava Easy CyteTM8, Millipore, Hayward, CA, USA).

2.18. Seahorse ua pa ntsuas

Mitochondrial ua pa muaj nuj nqi nyob rau hauv SH-SY5Y hlwb raug ntsuas los ntawm kev siv Seahorse extracellular flux (XFe96) tshuaj ntsuam xyuas (Agi-lent, Santa Clara, USA) los ntawm kev hloov pauv ntawm cov pa oxygen noj (OCR). SH-SY5Y cov noob qoob loo ntawm 3 × 103cells / zoo tau tso cai ua raws li Seahorse cell kab lis kev cai daim phiaj thiab kom ncav cuag li 80 feem pua ​​​​ntawm lub sijhawm ntawm kev sim. Hnub tom qab, cov hlwb raug pretreated nrog Ka thiab ces kis tau MPP ntxiv rau lwm 24 teev. OCR tau kuaj pom nyob rau hauv cov kab mob basal ua raws li qhov sib ntxiv ntawm 1 μM oligomycin, 1 μM FCCP, nrog rau 1 μM rotenone & antimycin A.

2.19 ib. Kev txheeb cais

Cov ntaub ntawv tau nthuav tawm raws li txhais tau tias ± SEM. Qhov tseem ceeb ntawm qhov sib txawv yog txiav txim los ntawm Tub Ntxhais Kawm T-test, Ob-txoj kev tsom xam ntawm qhov sib txawv, lossis ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) ua raws li Tukey's post hoc test. Qhov sib txawv tau suav tias yog qhov tseem ceeb ntawm P<>

3. Cov txiaj ntsig

Qhov tshwm sim 1. Ka restores lub cev muaj zog dysfunction thiab nce dopamine theem nyob rau hauv lub striatum ntawm MPTP/p PD qauv nas

Txhawm rau tshawb xyuas cov txiaj ntsig neuroprotective ntawm Kaon PD pathogenesis, 4-hli txiv neej C57BL/6 nas tau txhaj nrog neurotoxin MPTP los tsim MPTP/p PD qauv thiab tau kho nrog Ka (Daim duab la). Tom qab cov qauv induction, qhov kev xeem rotarod thiab cov ncej xeem tau siv los ntsuas lub cev muaj zog thiab kev coj tus cwj pwm ntawm cov nas hauv ntau pawg. Raws li tau pom, lub sijhawm ua haujlwm rotarod tau txo qis hauv MPTP-kho nas, thiab cov nyhuv no tau tiv thaiv los ntawm Ka (Fig.1b). Ka kuj tau kho cov kev coj cwj pwm tsis zoo uas tau tshwm sim los ntawm MPTP, raws li qhia los ntawm kev txo qis ntawm lub sijhawm tig thiab tag nrho lub sijhawm nyob rau hauv kev xeem ncej (Fig.lc thiab d), tsis cuam tshuam rau lub cev hnyav ntawm cov nas (Fig.1e). Tsis tas li ntawd, HPLC kev tshuaj xyuas tau pom tias qib ntawm dopamine hauv striatum tau txo qis hauv MPTP/p-cov nas sib tw, thiab qib no tau rov qab los ntawm kev kho Ka (Daim duab 1f). Ka kho kuj txo MPTP / p-vim txo qis hauv dihydroxy-phenyl acetic acid (DOPAC, metabolite ntawm dopamine) qib hauv striatum (Fig.1g). Cov txiaj ntsig no qhia tias Ka rov ua kom lub cev tsis muaj zog thiab nce qib dopamine hauv striatum hauv MPTP-induced PD nas.

Qhov tshwm sim 2. Ka alleviates qhov poob ntawm DA neurons hauv SNpc ntawm MPTP/p PD qauv nas

Tom ntej no, txhawm rau ntsuas cov txiaj ntsig neuroprotective ntawm Kaon MPTP / p-induced DA neuronal impairment, peb tau tshuaj xyuas tyrosine hydroxylase (THD ntxiv rau cov neurons hauv murine SNpc los ntawm kev tiv thaiv kab mob. Raws li qhia hauv daim duab 2a thiab b, MPTP / p induced ib tug tseem ceeb txo. Nyob rau hauv tus naj npawb ntawm TH plus neurons, uas tau alleviated los ntawm Ka kho. Tsis tas li ntawd, stereological suav ntawm tag nrho cov neurons hauv SNpc, raws li txhais los ntawm Nissl

Fig. 1. Ka kho mob txo lub cev tsis muaj zog thiab nce dopamine ntau ntau hauv striatum ntawm MPTP/p PD qauv nas.

(a) Schematic daim duab ntawm kev sim tsim. (b) Lub sijhawm ntawm tus pas ntsuas tau ntsuas rau qhov ntsuas rotarod peb hnub sib law liag. (cd) Lub sijhawm tig rov qab (lub sijhawm tig) thiab nqis ib tus ncej (lub sijhawm nce toj) tau sau tseg rau kev xeem ncej. (e) Nas lub cev hnyav tau ntsuas thaum kawg ntawm txoj kev tshawb no, (fg) Dopamine (DA) thiab dihydroxy-phenyl acetic acid (DOPAC) nyob rau hauv lub striatum tau soj ntsuam los ntawm high-performance kua chromatography. Cov ntaub ntawv raug qhia raws li qhov txhais tau tias ± SEM.n =9-10 rau txhua pawg hauv (b, C, d); n=6-8 rau txhua pab pawg hauv (f,g).ns, tsis tseem ceeb, *P<><><0.001, by="" one-way="" anova="" followed="" by="" tukey's="" post="" hoc="" test.="" mptp,="" 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;="" ka,="">

staining, txheeb xyuas tias qhov poob ntawm THt hlwb cuam tshuam txog kev tuag ntawm cellular tab sis tsis yog qhov txo qis ntawm TH qhia thiab pom tias Ka kho kho qhov txo qis ntawm Nissl-positive neurons hauv SNpc ntawm MPTP-kho nas los ntawm 43.3 feem pua ​​​​rau 25.4 feem pua ​​( Fig.2c thiab d). Tsis tas li ntawd, Ka tau txo qis MPTP vim qhov txo qis hauv TH thiab DAT cov protein qhia, raws li ntsuas los ntawm Western blotting (Fig.2e-g). Cov pov thawj no tau lees paub cov txiaj ntsig neuroprotective ntawm Ka ntawm tus qauv PD nas.

Qhov tshwm sim 3. Ka txo qhov sib xyaw ntawm LD vacuoles thiab oxidative kev nyuaj siab hauv SNpc ntawm MPTP/p-kho nas.

Cov pov thawj ntxiv tau pom tias muaj kev koom tes ntawm LDs hauv NDDs [10], tsis tu ncua, peb txoj haujlwm yav dhau los tau txheeb xyuas qhov nce LDs hauv SNpc ntawm MPTP-induced PD nas [12]. LDS tuaj yeem pom tau yooj yim thiab qhia cov puag ncig, cov qauv ntom ntom uas muaj homogenous amorphouds [28]. Nquag, LDs tuaj yeem degraded ntawm autophagy kom tsis txhob sib sau ua ke [29]. Txhawm rau tshawb xyuas seb Ka puas tuaj yeem tswj hwm LDs hauv PD, qhov sib xyaw ntawm LDs tau txheeb xyuas los ntawm kev xa hluav taws xob hluav taws xob (TEM) hauv SNpc ntawm kev tswj hwm, MPTP / p, thiab MPTP / p ntxiv rau Ka nas. Raws li tau pom hauv peb txoj haujlwm dhau los [12], tus naj npawb thiab qhov loj ntawm LDs tau nce hauv MPTP / p nas piv rau cov kev tswj hwm saline (Fig.3a thiab b) thiab tau txo qis hauv Ka-kho nas (Fig.3c- e). Ntxiv mus, nyob rau hauv Ka-kho nas, LDs tau nquag pom nyob ze rau, nyob rau hauv, los yog degraded los ntawm autolysosomes, uas tau txhais raws li electron-dense lipofuscin granules. Ntxiv histological staining ntawm TH ntxiv rau cov neurons nrog BODIPY, ib qho xim uas tshwj xeeb tshaj tawm cov lipids nruab nrab thiab feem ntau siv los kuaj LDs [30], pom tias ob qho tib si tus naj npawb thiab qhov loj ntawm BODIPY ntxiv rau LDs hauv SNpc tau siab dua hauv PD nas dua li hauv kev tswj hwm. , thiab tau txo qis los ntawm kev kho Ka (Daim duab 3f-h).

Yog tias LDs tsis tuaj yeem degraded raws sijhawm, cov LDs tuaj yeem raug peroxidation thiab pab txhawb rau mitochondrial ROS-mediated stress [4], uas ua rau muaj kev cuam tshuam rau neurotoxicity thiab kab mob kev loj hlob. Yog li ntawd, peb tau soj ntsuam ntxiv cov lus qhia ntawm cov noob cuam tshuam nrog kev tiv thaiv dawb FA toxicity (Gpx8), neutralizing oxidative hom, superoxide radicals (Sod3) thiab hydrogen peroxide (Cat), thiab FA metabolism (Acsbg1 thiab Dbi) raws li qhia [31] , tag nrho cov uas tau poob qis hauv SNpc ntawm MPTP-kho nas piv nrog cov tswj, thiab cov teebmeem no tau ua kom zoo dua hauv Ka-kho PD nas (Fig. 3i). Ib yam li ntawd, MPTP/p kuj nce qib mRNA qhia

ntawm NADPH oxidase subunits xws li Nox1, Nox2, thiab Nox4 hauv SNpc ntawm MPTP/p nas, uas tau ntxiv blunted los ntawm Ka kho (Fig.3j). Ua ke, cov ntaub ntawv no qhia tias Ka alleviates MPTP / p-induced LD sacumulation, peroxidation, thiab ROS-mediated stress.

Kab lus no yog muab rho tawm los ntawm Cov Ntsiab Lus muaj nyob ntawm ScienceDirect Redox Biology phau ntawv xov xwm homepage: www.elsevier.com/locate/redox