Kev kawm txog cov nyhuv ntawm cov kev ua yeeb yam (ib tug nquag muaj cov khoom xyaw hauv cistanche Showicola) ntawm nafld los ntawm inducing autophagy

Abstract:

 

Lub Hom Phiaj:Txhawm rau tshawb txog kev ua haujlwm autophagy-inducing muaj nuj nqis ntawm cov khoom noj muaj zog (ACT) hauv contanche Squaticola thiab nws cov nyhuv ntawm cov kab mob tsis muaj roj (nafld).

TxojkevTxhawm rau noj Echinacoside thiab ua raws li kev tshawb fawb cov khoom, 1 {}}}}}}} ·)} μm) A1 rau kev kho mob. Protein imbunoblotting tau siv los tshawb xyuas cov kev qhia txog autophagy cuam tshuam txog cov protein 1 (lc 3- ii). Hepg2 cells tau cuam tshuam nrog kev sib txawv ntawm kev ua nyob rau hauv Hepg2 cov tshuaj tiv thaiv ntsev.Qhov kev siv tshuaj tiv thaiv cov protein uas siv cov protein immunolotting thev naus laus zis. Ib qho qauv NAFDD tsim nyob rau hauv Vitro nrog kev cuam tshuam kev siv yeeb tshuaj yog siv los cov roj av liab thiab pom cov txheej txheem dav dav thiab tshawb xyuas cov txheej txheem intracellular. Saib xyuas lub zos ntawm LC3 protein thiab lipid tee los ntawm immunofluorescence thev naus laus zis. Cov txiaj ntsig tau ua rau muaj peev xwm ua rau muaj autophagy hauv Hepg2 cov protein, thiab {{21} {{{}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}} ntsig txog), nrog ntsiab lus tseem ceeb (P0.05). 50 μmol / u ua qhov tseem ceeb indophagy hauv Hepg2 hlwb, thiab qib kev qhia ntawm LC {25 ± 0.83, 0.35 ± 0..18, feem nrog). TG Theem ntawm Hepg2 hlwb hauv Pab Pawg Tiv Thaiv (19.11 ± qis dua li cov qauv ntawm cov tub ntxhais vitroell (34.15)<0.05). The number of positive lipid droplets in HepG2 cells in the ACT intervention group decreased significantly, and the number of autophagosomes inside the cells increased significantly, showing clear co localization with lipid droplets in space. Conclusion Cistanche deserticola ACT has significant autophagy induction ability, and it may have potential preventive and therapeutic effects on NAFLD.

 

Cov ntsiab lus:Kev ua kom tsis muaj roj ntsha ua kab mob;Cistanche Squaticola;Ua yeeb yam; Autophagy

 

TCM cov tshuaj ntsuab cistanche rau kev kho cov tsis muaj roj cawv ua kab mob (NAFLD)

Nyem Pic kom tau txais cov ntsiab lus ntxiv

 

Nyob rau hauv ob peb xyoo dhau los, tsis muaj roj cawvMob siab Kab Mob(NAFDLD) tau dhau los ua qhov feem ntauCov Kab Mob Siab Mob, nrog kev sib txuas thoob ntiaj teb ntawm kwv yees li 25% ntawm cov neeg laus, thiab yog suav hais tias yog sib cuam tshuam nrog metabolic syndrome [1]. Txawm hais tias qhov tshwm sim ntawm daim siab cov kev cuam tshuam hauv NAFLD cov kab mob tsawg dua li cov mob qog noj ntshav xws li mob siab rau cov neeg mob qog noj ntshav xws li mob siab rau cov neeg mob lub nra rau ntawm cov neeg mob [2-5]. Txawm hais tias muaj kev ua kom zoo dua them rau NAFDD, Nafld ua tus kab mob tseem ceeb tsis tau txais kev txaus siab txaus. Yog li ntawd, tseem muaj kev nkag siab ntxiv ntawm cov pathogenesis ntawm lub pathogenesis ntawm lub tswv yim zoo tau ua cov teeb meem kev kho kom txaus siab tau daws tau daws [{}].

Cov kev tshawb fawb tsis ntev los no tau qhia tias cellular autopyagy plays ib lub luag haujlwm tseem ceeb hauv daim siab thev naus laus zis thiab pathology [8]. Autophagy dysfunction los yog dysregulation yog txuam nrog ntau yam ntawm daim siab mob xws liCawv yog kab mob ntsig txog kab mob[9], NAFDD [10], tsis-cawv steatohepatitis [11], thiab hepatocellular carcinoma [12]. Cov yam ntxwv pathological ntawm NAFLD yog ntau qhov kev tso tawm ntawm Triglyerides (TG) thiab lwm yam lipid tee dej (LIP) hauv Hepatocytes nyob rau hauv kev txwv tsis pub haus cawv. Rau lub siab, ob txoj hauv kev tseem ceeb tswj cov catabolism hauv lds yog lipolysis thiab autophagy.
Lipolysis txoj kev yog tswj hwm los ntawm cytoplasmic nruab nrab lipases, xws li hormas-rhiab liipase thiab adipose tg lase. Hauv kev coj Lipoaugy Pathway, LDS yuav xaiv tau los ntawm autophagosomes tau xa mus rau Lysosomes thiab decomposed los ntawm acid lasroundase hauv lysosomes. Yog li ntawd, ua kom ua haujlwm lipoautophagy hauv Hepatocytes tau dhau los ua kev siv tau zoo rau kev tiv thaiv thiab kho NAFLD.
Cistanche Squaticola yog cov tshuaj ntsuab ntawm cov tshuaj ntsuab uas tau siv dav hauv kev kuaj mob hauv kev ua tshuaj suav tshuaj. Nyob rau tib lub sijhawm, nws tau ntev tau siv los ua khoom noj khoom haus kev noj qab haus huv. Cistanche Squaticola muaj ntau yam khoom xyaw bioactive, qhov tseem ceeb tshaj plaws uas yog cistanche Squaticola Phenycetanol Glycosides. Raws li cov neeg sawv cev ntawm phenylethanoid glycosides, echinacoside (xws li antioxosidant (Act) tau tshaj tawm tias muaj ntau yam kev tiv thaiv, thiab daim siab tiv thaiv [13]. Nyob rau hauv cov lus cog tseg ntawm daim siab tiv thaiv, kev tshawb fawb pom tau tias Ech tuaj yeem tiv thaiv lub siab mob-, ua rau muaj kev hloov pauv qib ntawm lub siab fibrosis-cuam tshuam cov noob. ACT has a significant protective effect on chemical and drug-induced liver damage, such as alcohol, carbon tetrachloride, and lipopolysaccharide. Ua kom txo cov oxidative kev ntxhov siab tshwm sim los ntawm cov tshuaj lom, tshem tawm cov pa lom neeg ntau dua hauv daim siab, thiab txo qis malondeshyde convenation. Nyob rau tib lub sijhawm, nws tseem nce siab dua daim siab superoxide dismutase kev ua si thiab txo cov ntsiab lus glutathione histopathological kev puas tsuaj thiab kev kuaj mob. Txawm li cas los xij, tsis muaj daim ntawv qhia txog cov ntawv sau tseem ceeb ntawm cov paenyletanoid glycosides ntawm cistanchoidingicola yog koom nrog autophagy hauv exerting daim siab tiv thaiv. Raws li qhov no, txoj kev kawm no siv cov qauv ntawm cov qauv ntawm cistophagy thiab tshawb xyuas nws cov chaw muag tshuaj, npaj kom muaj kev tshawb fawb txog yav tom ntej.

 

1 cov ntaub ntawv thiab cov hau kev

1.1 Khoom

1.1.1 Cov Ntsiab Lus Tshawb Fawb Ech thiab ACT tau raug siv raws li kev tshawb fawb.

1.1.2 Cov ntaub ntawv Hepg2 tau yuav los ntawm Wuhan Pungei Life Science (DMEM thiab trypsin tau yuav los ntawm hyClozone tuam txhab, Asmeskas, fetal bovine ntshav (batch)) TG (Batch Zauv: BC0625) Cov Khoom Siv Mus Nrhiav tau tau yuav los ntawm Oloubo Compints, Suav teb (ntau tus lej: BCBZIDE (ntau tus lej: Aldrich, Asmeskas; Lub autophagy-lysosome inhibitor Bafilomycin A1 (Bafa1, ntau tus naj npawb: Tlrl-Baf1) tau yuav los ntawm Invitrogen, Asmeskas; Lub autophagy activator rapamycin (ntau tus naj npawb: S17098) tau yuav los ntawm Shanghai Yuane Biotechnology Co., Ltd. MicroTove-txuam Protein 1 lub teeb saw 3 (LC3, ntau tus lej: L7543, {{33} {{33} {{33} {33} {{33} {{33} {{33} {{33} {33} Horseradish peroxidase-sau cov tshuaj tiv thaiv theem nrab (ntau tus kab mob, cov khoom siv Alexa: 546, Dilution Ratio: 1: 1000) yog Yuav los ntawm Sigma-Aldrich tuam txhab, Tebchaws Asmeskas, thiab lub ntsws 493/501 (ntau tus lej: D3922) tau yuav los ntawm cov tuam txhab Invitrogen, USA.

 

1.2 Txoj Kev

1.2.1 Cell Kev Cai

Hepg2 cell yog kev coj noj coj ua nyob rau hauv Dulbecco tau hloov kho lub siab zoo li 10% thiab 100 plhom botenomycin hauv 37 degree, 5% carbon dioxide incubator. Thaum lub cell sib cuag tau txog 85%, subculture, plating, kev cuam tshuam thiab lwm yam haujlwm tau ua.

 

1.2.2 cov protein immunolotting kom tau

1.2.2.1 ACT thiab ECH kho

LC {3-}}} Cov qib protein} Muaj protein tso rau hauv cov hlwb, yog li Ambifying lub peev xwm los ntawm kev muaj peev xwm. Yog li ntawd, kev siv kev sib xyaw ua ke ntawm Bafa1 tuaj yeem siv los ua ib qho kev saib xyuas automanagy los ntsuas cov peev xwm ntawm kev muaj peev xwm. Hepg2 hlwb hauv Logarithmic kev loj hlob theem tau pauv mus rau 3.5 cm cell kab lis kev cai lauj kaub tais diav rau yav tsaus ntuj kab lis kev cai. 100 μmol / l Act, Ech thiab Bafa1 tau siv rau kev cuam tshuam rau 12 h. Lub hlwb tau ntxuav nrog ua ntej txias phosphate phosphate buffer daws (PBS) thiab Lysed tam sim ntawd nyob rau hauv Ripa protein tsishu rau 10 min. Tus supernatant yog centrifuged thiab tag nrho cov protein ntau tau txiav txim siab siv cov protein revein concentration cov khoom siv. Tom qab kev ntsuas cov khoom nrog sodium dulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecyl sulecin Tom qab ntawd 30 μl ntawm cov qauv tau coj nrog micropipette thiab electrophoresed on ib polyacrylamide gel.
Tom qab electrophoresis, cov protein tau pauv los ntawm cov gel rau nitrocellulose mem duab, thiab muab cov tshuaj tiv thaiv kab mob rau ntawm chav sov ntawm chav kub rau 1 h. ECL txoj kev siv rau kev tsim xim xim. Lawv tau raug teeb tsa cov pab pawg tsis sib haum

 

1.2.2.2 Qhov ntau ntau qhov kev sib txuas ntawm ACT daws cov qib kev qhia ntawm LC {}}}} ⅱ ⅱ ⅱ ⅱ ⅱ ⅱ ⅱ ⅱ ⅱ ⅱ protein hauv Hepg2 hlwb

Lub logarithmic kev loj hlob theem hepg2 cell tau pauv mus rau 3.5 cm cen kab lis kev cai lauj kaub rau yav tsaus ntuj kev kho mob Pawg tswj hwm (pab pawg ua, kev tswj ntsug), kev tshaib plab pab pawg HEPG2, 25, 50, thiab 100 μMol / L Act pab pawg tau teeb tsa.

1.2.3 MTT txoj kev txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau muaj sia nyob ntawm HEPG2 cov hlwb kho nrog ntau qhov ntau ntawm ACT rau 24 thiab 48 teev
Hepg2 hlwb hauv Logarithmic kev loj hlob theem tau txais hauv {1}}}}} Kev ua kom muaj qhov ntom ntom ntom nti thiab kev coj ua hauv qhov tsim kom loj hlob rau 12 h. 2 {0}, 40, 60, 80, thiab 100 μmol / u Txoj Cai tau ntxiv rau 24 thiab 48 h. 0.5 mg / ml mtt kev daws teeb meem tau ntxiv rau hauv ib puag ncig tsaus nti thiab loj hlob hauv ib qho incubator. Tom qab 4 h, tus supernatant tau muab pov tseg, 100 μl ntawm Dulethyl SullSyl Sulfoxide tau ntxiv rau txhua qhov zoo, thiab cov hlwb tau shaken nyob rau hauv ib qho kub tsis tu ncua saker ntawm 37 degree rau 10 feeb degree rau 10 feeb Lub zog kho qhov muag tsis zoo (OD) tus nqi ntawm nthwv dej 490 nm tau kuaj pom los ntawm enzyme cim, thiab cov xov tooj ntawm tes muaj sia tau muab xam.

 

1.2.4 hauv vitro kev tsim kho ntawm NAFLD CELOR QAUV

Ib qho kev hnyav ntawm palmosic acid (PA) tau hnyav thiab yaj tag nrho cov khoom siv 100 mmol /}}} degree tub yees rau kev siv tom qab. Lub PA npaj daws tau diluted rau 100 μmol / u siv cov kab lis kev cai sib xyaw ua ke, thiab 1% fatty acid-dawb bovine Lub hlwb tau raug muab tso rau ntawm 37 degree rau 3 h los npaj kev daws teeb meem induction. Hepg2 hlwb hauv Logarithmic kev loj hlob theem tau pauv mus rau ib qho kev coj noj coj ua uas tsim nyog rau kev daws teeb meem thaum hmo ntuj, thiab tom qab ntawd kev npaj ua haujlwm tau siv rau kev cuam tshuam.

 

1.2.5 Act Kev cuam tshuam NaFld hauv vitro cor qauv tg kom pom thiab roj liab o staining

 

1.2.5.1 tg nrhiav

Hepg2 hlwb hauv Logarithmic kev loj hlob theem tau pauv mus rau ib pawg kab lis kev cai ntawm kev coj noj coj ua raws li pawg neeg tswj hwm dawb, qauv pab pawg thiab cov pab pawg cuam tshuam. Tus qauv pab pawg tau ntxias nrog 100 μmol / l pat truction daws teeb meem, thiab cov pab pawg tiv thaiv kev daws teeb meem, thiab cov kev daws teeb meem Lub sijhawm kev cuam tshuam yog 24 h. Maj mam tshem tawm cov kev coj noj coj ua qub, maj mam ntxuav cov xovtooj ntawm cov dej khov, nqa mus rau cov ntsiab lus ntawm tg Cov lus qhia TG Cov lus qhia, thiab thaum kawg kho cov qib TG ib mg ntawm cov protein.

1.2.5.2 roj liab o staining

Hepg2 hlwb hauv Logarithmic kev loj hlob theem tau pauv mus rau 3.5 cm kab lis kev cai phaj npog nrog lub tshuab qias neeg rau yav tsaus ntuj kab lis kev cai. 100 μmol / l PA raug ntxias lub hlwb los ua laspid tee dej. Nyob rau tib lub sijhawm, 50 μmol / u Tsab ntawv tau ntxiv rau kev cuam tshuam rau 24 h.
Cov kab hauv tes tau sau, ntxuav 3 zaug nrog cov roj ua ntej rau ntawm 10% ntim, thiab thaij duab nyob rau hauv lub tshuab tsom.

 

1.2.6 immunofluorescence kev soj qab ntawm autophagosomes thiab lipid tee tom qab kev kho mob

Hepg2 hlwb hauv Logarithmic kev loj hlob theem tau pauv mus rau 3.5 cm kab lis kev cai phaj npog nrog lub tshuab qias neeg rau yav tsaus ntuj kab lis kev cai. 1 {0 0 μmol / l PA tau siv los ntxias qhov tsim ntawm Lipid Dopsle hauv lub hlwb. Nyob rau tib lub sijhawm, ACT tau ntxiv rau kev pabcuam kev cuam tshuam rau 24 h. Lub cell slides tau sau, ntxuav 3 zaug nrog kev kub taub hau, tsau rau ntawm chav sov rau 1 h nrog cov tshuaj tiv thaiv theem pib, ntxuav 3 zaug nrog PBS, thiab ntxiv nrog ib qho fluorescent-sau cov tshuaj tiv thaiv theem nrab ntawm chav tsev, ua kom zoo ntawm qhov kub thiab txias thiab txav ntawm lub teeb rau 1 h. Tom qab cov kab mob antibodity, tau ntxuav tau 3 zaug nrog Lipid Drocy 4 air Pawg tswjfwm dawb, Rapamcin pab pawg kho thiab pab pawg neeg tsis paub tab tau teeb tsa.

 

1.3 Tsom xam

SPSS25. {{0}} coistical software tau siv rau cov ntaub ntawv tsom xam. Cov ntaub ntawv ntau tau qhia tias yog x ± s. Ib-txoj kev soj ntsuam ntawm kev sib txawv thiab lsd-t tes tau siv. P<0.05 was considered statistically significant.