Ntu 1: Acteoside Counteracts Interleukin-1 -Induced Catabolic Processes Los ntawm Kev Hloov Kho Ntawm Mitogen-Activated Protein Kinases Thiab NFκB Cellular Signaling Pathway

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

Hyang I Lim , 1 Do Kyung Kim , 1 Tae-Hyeon Kim , 1 Kyeong-Rok Kang

Osteoarthritis (OA) yog cov kab mob uas feem ntau degenerative sib koom ua ke nrog mob pob qij txha uas tshwm sim los ntawm kev loj hlob ntawm cov pob txha mos ntawm cov pob qij txha ntawm cov pob qij txha.Acteoside, caffeoylphenylethanoid glycoside, muaj ntau yam kev ua ub no xws li tshuaj tua kab mob, tshuaj tiv thaiv kab mob, tshuaj tiv thaiv kab mob, tshuaj tiv thaiv oxidative, cytoprotective, thiab neuroprotective effective. Ntxiv mus, qhov ncauj tswj hwm ntawmacteosidentawm qhov ntau npaum li cas tsis ua rau genotoxicity. Yog li ntawd, lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog txhawm rau txheeb xyuas cov txiaj ntsig ntawm anticatabolicacteosidetiv thaiv osteoarthritis thiab nws cov anticatabolic signaling txoj kev.Acteosidetsis txo qis qhov ua tau zoo ntawm nas fibroblast L929 hlwb siv los ua cov hlwb qub thiab thawj cov nas chondrocytes.Acteosidecounteracted IL-1 -induced proteoglycan poob nyob rau hauv chondrocytes thiab pob txha pob txha pob txha los ntawm suppressing qhia thiab ua kom cov pob txha mos-degrading enzyme xws li matrix metalloproteinase- (MMP-) 13, MMP-1, thiab MMP{ {6}}. Tsis tas li ntawd, acteoside inhibited qhov kev qhia ntawm inflammatory mediators xws li inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, thiab prostaglandin E2 nyob rau hauv thawj nas chondrocytes kho nrog IL-1 . Tom qab ntawd, kev qhia ntawm proinflammatory cytokines tau txo qis los ntawm acteoside hauv thawj nas chondrocytes kho nrog IL-1 . Ntxiv mus, acteoside suppressed tsis yog tsuas yog cov phosphorylation ntawm mitogen-activated protein kinases nyob rau hauv thawj nas chondrocytes kho nrog IL-1 tab sis kuj translocation ntawm NFκB los ntawm lub cytosol mus rau lub nucleus los ntawm suppression ntawm nws phosphorylation. Kev tswj hwm qhov ncauj ntawm 5 thiab 10mg / kg acteoside txo qis kev loj hlob ntawm cov pob txha pob txha hauv cov qauv nas osteoarthritic tsim los ntawm destabilization ntawm medial meniscus. Peb cov kev tshawb fawb qhia tiasacteosideyog ib qho kev cog lus muaj peev xwm anticatabolic tus neeg sawv cev los yog ntxiv rau attenuate los yog tiv thaiv kev loj hlob degeneration ntawm articular pob txha mos.

cistanche-acteoside

1. Taw qhia

Osteoarthritis (OA) yog cov kab mob uas feem ntau degenerative sib koom ua ke nrog mob pob qij txha uas tshwm sim los ntawm kev loj hlob ntawm pob txha pob txha pob txha ntawm cov pob qij txha [1]. Vim qhov nce hauv kev cia siab ntawm lub neej, qhov tshwm sim ntawm OA nrog kev poob ntawm kev txav mus los thiab mob pob qij txha ntev los ntawm cov pob txha mos ntawm cov pob txha mos ntawm cov pob qij txha tau kwv yees li ntawm 18 feem pua thiab 9.6 feem pua ntawm cov poj niam tom qab cev xeeb tub thiab hauv cov txiv neej, feem. ]. Txawm hais tias 2 Oxidative Medicine thiab Cellular Longevity thoob ntiaj teb kev nthuav dav ntawm OA nce txhua xyoo, cov kab mob pathophysiological ntawm OA tseem tsis tau paub. Nws tuaj yeem tshwm sim los ntawm qhov nyuaj heev thiab ntau yam kev pheej hmoo xws li kev laus, poj niam los txiv neej, kev xeeb tub xeeb ntxwv, kev raug mob sib koom tes, thiab kev sib koom ua ke hnyav. Tsis tas li ntawd, kev sib raug zoo ntawm neuro-pathological ntawm kev loj hlob degeneration ntawm pob txha pob txha thiab kev loj hlob ntawm kev mob sib koom tes tsis paub [3]. Yog li, lub hom phiaj ntawm kev tswj xyuas kev kho mob rau cov neeg mob OA yog kev saib xyuas lub cev txav mus los thiab kev ua haujlwm sib koom ua ke los ntawm kev kho mob los ntawm kev mob sib koom tes, siv cov tshuaj pharmacological thiab nonpharmacological thiab kev sib koom tes hloov pauv. Qhov kev thov rau kev txhim kho cov kev cuam tshuam zoo lossis kev pabcuam ntxiv, nrog rau kev nyab xeeb mus sij hawm ntev, los tiv thaiv lossis txo qis OA kom tswj tau lub neej zoo los ntawm kev saib xyuas cov neeg kho tshuab kev sib koom ua haujlwm hauv cov neeg laus yog nce.

Raws li pom nyob rau hauv daim duab 1, acteoside (CAS No. 61276-17-3; C29H36O15) yog ib qho caféoylphenylethanoid glycoside cais los ntawm ntau hom tshuaj ntsuab xws li Verbascum chlorides [4], Buddleja globosa [5], thiab Plantago australis [6]. Acteoside muaj ntau yam kev ua haujlwm lom neeg xws li tshuaj tua kab mob [5], tshuaj tiv thaiv kab mob [7], tshuaj tiv thaiv kab mob [8], tshuaj tiv thaiv kab mob [9], cytoprotective [9], thiab neuroprotective effect [10]. Tsis tas li ntawd, qhov ncauj kev tswj hwm ntawm acteoside ntawm ntau npaum li cas tsis ua rau genotoxicity [11].

Yog li, peb xav tias acteoside nrog kev tiv thaiv kab mob lom neeg muaj kev nyab xeeb muaj cov nyhuv anticatabolic cuam tshuam nrog kev tiv thaiv pob txha pob txha tiv thaiv kev loj hlob ntawm cov pob txha mos los ntawm kev tawm tsam ntawm cov kab mob catabolic xws li cov proinflammatory cytokines, inflammatory enzymes sib koom ua ke ntawm cartilage, . Yog li ntawd, lub hom phiaj ntawm txoj kev tshawb no yog los soj ntsuam cov acteoside-induced anticatabolic cov teebmeem thiab nws cellular signaling txoj kev nyob rau hauv vitro, siv thawj chondrocytes cais los ntawm articular pob txha mos ntawm nas lub hauv caug sib koom, thiab nyob rau hauv vivo, siv ib tug OA tsiaj qauv tsim los ntawm kev phais. destabilization ntawm medial meniscus nyob rau hauv lub hauv caug sib koom ntawm nas.

cistanche-acteoside

acteoside los ntawmcistanche

2. Cov txheej txheem

2.1. Cell Culture. Thawj cov nas chondrocytes raug cais tawm ntawm cov pob txha mos ntawm cov nas (5-hnub nyoog; Sprague–Daw-ley) pob qij txha hauv caug, raws li txoj cai (CIA- CUC2019-A0027) pom zoo los ntawm Lub Tsev Haujlwm Tsiaj Saib Xyuas thiab Siv Pawg ntawm Chosun University, Gwangju, Republic of Kauslim. cais thawj nas chondrocytes tau khaws cia hauv Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F12) (Thermo Scientific, Rock-ford, IL, USA) supplemented with 10 feem pua fetal bovine serum (FBSlogies) , Grand Island, NY, USA), tshuaj tua kab mob (50U/mL penicillin thiab 50ug/mL streptomycin), thiab 50ug/mL ascorbic acid. Cov nas ib txwm muaj fibroblast L-929 xov tooj ntawm tes tau yuav los ntawm American Type Culture Collection (ATCC). Raws li cov lus qhia tau muab los ntawm ATCC, L-929 cov hlwb tau coj los ua kab ke hauv Eagle qhov nruab nrab qhov tseem ceeb yam tsawg kawg nkaus, muaj 10 feem pua FBS, thiab tau loj hlob hauv qhov chaw humidified ntawm 37 degree nrog 5 feem pua CO2.

2.2. Cell Viability Assay. Qhov kev ntsuam xyuas dimethyl thiazolyl diphenyltetrazolium ntsev (MTT) tau ua los ntsuas qhov ua tau zoo ntawm nas fibroblast cell kab L929 hlwb siv los ua ib lub xov tooj ntawm tes thiab thawj nas chondrocytes kho nrog acteoside. Luv luv, L929 hlwb, thiab thawj cov nas chondrocytes tau coj los ntawm cov cell ceev ntawm 8 × 105 hlwb / mL hauv kab lis kev cai daim hlau rau 24h thiab tom qab ntawd kho nrog 2.5, 5, 10, 25, 50, thiab 100μM acteoside rau 24h. Tom qab kev kho mob nrog MTT tov, ob qho tib si L929 hlwb thiab chondrocytes tau txuas ntxiv mus rau 4h. Tom qab incubation, tsim MTT crystals raug tshem tawm tag nrho nyob rau hauv dimethyl sulfoxide thiab ntsuas rau absorbance ntawm 570nm siv lub spectrometer (Epoch microplate spectrophotometer, BioTek®, Winooski, VT, USA) los soj ntsuam cell viability.

2.3. Cell Live/Dead Assay. Cell ciaj sia taus yog ua los ntawm Cell Live/Dead assay kit (Molecular Probes, Carlsbad, CA, USA), uas yog tsim los ntawm ntsuab calcein-AM rau kev sau cov hlwb nyob (nrog ntsuab fluorescence) thiab ethidium homodimer-1 rau kev sau ntawv tuag cell (nrog liab fluorescence). Luv luv, L929 hlwb thiab thawj cov nas chondrocytes tau coj los ntawm cov cell ceev ntawm 8 × 105 hlwb / mL ntawm cov kab swb (Nunc® Lab-Tek® Chamber Slide™ system; Sigma- Aldrich; Merck KGaA) rau 24h thiab tom qab ntawd kho nrog 50 thiab 100μM acteoside rau 24h. Tom qab cog, ib qho kev ntsuam xyuas cell ciaj sia tau ua raws li cov neeg tsim khoom cov lus qhia. Tom qab ntawd, cov kab mob stained tau ua duab siv lub tshuab ntsuas fluorescence (Eclipse TE200; Nikon Instruments, Melville, NY).

2.4. Dimethylmethylene Blue (DMMB) Kev Ntsuam Xyuas. DMMB kev soj ntsuam tau ua los ntsuas qhov kev hloov pauv ntawm cov ntsiab lus proteoglycan hauv thawj nas chondrocytes kho nrog acteoside rau 21 hnub nyob rau hauv lub xub ntiag los yog tsis muaj IL-1 . Txhawm rau tswj hwm tus yam ntxwv ntawm thawj cov nas chondrocytes rau 21 hnub, thawj cov nas chondrocytes (2 × 106 hlwb) raug tshem tawm hauv 1mL ntawm 1.2 feem pua alginate thiab tom qab ntawd muab qhwv los ntawm kev tso tawm ntawm tes / alginate ncua mus rau kev daws ntawm 105mM CaCl2. Thawj nas chondrocytes encapsulated nyob rau hauv alginate yog kab lis kev cai rau 24h nyob rau hauv DMEM / F12 (muaj 10 feem pua FBS, 50U/mL penicillin, 50ug/mL streptomycin, thiab 50ug/mL ascorbic acid) thiab ces hloov rau EMF 24 feem pua. mini-insulin-transferrin-selenium (mini-ITS) thiab 50ug / mL ascorbic acid. Tom qab ntawd, cov chondrocytes tau kho nrog 50 lossis 100μM acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 1ng / mL IL-1 rau 21 hnub. Hnub 21, thawj nas chondrocytes tau sau rau kev soj ntsuam ntawm cov ntsiab lus proteoglycan siv DMMB kev soj ntsuam, raws li tau piav qhia yav dhau los [12]. Tsis tas li ntawd, txhawm rau txheeb xyuas cov ntsiab lus ntawm proteoglycan hauv ib lub xovtooj ntawm tes thiab ntsuas qhov kev loj hlob ntawm thawj tus nas chondrocytes, cov xovtooj ntawm tes tau ntsuas los ntawm DNA kev soj ntsuam siv PicoGreen (Molecular Probes, Carlsbad, CA), raws li cov chaw tsim khoom cov lus qhia.

2.5. Ex Vivo Organ Culture of Rat Articular pob txha mos. Cov ntaub so ntswg pob txha raug cais tawm ntawm lub hauv caug pob qij txha ntawm 5-hnub nyoog Sprague-Dawley nas thiab tom qab ntawd coj mus rau hauv DMEM/F12 ntxiv nrog 10 feem pua FBS. Tom ntej no, lub articular pob txha mos

cov qauv raug kho nrog 100μM acteoside hauvnyob los yog tsis muaj 10ng/mL IL-1 rau 7 hnub. Thaum kawg ntawm lub sijhawm kab lis kev cai, cov qauv tau sau thiab kho hauv 4 feem pua ntawm paraformaldehyde rau 72h rau kev tshuaj xyuas histological.

2.6. Histological Analysis. Kev tshuaj xyuas histological siv safranin-O thiab nrawm ntsuab staining tau ua kom paub tseeb tias qhov poob ntawm proteoglycan hauv pob txha pob txha kho nrog 100μM acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 10ng / mL IL-1 rau 7 hnub. Piv txwv li, cov qauv pob txha pob txha pob txha raug decalcificed nyob rau hauv ethylenediaminetetraacetic acid thiab ces embedded nyob rau hauv paraf. Tom qab ntawd, cov txheej txheem npaj ua ke uas muaj cov pob txha mos pob txha raug txiav mus rau 5μm tuab thiab muab tso rau hauv cov swb. Safranin-O thiab ceev ntsuab staining tau ua tom qab los soj ntsuam kev poob ntawm proteo-glycan hauv cov pob txha pob txha hauv av. Tsis tas li ntawd, hematoxylin thiab eosin staining tau ua los soj ntsuam cov qauv ntawm cov pob txha pob txha.

2.7. Western Blotting. Western blotting tau ua los tshawb xyuas qhov qhia ntawm catabolic yam xws li MMP-13, MMP-1, MMP-3, inducible nitric oxide synthase (iNOS), thiab cyclooxygenase-2 (COX -2) thiab kev hloov pauv ntawm cellular signaling molecules xws li mitogen-activated protein kinases thiab nuclear factor-kappa B (NFκB). Luv luv, nas thawj chondrocytes tau kho nrog 50 lossis 100μM acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 10ng / mL IL-1 rau 24 teev. Tom qab ntawd, nas thawj chondrocytes tau sau los ntawm centrifugation thiab tau lysed siv lysis buffer (Cell Signaling Technology, Danvers, MA, USA) raws li cov chaw tsim khoom cov lus qhia. Tsis tas li ntawd, txhawm rau txheeb xyuas qhov kev hloov pauv hauv nuclear ntawm NFκB, nas thawj chondrocytes tau kho nrog 50 lossis 100μM acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 10ng / mL IL-1 rau 24h. Tom qab ntawd, cytosolic thiab nuclear feem raug muab rho tawm siv NE-PER™ Nuclear thiab Cytoplasmic extraction reagents (Thermo Sci-entific, Rockford, IL, USA) raws li cov chaw tsim khoom cov lus qhia. Qhov concentration ntawm tag nrho cov protein rho tawm los ntawm thawj nas chondrocytes tau txiav txim siab siv cov khoom siv bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL, USA) raws li cov chaw tsim khoom cov lus qhia. Tsis tas li ntawd, qhov nruab nrab nruab nrab tau sau los txheeb xyuas cov qib ntawm pob txha mos-degrading enzymes secreted los ntawm chondrocytes. Qhov sib npaug ntawm cov protein thiab qhov nruab nrab txias tau electrophoresed ntawm sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) thiab tom qab ntawd xa mus rau nitrocellulose daim nyias nyias. Tom qab ntawd, sab hnub poob blotting tau ua tiav siv lub hom phiaj tiv thaiv lub cev tiv thaiv MMP-13, MMP-1, MMP-3, iNOS, COX-2, phosphoERK1/2, tag nrho-ERK1 /2, phosphop38, tag nrho-p38, phosphor JNK, tag nrho JNK, phosphoNFκB, tag nrho NFκB, - actin, thiab lamin B. Immunoreactive bands tau pom los ntawm kev siv cov tshuaj kho kom zoo dua qub (Thermo Sci- entificc, Rockford, IL, USA) raws li cov chaw tsim khoom cov lus qhia thiab tom qab ntawd ua duab los ntawm Microchemi ntaus ntawv (DNR Bioimaging Systems, Jerusalem, Israel).

2.8. Quantitative Polymerase Chain Reaction (qPCR) thiab Quantitative Real-Time PCR (qRT-PCR). Thawj nas chondrocytes tau kho nrog 50 lossis 100μM acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 10ng / mL IL-1 rau 24h. Tom qab ntawd, tag nrho RNA raug cais tawm ntawm thawj nas chondrocytes siv TRIzol reagent (Invitrogen, Carlsbad, CA, USA) raws li cov chaw tsim khoom cov lus qhia. Tag nrho RNA concentration tau ntsuas siv NanoDrop 2000 (Thermo Scientific, Rockford, IL, USA). Txhawm rau tsim cov cDNA, 1ug RNA tau thim rov qab los ntawm kev siv ThermoScript thim rov qab-PCR system (Invitrogen, Carlsbad, CA, USA) raws li cov chaw tsim khoom cov lus qhia. qPCR ntawm cDNA tau ua tiav siv 2 × TOPsimple™ DyeMIX-Taq (Enzynomics, Seoul, Republic of Kauslim) thiab cov primers tshwj xeeb ntawm TaKaRa PCR Thermal Cycler Device (TaKaRa Bio Inc., Shiga, Nyiv). Tom qab ntawd, PCR cov khoom lag luam tau electrophoresed ntawm agarose gel los txiav txim siab theem ntawm cov hom phiaj. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) tau siv los ua kev tswj hwm endogenous. Ntxiv rau, rau qRT-PCR, cDNA tau nthuav dav siv Eco™ Real-Time PCR system (illumine Inc., San Diego, CA, USA). -Actin tau siv los ua kev tswj hwm endogenous. Cov kab ke ntawm cov primers siv nyob rau hauv qPCR thiab qRT-PCR yog cov ntsiab lus nyob rau hauv Tables 1 thiab 2, feem.

cistanche (acteoside) txiaj ntsig: txhim kho kev tiv thaiv

3. Cov txiaj ntsig

3.1. Acteoside tsis cuam tshuam rau L929 Cell thiab Primary Rat Chondrocyte Viability. Tus nas fibroblast cell kab L929 siv raws li cov hlwb ib txwm raug kho nrog 2.5, 5, 10, 25, 50, thiab 100μM acteoside rau 24h. Tom qab ntawd, qhov kev ntsuam xyuas MTT tau ua los ntsuas cytotoxicity ntawm acteoside ntawm L929 hlwb. Raws li pom nyob rau hauv daim duab 2(a), txheeb ze viabilities ntawm L929 hlwb tau txiav txim siab yog 94:8 ± 8 feem pua, 93:6 ± 7 feem pua, 100 ± 5 feem pua, 103:9 ± 5 feem pua, 126:1 ± 8 feem pua. , thiab 122: 7 ± 4 feem pua ntawm 2.5, 5, 10, 25, 50, thiab 100μM acteoside, feem, piv nrog kev tswj hwm (100: 02 ± 3%). Tsis tas li ntawd, txhawm rau txheeb xyuas qhov cytotoxicity ntawm acteoside ntawm thawj nas chondrocytes, MTT kev soj ntsuam tau ua raws li qhia hauv daim duab 2(b). Cov viabilities ntawm thawj nas chondrocytes kho nrog 2.5, 5, 10, 25, 50, thiab 100μM acteoside raug txiav txim siab li 114 ± 4 feem pua, 117: 8 ± 6 feem pua, 123: 9 ± 5 feem pua, 132: 6 ± 4 feem pua, 153: 1 ± 7 feem pua, thiab 142: 1 ± 6 feem pua, feem, piv nrog kev tswj hwm (100: 4 ± 5%). Tsis tas li ntawd, txhawm rau txheeb xyuas qhov cuam tshuam ntawm acteoside ntawm kev muaj peev xwm ntawm L929 hlwb thiab thawj nas chondrocytes, Cell Live/Dead assay tau ua raws li qhia hauv daim duab 2(c). Tus naj npawb ntawm cov hlwb tuag stained li liab fluorescence tsis nce rau L929 hlwb thiab thawj nas chondrocytes kho nrog 50 thiab 100μM acteoside rau 24h. Cov ntaub ntawv no tsis tu ncua qhia tau hais tias kev siv ntau npaum li cas ntawm acteoside tsis cuam tshuam rau kev muaj peev xwm ntawm L929 hlwb thiab thawj nas chondrocytes. Yog li, 50μM acteoside thiab 100μM acteoside, uas yog cov tshuaj tsis muaj tshuaj lom nyob rau hauv ob qho tib si L929 hlwb thiab thawj tus nas chondrocytes, tau siv los txheeb xyuas nws cov tshuaj tiv thaiv catabolic hauv vitro kev tshawb fawb siv thawj nas chondrocytes.

3.2. Acteoside Counteracts IL-1 -Induced Proteoglycan Loss nyob rau hauv Primary Rat Chondrocytes. Thawj nas chondrocytes nyob rau hauv alginate hlaws dai tau kho nrog 50 thiab 100μM acteoside nyob rau hauv lub xub ntiag los yog tsis muaj 1ng / mL IL- 1 rau 21 hnub. Tom qab ntawd, qhov kev ntsuam xyuas DMMB tau ua los ntsuas qhov kev hloov pauv hauv cov ntsiab lus proteoglycan raws li qhia hauv daim duab 3(a). Cov ntsiab lus proteoglycan txheeb ze tau txiav txim siab li 88: 3 ± 18: 1 feem pua thiab 86: 8 ± 16: 3 feem pua hauv thawj nas chondrocytes kho nrog 50 thiab 100μM acteoside, feem, piv nrog kev tswj (103: 8 ± 32: 3%). Txawm hais tias tus txheeb ze cov ntsiab lus proteoglycan tau txo qis los ntawm acteoside, cov txiaj ntsig no tsis tseem ceeb. Txawm li cas los xij, tus txheeb ze cov ntsiab lus proteoglycan txo qis los ntawm 37: 1 ± 14: 7 feem pua hauv thawj nas chondrocytes kho nrog 1ng / mL IL-1 , tab sis 50 thiab 100μM acteoside significantly txo cov proteoglycan cov ntsiab lus los ntawm 1 27: feem pua thiab 64 ± 14: 5 feem pua , feem, nyob rau hauv muaj 1ng / mL IL-1 . Tom qab ntawd, txhawm rau txheeb xyuas seb acteoside suppresses IL -1 - induced proteoglycan poob, articular pob txha dissected los ntawm nas lub hauv caug pob qij txha tau kho nrog 100μM acteoside nyob rau hauv lub xub ntiag los yog tsis muaj 10ng / mL IL-1 rau 7 hnub. Tom qab ntawd, kev soj ntsuam histological siv H&E staining thiab safranin-O thiab ceev ntsuab staining tau ua raws li qhia hauv daim duab 3(b). Kev hloov pauv morphological tsis tau pom siv H&E staining; Txawm li cas los xij, safranin-O thiab nrawm ntsuab staining qhia tias cov proteoglycan stained raws li xim liab tsis hloov hauv cov pob txha pob txha kho nrog 100μM acteoside piv nrog rau hauv kev tswj. Txawm li cas los xij, kev poob qis proteoglycan hnyav tau tshwm sim los ntawm 10ng / mLIL-1 hauv pob txha pob txha, thiab 100μM acteoside tseem ceeb cuam tshuam cov proteoglycan poob hauv pob txha pob txha kho nrog 10ng / mL IL - 1 . Sib sau ua ke, cov ntaub ntawv no tsis tu ncua qhia tias acteoside muaj cov nyhuv anticatabolic uas ua rau cov pob txha mos degeneration los ntawm kev tawm tsam IL-1 -induced proteoglycan poob.

3.3. Acteoside muaj cov nyhuv Anticatabolic uas cuam tshuam MMP kev nthuav qhia thiab ua kom muaj nyob rau hauv thawj tus nas Chondrocytes kho nrog IL-1 . Txhawm rau tshawb xyuas seb qhov tshwm sim ntawm acteoside-induced anticatabolic cuam tshuam puas cuam tshuam nrog kev cuam tshuam ntawm MMP kev qhia thiab kev ua haujlwm, thawj nas chondrocytes tau kho nrog 50 thiab 100μM acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 10ng / mL IL-1 rau 24h. Tom qab ntawd, cov kev hloov pauv hauv MMPs tau tshawb xyuas. Raws li pom hauv daim duab 4(a), txawm hais tias qhov kev qhia ntawm cov pob txha mos-degrading enzymes xws li MMP-13, MMP-1, thiab MMP-3 tau nce ntau hauv cov xov xwm kho mob ntawm thawj nas. chondrocytes kho nrog 10ng / mL IL-1, nws tau txo qis los ntawm acteoside nyob rau hauv koob tshuaj. Tsis tas li ntawd, cov txiaj ntsig ntawm ob qho tib si qPCR (Daim duab 4(b)) thiab qRT-PCR (Daim duab 4(c)) tau qhia tias IL-1 qhov tseem ceeb nce qib mRNA ntawm MMPs xws li MMP-13, MMP{ {22}}, thiab MMP-3 hauv thawj nas chondrocytes. Txawm li cas los xij, lawv txo koob tshuaj-nyob ntawm thawj nas chondrocyteskho nrog 50 thiab 100μM acteoside. Ntxiv mus, 50 thiab 100μM acteoside muaj txiaj ntsig zoo rau kev ua kom MMPs hauv cov nas thawj chondrocytes kho nrog 10ng / mL IL-1 (Daim duab 4(d)). Ua ke, cov ntaub ntawv no tsis tu ncua qhia tias acteoside muaj cov nyhuv anticatabolic uas inhibits kev qhia thiab ua kom cov pob txha mos-degrading enzymes.

3.4. Acteoside txwv tsis pub tshaj tawm thiab tsim tawm ntawm IL1 -Induced Catabolic Inflammatory Mediators thiab Proinflammatory Cytokines hauv Primary Rat Chondrocytes. Txhawm rau txiav txim siab seb acteoside puas muaj kev tiv thaiv tiv thaiv OA, thawj cov nas chondrocytes tau kho nrog 50 thiab 100μM acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 10ng / mL IL-1 rau 24h. Tom qab ntawd, cov kev hloov pauv hauv cov kab mob sib kis, cov neeg sawv cev catabolic xws li iNOS, COX-2, thiab PGE2, tau tshawb xyuas. Cov qib mRNA ntawm iNOS, COX-2, thiab PTGS{11}} tau nce ntxiv los ntawm IL{12}} hauv thawj nas chondrocytes.

Txawm li cas los xij, lawv txo koob tshuaj-nyob ntawm tus thawj nas chondrocytes kho nrog acteoside (Daim duab 5(a)). Tsis tas li ntawd, acteoside tsis tsuas yog txwv tsis pub muaj kev qhia ntawm iNOS thiab COX-2 hauv thawj nas chondrocytes kho nrog IL-1 (Daim duab 5(b)) tab sis kuj tseem txo qis qhov txheeb ze ntawm NO thiab PGE2 raws li qhia. hauv daim duab 5(c) thiab 5(d), feem. Cov ntaub ntawv no qhia tias acteoside inhibits qhov kev qhia ntawm inflammatory mediator-induced proinflammatory cytokines uas ua raws li catabolic yam ua rau induce lub zuj zus degeneration ntawm articular pob txha mos. Yog li, txhawm rau tshawb xyuas qhov kev hloov pauv ntawm proinflammatory cytokines los ntawm 50μM acteoside hauv thawj nas chondrocytes kho nrog 10ng / mL IL- 1 , ib qho cytokine array tau ua raws li qhia hauv daim duab 6. Acteoside suppressed cov kev qhia ntawm netropu cytokine. chemoattractant- (CINC-) 2, CINC-3, ciliary neurotrophic factor (CNTF), fractalkine (CX3CL1), IL-1 , IL- 1 , leptin, monocyte chemoattractant protein{{22} } (MCP-1), macrophage inflammatory protein- (MIP-) 3 , thiab -nervekev loj hlob zoo (NGF) hauv thawj nas chondrocytes kho nrog IL-1 piv nrog IL-1 ib leeg. Ua ke, cov ntaub ntawv no qhia tsis tu ncua hais tias acteoside tiv thaiv qhov kev loj hlob ntawm cov pob txha mos los ntawm kev tawm tsam ntawm cov kab mob sib kis thiab cov cytokines proinflammatory tawm tsam IL-1 -induced catabolic effects hauv thawj nas chondrocytes.

3.5. Acteoside Suppresses MAPK thiab NFκB Phosphorylation hauv Primary Rat Chondrocytes Kho nrog IL-1 . Txhawm rau tshawb xyuas cov xov tooj ntawm tes taw qhia txoj hauv kev cuam tshuam nrog acteoside-induced anticatabolic cuam tshuam tawm tsam proinflamma-tory cytokine IL-1 hloov pauv ntawm MAPK thiab NFκB, thawj nas chondrocytes tau kho nrog 50 thiab 100μM acteoside ntawm IL{8{8} lossis tsis tuaj. }} rau 24 h. Tom qab ntawd, tag nrho cov protein tau muab rho tawm thiab electrophoresed ntawm SDS-PAGE gel los ua cov western blot. Raws li pom nyob rau hauv daim duab 7, MAPK xws li ERK1/2, p38, thiab JNK yog qhov tseem ceeb phosphorylated nyob rau hauv thawj nas chondrocytes kho nrog IL-1 , whereas 50 thiab 100μM acteoside tsis significantly phosphorylation ntawm MAP. tswj hauv thawj nas chondrocyte. Txawm li cas los xij, 50 thiab 100μM acteoside koob tshuaj-dependently suppressed IL-1 -induced MAPK phosphorylation hauv thawj nas chondrocytes. Tsis tas li ntawd, phosphorylation ntawm NFκB hauv thawj nas chondrocytes kho nrog 10ng / mL IL-1 tau maj mam txo los ntawm acteoside nyob rau hauv koob tshuaj. Cov ntaub ntawv no qhia tau hais tias MAPK thiab NFκB cellular signaling pathways muaj kev koom tes nrog cov acteoside-induced anticatabolic effects tawm tsam IL-1 hauv thawj nas chondrocytes.

3.6. Acteoside Suppresses Translocation ntawm NFκB los ntawm Cytosol mus rau Nucleus los ntawm Kev Tawm Tsam ntawm IL-1 -Induced NFκB Phosphorylation hauv Primary Rat Chondrocytes. Txhawm rau tshawb xyuas seb acteoside txwv tsis pub hloov pauv ntawm NFκB los ntawm cytosol mus rau lub nucleus, thawj cov nas chondrocytes tau kho nrog 50 thiab 100ug / mL acteoside nyob rau hauv lub xub ntiag lossis tsis muaj 10ng / mL IL-1 . Raws li pom hauv daim duab 8(a), NFκB tau hloov pauv mus rau lub nucleus los ntawm cytosol ntawm thawj nas chondrocytes kho nrog 10ng / mL IL-1 . Txawm li cas los xij, nws tseem ceeb heev inhibited los ntawm acteoside nyob rau hauv koob tshuaj. Tsis tas li ntawd, txawm hais tias qib NFκB tau nce ntxiv hauv cov khoom siv hluav taws xob tau muab rho tawm los ntawm thawj cov nas chondrocytes kho nrog 10ng / mL IL-1 , nws yog koob tshuaj-dependently txo los ntawm acteoside raws li qhia hauv daim duab 8(b). NFκB qib tau txo qis hauv cytosolic feem muab rho tawm los ntawm thawj cov nas chondrocytes kho nrog 10ng / mL IL-1 , tab sis nws yog koob tshuaj ntau ntxiv los ntawm acteoside. Muab ua ke, cov ntaub ntawv no tsis tu ncua qhia tias acteoside-induced anticatabolic cuam tshuam tawm tsam IL -1 yog koom nrog hauv kev cuam tshuam ntawm kev hloov pauv ntawm cytosol mus rau lub nucleus ntawm kev hloov pauv ntawm NFκB txoj kev taw qhia hauv thawj nas chondrocytes.

3.7. Acteoside Attenuates Progressive Degeneration ntawm Articular pob txha mos nyob rau hauv phais DMM-Induced Knee Joint OA Tsiaj. Txhawm rau nthuav tawm cov txiaj ntsig ntawm acteoside-induced anticatabolic hauv vivo, OA-induced tsiaj tsim los ntawm kev phais DMM ua rau ntawm lub hauv caug sib koom ntawm BALB/c nas tau hais lus 5 thiab 10mg / kg acteoside daws hauv 5 feem pua ethanol txhua hnub rau 8 lub lis piam. Tom qab ntawd, lub hauv caug pob qij txha tau soj ntsuam histologically siv safranin-O thiab ceev ntsuab staining raws li qhia nyob rau hauv daim duab 9. Cov proteoglycan poob thiab raug mob ntawm cov pob txha pob txha pob txha pob txha loj heev nyob rau hauv lub hauv caug ob leeg dissected los ntawm DMM-induced OA tsiaj. Txawm li cas los xij, kev tswj hwm qhov ncauj ntawm acteoside inhibited proteoglycan poob thiab kev raug mob tsawg dua ntawm cov pob txha mos piv nrog lub tsheb nkaus xwb. Tsis tas li ntawd, Mankin qhov qhab nia qhab nia tau nce ntxiv hauv OA tsiaj pawg (n =5, 3± 0}: 7) muab tsheb tsuas yog piv nrog Naïve (n =5, 0) :67 ± 0:5). Txawm li cas los xij, kev tswj hwm qhov ncauj ntawm 5 thiab 10mg / kg acteoside rau hauv OA tsiaj pawg (n =5) txo qis Mankin qhab nia los ntawm 2 ± 0: 7 thiab 1: 67 ± 0: 5 , feem, piv nrog lub tsheb nkaus xwb. Ua ke, cov ntaub ntawv no qhia tau hais tias qhov ncauj tswj hwm ntawm acteoside txo qis kev loj hlob ntawm cov pob txha mos hauv cov pob qij txha nrog cov kab mob catabolic.