Amavuta meza ya Saposhnikovia divaricata ya buji nisabune akora amavuta menshi ya diffuser amavuta yingenzi kubitsa urubingo.

Amavuta meza ya Saposhnikovia amavuta ya divaricata ya buji nisabune akora amavuta menshi ya diffuzeri yamavuta mashya kubitwikwa byo gutwika urubingo.

ibisobanuro bigufi:

2.1. Gutegura SDE

Inkeri ya SD yaguzwe nk'icyatsi cyumye muri Hanherb Co (Guri, Koreya). Ibikoresho by'ibihingwa byemejwe na tagisi na Dr. Go-Ya Choi wo mu kigo cya Koreya gishinzwe ubuvuzi bw’iburasirazuba (KIOM). Icyitegererezo cyerekana inomero (nimero 2014 SDE-6) yashyizwe muri Herbarium yo muri Koreya y’umutungo kamere w’ibimera. Inkeri zumye za SD (320 g) zavanywemo kabiri hamwe na 70% Ethanol (hamwe na 2 h yagarutse) hanyuma ibiyikuramo noneho byibanda kumuvuduko muke. Decoction yarayungurujwe, lyofilize, ibikwa kuri 4 ° C. Umusaruro wibikoresho byumye biva mubikoresho bitangiye byari 48.13% (w / w).

2.2. Isesengura ryinshi-Imikorere ya Liquid Chromatografiya (HPLC) Isesengura

Isesengura rya Chromatografique ryakozwe hamwe na sisitemu ya HPLC (Amazi Co, Milford, MA, USA) hamwe na deteri ya Photodiode. Kubisesengura HPLC ya SDE, prima-O-glucosylcimifugin yaguzwe mu kigo cya Koreya gishinzwe guteza imbere inganda z’ubuvuzi gakondo (Gyeongsan, Koreya), nasec-O-glucosylhamaudol na 4′-O-β-D-glucosyl-5-O-methylvisamminol yitaruye muri laboratoire yacu kandi igaragazwa nisesengura ryerekanwa, cyane cyane NMR na MS.

Ingero za SDE (0.1 mg) zashongeshejwe muri 70% Ethanol (10 mL). Gutandukanya Chromatografique byakozwe hamwe na XSelect HSS T3 C18 inkingi (4,6 × 250 mm, 5μm, Amazi Co, Milford, MA, Amerika). Icyiciro kigendanwa cyari kigizwe na acetonitrile (A) na 0.1% acide acetike mumazi (B) kumuvuduko wa 1.0 mL / min. Porogaramu ya Multistep gradient yakoreshejwe kuburyo bukurikira: 5% A (0 min), 5-20% A (0-10 min), 20% A (10-23 min), na 20-65% A (23-40 min) ). Uburebure bwumurambararo bwerekanwe kuri 210-400 nm hanyuma bwandikwa kuri 254 nm. Ingano yo gutera inshinge yari 10.0μL. Ibisubizo bisanzwe byo kumenya chromone eshatu byateguwe kumurongo wanyuma wa 7,781 mg / mL (prim-O-glucosylcimifugin), 31.125 mg / mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), na 31.125 mg / mL (sec-O-glucosylhamaudol) muri methanol kandi ikabikwa kuri 4 ° C.

2.3. Isuzuma ryibikorwa byo kurwanya ibicanwaMuri Vitro

2.3.1. Umuco w'akagari no kuvura icyitegererezo

RAW 264.7 ingirabuzimafatizo zabonetse mu cyegeranyo cy’umuco w’Abanyamerika (ATCC, Manassas, VA, Amerika) kandi gikura mu buryo bwa DMEM burimo antibiyotike 1% na 5.5% FBS. Ingirabuzimafatizo zashyizwe mu kirere cyuzuye cya 5% CO2 kuri 37 ° C. Mu gukangura ingirabuzimafatizo, uburyo bwasimbujwe uburyo bushya bwa DMEM, na lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co, St. Louis, MO, USA) kuri 1μg / mL yongeyeho SDE ihari cyangwa idahari (200 cyangwa 400μg / mL) hiyongereyeho 24 h.

2.3.2. Kumenya Oxide ya Nitric (OYA), Prostaglandine E2 (PGE2), Tumor Necrosis Factor-α(TNF-α), na Interleukin-6 (IL-6) Umusaruro

Ingirabuzimafatizo zavuwe na SDE kandi zikangurwa na LPS kuri 24 h. NTA musaruro wasesenguwe no gupima nitrite ukoresheje reagent ya Griess ukurikije ubushakashatsi bwabanje [12]. Ibanga rya cytokine ikongora PGE2, TNF-α, na IL-6 byariyemeje gukoresha ibikoresho bya ELISA (sisitemu ya R&D) ukurikije amabwiriza yabakozwe. Ingaruka za SDE kuri OYA na cytokine byakozwe kuri 540 nm cyangwa 450 nm ukoresheje Wallac EnVision™umusomyi wa microplate (PerkinElmer).

2.4. Isuzuma ryibikorwa bya AntiosteoarthritisMuri Vivo

2.4.1. Inyamaswa

Imbeba z'umugabo Sprague-Dawley (zifite ibyumweru 7) zaguzwe na Samtako Inc. (Osan, Koreya) kandi zicumbikirwa mubihe byagenzuwe hamwe na 12-h yumucyo / umwijima kuri° C naUbushuhe. Imbeba zahawe indyo ya laboratoire n'amaziad libitum. Inzira zose zigeragezwa zakozwe hubahirijwe amabwiriza y’ikigo cy’igihugu cy’ubuzima (NIH) kandi zemezwa na komite ishinzwe kwita ku nyamaswa n’imikoreshereze ya kaminuza ya Daejeon (Daejeon, repubulika ya Koreya).

2.4.2. Kwinjiza OA hamwe na MIA mu mbeba

Inyamaswa zatoranijwe kandi zishyirwa mumatsinda yo kuvura mbere yo gutangira ubushakashatsi (kuri buri tsinda). Igisubizo cya MIA (3 mg / 50μL ya 0,9% saline) yatewe mu buryo butaziguye mu mwanya w’imbere y’amavi y’iburyo munsi ya anesthesia yatewe no kuvanga ketamine na xylazine. Imbeba zagabanyijwemo amatsinda ane: ) indomethacine- (IM-) itsinda ryavuwe (2 mg / kg) hamwe no guterwa MIA. Imbeba zatanzwe mu kanwa hamwe na SDE na IM icyumweru 1 mbere yo guterwa MIA ibyumweru 4. Igipimo cya SDE na IM cyakoreshejwe muri ubu bushakashatsi cyari gishingiye ku bakoreshejwe mu bushakashatsi bwabanje [10,13,14].

2.4.3. Ibipimo bya Hindpaw Gukwirakwiza Ibiro

Nyuma yo kwinjizwa kwa OA, impirimbanyi yumwimerere mubushobozi bwo gutwara ibiro bya hindpaws yarahungabanye. Ikizamini kidafite ubushobozi (ibikoresho bya Linton, Norfolk, UK) byakoreshejwe mugusuzuma impinduka zo kwihanganira ibiro. Imbeba zashyizwe mubyumba byapimwe. Imbaraga zo gutwara ibiro zashyizwe mu gihimba cy'inyuma zagereranijwe mu gihe cya 3 s. Ikigereranyo cyo kugabana ibiro cyabazwe nuburinganire bukurikira:15].

2.4.4. Ibipimo bya Serumu Cytokine Urwego

Ingero zamaraso zashyizwe kuri g 1.500 kuminota 10 kuri 4 ° C; noneho serumu yakusanyirijwe ikabikwa kuri −70 ° C kugeza ikoreshejwe. Urwego rwa IL-1β, IL-6, TNF-α, na PGE2 muri serumu byapimwe hifashishijwe ibikoresho bya ELISA biva muri R&D Sisitemu (Minneapolis, MN, USA) ukurikije amabwiriza yabakozwe.

2.4.5. Isesengura-Igihe Cyuzuye RT-PCR Isesengura

RNA yose yakuwe mu ngingo zifatanije zivi ikivi ikoresheje TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), ihindurwa-yandikwa muri cDNA na PCR-yongerewe hakoreshejwe ibikoresho bya TM One Step RT PCR hamwe nicyatsi cya SYBR (Ikoreshwa rya Biosystems , Ikirwa Kinini, NY, Amerika). PCR nyayo-nyayo yakozwe hakoreshejwe Sisitemu ya Biosystems 7500 Real-Time PCR (Ikoreshwa rya Biosystems, Grand Island, NY, USA). Urutonde rwa primer hamwe na probe-ikurikiranye byerekanwe kumeza1. Aliquots z'icyitegererezo cDNAs hamwe na GAPDH cDNA zingana zingana na TaqMan® Universal PCR ivanze irimo ADN polymerase ukurikije amabwiriza yabakozwe (Applied Biosystems, Foster, CA, USA). Imiterere ya PCR yari min 2 kuri 50 ° C, min 10 kuri 94 ° C, 15 s kuri 95 ° C, na min 1 kuri 60 ° C kuri cycle 40. Ubwinshi bwa gene yintego bwagenwe hakoreshejwe igereranya rya Ct (umubare wikigereranyo cyumubare wambukiranya imipaka hagati yuburyo bwongerewe imbaraga), ukurikije amabwiriza yabakozwe.

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Ubushobozi bwo gutanga:10000 Igice / Ibice buri kwezi

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Ibicuruzwa birambuye

Ibicuruzwa

Osteoarthritis (OA) ni indwara ya musculoskeletal ikunze kugaragara kandi ni indwara ikunze kwibasira abasaza [1]. OA ni indwara iterwa igice kimwe no gukomeretsa, gutakaza imiterere n'imikorere ya karitsiye, hamwe no kugabanya inzira ya proinflammatory na anti-inflammatory [2,3]. Ifata cyane cyane karitsiye ya articular hamwe namagufa ya subchondral yamagambo yingingo ya synovial kandi bikaviramo kunanirwa hamwe, biganisha kububabare bwo kwikorera ibiro harimo kugenda no guhagarara [4].

Nta muti wa OA, kuko biragoye cyane kugarura karitsiye imaze gusenywa [5]. Intego zo kuvura nugukuraho ububabare, kubungabunga cyangwa kunoza urujya n'uruza, kongera imbaraga zingingo, no kugabanya ingaruka mbi zindwara. Ubuvuzi bwa farumasi bwa OA bugamije kugabanya ububabare hagamijwe kongera imikorere yumurwayi hamwe nubuzima bwiza. Nubwo gusenya karitsiye aricyo kintu cyingenzi muri OA, iyangirika rya kolagen nicyo kintu cyibanze kigaragaza iterambere ridasubirwaho rya OA ifatanije n’umuriro [6,7]. Ubuvuzi hamwe nibikorwa byo kurwanya inflammatory na chondroprotective byitezwe kugabanya ububabare no gukomeza uburinganire bwa matrix kubarwayi ba OA.

Kubwibyo, kugabanya umuriro bishobora kuba ingirakamaro mubuyobozi bwa OA. Ubushakashatsi buherutse gukorwa bwerekana uruhare rukingira umutungo w’ibimera ku iterambere rya OA, mu rwego rwo kugabanya uburibwe bwa chondrocyte no kurimbuka kwa karitsiye, binyuze mu bushobozi bwabo bwo gukorana n’imitsi ifitanye isano, bikaviramo kugabanya ububabare bufatanije [8].

Umuzi waSaposhnikovia divaricataSchischkin (Umbelliferae) yakoreshejwe cyane mu buvuzi gakondo mu kuvura umutwe, ububabare, uburibwe, na rubagimpande muri Koreya no mu Bushinwa [9,10]. Ingaruka zitandukanye za farumasi yaSaposhnikovia divaricata(SD) ikubiyemo kandi anti-inflammatory, analgesic, antipyretic, na antiarthritic [9,11]. Ubushakashatsi buherutse gukorwa bwerekanye ko SD chromone ikuramo ingaruka ziterwa na antirheumatoide arthritis muburyo bwimbeba ya kolagen iterwa na artrite.10]; icyakora, ubushakashatsi buke bwakozwe kugirango bunganire ibikorwa byo kurwanya inflammatory na antiarthritis yaSaposhnikovia divaricatagukuramo (SDE).

Kubwibyo, ubu bushakashatsi bwakoze iperereza ku bikorwa byo kurwanya inflammatory na antiosteoarthritis ya 70% ya Ethanol ya SD. Ubwa mbere, hasuzumwe ingaruka zo kurwanya inflammatory ya SDEmuri vitromuri LPS iterwa na RAW 264.7. Ibikurikira, ingaruka zo kurwanya antosteoarthritis ya SDE zapimwe mugusuzuma ikwirakwizwa ryikiremereye, iyangirika rya karitsiye ya articular, hamwe nigisubizo cyakongejwe muburyo bwimbeba ya monosodium iodoacetate- (MIA-) yateje OA.