Ntu Ⅱ: Mechanism Of Autophagy Regulation in MPTP-Induced PD nas Via MTOR Signaling Pathway Los ntawm Echinacoside

Kev txhim kho cov tsos mob Neurobehavioral hauv MPTP-Induced PD nas los ntawm Echinacoside

Echinacoside txo lub sijhawm nce toj hauv MPTP-Induced PD nas

T-tig thiab T-Tag nrho hauv pawg M tau pom ntev dua li cov hauv pawg N (P < 0="" 01).="" t-tig="" thiab="" t-tag="" nrho="" hauv="" pawg="" eh="" poob="" qis="" dua="" piv="" nrog="" cov="" hauv="" pawg="" m="" (p="">< 0="" 01).="" piv="" nrog="" rau="" pawg="" eh="" thiab="" m="" pawg,="" ob="" qhov="" kev="" ntsuas="" saum="" toj="" no="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" tau="" nce="" ntau="" (p=""><0.01 thiab="" p=""><0.05, feem).="" kev="" sib="" piv="" ntawm="" pawg="" eh="" thiab="" ra="" pab="" pawg="" pom="" tias="" tsis="" muaj="" qhov="" sib="" txawv="" tseem="" ceeb="" hauv="" t-tig="" thiab="" t-tag="" nrho="" (raws="" li="" qhia="" hauv="" daim="" duab="">

Echinacoside in cistanche (2)

Echinacoside yog qhov zoo rau kev noj qab haus huv

Echinacoside nce qhov kev ua haujlwm tsis tu ncua hauv MPTP-Induced PD nas

Tus naj npawb rov qab nyob rau hauv tsib feeb, thiab tag nrho qhov kev ncua deb li ntawm 30 feeb txhua qhov poob qis hauv pawg M piv nrog cov hauv pawg N (P < 0.01).="" qhov="" saum="" toj="" no="" ob="" qhov="" ntsuas="" tau="" nce="" ntau="" hauv="" pawg="" eh="" thiab="" pawg="" ra="" piv="" nrog="" cov="" hauv="" pawg="" m="" (p="">< 0.01).="" piv="" nrog="" rau="" pawg="" eh,="" tus="" lej="" txhawb="" nqa="" thiab="" tag="" nrho="" qhov="" kev="" ncua="" deb="" tau="" txo="" qis="" hauv="" pawg="" cq="" thiab="" pawg="" wo="" (p=""><0.01). tsis="" muaj="" qhov="" sib="" txawv="" tseem="" ceeb="" hauv="" qhov="" ntsuas="" saum="" toj="" no="" ntawm="" pawg="" eh="" thiab="" ra="" pawg="" (p=""> 0.05) (raws li qhia hauv daim duab 1B).

Daim duab kuv Cov neurobehavior hauv nas ntawm txhua pab pawg.

(A) Kev xeem ncej hauv cov nas ntawm txhua pab pawg∶ Lub sijhawm tig (T-Tumn); Tag nrho lub sijhawm dimping (下 Totl).

(B) Kev ua ub ua no nyob rau hauv nas ntawm txhua pab pawg: Lub sij hawm erection nyob rau hauv tsib feeb (Rearing tooj); Kab rov tav txav deb li ntawm 30 feeb (tag nrho nrug).

(D) Lub rotarod nyob rau hauv nas ntawm txhua pab pawg: Lub sij hawm nyob rau hauv lub rotarod (tseem hu ua Latency rau poob)

Notes: *P<0.05,**p><0.0i, compared="" with="" the="" model=""><0.0l, compared="" with="" the="" high-dose="">echinacoside cov tshuajpab pawg, n =6.

Echinacoside ncua lub Fore thiab Hind Limb Strides hauv MPTP-Induced PD nas

Lub hauv ntej thiab nraub qaum stride txo qis hauv pawg M piv nrog cov hauv pawg N (P < {{0}}}01).="" lub="" fore="" thiab="" hind="" limb="" stride="" tau="" nce="" ntxiv="" hauv="" pawg="" eh="" thiab="" ra="" pawg="" piv="" nrog="" cov="" hauv="" pawg="" m="" (p="">< 0.01).="" piv="" nrog="" rau="" pawg="" eh="" thiab="" pawg="" m,="" lub="" hauv="" ntej="" thiab="" nraub="" qaum="" strides="" tau="" txo="" qis="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" (p=""><0.01 thiab="" p=""><0.05, raws="" li).="" tsis="" muaj="" qhov="" sib="" txawv="" tseem="" ceeb="" hauv="" kev="" ua="" ntej="" thiab="" hind="" limb="" strides="" ntawm="" pawg="" eh="" thiab="" ra="" pawg="" (raws="" li="" pom="" hauv="" daim="" duab="">

Echinacoside ncua lub latency kom poob hauv MPTP-Induced PD nas

Lub sijhawm nyob ntawm rotarod, tseem hu ua latency kom poob, tau txo qis hauv pawg M piv nrog N pawg (P < {{0}}.01).="" lub="" latency="" kom="" poob="" ntau="" ntxiv="" hauv="" pawg="" eh="" thiab="" pawg="" ra="" piv="" nrog="" pawg="" m="" (p="">< 0.01).="" lub="" latency="" poob="" qis="" heev="" nyob="" rau="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" (p=""><0.01 thiab="" p=""><0.05, ntsig="" txog)="" piv="" nrog="" pawg="" eh="" thiab="" m="" pawg.="" tsis="" muaj="" qhov="" sib="" txawv="" tseem="" ceeb="" hauv="" qhov="" latency="" kom="" poob="" ntawm="" pawg="" eh="" thiab="" ra="" pawg="" (p=""> 0.05) (raws li qhia hauv daim duab 1D).

Echinacoside in cistanche (15)

Echinacosideyog qhov zoo rau kev noj qab haus huv

Echinacoside nce cov ntsiab lus ntawm DA thiab Metabolite hauv Striatum hauv MPTP-Induced PD nas

Piv nrog rau pawg N, cov ntsiab lus ntawm DA, DOPAC, thiab HVA hauv pawg M tau txo qis (P < {{0}}}01).="" cov="" ntsiab="" lus="" ntawm="" da,="" dopac="" thiab="" hva="" tau="" nce="" ntau="" hauv="" pawg="" eh="" thiab="" pawg="" ra="" piv="" nrog="" cov="" hauv="" pawg="" m="" (p="">< 0.01).="" piv="" nrog="" rau="" pawg="" eh="" thiab="" m="" pawg,="" cov="" ntsiab="" lus="" ntawm="" da,="" dopac,="" thiab="" hva="" txo="" qis="" hauv="" pawg="" cq="" thiab="" pawg="" wo="" (p="">< 0="" 0}1="" thiab="" p="">< 0.05,="" raws="" li="" )="" .="" tsis="" muaj="" qhov="" sib="" txawv="" tseem="" ceeb="" hauv="" cov="" ntsiab="" lus="" ntawm="" da,="" dopac,="" thiab="" hva="" ntawm="" pawg="" eh="" thiab="" pawg="" ra="" (p=""> 0.05) (raws li pom hauv daim duab 2A–C). Piv nrog rau pawg N, cov ntsiab lus ntawm 5-HT poob qis hauv pawg M (P < 0.01),="" thiab="" cov="" ntsiab="" lus="" ntawm="" 5-ht="" hauv="" pawg="" eh="" tau="" nce="" ntau="" (p="">< 0.01).="" piv="" nrog="" rau="" pawg="" eh,="" cov="" ntsiab="" lus="" ntawm="" 5-ht="" poob="" qis="" hauv="" pawg="" cq="" thiab="" wo="" pawg="">< 0.01)="" (as="" demonstrated="" in="" figure="">

Daim duab 2 Kev kuaj pom cov ntsiab lus neurotransmitter hauv striatum los ntawm High-performance kua theem electrochemical method (HPLC-ECD).

(A) Dopamine (DA):

(B) dihydroxsyphenybacetic acid (DOPAC);

(D) 5-hydroxytryptamine (5-HT).

Notes:*P < 0.05.*P<00 i.="" compared="" with="" the="" model="" group;=""><><001.compared with="" the="" high-dose="">echinacoside cov tshuajpab pawg

Echinacoside nce qhov kev nthuav qhia ntawm TH hauv Substantia Nigra thiab txo qis kev nthuav qhia ntawm -Synuclein hauv PD nas.

Ua ntej, ob chav immunofluorescence staining tau ua rau TH thiab -synuclein. Piv nrog rau pawg N, qhov kev qhia ntawm TH hauv substantia nigra txo qis hauv pawg M, thaum qhov kev qhia ntawm -synuclein nce ntxiv. Txawm li cas los xij, piv nrog pawg M, kev qhia ntawm - synuclein txo qis hauv pawg EH thiab RA pawg. Cov kab lus ntawm -synuclein tau nce ntxiv hauv pawg CQ thiab WO pawg, uas yog ntau dua li ntawm pawg M (raws li pom hauv daim duab 3).

Daim duab 3 immunofluorescence ob chav staining ntawm TH thiab a-synuclein hauv cov nigra loj hauv cov nas ntawm txhua pawg.

Daim duab qhia pom qhov immunofluorescence ob chav staining ntawm tyrosine hydroxylase (TH)

-synuclein hauv substantia nigra hauv nas ntawm txhua pab pawg n =6

Kev nthuav dav ntawm fluorescence microscope: 200 × .TH tau los ntshav nrog ntsuab fluorescence,

2nd a-tub kawm ntawv tau los ntshav nrog liab fluorescence, xiav DAPI sawv cev rau cov liab qab.

Qhov sib tshooj ntawm ntsuab 2nd liab yog ob-stained zoo cell (daj).

Tom qab ntawd, Western blot tau txais yuav los ntsuas ob qhov ntsuas saum toj no. Cov txiaj ntsig tau pom tias cov protein qhia ntawm TH hauv pawg M tau qis dua li ntawm pawg N (P < {{0}}}01).="" cov="" lus="" qhia="" cov="" protein="" ntawm="" th="" hauv="" pawg="" eh="" thiab="" ra="" pawg="" tau="" nce="" ntau="" dua="" piv="" nrog="" rau="" hauv="" pawg="" m="" (p="">< 0.{{10}}1).="" cov="" protein="" qhia="" ntawm="" th="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" tau="" poob="" qis="" dua="" piv="" nrog="" rau="" hauv="" pawg="" eh="" (p=""><0.01). cov="" protein="" qhia="" ntawm="" -synuclein="" nyob="" rau="" hauv="" pawg="" m="" kuj="" nce="" piv="" nrog="" rau="" pawg="" n="" (p=""><0.01). txawm="" li="" cas="" los="" xij,="" cov="" ntsiab="" lus="" ntawm="" cov="" protein="" ntawm="" -="" synuclein="" hauv="" pawg="" eh="" thiab="" pawg="" ra="" tau="" poob="" qis="" dua="" piv="" nrog="" rau="" hauv="" pawg="" m,="" thiab="" qhov="" sib="" txawv="" tseem="" ceeb="" (p=""><0.01). piv="" nrog="" rau="" pawg="" eh="" thiab="" pawg="" m,="" cov="" lus="" qhia="" protein="" ntawm="" -synuclein="" hauv="" cq="" pawg="" thiab="" wo="" pawg="" tau="" nce="" ntau,="" thiab="" qhov="" sib="" txawv="" tseem="" ceeb="" (p=""><0.01) (raws="" li="" qhia="" hauv="" daim="" duab="">

Daim duab 4 Cov kev hloov pauv ntawm cov protein qhia ntawm TH thiab -synuclein hauv cov nigra ntau hauv cov nas ntawm txhua pab pawg.

Notes∶甲 P<><00i,compared with="" the="" model="" group;=""><><00i,compared with="" the="" high-dose="">echinacoside cov tshuajpab pawg n=6.

Echinacoside txo qis kev nthuav qhia ntawm P62 hauv Substantia Nigra hauv MPTP-Induced PD nas

Ua ntej, Western blot tau txais yuav los kuaj xyuas cov protein qhia ntawm P62. Cov txiaj ntsig tau qhia tias cov protein qhia ntawm P62 hauv pawg M tau nce pom zoo piv nrog N pawg (P < 0.01).="" cov="" lus="" qhia="" protein="" ntawm="" p62="" hauv="" pawg="" eh="" thiab="" ra="" pawg="" tau="" poob="" qis="" dua="" piv="" nrog="" pawg="" m,="" thiab="" qhov="" sib="" txawv="" yog="" qhov="" tseem="" ceeb="" (p="">< 0.01).="" piv="" nrog="" rau="" pawg="" eh,="" cov="" lus="" qhia="" protein="" ntawm="" p62="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" tau="" nce="" ntxiv="" (p=""><0.01) (raws="" li="" qhia="" hauv="" daim="" duab="" 5c).="" tom="" qab="" ntawd,="" immunofluorescence="" staining="" tau="" ua="" rau="" p62="" thiab="" dapi.="" nws="" tau="" pom="" tias="" piv="" nrog="" n="" pab="" pawg,="" p62="" hauv="" substantia="" nigra="" nce="" ntau="" pom="" tseeb="" hauv="" pawg="" m,="" cov="" lus="" ntawm="" p62="" poob="" qis="" hauv="" pawg="" eh="" thiab="" pawg="" ra,="" thiab="" cov="" lus="" qhia="" ntawm="" p62="" nce="" hauv="" pawg="" cq="" thiab="" wo.="" pab="" pawg="" (raws="" li="" qhia="" hauv="" daim="" duab="">

Daim duab 5 Cov kev hloov pauv ntawm cov protein qhia ntawm Becin I, LC3ll P62, thiab P62 immunofluorescence staining nyob rau hauv substantia nigra nyob rau hauv nas ntawm txhua pawg.

Notes: *P<><00l, compared="" with="" the="" model=""><>< 0.0i,="" compared="" with="" the="" high-dose="">echinacoside cov tshuajpab pawg

Qhov loj ntawm lub fluorescence microscope: 200 ×.P62 tau sau nrog cov fluorescence liab. DAPI xiav sawv cev rau liab qab, n= 6.

Echinacoside nce kev nthuav qhia ntawm Beclin 1 thiab LC3II hauv Substantia Nigra hauv MPTP-Induced PD nas

Ua ntej, Western blot tau txais los txiav txim siab cov protein qhia ntawm Beclin 1 hauv substantia nigra hauv nas ntawm txhua pawg. Nws tau pom tias cov protein qhia ntawm Beclin 1 hauv pawg M tau nce ntau dua piv nrog N pawg (P < 0.01).="" txawm="" li="" cas="" los="" xij,="" qhov="" nce="" ntawm="" cov="" protein="" qhia="" ntawm="" beclin="" 1="" hauv="" pawg="" eh="" thiab="" pawg="" ra="" tau="" pom="" tseeb="" dua="" (p="">< 0.01).="" piv="" nrog="" rau="" pawg="" eh="" thiab="" pawg="" m,="" cov="" lus="" qhia="" protein="" ntawm="" beclin="" 1="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" tau="" poob="" qis,="" thiab="" qhov="" sib="" txawv="" tseem="" ceeb="" (p=""><0.01) (raws="" li="" qhia="" hauv="" daim="" duab="">

Vim tias LC3 yog qhov taw qhia tseem ceeb ntawm autophagy, ob chav staining tau ua rau TH thiab LC3. Cov txiaj ntsig tau pom tias tus lej ntawm LC3 cov hlwb zoo hauv pawg M tau nce ntau ntxiv piv nrog rau hauv pawg N. Tus naj npawb ntawm LC3 zoo hlwb hauv pawg EH thiab pawg RA tau nce ntau dua li hauv pawg M. Txawm li cas los xij, thaum siv cov autophagy inhibitor, tus naj npawb ntawm LC3 cov hlwb zoo hauv WO pawg tau qis dua li hauv pawg M, thaum tus naj npawb ntawm LC3 zoo hlwb tsis txo qis hauv pawg CQ (raws li qhia hauv daim duab 6) . Tom qab ntawd, Western blot tau siv los kuaj pom cov protein qhia ntawm LC3 hauv substantia nigra hauv cov nas ntawm txhua pawg. Cov txiaj ntsig tau qhia tias cov protein qhia ntawm LC3II hauv pawg M tau nce ntau ntxiv piv nrog N pawg (P < 0.01).="" protein="" expressions="" ntawm="" lc3ii="" nyob="" rau="" hauv="" pawg="" eh,="" pawg="" ra,="" thiab="" cq="" pab="" pawg="" tau="" nce="" ntau="" npaum="" li="" cas="" piv="" nrog="" rau="" pawg="" m="" (p="">< 0.01).="" hauv="" pawg="" wo,="" cov="" protein="" qhia="" ntawm="" lc3ii="" poob="" qis="" dua="" piv="" nrog="" eh="" pawg="" thiab="" m="" pawg,="" thiab="" qhov="" sib="" txawv="" yog="" qhov="" tseem="" ceeb="" (p="">< 0.01="" thiab="" p="">< 0.05,="" feem)="" (raws="" li="" qhia="" hauv="" daim="" duab="">

Echinacoside in cistanche (11)

Echinacosideyog zoo rau khoth

Cov kev hloov pauv ntawm Substantia Nigra Ultrastructure thiab Autophagosomes Pom los ntawm Transmission Electron Microscope

Los ntawm kev soj ntsuam ntawm cov substantia nigra ultrastructure, nws tau pom tias, tshwj tsis yog rau pawg N, cov nas tag nrho pom qhov sib txawv ntawm substantia nigra ultrastructure raug mob, tshwm sim raws li o ntawm mitochondria, nthuav ntawm ntxhib endoplasmic reticulum, expansion ntawm Golgi, nce. lysosomes, thiab tsim ntawm autophagosomes nrog ob chav membrane qauv. Tus naj npawb ntawm autophagosomes hauv EH pawg, RA pab pawg, thiab CQ pawg tau nce ntau dua piv nrog lwm pab pawg. Tus naj npawb ntawm autophagosomes hauv pawg CQ yog siab dua li hauv pawg M, uas tau ua raws li cov txiaj ntsig ntawm kev nce hauv cov protein qhia ntawm LC3II hauv fluorescence ob chav staining thiab Western blot. Nws kuj tau hais tias tus naj npawb ntawm LC3II thiab autophagosomes tau sib raug zoo rau qee yam. Tsis muaj qhov pom tseeb nce hauv autophagosomes hauv WO pawg, qhia tias hauv pawg no, qhov nce ntawm autophagy tshwm sim los ntawmechinacoside cov tshuajtau inhibited (raws li qhia hauv daim duab 7).

Daim duab 7 Cov kev hloov pauv ntawm ultrastructural thiab tsim ntawm autophagosome hauv cov nigra loj hauv cov nas ntawm txhua pawg.

(A) Pab pawg tswj hwm saline ib txwm, nrog rau cov qauv ntawm cov substantia nigra;

(B) pawg qauv, nrog rau kev tshwm sim ntawm mitochondria o o, ntxhib endoplasmic reticulum expansion, thiab tsim ntawm lysosome thiab autophagosome:

(C thiab D).echinacoside cov tshuajua haujlwm. Nrog kev tsim ntau dua ntawm lysosome thiab autophagosome:

(E) pawg rapamycin, nrog kev tshwm sim ntawm mitochondria o thiab tsim ntawm lysosome thiab autophagosome;

(F) pawg chloroquine, nrog kev tshwm sim ntawm mitochondria o thiab tsim ntawm lysosome thiab autophagosome:

(G thiab H) pawg wortmannin. nrog pom o mitochondria, nthuav rouch edopbs mik reticulum

(G), thiab o ntawm mitochondri2. Kev nthuav dav ntawm ntxhib endoplasmic reticulum, pom tseeb nthuav dav df Gds thiab nce ntawm Mysosomes.

Cov nyhuv ntawm Echinacoside ntawm Protein Qhia ntawm AKT thiab mTOR hauv Substantia Nigra hauv MPTP-Induced PD nas

Western blot tau siv los txheeb xyuas cov protein qhia ntawm AKT thiab mTOR hauv txhua pab pawg. Cov txiaj ntsig tau qhia tias piv nrog N pab pawg, cov protein qhia ntawm p-AKT / AKT poob qis hauv M pawg (P < 0.01).="" cov="" lus="" qhia="" protein="" ntawm="" p-akt/akt="" hauv="" pawg="" eh="" thiab="" ra="" pawg="" tau="" nce="" pom="" zoo="" piv="" nrog="" pawg="" m="" (p="">< 0="" {{10}}}1),="" thiab="" cov="" protein="" qhia="" ntawm="" p-akt/akt="" hauv="" pawg="" eh="" tau="" siab="" dua="" li="" hauv="" pawg="" ra="" (p="">< 0="" 01).="" piv="" nrog="" rau="" pawg="" eh,="" cov="" lus="" qhia="" protein="" ntawm="" p-akt="" akt="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" tau="" poob="" qis="" (p=""><0.01) (raws="" li="" qhia="" hauv="" daim="" duab="" 8).="" tsis="" tas="" li="" ntawd,="" nws="" tau="" pom="" tias="" cov="" protein="" qhia="" ntawm="" p-mtor="" mtor="" hauv="" pawg="" m="" tau="" qis="" dua="" me="" ntsis="" hauv="" pawg="" n="" (p=""><0.05), tab="" sis="" qhov="" txo="" qis="" ntawm="" cov="" protein="" qhia="" ntawm="" p-mtor="" mtor="" yog="" qhov="" tseem="" ceeb="" hauv="" pawg="" eh="" thiab="" pawg="" ra="" ntau="" dua="" hauv="" pawg="" m="" (p=""><0.01). piv="" nrog="" rau="" pawg="" eh,="" cov="" protein="" qhia="" ntawm="" p-mtor="" mtor="" tau="" nce="" hauv="" pawg="" cq="" thiab="" wo="" pawg="" (p="">< 0.01)="" (raws="" li="" pom="" hauv="" daim="" duab="" 8,="" sab="">

Daim duab 8 Kev hloov pauv ntawm cov protein qhia ntawm AKT thiab mTOR hauv cov nigra ntau hauv nas ntawm txhua pab pawg.

Lus Cim: *P < {{0}}.05,="" **p="">< 0.01,="" piv="" nrog="" rau="" pawg="" qauv;="">< 0.01,="" compared="" with="" the="" high-dose="">echinacoside cov tshuajpab pawg, n= 6.

Echinacoside in cistanche