Dietary Cistanche Alleviated Acute Ileum Damage Of Duck

Cov ntsiab lus: Aflatoxin B1 (AFB1) yog cov tshuaj lom neeg lom neeg ruaj khov uas ua rau muaj kev phom sij rau tib neeg thiab tsiaj txhu noj qab haus huv thiab kis kab mob ntau yam tsiaj thiab tib neeg cov zaub mov. Qhov kev tshawb fawb tam sim no tsom los tshawb xyuas qhov cuam tshuam ntawmnoj cistanchentawm ileum raug mob nyob rau hauv ducks ntxias los ntawm AFB1 kev tswj hwm thiab tshawb xyuas nws cov txheej txheem hauv qab. Ducks (N=450, txiv neej hnub nyoog ib hnub) nrog qhov hnyav zoo sib xws tau muab tso rau 3 pawg, uas muaj pawg tswj hwm, AFB1 pawg (60 µg AFB1 kg-1 lub cev hnyav), thiab cistanche (500 mg. cistanche kg-1 noj) ntxiv rau AFB1 pawg. AFB1 kev tswj hwm tau ua rau muaj kev puas tsuaj ileum, AFB1-DNA adducts hauv cov ntshav, thiab oxidation kev nyuaj siab thiab mob. Ntxiv cistanche rau hauv cov zaub mov tiv thaiv ileum tiv thaiv kev puas tsuaj los ntawm AFB1 kev tswj hwm, txo AFB1-DNA adducts hauv cov ntshav, thiab tshem tawm oxidation kev nyuaj siab thiab mob hauv ileum ntawm ducks.Anti-oxidationthiabanti-inflammatory cov teebmeem ntawm cistanchetuaj yeem tiv thaiv lub ileum tiv thaiv kev puas tsuaj loj los ntawm kev ua kom Nrf2-YOG txoj kev taw qhia thiab inhibiting NF-κB txoj hauv kev. Kom meej meej,cistancheyog kev noj haustiv thaiv oxidationthiabtiv thaiv kab mobtus neeg sawv cev los ntawm kev ua kom Nrf2-YOG txoj kev taw qhia thiab cuam tshuam rau NF-κB qhov taw qhia txoj hauv kev mob kev puas tsuaj los ntawm AFB1 kev tswj hwm.

Ntsiab lus:cistanche; mob ileum; AFB1-DNA adducts; Nrf2- YOG;

Nyem qhov no Rau Cistanche Anti-aging khoom

Nug ntxiv:

Email: wallence.suen@wecistanche.com Whatsapp ntxiv rau 86 15292862950

1. Taw qhia

Nqaij yog ib qho tseem ceeb ntawm cov protein zoo rau tib neeg noj zaub mov. Duck nqaij yog abundantly noj thoob ntiaj teb, tshwj xeeb tshaj yog nyob rau hauv Asia vim hais tias ntawm nws cov khoom noj khoom haus zoo yam ntxwv [1]. Yog li ntawd, kev yug me nyuam ntawm ducks tau nyiam cov neeg nyiam. Rau cov ducks, muaj ntau yam tsis zoo hauv cov txheej txheem yug me nyuam, xws li AFB1 uas cuam tshuam kev noj qab haus huv ntawm ducks. Aflatoxin B1 (AFB1) yog ib qho ntawm cov tshuaj lom metabolites tsim los ntawm Aspergillus hom. AFB1 tau lees paub tias yog cov tshuaj lom neeg tshaj plaws ntawm aflatoxin (AF) pawg, nrog rau ntau yam tshuaj lom neeg los hem kev noj qab haus huv ntawm tib neeg thiab tsiaj txhu [2].

Rau tib neeg lossis tsiaj txhu, zaub mov lossis kev pub noj yog ib qho tseem ceeb thiab tseem ceeb rau kev kis tus kab mob AFB1, tab sis kev nqus pa thiab kev sib cuag ncaj qha rau ntawm daim tawv nqaij lossis mucosa kuj raug suav thiab tsis quav ntsej [3]. Cov kev tshawb fawb yav dhau los tau ua pov thawj tias AFB1 ua rau muaj zog toxicity uas nyuaj heev thiab muaj zog, ua rau muaj kev loj hlob qeeb, kev ua tsis zoo ntawm lub cev, lub siab toxicity, plab zom mov thiab txawm tias mob qog noj ntshav [4,5]. AFB1 tau txais los ntawm cov khoom noj los yog kev sib cuag mucosa muaj qhov cuam tshuam tsis zoo rau cov kab mob ua pa, plab hnyuv thiab cov ntaub so ntswg thiab kev loj hlob [3,6,7]. Cov ntaub so ntswg thiab lub cev puas tsuaj tshwm sim los ntawm AFB1 kev tswj hwm ntsig txog oxidation kev nyuaj siab thiab mob. AFB1 kev tswj hwm tau pom tias nce AFB1-DNA adducts cov ntsiab lus hauv lub cev raug mob [8]. Kev nqus thiab hloov pauv ntawm cov as-ham thiab co toxins tau tshwm sim hauv lub plab thiab cov hnyuv, yog li qhov me me

Txoj hnyuv mucosal tiv thaiv kab mob yog thawj kab los tiv thaiv lub cev tiv thaiv kev raug mob [9]. Lub functionality thiab morphology ntawm kev noj qab haus huv plab hnyuv ib ntsuj av yuav raug puas los ntawm unfavorable yam xws li toxicants, kab mob thiab kab mob [10,11]. AFB1 induced ntawm kev puas tsuaj rau txoj hnyuv qauv, manifested los ntawm shedding ntawm epithelial hlwb nyob rau hauv jejunal villus thiab lymphocytic cell infiltration nyob rau hauv cov hnyuv ntawm nqaij qaib [12,13]. Cov kev ua haujlwm sib txawv thiab kev ua haujlwm ntawm lub cev tuaj yeem raug ntaus nqi los ntawm cov txheej txheem ntawm cov tshuaj lom neeg metabolism uas feem ntau nrog oxidative stress Inflammation. Nws yog ib qho tseem ceeb kom nrhiav tau ib qho tshuaj tua kab mob kom txo tau thiab txo qhov kev hem thawj ntawm AFB1 rau tib neeg thiab tsiaj txhu.

Yog li ntawd, AFB1 yog ib qho kev txhawj xeeb tshaj plaws hauv kev lag luam nqaij qaib vim nws muaj zog toxicity. cistanche yog ib hom polyphenol tivthaiv tshwm sim nyob rau hauv turmeric rhizomes (Cur cuma Longa Linn) raws li ib tug functional feed additive siv nyob rau hauv cov tsiaj nyeg pub [14], thiab dav siv.

raws li ib tug ntsuab thiab ntuj txuj lom, colorant, preservative thiab flavoring nyob rau hauv cov zaub mov kev lag luam. Cov nroj tsuag rho tawm xws li cistanche uas muaj polyphenols txhim kho lub cev tiv thaiv kab mob [15]. cistanche plays lub luag haujlwm tseem ceeb ntawm oxidative stress-mediated pathological-pathological tej yam kev mob thiab kev ua ub no thaum siv los ua kev kho mob thiab tiv thaiv kab mob ntev [16,17]. Cov kev sim tshuaj ntsuam xyuas tau qhia tias cistanche tsis muaj tshuaj lom lossis muaj kev phiv loj, thiab nrog cov khoom tiv thaiv carcinogenic thiab anti-toxicogenic, uas ua rau cistanche yog ib qho tshuaj tiv thaiv zoo rau AFB1-vim cov ntaub so ntswg puas [18–20]. Cov ntaub ntawv qhia tau hais tias cistanche muaj peev xwm los tiv thaiv kab mob, tiv thaiv apoptosis thiab antioxidation los ntawm activating Nrf2/HO-1 txoj kev, upregulated antiox muaj peev xwm mus tshem tawm o thiab oxidative kev nyuaj siab nyob rau hauv lub cev thiab tshem tawm AFB1-induced oxidative kev nyuaj siab [21–25]. cistanche suppressed oxidation kev nyuaj siab thiab inflammasome tsim los ntawm activating Nrf2-YOG thiab inhibiting NF-κB signaling txoj hauv kev nyob rau hauv lub raum demoning [26], anti-oxidation thiab anti-inflammatory khoom. Tsis tas li ntawd, kev noj zaub mov cistanche tau txhim kho lub peev xwm antioxidant hauv broilers [27] thiab ducks [28]. Tsis muaj kev sim los cuam tshuam lub luag haujlwm ntawm cistanche hauv Nrf2-ARE thiab NF-κB taw qhia txoj hauv kev thiab hauv tsiaj qauv ntawm kev tswj hwm tus mob AFB1.

Txoj kev tshawb no tso lub teeb pom kev ntawm qhov teeb meem no thiab muab lub hauv paus theoretical rau cistanche ua cov khoom noj ntxiv los tiv thaiv duck txoj kev noj qab haus huv tiv thaiv AFB1 kev tswj hwm thiab txo qis kev lag luam poob ntawm kev noj zaub mov thiab kev lag luam yug me nyuam los ntawm AFB1 cov pa phem.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Tshuaj

cistanche tau yuav los ntawm Nanjing Nutri-herb Biotech Co., Ltd. (Nanjing, Jiangsu, Tuam Tshoj, CAS: 458-37-7), nws purity yog ntau tshaj 98 feem pua ​​​​los ntawm HPLC tsom xam. AFB1 (purity Ntau dua lossis sib npaug li 98 feem pua, CAS NO . 1162-65-8) tau yuav los ntawm Shanghai Yuan ye Bio-Technology Co., Ltd. (Shanghai, Suav). Cov tshuaj tiv thaiv uas siv rau hauv txoj kev tshawb no tau yuav los ntawm Bey time Biotechnology, Shanghai, Suav teb, suav nrog GAPDH (Catalog naj npawb: AG019), Nrf2 (Catalog naj npawb: AF7623), Phospho-NF-κB p65 (Ser276) (Catalog naj npawb: AF5875), HRP labeled Tshis Anti-Lavbit IgG (H plus L) (Catalog naj npawb: A0208) thiab HRP-labeled tshis

Anti-Mouse IgG (H plus L) (Catalog naj npawb: A0216).

2.2. Ducks thiab Husbandry

Cov txheej txheem sim tau ua raws li cov kev coj ua tau teev tseg hauv Phau Ntawv Qhia Txog Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Tsiaj Agricultural Hauv Kev Tshawb Fawb Txog Kev Ua Liaj Ua Teb thiab Kev Qhia ntawm Northeast Agricultural University (Txoj Cai Tus lej: NEAU-[2011]-9).

Ducks (n {{0}}, ib-hnub-txiv neej Anas platyrhynchos, 33.8 ± 0.2 g) uas tsis muaj qhov sib txawv hnyav tau yuav los ntawm kev lag luam hatchery thiab random muab rau 3 pawg (Table 1), nrog 10 replicate cwjmem (khob) rau ib pab pawg thiab 15 ducks rau ib tug cwj mem rau ib hnub 70-

kev sim pub mis. Cov zaub mov basal tau tsim raws li National Research Council (1994). Ducks nyob rau hauv T0 thiab T0 pab pawg neeg AFB1 tau noj cov pob kws-soybean basal noj zaub mov (Table 2), ducks hauv pawg T500 ntxiv rau AFB1 tau noj cov zaub mov basal ntxiv nrog 500 mg ntawm

cistanche kg−1 ntawm kev noj haus (T500) ib 70- hnub sim. Hauv 70 hnub, ducks nrog lub cev hnyav zoo sib xws hauv T0 ntxiv rau AFB1 thiab T500 ntxiv rau AFB1 pawg tau noj 60 µg ntawm AFB1 kg−1 ntawm lub cev hnyav, thiab ducks hauv T0 pab pawg tau noj cov khoom sib npaug ntawm cov tshuaj PBS. Cov Ducks tau pub rau hauv Acheng Experimental Base ntawm Northeast Agricultural University thiab muab nrog ad libitum nkag mus rau dej thiab hmoov noj. Kaum tsib ducks nrog lub cev hnyav (1.4 ± 0.3 kg) los ntawm txhua pab pawg tau raug xaiv thiab yoo mov rau 12 h, tom qab ntawd pub PBS tov rau ducks hauv T0 pawg thiab 60 µg ntawm AFB1 kg-1 ntawm lub cev qhov hnyav rau ducks hauv T0 ntxiv rau AFB1 thiab T500 ntxiv rau AFB1 pawg tib lub sijhawm. Tom qab 12 teev, kaum tsib ducks hauv txhua pab pawg tau txais cov qauv os.

2.3. Qauv Sau

Cov ntshav kuaj (10 mL) tau txais los ntawm kev siv cov hlab heparin los ntawm cov hlab ntsha ntawm cov tis qaib thiab centrifuged ntawm 1000 × g rau 15 feeb ntawm 4 ◦C. Cov plasma tau muab cais tam sim ntawd thiab khaws cia ntawm −80 ◦C rau kev tshuaj xyuas. Ducks tau tshuaj loog los ntawm kev nqus ether thiab tua kom tau txais ileum. Lub ileum tau ntxuav 3 zaug hauv PBS, tam sim ntawd, thiab ib tus zuj zus khaws cia rau hauv lub tank kua nitrogen tom qab ntawd ntawm −80 ◦C rau qRT-PCR thiab tshuaj tiv thaiv antioxidant. Tom qab ntawd, kwv yees li 0.125 cm3 ileum tau txais thiab muab tso rau hauv 4 feem pua ​​​​cov tshuaj paraformaldehyde rau cov ntaub so ntswg thiab txog 1 mm3 ileum tau muab tso rau hauv electron microscope.

daws ntawm 4 ◦C rau tom qab kev soj ntsuam ultrastructural.

2.4. Kev soj ntsuam ntawm Antioxidant Qib hauv Plasma

Plasma qib ntawm tag nrho cov superoxide dismutase (T-SOD), glutathione peroxidase (GSH Px), Glutathione S-transferase (GSH-ST), thiab malondialdehyde (MDA) tau ntsuas los ntawm cov khoom siv tshuaj ntsuam (Nanjing Jiancheng Bioengineering Institute, Nanjing, Tuam Tshoj), raws, nrog

UV-VIS Spectrophotometer (UV1100, MAPADA, Shanghai, Suav).

2.5. Kev soj ntsuam ntawm AFB1-DNA Adducts Levels hauv Plasma

Lub cim ntawm AFB1-DNA adducts hauv cov ntshav tau txiav txim siab siv cov khoom siv ELISA raws li cov khoom siv tshwj xeeb (Nanjing Jiancheng Bioengineering Institute, Nan jing, Suav).

2.6. Kev soj ntsuam ntawm Antioxidant muaj peev xwm hauv Ileum

Ileum (100). -7.4) kom tau 10 feem pua ​​​​ileum/SPSS homogenate. Kev ua los yog cov ntsiab lus ntawm tag nrho superoxide dismutase (T-SOD U / mg Protein), reductive Glu glutathione (GSH-PX µmol / mg Protein), Glutathione S-transferase (GSH-ST U / mg Protein) thiab Malondialdehyde (MDA nmol / mg Protein) hauv ileum tau soj ntsuam siv cov khoom siv Assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, Tuam Tshoj), feem, nrog rau UV-VIS

spectrophotometer (UV1100, MAPADA, Shanghai, Tuam Tshoj).

2.7. RNA Isolation thiab Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR)

Tag Nrho RNA ntawm duck ileum (100.00 mg) raug cais tawm siv cov khoom siv reagent (TaKaRa, Nyiv) raws li cov txheej txheem pom zoo los ntawm cov tuam txhab. Qhov concentration thiab purity ntawm tag nrho RNA tau kuaj pom ntawm A260 / A280 piv nrog spectrophotometer (IMPLEN, Lub teb chaws Yelemees). 1 µg tag nrho RNA hauv txhua tus qauv tau hloov mus rau hauv cDNA nrog Prime Script™ RT reagent cov khoom siv nrog gDNA Eraser (TaKaRa, Dalian, Tuam Tshoj) raws li

rau raws tu qauv pom zoo los ntawm manufacturers. Cov tau txais cDNA los ntawm txhua lub duck ileum tau siv los ua tus qauv rau TB Green™ Premix Ex Taq™ (TaKaRa, Dalian, Tuam Tshoj) RT-PCR (qRT-PCR) cov khoom siv. Cov gene accession naj npawb ntawm ducks tau txais los ntawm NCBI thiab

cov duck gene primers tau synthesized los ntawm Sangon Biotech Co., Ltd. (Shanghai, Suav teb) (Table 3). Qhov cuam tshuam gene qhia nyob rau hauv duck ileum tau txiav txim siab los ntawm Quanta gene Q225 thermal cycler apparatus. QRT-PCR tau khiav hauv Monad Xaiv Real-Time

PCR System (ABI 7500 real-time PCR instrument (USA)) ntws mus rau qhov xwm txheej: ib lub voj voog ntawm 95 ◦C rau 30 s, 40 cycles ntawm 95 ◦C rau 5 s thiab ntawm 60 ◦C rau 30 s. Cov txheeb ze ntawm cov noob qhia piv ntawm kev kuaj pom mRNA tau kuaj pom siv 2-∆∆Ct txoj kev thiab normalized rau -actin qhia.

2.8. Western Blotting

Cov qaib ileum tau pulverized thiab lysed hauv RIPA tsis muaj 1 mmol / L PMSF (Beyotime, Shanghai, Tuam Tshoj) hauv dej khov. Tag nrho cov protein ntau ntawm cov ileum tau txiav txim siab los ntawm cov khoom siv tshuaj bicinchoninic acid (BCA) (Nanjing Jiancheng Bioengineering Institute, Nanjing, Suav). 12% thiab 10% SDS-polyacrylamide gel rau electrophoresis yog

siv kom tau cov hom phiaj protein nrog qhov sib txawv molecular hnyav. Tom qab ntawd, lub hom phiaj cov protein tau raug xa mus rau polyvinylidene-difluoride (PVDF) daim nyias nyias (Beyotime, Shanghai, Tuam Tshoj) rau blots nrog trans blotting apparatus. PVDF daim nyias nyias tau ntxuav 3 zaug rau 10 feeb txhua zaus hauv 1 × PBST thiab tom qab ntawd thaiv 2 h hauv 5 feem pua ​​​​ntawm cov mis nyuj. PVDF mem brane raug ntxuav 3 zaug dua, thiab incubated nrog GAPDH, Nrf2 thiab P-P65 (Beyotime Biotechnology, Shanghai, Suav teb) thawj cov tshuaj tiv thaiv rau 8 -12 teev ntawm 4 ◦C, raws li. Hnub tom qab, PVDF daim nyias nyias tau ntxuav 3 zaug ntxiv, thiab incubated nrog cov sib thooj horseradish peroxidase-labeled antibody ntawm 37 ◦C rau 1 h, ces ntxuav 3 zaug dua.

Lub hom phiaj protein bands tau kuaj pom thiab pom nyob rau hauv qhov kev txiav txim ntawm kev txhim kho cov khoom siv tshuaj fluorescence BeyoECL Star (Beyotime Biotechnology, Shanghai, Suav). Cov duab ntawm blots tau sau tseg thiab txheeb xyuas los ntawm Essential V6 imaging platform (UVITEC, Cambridge,

England). GAPDH protein tau txais txiaj ntsig los ntawm kev tswj hwm sab hauv protein. Tag nrho cov txiaj ntsig ntawm kev sim tau rov ua dua hauv triplicate. Cov txheeb ze nthuav tawm ntawm cov hom phiaj proteins tau nthuav tawm raws li qhov piv ntawm band siv ntawm cov proteins rau GAPDH.

2.9. Kev txheeb cais

Cov ntaub ntawv sim tau txais los ntawm tsawg kawg yog rau lub sijhawm thiab txhua tus qauv yog mea sured peb zaug. Kev soj ntsuam ntawm cov ntaub ntawv tshawb fawb siv Independent-Samples T Test los ntawm SPSS (Version 22.0, SPSS Inc., Chicago, IL, USA) nrog 5 feem pua ​​​​Qhov tshwm sim ntawm qhov yuam kev thiab qhov tseem ceeb ntawm cov ntaub ntawv yog p < {{4} }.05 hauv qhov kev tshawb fawb no