Ntu 2: Echinacoside nce cov phev kom muaj nuj nqis hauv nas Los ntawm Targeting Hypothalamic Androgen Receptor

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Zhihui Jiang 1,2, Bo Zhou 2, Xinping Li2, Gordon M. Kirby3 & Xiaoying Zhang 1,2

Pls nyem qhov no mus rau Part 1

Kev sim kev kho mob.

Kev sim 1. Cov nas tau muab faib ua rau pawg, xya nas nyob rau hauv txhua pab pawg (n=7).ECH(Echinacoside los ntawm cistanche)(CAS: 82854-37-3; Chengdu Preferred Biotechnology Co., Ltd, Sichuan, Suav teb) tau muab siv lub raj intragastric, thiab testosterone propionate (TP, CAS: 57-85-2; KingYork, Tianjin, Suav teb) tau muab los ntawm kev txhaj tshuaj intramuscular ib zaug ib hnub rau 14 hnub raws li cov qauv kev sim hauv qab no: Cov pab pawg tswj hwm: ib txwm saline (10 mL / kg), ECH_(L)pawg (5 mg/kg), ECH_(M)pawg (20 mg/kg), ECH_(H)pawg (80 mg/kg), TP (15 mg/kg) thiab enzalutamide (AR inhibitor, ib zaug ib hnub twg rau 14 hnub; 20 mg/kg; CAS: 915087- 33-1; Aladdin, Shanghai, Suav teb).

Tom qab 2 lub lis piam ntawm kev kho mob, cov nas tau muab tshuaj loog nrog diethyl ether los sau cov ntshav kuaj rau kev ntsuam xyuas cov qib tshuaj hormones. Tom qab ntawd cov nas tau muab cais tawm kom cais cov hypothalamus, encephalon ntxiv rau pituitary, testis thiab cauda epididymis. Cov qauv ntawm cauda epididymis tau muab tso rau hauv ib txwm saline nrog 5 feem pua ​​​​BSA rau kev soj ntsuam cov phev zoo, thiab cov hypothalamus, encephalon ntxiv rau pituitary thiab testes tau khov hauv cov kua nitrogen thiab khaws cia ntawm -80 degree rau kev tshawb nrhiav ntxiv.

Kev sim 2. Cov nas tau muab faib ua 10 pawg, tsib tsiaj rau ib pab. ECH (20 mg / kg) tau muab tshuaj rau qhov ncauj thiab cov nas hauv txhua pawg tau muab tshuaj loog nrog diethyl ether ntawm lub sijhawm cov ntsiab lus 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, 9 h thiab 12 teev tom qab kev tswj hwm rau kev sau cov qauv.

Plasma tau sau thiab lub plawv tau nthuav tawm. Perfusion ntawm ib txwm saline mus rau sab laug ventricles yog ua los ntawm ib tug infusion apparatus kom txog rau thaum lub siab thiab ntsws tau blanched. Tom qab ntawd cov hypothalamus thiab testis tau sau los txiav txim siab cov ntaub so ntswg ntawm ECH(Echinacoside los ntawm cistanche).

Kev sim 3. Cov nas tau muab faib ua 4 pawg, xya hauv ib pab. ECH(Echinacoside los ntawm cistanche)thiab BPA (CAS: 80-05-7; Aladdin, Shanghai, Tuam Tshoj) tau muab siv rau hauv lub raj intragastric ib hnub ib zaug rau 6 lub lis piam raws li cov qauv kev sim hauv qab no: pawg ib txwm (pob kws roj, 10 mL / kg, bw / d), pawg qauv: BPA pawg (BPA 200 mg / kg, bw / d, pob kws roj), thiab pawg sim: BPA ntxiv rau ECH pawg (BPA 200 mg / kg; ECH 20 mg / kg).

Tom qab 42 hnub ntawm kev kho mob, cov nas tsuag tau muab tshuaj loog nrog diethyl ether thiab cov ntshav tau sau rau kev tshuaj ntsuam xyuas qib hormone. Lub testis thiab cauda epididymis tau muab cais thiab sau. Lub cauda epididymis tau siv rau kev soj ntsuam cov phev zoo thiab cov testis tau khov rau hauv cov kua nitrogen thiab khaws cia ntawm -80 degree rau kev tshawb nrhiav ntxiv.

Daim duab 7. Txoj hauv kev tswj hwm los ntawm kev xaiv HPG axis-related hormone thiab protein nyob rau hauv txiv neej yug me nyuam raws li kwv yees los ntawm Pathway Ontology Database thiab KEGG Pathway Database.

Lus Cim: Cov txheej txheem tswj hwm yog sawv cev los ntawm keeb kwm ntawm cov xim sib txawv thiab cov duab, thiab cov xwm txheej tswj tau tshwm sim siv cov xub thiab kab.

Cov ntaub ntawv xa mus rau txhua qhov protein tau ua raws li Pathway Ontology Database thiab KEGG Pathway Database, thiab tsis tau teev tseg.

Kev txiav txim siab ntawm cov phev zoo. Kev npaj phev suspension. Cauda epididymides tau minced rau hauv 5 mL ntawm ib txwm saline nrog 5 feem pua ​​BSA thiab incubated rau 5 min ntawm 37 degree kom cia lawv cov ntsiab lus mus rau hauv nruab nrab.

Tus nab npawb phev: Raws li ib txoj kev piav qhia los ntawm Yokoi (2003), cov phev diluted suspension (1:10; v / v; phev ncua / 10 feem pua ​​​​methanol) raug xa mus rau txhua chav suav ntawm hemocytometer thiab tau tso cai sawv ntsug rau 5. min thiab tom qab ntawd suav nrog lub teeb pom kev zoo (Nikon, Instruments Inc., Nyiv) ntawm X200 magnification.

Sperm viability: Tag nrho ntawm 20 μL ntawm cov phev raug tshem tawm tau tov nrog qhov sib npaug ntawm eosin-nigrosin stain rau 2 min; cov phev tsis muaj xim raug suav nrog hauv lub teeb tsom iav ntawm 200x magnification.

Sperm motility: Tag nrho ntawm 10 μL ntawm cov phev raug tshem tawm tau muab tso rau hauv ib lub iav swb thiab tau kaw cia li txawb los yog immobile nyob rau hauv lub teeb tsom iav ntawm 200x magnification.

Kev txiav txim siab ntawm cov tshuaj hormones. Qib ntawm Testosterone (T) thiab LH raug ntsuas hauv cov ntshav, encephalon ntxiv rau pituitary thiab testis homogenates siv cov khoom siv radioimmunoassay (RIA) (Beijing Sino-UK Institute of Biological Technology, Beijing, Suav). Luv luv, anti-testosterone los yog anti-LH IgG antibody (100 μL) thiab 125-I-conjugated anti-nas antibody tau ntxiv rau cov qauv lossis tus qauv (100 μL), thiab sib tov rau ntawm lub pob zeb thaum hmo ntuj ntawm 4 degree . Tom qab ntxuav peb zaug nrog PBS-Tween 20 (500 μL), cov khoom sib tov tau centrifuged (3500 rpm / min) ntawm 4 degree rau 15 min. CPM qhov tseem ceeb ntawm cov nag lossis daus tau raug soj ntsuam los ntawm cov khoom siv hluav taws xob hauv xov tooj cua (Beijing Sino-western Technology Co. Ltd, CN202M/KZ4GC-1200, Beijing, Tuam Tshoj) thiab cov concentrations ntawm T thiab LH raug xam raws li cov qauv ntawm tus qauv. nkhaus. T thiab LH coefficient ntawm kev sib txawv yog 2.8 feem pua ​​thiab 3.2 feem pua ​​​​hauv cov qauv pawg, thiab 2.1 feem pua ​​​​thiab 2.8 feem pua ​​​​hauv cov qauv, raws li.

Kev txiav txim siab ntawm cov noob caj noob ces los ntawm lub sijhawm tiag tiag ntawm PCR. Tag Nrho RNA raug cais tawm los ntawm cov noob qes khov thiab encephalon ntxiv rau cov ntaub so ntswg pituitary siv cov khoom siv RNA Simple Total RNA (Tiangen, Beijing, Suav). Quantitative real-time PCR (q RT-PCR) tau ua tiav rau kev nthuav dav ntawm cDNA siv 2 × SYBR Green I PCR Master Mix (Vazyme, Nanjing, Suav). Cov txheej txheem PCR muaj 95 degree rau 30 vib nas this tom qab 35 lub voj voog ntawm 95 degree rau 15 vib nas this, 58 degree rau 30 vib nas this thiab 72 degree rau 30 vib nas this. Kev soj ntsuam kev sib tw melting tau ua rau ntawm PCR cov khoom lag luam txhawm rau txheeb xyuas qhov tshwj xeeb ntawm primer thiab cov khoom purity. Ib qho nkhaus dissociation tau ua rau txhua lub phaj kom paub meej tias kev tsim khoom ntawm ib qho khoom. Qhov txheeb ze ntau ntawm txhua mRNA raug xam. PCR primers siv rau txoj kev tshawb no muaj nyob rau hauv Table 2.

Western blot. Rau AR kev tshuaj ntsuam xyuas, kev rho tawm thiab cais tawm ntawm cytoplasmic thiab nuclear protein tau ua los ntawm Cytoplasmic thiab Nuclear Protein Extraction Kit (Beyotime, Nanjing, Tuam Tshoj) raws li cov chaw tsim khoom cov lus qhia. Cov concentrations ntawm cytoplasmic thiab nuclear proteins tau soj ntsuam siv Bradford Protein Assay Kit (Beyotime, Jiangsu, Tuam Tshoj). Cov qauv protein (80 ug) tau khiav ntawm 12 feem pua ​​​​thiab 5 feem pua ​​​​SDS-PAGE gel thiab xa mus rau PVDF daim nyias nyias. Tom qab thaiv, daim nyias nyias tau incubated nrog anti-AR IgG cov tshuaj tiv thaiv (1: 1, 000; Bioss; Beijing Tuam Tshoj), thiab nas polyclonal anti-GAPDH cov tshuaj tiv thaiv (1: 1, 000; Wuhan Boster Tshuaj lom neegTechnology, Wuhan, Suav teb) los yog nas polyclonal anti-Lamin b1 tshuaj tiv thaiv rau 2 h. Lub membrane tau ntxuav peb zaug nrog TBST thiab incubated nrog ib tug HRP-conjugated luav anti-nas IgG antibody (1:5, 000; Bioss; Beijing Tuam Tshoj). Lub teeb liab tau pom los ntawm kev siv ChemiDoc Imaging System (Tanon-3,500, Shanghai, Suav).

High-Performance Liquid Chromatography (HPLC) kev soj ntsuam. Cov ntshav tau sau rau hauv cov iav heparinized. Lub plasma tau sib cais los ntawm centrifugation tam sim ntawd ntawm 6, 000 rpm rau 10 min thiab khaws cia ntawm −20 degree rau kev sim ntxiv. Cov qauv hypothalamus thiab testis los ntawm txhua lub sij hawm taw tes tau sib sau ua ke thiab homogenized nrog methanol. Tom qab centrifugation ntawm 10, 000 rpm ntawm 4 degree rau 10 min, lub supernatant yog concentrated los ntawm N2 thiab cov residue yog yaj nyob rau hauv 50 μL ntawm methanol thiab lim los ntawm ib tug 0.45 μm flter. Kaum μL ntawm cov qauv lim dej tau txhaj rau hauv HPLC system rau kev tshuaj xyuas.

Tus qauv nkhaus muaj cov qauv uas muaj 50, 100, 250, 500 thiab 750 ng / mL ntawm ECH(Echinacoside los ntawm cistanche)(Chengdu Pufei De Biotech Co., Ltd, Sichuan, Suav teb). Plasma zoo tswj cov qauv spiked nrog 75ng / mL (tsawg), 150 ng / mL (nrab) thiab 300 ng / mL (siab) ntawm ECH tau npaj raws li kev ntsuas qhov tseeb thiab qhov tseeb ntawm txoj kev.

Chromatography tau ua nrog HPLC system (D2{{10}}}00 Cov neeg tseem ceeb series, Hitachi, Nyiv) ua ke nrog lub ntsuas UV (L-2400, Hitachi, Nyiv). Kev sib cais tau ua tiav ntawm Thermo-C18 (250 mm × 4.6 mm id, 4.6 μm hais) kem khaws cia ntawm 25 degree. Cov theem ntawm lub xov tooj ntawm tes yog ib qho gradient npaj los ntawm 0.1 feem pua ​​​​phosphoric acid uas muaj 0.04 feem pua ​​trimethylamine (cov khoom A) thiab methanol (cov khoom B). Cov kab gradient yog raws li hauv qab no: 70–90 feem pua ​​A dhau 0-2 min, 60–70 feem pua ​​A dhau 2–6 min, 55–60 feem pua ​​B dhau 6–8 feeb thiab rov qab mus rau 90 feem pua ​​A ntawm 8 min tam sim ntawd. Tus nqi ntws yog 0.8 mL / min. UV ntes tau ua haujlwm ntawm 332 nm. Peak cheeb tsam raug ntsuas raws li kev ntsuas ntsuas.

Pharmacokinetic kev tshawb fawb hauv nas. Kev nkag mus ntawm ECH(Echinacoside los ntawm cistanche)mus rau hypothalamus thiab testis tau raug tshuaj xyuas pharmacokinetic nrog Tshuaj thiab Statistics software (Drug and Statistics, Mathematical Pharmacology Professional Committee of China). Pharmacokinetic tsis tau txiav txim siab siv txoj kev uas tsis yog-compartmental raws li kev tshawb fawb lub sijhawm. Cov tshuaj pharmacokinetic ua ntej suav nrog lub sijhawm kom ncov tas li (Tmax), ncov siab (Cmax), tshem tawm tus nqi tas li (Ke), tshem tawm ib nrab-lub neej (T0.5), thaj tsam hauv qab nkhaus (AUC{ {4}}–12), pom meej qhov ntim (Vc), thiab tshem tawm (CL) tau txais los tshuaj xyuas ntxiv. Lub paj hlwb/plasma ratios raug xam raws li AUC0-t qhov tseem ceeb rau ntshav thiab lub hlwb.

ECH-Ovalbumin (ECH-OVA) synthesis thiab kev txheeb xyuas. Tag nrho ntawm 9.8 mg ntawm ECH(Echinacoside los ntawm cistanche)thiab 1. Cov sib tov yog concentrated los ntawm N2 thiab cov residue tau ua ke nrog 10.8 mg ntawm N-hydroxysuccinimide (NHS) thiab 19.3 mg ntawm dicyclohexylcarbodiimide (DCC) thiab yaj nyob rau hauv 4 mL N, N-dimethylformamide (DMF) rau 12 h nrog stirring ntawm chav tsev kub. . Tom qab centrifugation ntawm 2, 000 g rau 5 min ntawm 4 degree, tus supernatant tau ntxiv rau 5 mL ntawm PBS uas 14.4 mg ovalbumin (OVA) tau yaj. Cov sib tov tshiab tau nplawm rau 24 teev ntawm 4 degree. Tom qab ntawd cov tshuaj tiv thaiv tau dialyzed tawm tsam PBS rau peb hnub. Lub xub ntiag ntawm ECH-OVA tau lees paub los ntawm UV spectra (Shimadzu Scientifc Instruments, Inc. Columbia, MD USA) ntawm lub wavelength xws li 190 txog 400 nm thiab los ntawm denaturing PAGE.

Indirect ELISA (iELISA). Lub phaj microtiter tau coated nrog ECH-OVA (2 ug / 100 μL) thiab incubated hmo ntuj ntawm 4 degree. Lub phaj tau ntxuav peb zaug nrog PBST thiab ob zaug nrog PBS; thiab 5 feem pua ​​​​PBSM (PBS uas muaj 5 feem pua ​​​​skimmed mis nyuj) tau ntxiv los thaiv cov chaw tsis sib haum ntawm 37 degree rau 2 teev. Tom qab cov phiaj tau ntxuav nrog PBST thiab PBS, qhov dej tau muab faib ua 4 pawg; pab pawg sim, 1 ug AR tag nrho cov protein / 100 μL; pawg zoo, luav anti-OVA antibody (1: 1, 000); pawg tsis zoo, 1 ug ntawm bovine serum albumin; thiab ib pab pawg dawb huv uas muaj PBS. Tom qab incubation rau 1.5 h, daim hlau raug ntxuav, thiab 100 μL / qhov dej ntawm luav HRP-conjugated IgG (1: 1,{22}}) tau ntxiv rau cov pab pawg zoo, 100 μL / qhov dej ntawm anti-AR tau ntxiv. (1:1,{27}}) rau lwm pab pawg. Tom qab incubation rau 1.5 teev, luav HRP-conjugated IgG (1:5,{33}}) dilution tau ntxiv rau hauv daim hlau. Tom qab incubation ntawm 37 degree rau 1 h, cov phaj tau ntxuav peb zaug nrog PBST thiab ob zaug nrog PBS. Tom qab ntawd TMB tau ntxiv thiab incubated rau 10 min nyob rau hauv qhov tsaus ntuj ntawm chav tsev kub thiab 2 M H2SO4 ntxiv kom tsis txhob cov tshuaj tiv thaiv. Lub absorbance tau nyeem ntawm lub wavelength ntawm 450 nm.

Molecular docking. Kev tshawb nrhiav molecular docking tau ua los tshawb xyuas hom kev khi ntawm cov khoom sib xyaw ECH(Echinacoside los ntawm cistanche)rau tib neeg androgen receptor (AR) siv Autodock vina 1.1.2 (http://vina.scripps.edu). Cov qauv peb-dimensional (3D) ntawm AR (PDB ID: 2YHD) tau rub tawm los ntawm Protein Data Bank (http://www.rcsb.org/pdb/home/hone.do). Tus qauv 3D ntawm ECH tau txais los ntawm ChemBioDraw Ultra 14.0 thiab ChemBio 3D Ultra 14.0 software. Lub AutoDockTools 1.5.6 pob (http://mgltools.scripps.edu) tau ua haujlwm los tsim cov ntaub ntawv docking input. Daim phiaj tshawb nrhiav ntawm AR tau txheeb xyuas qhov chaw x: 36.141, chaw y: 8.513, thiab chaw z:

21.304 nrog qhov loj me x: 15, qhov loj me y: 15, thiab qhov loj me z: 15. Tus nqi ntawm kev ua kom tiav tau teeb tsa rau 20. Rau Vina docking, lub default parameters tau siv yog tias nws tsis tau hais. Qhov qhab nia zoo tshaj plaws raws li kev txiav txim los ntawm Vina docking qhab nia raug xaiv thiab tsom xam pom siv PyMOL 1.7.6 software (http://www.pymol.org).

Kev txheeb xyuas cov ntaub ntawv thiab cov txheej txheem txheeb cais. Cov ntaub ntawv tau raug tshuaj xyuas siv cov ntaub ntawv txheeb xyuas software SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Ib txoj kev ANOVA tau ua haujlwm rau kev sib piv ntawm cov pab pawg. Tukey qhov kev sib piv ntawm qhov sib txawv tseem ceeb ntawm cov pab pawg tau txiav txim siab. Cov txiaj ntsig tau qhia tias txhais tau tias ± tus qauv sib txawv (SD) siv Graph Pad Prism software v.7 (GraphPad Software, Inc, California, USA).

Kev pom zoo ntawm kev ncaj ncees thiab kev tso cai los koom. Kev pom zoo rau kev ncaj ncees rau txoj kev kawm no tau txais los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Anyang Institute of Technology.

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