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2.1. Kev npaj SDE

Cov rhizomes ntawm SD tau yuav los ua tshuaj ntsuab qhuav los ntawm Hanherb Co. (Guri, Kauslim). Cov ntaub ntawv cog qoob loo tau lees paub taxonomically los ntawm Dr. Go-Ya Choi ntawm Kaus Lim Kauslim Lub Tsev Haujlwm Saib Xyuas Tshuaj Oriental (KIOM). Ib daim voucher specimen (tus naj npawb 2014 SDE-6) tau muab tso rau hauv Korean Herbarium ntawm Standard Herbal Resources. Qhuav rhizomes ntawm SD (320 g) tau muab rho tawm ob zaug nrog 70% ethanol (nrog rau 2 h reflux) thiab cov extract yog ces concentrated nyob rau hauv txo siab. Lub decoction tau lim, lyophilized, thiab khaws cia ntawm 4 ° C. Cov txiaj ntsig ntawm qhuav extract los ntawm cov ntaub ntawv nyoos pib yog 48.13% (w / w).

 

2.2. Quantitative High-Performance Liquid Chromatography (HPLC) Kev Ntsuam Xyuas

Kev soj ntsuam chromatographic tau ua nrog HPLC system (Waters Co., Milford, MA, USA) thiab lub ntsuas hluav taws xob photodiode array. Rau HPLC tsom xam ntawm SDE, qhov tseem ceeb-O-glucosylcimifugin tus qauv tau yuav los ntawm Kaus Lim Kauslim Lub Tsev Haujlwm Saib Xyuas Kev Lag Luam Kev Lag Luam (Gyeongsan, Kaus Lim Qab Teb), thiabsec-O-glucosylhamaudol thiab 4'-O-βD-glucosyl-5-O-methylvisamminol raug cais tawm hauv peb lub chaw kuaj mob thiab txheeb xyuas los ntawm kev tshuaj ntsuam spectral, feem ntau los ntawm NMR thiab MS.

Cov qauv SDE (0.1 mg) tau yaj hauv 70% ethanol (10 mL). Chromatographic sib cais tau ua nrog XSelect HSS T3 C18 kem (4.6 × 250 mm, 5μm, Waters Co., Milford, MA, USA). Cov theem txawb muaj xws li acetonitrile (A) thiab 0.1% acetic acid hauv dej (B) ntawm tus nqi ntws ntawm 1.0 mL / min. Qhov kev pab cuam gradient multistep tau siv raws li hauv qab no: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), thiab 20–65% A (23–40 min. ). Kev tshawb nrhiav wavelength tau luam tawm ntawm 210-400 nm thiab kaw ntawm 254 nm. Kev txhaj tshuaj yog 10.0μL. Cov qauv kev daws teeb meem rau kev txiav txim siab ntawm peb chromones tau npaj ntawm qhov kawg siab ntawm 7.781 mg / mL (prim-O-glucosylcimifugin), 31.125 mg / mL (4'-O-βD-glucosyl-5-O-methylvisamminol), thiab 31.125 mg / mL (sec-O-glucosylhamaudol) hauv methanol thiab khaws cia ntawm 4 ° C.

2.3. Kev ntsuam xyuas ntawm Kev Tiv Thaiv Kab MobHauv Vitro

2.3.1. Cell Kab lis kev cai thiab qauv kev kho mob

RAW 264.7 hlwb tau txais los ntawm American Type Culture Collection (ATCC, Manassas, VA, USA) thiab loj hlob hauv DMEM nruab nrab uas muaj 1% tshuaj tua kab mob thiab 5.5% FBS. Cells raug incubated nyob rau hauv ib tug humidified huab cua ntawm 5% CO2 ntawm 37 ° C. Txhawm rau txhawb cov hlwb, qhov nruab nrab tau hloov nrog DMEM tshiab nruab nrab, thiab lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) ntawm 1μg/mL tau ntxiv rau qhov muaj lossis tsis muaj SDE (200 lossis 400μg / mL) ntxiv rau 24 teev.

2.3.2. Kev txiav txim siab ntawm Nitric Oxide (NO), Prostaglandin E2 (PGE2), Tumor Necrosis Factor-α(TNF-α), thiab Interleukin-6 (IL-6) Production

Cov hlwb raug kho nrog SDE thiab txhawb nqa nrog LPS rau 24 teev. TSIS MUAJ ntau lawm tau soj ntsuam los ntawm kev ntsuas nitrite siv Griess reagent raws li kev tshawb fawb yav dhau los [12]. Kev tso tawm ntawm inflammatory cytokines PGE2, TNF-α, thiab IL-6 tau txiav txim siab siv cov khoom siv ELISA (R&D systems) raws li cov lus qhia ntawm cov chaw tsim khoom. Cov teebmeem ntawm SDE ntawm TSIS MUAJ thiab cytokine ntau lawm tau txiav txim siab ntawm 540 nm lossis 450 nm siv Wallac EnVision™Microplate nyeem ntawv (PerkinElmer).

2.4. Kev ntsuam xyuas ntawm Antiosteoarthritis ActivityHauv Vivo

2.4.1. Tsiaj

Txiv neej Sprague-Dawley nas (7 lub lis piam) tau yuav los ntawm Samtako Inc. (Osan, Kaus Lim Kauslim) thiab nyob hauv kev tswj xyuas nrog 12-h lub teeb / tsaus voj voog ntawm°C thiab% av noo. Cov nas tsuag tau muab cov zaub mov thiab dejad libitum. Tag nrho cov txheej txheem sim tau ua raws li National Institutes of Health (NIH) cov lus qhia thiab pom zoo los ntawm Pawg Saib Xyuas Tsiaj thiab Siv Khoom ntawm Daejeon University (Daejeon, Republic of Kauslim).

2.4.2. Induction ntawm OA nrog MIA hauv nas

Cov tsiaj tau muab faib ua randomized thiab muab rau cov pab pawg kho mob ua ntej pib qhov kev kawm (ib pab pawg). MIA daws (3 mg/50μL ntawm 0.9% saline) tau txhaj ncaj qha rau hauv qhov chaw intra-articular ntawm lub hauv caug sab xis hauv qab tshuaj loog induced nrog kev sib xyaw ntawm ketamine thiab xylazine. Cov nas tsuag tau muab faib ua plaub pawg: (1) pab pawg saline uas tsis muaj kev txhaj tshuaj MIA, (2) pab pawg MIA nrog kev txhaj tshuaj MIA, (3) pab pawg kho SDE (200 mg / kg) nrog kev txhaj tshuaj MIA, thiab (4) ) cov pab pawg kho mob indomethacin- (IM-) (2 mg / kg) nrog kev txhaj tshuaj MIA. Cov nas tsuag tau muab qhov ncauj nrog SDE thiab IM 1 lub lis piam ua ntej MIA txhaj rau 4 lub lis piam. Qhov ntau npaum li cas ntawm SDE thiab IM siv nyob rau hauv txoj kev tshawb no yog raws li cov neeg ua hauj lwm hauv cov kev tshawb fawb yav dhau los [10,13,14].

2.4.3. Kev ntsuas ntawm Hindpaw Weight-Bearing Distribution

Tom qab OA induction, thawj qhov sib npaug hauv qhov hnyav-kev muaj peev xwm ntawm hindpaws raug cuam tshuam. Tus ntsuas ntsuas tsis muaj peev xwm (Linton instrumentation, Norfolk, UK) tau siv los ntsuas qhov kev hloov pauv ntawm qhov hnyav-kev kam rau siab. Cov nas tau ua tib zoo muab tso rau hauv chav ntsuas ntsuas. Qhov hnyav-kev quab yuam tawm los ntawm lub hind limb yog nruab nrab ntawm 3 s lub sijhawm. Qhov hnyav faib piv tau suav los ntawm qhov sib npaug hauv qab no: [qhov hnyav ntawm sab xis hind limb / (qhov hnyav ntawm sab xis hind limb + qhov hnyav ntawm sab laug hind limb)] × 100 [15].

2.4.4. Kev ntsuas ntawm Serum Cytokine Levels

Cov ntshav kuaj tau centrifuged ntawm 1,500 g rau 10 min ntawm 4 ° C; Tom qab ntawd cov ntshav tau sau thiab khaws cia ntawm -70 ° C kom txog thaum siv. Qib IL-1β, IL-6, TNF-α, thiab PGE2 hauv cov ntshav tau ntsuas siv ELISA cov khoom siv los ntawm R&D Systems (Minneapolis, MN, USA) raws li cov lus qhia ntawm cov chaw tsim khoom.

2.4.5 ib. Real-Time Quantitative RT-PCR Analysis

Tag Nrho RNA tau muab rho tawm los ntawm lub hauv caug cov ntaub so ntswg siv TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), thim rov qab mus rau cDNA thiab PCR-amplified siv TM Ib Kauj Ruam RT PCR cov khoom siv nrog SYBR ntsuab (Applied Biosystems , Grand Island, NY, USA). Real-time quantitative PCR tau ua tiav siv Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Grand Island, NY, USA). Cov primer sequences thiab probe-sequence yog qhia nyob rau hauv Table1. Aliquots ntawm cov qauv cDNAs thiab qhov sib npaug ntawm GAPDH cDNA tau nthuav dav nrog TaqMan® Universal PCR tus tswv sib xyaw uas muaj DNA polymerase raws li cov lus qhia chaw tsim khoom (Applied Biosystems, Foster, CA, USA). PCR cov xwm txheej yog 2 min ntawm 50 ° C, 10 min ntawm 94 ° C, 15 s ntawm 95 ° C, thiab 1 min ntawm 60 ° C rau 40 cycles. Qhov siab ntawm lub hom phiaj noob tau txiav txim siab siv qhov sib piv Ct (threshold cycle number ntawm cross-point of amplification plot and threshold), raws li cov chaw tsim khoom cov lus qhia.

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