Cov nyhuv ntawm Cistanche Tubulosa Extracts Rau Txiv neej Kev Ua Haujlwm Kev Ua Haujlwm hauv Streptozotocin-Nicotinamide-Induced Diabetic Rats-Ⅰ

1. Taw qhia

Mob ntshav qab zib mellitus (DM) yog ib qho mob uas muaj ntau ntxivqib qabzibnyob rau hauv cov ntshav vim qhov tsis muaj peev xwm ntawm pancreas hauv kev tsim cov tshuaj insulin txaus, lossis lub cev tsis muaj peev xwm siv tau zoo. Raws li WHO, tus naj npawb ntawm cov neeg mob ntshav qab zib tau nce los ntawm 108 lab hauv 1980 txog 422 lab hauv 2014 [1]. Muaj plaub yam tseem ceeb: mob ntshav qab zib ua ntej (ib theem ua ntej ntshav qab zib), hom 1 (qhov twg pancreas tsis tsim cov tshuaj insulin), hom 2 (lub cev tsis siv tshuaj insulin), thiab ntshav qab zib gestational (uas tshwm sim thaum cev xeeb tub). Oxidative stress tshwm sim vim qhov tsis sib xws ntawm kev tsim cov pa oxygen reactive (ROS) thiab antioxidants [2] thaum kawg ua rau mob ntshav qab zib. Cov teeb meem ntawm ntshav qab zib muaj xws li lub raum tsis ua haujlwm, paj hlwb puas, dig muag, mob stroke, plawv nres, fetal tuag, thiab infertility. [1]. Cov kev tshawb fawb pom tau tias phev nuclei, deoxyribonucleic acid (DNA), thiab mitochondria raug puas tsuaj rau cov txiv neej mob ntshav qab zib [3]. Oxidative stress thaum lub sij hawm thauj phev yuav hloov cov txheej txheem ntawm cov txiv neej reproductive system [4,5].

NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev sib deev PHGS75% ECH 30% ACT 12%

Cov txheej txheem ntawm spermatogenesis yog tswj los ntawm hypothalamus-pituitary-gonadal (HPG) axis. Lub gonadotropin-tso hormone (GnRH) tsim los ntawm hypothalamus txhawb kev tsim cov tshuaj luteinizing hormone (LH) thiab follicle-stimulating hormone (FSH). Leydig hlwb pom nyob ib sab ntawm lub seminiferous tubule thiabtsim testosteronenyob rau hauv kev tswj ntawm LH. FSH ua rau kev tsim cov tshuaj tiv thaiv kab mob antigen-binding protein (ABP) uas pab kom dhau cov txiv neej cov tshuaj hormones. Yog li, kev xeeb tub thiab lwm yam teeb meem feem ntau tshwm sim los ntawm kev cuam tshuam uas tshwm sim hauv HPG axis. Kev tswj hwm ntawm kev sib xyaw ntawm streptozotocin-nicotinamide induces ntshav qab zib hauv cov nas sim. Streptozotocin yog cov tshuaj lom neeg ua rau cov hlwb tsim cov tshuaj insulin hauv pancreas thiab nicotinamide yog cov vitamin dej-soluble uas tiv thaiv cov hlwb los ntawm kev puas tsuaj tag [6].

Cistanche tubulosa yog cov nroj tsuag suab puam hu ua "Rou Cong-Rong" [7]. Nws yog cov nroj tsuag uas tsis yog chlorophyllic parasitic uas loj hlob nyob rau hauv paus ntawm Calotropis procera tsob ntoo thiab dav faib nyob rau hauv arid av ntawm Gansu, Qinghai, Xinjiang, Mongolia, Iran, thiab Is Nrias teb. Lub npe ntawm Cistanche tubulosa yog "Desert Hyacinth". Nws tau txais dav hauv Suav tshuaj thiab tau muab lub npe "Ginseng ntawm cov suab puam". Nws yog dav siv nyob rau hauv tshuaj teb los kho morbid leucorrhea, profuse metrorrhagia, mob raum kab mob, cem quav, impotence, thiab infertility. Chemical constituents ntawmCistanche tubulosa muaj cov uas tsis yog-volatile phenylethanoid glycosides(PHGs), iridoids, lignans, volatile roj, alditols, oligosaccharides, thiab polysaccharides [8]. Kev tshawb fawb qhia tiasechinacoside cov tshuajlos ntawm cov tshuaj ntsuab no tiv thaiv cov fibroblast puas los ntawm kev tswj ROS qib [9]. Cov tshuaj no tsim cov tshuaj tiv thaiv kab mob antioxidant, vasorelaxation, thiab kev ua haujlwm tiv thaiv kab mob ua ke nrog neuroprotection thiab kev tiv thaiv osteoporosis [10,11].

Txoj kev tshawb no tsom los tshawb xyuas cov txiaj ntsig ntawmCistanche tubulosa extractmuaj ECH ntawm kev ua me nyuam tsis ua haujlwm ntawm streptozotocin-nicotinamide-induced ntshav qab zib nas.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Khoom siv

ECH thiab CTE tau yuav los ntawm Sinphar Pharmaceutical Co., Ltd (Yilan, Taiwan). Advanced glycation kawg khoom (AGEs) tau yuav los ntawm Biovision (San Francisco, CA, USA). LC-540 thiab TM3 kab ntawm tes tau yuav los ntawm Food Industry Research and Development Institute (Hsinchu, Taiwan). Tsib-lub lim tiam-laus Sprague-Dawley (SD) txiv neej nas tau yuav los ntawm National Laboratory Animal Center (Taipei, Taiwan). Pub Lab Diet® tau yuav los ntawm PMI Nutrition International, Inc. (Taipei, Taiwan). 2,2-diphenyl-1-picrylhydrazyl (DPPH) tau los ntawm Sigma (St. Louis, MO, USA). Methanol thiab 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) kuj tau yuav los ntawm Sigma. Dulbecco qhov hloov kho Eagle nruab nrab / F12 thiab trypsin-EDTA tau txais los ntawm GIBCO (New York, NY, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dichloro-dihydro-fluorescein diacetate (DCFH-DA), Dimethyl sulfoxide (DMSO), thiab nitroblue tetrazolium (NBT) tau yuav los ntawm Sigma. Glucose enzymatic cov khoom siv tau los ntawm Kyokutoseiyaku, Tokyo, Nyiv. Cov khoom siv Insulin ELISA tau yuav los ntawm Mercodia AB Inc., Sylveniusgatan 8A (Uppsala, Sweden). T-PER Tissue Protein Extraction Reagent tau txais los ntawm Thermo Scientific (Chicago, IL, USA). Anti-tib neeg / Nas / Rat nuclear factor-kappa B NF-κB polyclonal antibodies tau muas los ntawm Santa Cruz Biotechnology, Santa Cruz, CA, USA. Anti-Mouse/Rat receptor rau cov khoom siv qib siab glycation kawg (RAGE) polyclonal antibodies, anti-Human/nas/rat StAR polyclonal antibodies, thiab anti-Human/Mouse/rat Cytochrome P450 17A1 monoclonal antibodies tau muas los ntawm Gene Tex (Irvine, CA, USA). Anti-Human/Mouse/Rat CYP11A1 polyclonal antibodies tau txais los ntawm Cell Signaling Technology (Beverly, PA, USA). Ib qho yooj yim-Blue reagent tau yuav los ntawm Invitrogen, Thermo Fisher Scientific (Carlsbad, CA, USA). Agarose thiab DNA marker 100 bp tau txais los ntawm Promega, Corporation (Madison, WI, USA). RNeasy Lipid Tissue Mini Cov Khoom Siv tau yuav los ntawm QIAGEN, Hilden, Lub Tebchaws Yelemees.

NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev sib deev PHGS75% ECH 30% ACT 12%

2.2. Cov txheej txheem

2.2.1. Hauv Vitro Analysis

LC{{0}} thiab TM3 Cell Kab lis kev cai: LC-540 kab xov tooj ntawm tes tau coj los ntawm 37 ◦C hauv Earle's Balanced Salt Solution (EBSS) nruab nrab ntxiv nrog sodium bicarbonate (1.5 g / L), L-glutamine (2 mM), tsis tseem ceeb amino acids (0.1 mM), sodium pyruvate (1.0 mM), thiab fetal bovine serum (FBS) (10%) hauv ib 5% CO2 incubator (CO2 incubator, Napco 5410, Taiwan). Lub TM3 cell kab yog kab lis kev cai hauv Dulbecco's modified Eagle medium (DMEM) supplemented nrog qabzib (4.5 g / L), sodium pyruvate (0.5 mM), L-glutamine (2.5 mM), sodium bicarbonate (1.2 g / L), {{ 28}}(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 15 mM), thiab fetal calf serum (FCS, 10%) los yog horse serum (5%) ntawm 37 ◦C hauv 5% CO2 incubator.

Kev txiav txim siab ntawm Cell Viability ntawmEchinacoside (ECH): 3-(4, 5-dimethylthiazol-2- yl)-2, 5- diphenyltetrazolium bromide (MTT) reagent tau siv rau kev soj ntsuam cell viability. Lub cell concentration tau hloov mus rau 2 × 105 hlwb / mL hauv 96-zoo phaj. Tom qab ntawd, 20 µL ntawm ECH (diluted nyob rau hauv 2% ntawm nruab nrab) yog ntxiv thiab incubated rau 24 teev ntawm 37 ◦C hauv 5% CO2 incubator. Tom qab ntawd, 100 µL ntawm MTT reagent tau ntxiv thiab incubated rau 4 h ntawm 37 ◦C nyob rau hauv qhov tsaus ntuj. Qhov nqus tau ntsuas ntawm 570 nm (ELISA nyeem, Dynatech MR5000, Kloten, Switzerland).

Cov txheeb ze ntawm tes viability (%)=[(Asample ntawm 570 nm − Ablank ntawm 570 nm)]/[(Acontrol ntawm 570 − Ablank ntawm 570)] × 100.

Nitroblue Tetrazolium (NBT) Reduction Assay thiab 2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay: Tus naj npawb ntawm cov hlwb raug kho rau 1 × 1{{10}}6 hlwb/ mL. Tom qab ntawd, 50 mg / mL ntawm ECH, resveratrol (RES), thiab qib siab glycation end-products (AGEs) (tswj) tau ntxiv thiab co-cultured rau 18 teev ntawm 37 ◦C. Tom qab ntawd, 0.3 mL ntawm NBT tov (0.1 mg / mL NBT, 5% FBS, thiab 3% dimethyl sulfoxide (DMSO) yaj hauv 10 mL DMEM) tau ntxiv thiab incubated ntawm 37 ◦C hauv 5% CO2 rau 1 h. Tom qab centrifugation (ntawm 800 × g rau 3 min) lub supernatant raug tshem tawm thiab 200 µL DMSO tau ntxiv thiab shaken rau 5 min hauv ib qho ultrasonic oscillator (Delta Ultrasonic Cleaner D 200, Keelung, Taiwan). Lub absorbance yog ntsuas ntawm 630 nm. Rau DPPH radical assay, Trolox (hauv 95% cawv) raug coj los ua tus qauv. Tom qab ntawd, 75 µL ntawm 0.5 mM DPPH tov yog tov nrog 25 µL ntawm cov qauv thiab tso cai rau hnov ​​​​mob ntawm chav tsev kub hauv qhov chaw tsaus rau 30 min. Qhov sib tov tau shaken thiab qhov nqus tau ntsuas ntawm 517 nm.

Reactive Oxygen Species (ROS) Cov Ntsiab Lus Kev Ntsuam Xyuas: Cov xov tooj ntawm tes tau hloov kho rau 2 × 105 hlwb / mL hauv 12- cov phaj zoo uas muaj 2% FBS. Tom qab ntawd, 50 µL / mL ntawm ECH, RES, thiab AGEs tau ntxiv rau hauv phaj thiab incubated ntawm 37 ◦C rau 24 teev. Tom qab 24 teev, 20, 70 -dichlorofluorescein-diacetate (DCFH-DA) tau ntxiv thiab incubated ntawm 37 ◦C rau 30 feeb. Tom qab centrifugation (400 × g, 5 min), lub supernatant raug tshem tawm thiab cov hlwb raug ntxuav ob zaug nrog phosphate-buffered saline (PBS). Tom qab ntawd, cov hlwb raug tshem tawm hauv 1 mL ntawm PBS thiab cov qib ROS tau txiav txim siab los ntawm kev siv lub cytometer ntws (Flow Cytometer, Becton Dickinson, CA, USA).

Kev txheeb xyuas thiab Kev Ntsuam Xyuas Ntau Yam ntawm Protein: Cov protein tau muab rho tawm los ntawm kev siv cov protein extraction reagent (T-PER). Lub cell ceev tau hloov mus rau 2 × 105 hlwb / mL nyob rau hauv ib tug 12-zoo phaj thiab 50 µL / mL ntawm ECH, RES, thiab ib tug receptor rau cov advanced glycation endproduct (RAGE) antagonist, thiab AGEs tau ntxiv thiab incubated ntawm 37 ◦C hauv 5% CO2 incubator rau 24 teev. Tom qab ntawd, 400 µL ntawm T-PER tau ntxiv, tshem tawm cov hlwb, thiab centrifuged ntawm 125, 000 × g rau 20 min (4 ◦C). Lub supernatant tau sau thiab khaws cia ntawm −80 ◦C (−80 ◦C lub freezer, Nuaire, Plymouth, MN, USA) rau kev tshuaj xyuas ntxiv. Cov khoom siv bicinchoninic acid (BCA) tau siv los ntsuas cov protein ntau. Tom qab ntawd, 10 µL ntawm cov txheej txheem cell tov thiab 200 µL ntawm BCA reagent tau ntxiv rau 8- cov phiaj zoo thiab incubated ntawm 37 ◦C hauv 5% CO2 incubator rau 30 min. Lub absorbance yog ntsuas ntawm 562 nm. Kev txheeb xyuas cov protein tau ua los ntawm sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS-PAGE) electrophoresis. Cov qauv tau npaj los ntawm kev ntxiv cov protein lysate rau cov protein thauj khoom tsis txaus thiab kev tsom xam sab hnub poob tau ua los xyuas cov proteins tshwj xeeb.

2.2.2. Hauv Vivo Analysis

Tsiaj Qauv Tsim: Rau caum {{0}} tus txiv neej hnub nyoog ib lim piam Sprague–Dawley (SD) nas tau yuav los ntawm National Laboratory Animal Center (Taipei, Taiwan). Txhua tus nas tau nyob hauv ib tus zuj zus hauv cov tshuaj tua kab mob stainless hlau tawb nyob rau hauv qhov ntsuas kub (23 ± 1 ◦C) thiab av noo (40-60%) nrog lub teeb 12 teev / 12 teev tsaus ntuj. Khoom noj thiab dej tau muab rau libitum. Txhua tus nas tau nyob hauv thawj lub lim tiam thiab muab cov nas noj zaub mov 5001 ua cov zaub mov tseem ceeb. Tag nrho cov txheej txheem ua raws li tus qauv ntawm Pawg Saib Xyuas Tsiaj Tsiaj thiab Siv Khoom Siv (IACUC Pom Zoo No. 101026) ntawm National Taiwan Ocean University, Taiwan. Tom qab yug me nyuam, nas tau muab faib ua ob pawg: pawg tswj hwm (Con) uas tau pub mis noj 5001, thiab pab pawg neeg mob ntshav qab zib uas tau noj cov zaub mov muaj roj ntau (HFD, 40%) rau tag nrho cov kev sim. Tom qab tau noj nrog HFD rau 4 lub lis piam, cov pab pawg neeg mob ntshav qab zib tau txhaj tshuaj streptozotocin (65 mg / kg) (STZ) txhawm rau ua rau mob ntshav qab zib mellitus (DM). Hauv 15 feeb ntawm kev txhaj tshuaj STZ, nicotinamide (230 mg / Kg Lub Cev Qhov hnyav) tau txhaj. Ib lub lim tiam tom qab txhaj tshuaj, qhov kev ntsuas qhov ncauj qhov ncauj (OGTT) tau ua los txiav txim siab qhov ua tiav ntawm cov ntshav qab zib mellitus (DM). Tom qab ntawd, cov tsiaj tau muab faib ua 6 pawg: kev tswj hwm (piv nrog kev sim zaub mov 5001); DM pawg (DM + 45% HFD); DMR pawg (DM + rosiglitazone: 0.571 mg/kg BW) + 45% HFD; DME1 pawg (DM + CTE: 80 mg/kg BW) + 45% HFD; DME2 pawg (DM + CTE: 160 mg/kg BW) + 45% HFD; thiab DME4 pawg (DM + CTE: 320 mg/kg BW) + 45% HFD. Rosiglitazone (RSG) raug coj los ua kev tswj hwm zoo. Peb koob tshuaj CTE (80, 160, thiab 320 mg / kg) tau siv raws li kev pom zoo ntawm Taiwan Food and Drug Administration (TFDA) cov lus qhia kev soj ntsuam kev noj qab haus huv. Kev kho mob tau muab mus txog thaum kawg ntawm qhov kev sim. Tag nrho cov nas sim tau muab txi tom qab 6 lub lis piam. Cov ntshav sau los ntawm nas yog centrifuged ntawm 3000 rpm rau 15 min ntawm 4 ◦C. Lub supernatant tau tshuaj xyuas rau cov hauv qab no tsom xam:

Tag Nrho Glucose, Triglyceride, thiab Cholesterol Kev Txiav Txim Siab: Kev txiav txim siab cov piam thaj tau ua los ntawm cov khoom siv qabzib enzymatic. Tom qab ntawd, 20 µL ntawm cov ntshav plasma kuaj tau ntxiv rau cov tshuaj reagents thiab khaws cia ntawm 37 ◦C rau 5 feeb. Tag nrho cov triglycerides thiab tag nrho cov roj cholesterol concentration tau txiav txim siab los ntawm kev siv cov khoom siv triglyceride thiab cov roj cholesterol enzymatic. Tom qab ntawd, 10 µL ntawm cov ntshav ntshav tau ntxiv rau cov tshuaj reagents thiab kev sim tau ua raws li cov chaw tsim khoom cov lus qhia. Lub absorbance yog ntsuas ntawm 510 nm.

Plasma tag nrho cov piam thaj / triglyceride / cholesterol (mg / dL)=(Asample − ABlank)/(Astandard − ABlank) × 200.

Asample: Absorbance of blood samples, ABlank: Absorbance value of kits without a sample, Astandard: Absorbance value of the standard reagent, 200: standard reagents at a concentration of 200 mg/dL.

Insulin, Leptin, thiab Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) Kev Txiav Txim:Cov qib insulin yog txiav txim los ntawm cov khoom siv insulin ELISA. Ntawm no, 25µL ntawm cov ntshav plasma tau ntxiv raureagents, thiab qhov nqus tau ntsuas ntawm 450 nm. Tag nrho cov ntsiab lus leptin tau txiav txim siab los ntawm kev siv aleptin enzyme immunometric assay kit.Tom qab ntawd, 100µL ntawm plasma tau txheeb xyuas thiab nqus tautau ntsuas ntawm 450 nm siv ELISA nyeem ntawv. Tag nrho cov kev sim tau ua raws licov neeg tsim khoom cov lus qhia. Tus nqi HOMA-IR tau txiav txim los ntawm tus qauv homeostasisKev ntsuas qhov sib npaug=Fasting plasma insulin concentration (mg/mL)× ceev ceev plasma qabzibQhov siab tshaj plaws (mmol / L) / 22.5.

Plasma Lipid Peroxidation: Ntawm no, {{0}}.5 mL ntawm cov ntshav ntshav tau tov nrog 1 mL ntawm reagent (15%, w/v trichloroacetic acid hauv 0}.25 N hydrochloric acid (HCl) ) thiab 0.375%, w/v thiobarbituric acid nyob rau hauv 0.25 N HCl) thiab muab tso rau hauv ib da dej da dej (Water Bath, BUCHI 461, Zürich, Switzerland) ntawm 100 ◦C rau 15 min. Tom qab txias, 1 mL ntawm n-butanol tau ntxiv, shaken, thiab centrifuged ntawm 1500 × g rau 10 min. Lub supernatant tau sau thiab qhov nqus tau ntsuas ntawm 532 nm [12].

Malondialdehyde (MDA) concentration (nM/mL)=[(A qauv ntawm 532 nm − Ablank ntawm 532 nm) / (Ib tus qauv ntawm 532 nm − Ablank ntawm 532 nm)] × 5.

tus qauv ntawm 532 nm − Ablank ntawm 532 nm)] × 5. Kev txiav txim siab ntawm Plasma Testosterone thiab Luteinizing Hormone (LH) Qib: Cov khoom siv testosterone ELISA tau siv los ntsuas qib ntawm testosterone hauv cov ntshav plasma. Cov khoom siv RIA tau siv los txiav txim siab LH concentration. Tom qab ntawd, 50 µL ntawm plasma tau ntxiv thiab cov kev suav ua raws li qhov concentration ntawm cov tshuaj hormone LH-RP-3 (tus qauv). Cov kauj ruam ntxiv tau ua raws li cov neeg tsim khoom cov lus qhia.

Kev txiav txim siab ntawm Tumor Necrosis Factor (TNF)- thiab Interleukin-6 (IL-6) Kev Cuam Tshuam: Cov tshuaj tiv thaiv uas ntes tau raug diluted 250 zaug hauv txheej txheej, ntxiv rau 96- Cov phaj zoo (100 µL / qhov dej), thiab khaws cia ntawm 4 ◦C rau ib hmos. Tus supernatant tau aspirated thiab ntxuav tsib zaug nrog ntxuav tsis (1 zaug PBS, 0.05% Tween 20). Tom qab incubation (1 h) nrog 200 µg ntawm kev soj ntsuam diluent, lub hlwb raug ntxuav 5 zaug nrog ntxuav tsis thiab 100 µL ntawm TNF- qauv tov los yog cov qauv kuaj tau ntxiv rau txhua qhov dej thiab incubated rau 2 h. Tom qab ntxuav, 100 µL ntawm IL-6- kuaj pom cov tshuaj tiv thaiv tau ntxiv thiab incubated rau 1 h ntawm chav tsev kub. Tus supernatant tau aspirated thiab ntxuav 5 zaug. Tom qab ntawd, 480 µL ntawm enzyme avidin-horseradish peroxidase (HRP) tau ntxiv thiab incubated rau 30 min ntawm chav tsev kub. Tus supernatant tau aspirated thiab ntxuav tsib zaug nrog ib tug ntxhua tsis. Tom qab ntawd, 100 µL ntawm substrate tov yog ntxiv thiab incubated rau 15 min ntawm chav tsev kub. Tom qab ntawd, 50 µL ntawm cov tshuaj nres (1M phosphoric acid) tau ntxiv rau txhua qhov dej thiab qhov nqus tau ntsuas ntawm 450 nm.

NATURAL CISTANCHE TUBULOSA FOR REPAIRING SEXUAL FUNCTION PHGS75% ECH 30% ACT 12%

Kev txheeb xyuas cov phev thiab cov qe ntshav tsis muaj zog

Sperm Sample Collection: Cov phev coj mus kuaj tau ua raws li txoj kev tau qhia yav dhau los hauv [13]. Cov phev tau sau los ntawm supernatant thiab siv rau kev tshuaj xyuas ntxiv.

Tus naj npawb ntawm cov phev, phev txav, thiab cov phev txawv txav: Tus naj npawb ntawm cov phev raug suav nrog cov hemocytometer thiab trypan xiav kua. Tom qab ntawd, 100 µL ntawm cov phev kua yog tov nrog trypan xiav tov thiab cov phev, motility, thiab txawv txav phev raug xam siv lub hemocytometer thiab microscope [14].

Nitro xiav Tetrazolium NBT Txo thiab Lipid Peroxidation ntawm Sperm thiab Testis: Cov lipid peroxidation thiab NBT txo tau raug tshuaj xyuas raws li cov txheej txheem ntawm kev tshuaj ntsuam plasma tib yam.

Kev txiav txim siab ntawm Superoxide Dismutase (SOD) thiab Catalase Activity: Superoxide dismutase (SOD) kev ua haujlwm tau txheeb xyuas los ntawm kev siv cov khoom siv RANSEL. Ntawm no, {{0}}. {9}}iodophenyl)- 3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT)). Tom qab sib tov, 0.25 mL xanthine oxidase (80 U / L) tau ntxiv thiab tshwm sim ntawm chav tsev kub rau 30 s. Lub absorbance yog ntsuas ntawm 505 nm. Cov protein ntau ntawm cov noob qes homogenate tau txiav txim los ntawm Bio-Rad DC cov khoom siv tshuaj ntsuam xyuas protein thiab nws cov haujlwm tshwj xeeb (U / mg protein) raug xam. Txoj haujlwm catalase (CAT) tau txiav txim siab raws li txoj kev qhia yav dhau los [15].

H&E (Hematoxylin thiab Eosin) Staining

Cov noob qes tau muab tso rau hauv 10% ntawm formalin rau 24 teev thiab khaws cia ntawm 4 ◦C. Cov ntaub so ntswg tau nyob rau hauv qhov loj me thiab txuas nrog tus swb. Tom qab kho (95% methanol + 5% acetic acid), cov swb tau muab tso rau hauv hematoxylin rau 3 min, ntxuav nrog dej ntws rau 5 feeb, thiab tom qab ntawd tsau lawv nrog 50%, 70%, thiab 90% cawv rau ib feeb. Tom qab ntawd, cov slides tau stained nrog eosin rau 10 s thiab soaked nyob rau hauv 100% cawv rau ib feeb kom txog thaum nws faded. Thaum kawg, tus swb tau tsau rau hauv xylene rau ib feeb. Tom qab ntawd, lub swb tau qhuav thiab kaw. Lub raj stained tau muab tso rau hauv ib qho inverted theem contrast microscope (Inverted Phase Difference Microscope, Olympus CK-2, Tokyo, Nyiv) los soj ntsuam morphology.

Analysis ntawm Hypothalamus

Tus nas KISS1, G-protein coupled receptor (GPR) 54, suppressor ntawm cytokine signaling 3 (SOCS-3), thiab sirtuin 1(SIRT1) gene sequences tau txheeb xyuas los ntawm NCBI (National Center for Biotechnology Information) gene database. thiab cov primer tshwj xeeb tau tsim los siv Primer 5.0 PREMIER Biosoft, Palo Alto, CA, USA.). Cov primers siv nyob rau hauv qhov kev sim yog teev nyob rau hauv Table 1.

Extraction ntawm Ribonucleic acid (RNA) los ntawm Hypothalamus. Kev rho tawm ntawm RNA los ntawm hypothalamus ntawm lub hlwb tau ua los ntawm kev siv RNeasy Lipid Tissue Mini Kit. Cov mRNAs tau txais tau raug ntsuas ntau los ntawm kev hloov pauv rov qab (RT) thiab polymerase saw cov tshuaj tiv thaiv (PCR). Kev qhuas DNA tau los ntawm RT tsom xam tau khaws cia ntawm −20 ◦C rau kev siv tom qab. Cov DNA rho tawm tau raug soj ntsuam los ntawm polymerase chain reaction (PCR) siv Taq polymerase. Tom qab ntawd, 5 ~ 10 µL ntawm PCR cov khoom raug coj mus kuaj thiab tshuaj xyuas los ntawm electrophoresis ntawm 1.5% agar colloid. Cov duab tau raug coj mus rau hauv UVP BioDoc-It imaging system. Reverse-transcribed complementary deoxyribonucleic acid (cDNA) raug coj thiab PCR tau ua tiav siv IQ SYBR Green Supermix Kit. Tom qab ntawd, nws tau txheeb xyuas nrog iQTM 5 Optical System Software. Cov protein tau muab rho tawm los ntawm kev siv cov protein extraction reagent (T-PER). Tom qab ntawd, 20 mg ntawm testicular homogenate tau ntxiv rau 400 µL ntawm T-PER thiab centrifuged (High-Speed ​​Centrifuge, Hettich CR-12, Tuttlingen, Lub teb chaws Yelemees) ntawm 4 ◦C (125,{16}} × g ) rau 20 min. Cov txheej txheem hauv qab no zoo ib yam li "kev txheeb xyuas thiab ntsuas qhov muaj pes tsawg leeg ntawm cov protein" hauv kev tshuaj ntsuam vitro.

2.3. Kev txheeb cais

Cov txiaj ntsig kev sim tau qhia raws li qhov nruab nrab ± tus qauv sib txawv (Mean ± SD) thiab cov ntaub ntawv tau txheeb xyuas los ntawm Kev Tshawb Fawb Cov Khoom Siv & Kev Pabcuam (SPSS) 11.0 software, IBM, Armonk, New York, NY, USA. Qhov sib txawv ntawm pawg tau txheeb xyuas los ntawm ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA). Ntau qhov sib piv ntawm cov pab pawg sib txawv tau txheeb xyuas los ntawm Duncan qhov kev sim ntawm tus nqi ntawm p < 0.05 raws li qib tseem ceeb.

3. Cov txiaj ntsig

3.1. Hauv Vitro Analysis

3.1.1. Kev sib piv ntawm Antioxidant Activities ntawm ECH, CTE, thiab RES

Lub sij hawm ntev high oxidative kev nyuaj siab thiab mob ntev yog qhov ua rau mob ntshav qab zibteeb meem [16]. CTEs muaj ntau yamphenylethanoid glycosides xws li echinacoside(ECH) thiab acteoside, nrog lub peev xwm rau kev ua haujlwm antioxidant [17]. DPPH radical scavengingKev soj ntsuam tau ua los ntsuas cov tshuaj tiv thaiv antioxidant ntawm ECH. Trolox thiab resveratrol(3,5,4'-trihydroxystilbene, RES) raug coj los ua tus qauv thiab kev tswj hwm zoo raws li. Raws li qhiahauv daim duab1, ECH tau pom zoo dua radical scavenging kev ua si thiab kev ua haujlwm tau ntau duatshaj qhov kev tswj zoo (RES).

3.1.2. Cell Viability ntawm ECH ntawm LC-540 thiab TM3 Leydig Cells

MTT kev soj ntsuam tau ua los ntsuas cov cell muaj peev xwm ntawm qhov sib txawv ntawm ECH. LC-540 Leydig hlwb thiab TM3 Leydig hlwb tau pom ntau dua 80% ntawm kev ua tau zoo tom qab tau kho nrog ECH (Daim duab 2). Raws li piv rau lwm cov concentrations, ib tug 100-µM concentration (dilutions ntawm 100 µM nyob rau hauv 2% FBS) ntawm ECH pom zoo cell viability nyob rau hauv TM3 Leydig hlwb. Cov txiaj ntsig tau qhia tias qhov nce ntxiv ntawm ECH tsis ua rau muaj kev cuam tshuam loj rau lub hlwb.

3.1.3. Cov nyhuv ntawm ECH ntawm AGE-Induced Superoxide Production los ntawm NBT Assay hauv LC-540 thiab TM3 Leydig Cells

AGEs ua rau oxidative puas hauv lub cev vim oxidation ntawm qabzib thiab tsim cov dawb radicals xws li O2-, hydroxyl radicals, thiab carbonyl pawg [18]. Tom qab raug txhawb nrog 50 ug / mL ntawm AGEs, LC -540 (Daim duab 3a) thiab TM3 (Daim duab 3b) hlwb tau kho nrog ECH thiab RES (5 μM thiab 10 μM txhua). Cov txiaj ntsig tau pom tias kev tsim cov superoxide anion tau nce hauv pawg tswj hwm (stimulated AGEs), tab sis kev tsim cov superoxide anion poob qis tom qab pawg tswj hwm (stimulated AGEs), tab sis kev tsim cov superoxide anion txo qis tom qab superoxide ntau lawm thiab tiv thaiv. cov hlwb los ntawm kev puas tsuaj oxidative. Nyob rau hauv cov ntaub ntawv ntawm LC-540 hlwb, 10 µM ECH qhia yuav luag zoo ib yam li cov pab pawg neeg. Kev tsim cov superoxide hauv ob lub hlwb tau txo qis nrog kev nce ntxiv ntawm ECH thiab RES.

NATURAL CISTANCHE TUBULOSA rau kev txhim kho kev sib deev PHGS75% ECH 30% ACT 12%

3.1.4. Qhov cuam tshuam ntawm ECH ntawm H2O2 Ntau Lawm hauv AGE-Stimulated LC-540 Leydig Cells

DCFH-DA kev soj ntsuam tau raug coj los kuaj pom muaj cov kab mob oxidative. Tom qab nkag mus rau hauv lub hlwb, fluorescent zas DCFH-DA yog oxidized los ntawm intracellular H2O2 thiab cov ntaub ntawv dichlorofluorescein (DCF). Raws li pom nyob rau hauv daim duab 4, muaj ib tug tseem ceeb nce nyob rau hauv intracellular H2O2 ntau lawm nyob rau hauv lub AGE-stimulated pab pawg neeg (tswj) whereas ntxiv ntawm 10 µM ECH thiab RES txo H2O2 ntau lawm hauv hlwb. Kev tswj hwm ntawm 10 µM ECH ua rau tsuas yog kwv yees li 47.1% ntawm H2O2 ntau lawm, qis dua hauv pawg tswj hwm.

3.1.5. Qhov cuam tshuam ntawm ECH ntawm RAGE thiab NF-κB Protein Expression Levels hauv AGE-Stimulated LC-540 Leydig Cells

Western blot tsom xam tau ua kom paub meej tias muaj RAGE hauv LC-540 Leydig hlwb. Cov nyhuv ntawm ECH ntawm RAGE (Daim duab 5a) thiab NF-κB (Daim duab 5b) protein qhia theem hauv AGEs-stimulated Leydig hlwb tau pom hauv daim duab 5. Cov txiaj ntsig tau pom tias 50 µg / mL concentration ntawm AGEs induced RAGE thiab NF- κB qhia hauv LC-540 Leydig hlwb thiab 10 µM concentration ntawm ECH thiab RES txo qhov kev qhia ntawm RAGE thiab NF-κB. Qhov kev qhia ntawm NF-κB tau qis dua hauv RES thiab ECH dua li hauv RAGE antagonists. Yog li, cov txiaj ntsig tau lees paub tias ECH txo qis qhov mob los ntawm kev txo qis RAGE thiab NF-κB.

3.1.6. Cov nyhuv ntawm ECH ntawm Testosterone Synthesis txoj hauv kev AGE-Stimulated LC-540 Leydig hlwb.

Cov txheej txheem ntawm spermatogenesis thiab txiv neej infertility nyob ntawm seb muaj testosterone. Raws li pom hauv daim duab 6, cov kab lus ntawm StAR (Daim duab 6a), CYP11A1 (Daim duab 6b), CYP17A1 (Daim duab 6c), thiab HSD17 3 (Daim duab 6d) cov protein tau txo qis hauv AGE-stimulated LC{{11 }} Leydig hlwb (tswj pab pawg). Cov kab lus ntawm Star, CYP11A1, CYP17A1, thiab HSD{16}} cov proteins tau nce ntxiv thaum RAGE antagonist, RES, thiab ECH tau ntxiv. Kev nthuav qhia ntau ntxiv ntawm Star thiab CYP11A1 proteins tau pom nyob rau hauv ob pawg ECH- thiab RES-kho. Kev qhia theem ntawm CYP17A1 yuav luag zoo sib xws hauv pawg RES thiab ECH. Cov kab lus ntawm HSD17 3 protein nyob rau hauv ECH-kho Leydig hlwb tau ntau dua li cov RES thiab RAGE antagonist-kho hlwb. Kev nthuav qhia ntau ntxiv ntawm Star, CYP11A1, CYP17A1, thiab HSD{30}} cov proteins qhia txog kev tsim tawm ntawm testosterone.