Anti-aging Effects Of Piper Cambodianum P. Plaub. Extract Ntawm Tib Neeg Dermal Fibroblast Cells Thiab Ib Qhov Mob-kho

Keeb kwm:

Kev laus ntawm daim tawv nqaijyog txuam nrog ib puag ncig yam xws li ultraviolet rays, huab cua paug, lub ntiajteb txawj nqus, thiab genetic yam, tag nrho cov uas yuav ua rau wrinkling ntawm daim tawv nqaij. Cov ntawv ceeb toom dhau los qhia tias qhov kho qhov txhab raug cuam tshuam los ntawm cov txheej txheem kev laus thiab cov tswv yim los tswj cov kab mob.muaj hnub nyoog txog qhov txhab khoyog tsim nyog los txhawb kev kho.Lub hom phiaj: Ntau cov nroj tsuag tshuaj ntsuab tau paub zoo rau lawv cov khoom ntawm kev tiv thaiv tawv nqaij thiab kev saib xyuas.Cistanche Tubulosa Extract(Cistanche Extract), ib tug tswv cuab ntawm Piperacecae yog cov nroj tsuag pom nyob rau hauv Nyab Laj uas tej zaum yuav muaj cov khoom kho mob. Yog li ntawd, cov teebmeem ntawm Cistanche Extract qia thiab nplooj extract ntawm lubkev laustau tshawb xyuas hauv vitro thiab hauv vivo.Cov txheej txheem: Cistanche Extract yaj yaj nyob rau hauv methanol tau tshawb xyuas siv Western blotting, real-time quantitative reverse transcription-polymerase saw cov tshuaj tiv thaiv, ntws cytometry, thiab cell mob-kho kev soj ntsuam. Peb soj ntsuam covAnti-aging nyhuv ntawm Cistanche Extractnyob rau hauv cov nas uas siv lub qhov txhab kho mob. Cov txiaj ntsig tau raug tshuaj xyuas los ntawm Tub Ntxhais Kawm qhov kev xeem t-test; *P,0. Cov txiaj ntsig: Cistanche Extractkev kho mobua qauv qhia hauv vitro thiab hauv vivo tiv thaiv kev laus. Western blot tsom ntawm Cistanche Extract-kho ib txwm tib neeg dermal fibroblast hlwb pom akoob tshuaj-raws li nce nyob rau hauv kev qhia ntawm extracellular matrix noobxws li collagen thiab elastin, tab sistxo qis kev qhia ntawm cov noob laus matrix metalloproteinase-3. Quantitative polymerase saw cov tshuaj tiv thaiv tau pom tias Cistanche Extract-kho cov hlwb tau nthuav tawm koob tshuaj nce ntxiv hauv tus xa xov RNA qhia qib ntawm collagen, elastin, thiab hyaluronan synthase -2 thiab txo qis qhia qib ntawm matrix metalloproteinase -1 gene aging. Cistanche ExtractAnti-aging khoua rau txo qis ntawm cov pa oxygen reactive hauv cov hlwb raug rau ultraviolet irradiation. Tsis tas li ntawd, Cistanche Extract tau pom qhov mob zoo-kho cov teebmeem hauv nas.Xaus: Txoj kev tshawb no qhia tau hais tiasAnti-aging thiab qhov txhab-kho los ntawm Cistanche Extract Extract. Yog li ntawd, Cistanche Extract Extractsawv cev rau ib qho kev cog lus tshiab kho tus neeg sawv cev rau kev tiv thaiv kev laus thiab kev kho qhov txhab. 

Ntsiab lus:Cistanche Extract, anti-aging, kho qhov txhab, antioxidant, ROS, tib neeg dermal fibroblasts

Nyem qhov no Kom Tau Txais Cov Lus Qhia Ntxiv Txog Cistanche Extract Rau Muag

Taw qhia

Tib neeg daim tawv nqaij muaj epidermal, dermal, thiab cov ntaub so ntswg subcutaneous. Epidermis cuam tshuam tsis zoo los ntawm cov kab mob abiotic, 1 thiab kev laus muaj feem cuam tshuam nrog cov qauv, kev ua haujlwm, thiab kev hloov pauv biochemical.2 Kev laus ntawm daim tawv nqaij yog cuam tshuam nrog ib puag ncig xws li ultraviolet (UV) rays, huab cua muaj kuab paug, lub ntiajteb txawj nqus, thiab caj ces, 3 tag nrho ntawm uas tuaj yeem ua rau tawv nqaij tawv.4,5 Reactive oxygen hom (ROS), suav nrog superoxide anion radical (⋅O2 −), hydrogen peroxide (H2 O2), hydroxyl radical (⋅OH), singlet oxygen (1 O2), lipid peroxides (LOOH), thiab lawv cov radicals (LOO⋅) yog tsim nyob rau hauv daim tawv nqaij raug UVA (320–400 nm) thiab UVB (290–320 nm). Cov yam ntxwv no ua rau cov tawv nqaij laus, phototoxicity, o, tsim cov qog nqaij hlav, thiab tawg ntawm cov cell membranes.6-8 Ob peb cov tshuaj cog qoob loo muaj txiaj ntsig zoo rau kev tiv thaiv tawv nqaij thiab kev saib xyuas. Piper cambodianum P. Plaub. (Cistanche Extract), ib tug tswv cuab ntawm Piperacecae yog cov nroj tsuag pom nyob rau hauv Nyab Laj uas tej zaum yuav muaj cov khoom kho mob, txij li Piper betel Linn., uas belongs rau tib tsev neeg, yog siv ntau yam decoctions rau kho qhov txhab, kub hnyiab, lymphangitis, thiab eczema. Tsis tas li ntawd, kua txiv hmab txiv ntoo ntawm nplooj ntawm Kammaru, uas yog ntau yam Piper beetle, muaj peev xwm kho mob pharyngitis, mob plab, thiab o. Nws cov hauv paus hniav thiab txiv hmab txiv ntoo yog cov paub zoo rau kev siv hauv kev kho mob malaria thiab mob hawb pob.9,10 Lub tshuab ua haujlwm thiab kev ua haujlwm tshuaj ntawm Cistanche Extract hauv kev tiv thaiv daim tawv nqaij tseem tsis tau tshawb xyuas kom meej. Txoj kev tshawb no tau tshawb xyuas qhov muaj peev xwm tiv thaiv kev laus thiab qhov txhab kho qhov txhab ntawm Cistanche Extract qia thiab nplooj extract hauv tib neeg dermal fibroblast (NHDF) hlwb thiab tus qauv nas ntawm kev kho qhov txhab.

Cov ntaub ntawv thiab cov txheej txheem

Kev npaj ntawm PFF extract

PFF extract yog muab los ntawm Kauslim Kev Tshawb Fawb Lub Tsev Haujlwm ntawm Bioscience & Biotechnology (KRIBB). Cov hmoov qhuav qhuav tau muab rho tawm hauv methyl ethanol 99.9 feem pua ​​​​rau 3 hnub nrog SD-Ultrasonic Cleaner (Seoul, South Kauslim) ntawm 45 degree rau 72 teev. Cov extract tau lim thiab concentrated ntawm 45 degree (Rotary Evaporator, N-1000SWD-EYELA, Tokyo Rikakikai Co., Ltd., Bohemya, NY, USA), thiab qhuav ntawm 70 degree rau 24 teev nrog Modul spin 40 (Biotron Corporation, Alberta, Canada). Extracted Cistanche Extract tau diluted nrog dej ntshiab ntawm ntau koob tshuaj rau txhua qhov kev sim.

Antibodies thiab reagents

Cov tshuaj tiv thaiv hauv qab no tau siv: anti-elastin (Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-matrix metalloproteinase (MMP) -3 (Santa Cruz), anti-extracellular signal-regulated kinase (ERK) (Santa Cruz), anti-collagen (Abcam, Cambridge, UK), anti-actin (Sigma-Aldrich Co., St Louis, MO, USA), anti-tumor necrosis factor receptor (TNFR) -1 (Thermo Fisher Scientific, Waltham, MA, USA), anti-epidermal growth factor receptor (EGFR) (Thermo Fisher Scientific), anti-pp38 (Thr180/Tyr182; Cell Signaling Tech, Danvers, MA, USA), anti-c-Jun ( Santa Cruz), anti-p53 (Cell Signaling Tech), thiab lwm yam tshuaj tiv thaiv kab mob (tiv thaiv nas lossis los tiv thaiv luav) los ntawm Komabiotech (Seoul, Kaus Lim Qab Teb).

Cell kab lis kev cai

Cov Neeg Laus Ib txwm Tib Neeg Lub Cev Muaj Zog Dermal Fibroblasts (NHDF) hlwb tau yuav los ntawm ATCC (PCS-201-012, Manassas, VA, USA). NHDF hlwb tau khaws cia hauv kab lis kev cai hauv Dulbecco's Modified Eagle's Media (1: 1) muaj 10 feem pua ​​​​fetal bovine serum thiab 1 feem pua ​​​​cov tshuaj tua kab mob. NHDF hlwb tau loj hlob ntawm 37 degree hauv humidified 5 feem pua ​​CO2 .

Analysis rau cell viability

NHDF hlwb tau plated ntawm qhov ceev ntawm 1.0 × 104 hlwb / zoo hauv 96-cov kab lis kev cai zoo rau kev ua tiav ntawm 37 degree nrog 5 feem pua ​​CO2 rau 24 teev. Cov hlwb raug kho nrog Cistanche Extract ntawm koob tshuaj 1, 10, thiab 50 µg / mL rau 24 teev. Cov kab lis kev cai nruab nrab raug tshem tawm, tom qab ntawd incubation nrog 90 mL ntawm EXCyto (Lucigen Corporation, Middleton, WI, USA) 10 μL ntawm 37 degree rau 3 teev. Lub absorbency tau ntsuas ntawm 450 nm (xws li 659 nm) nrog cov enzyme-linked immunosorbent assay reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). Cov txiaj ntsig tau nthuav tawm raws li kev txheeb ze ntawm cov cell viability ntawm cov tshuaj kho mob tawm tsam cov kev tswj hwm.

Cell mob-kho assay (cell migration assay)

Rau qhov kev ntsuam xyuas kev tsiv teb tsaws ntawm tes, cov monolayers tau ua tib zoo khawb los ntawm 10 μL pipette taub. Cov hlwb tau raug kho nrog Cistanche Extract ntawm koob tshuaj 1, 10, thiab 50 mg / mL rau 24 teev thiab tom qab ntawd qhov chaw raug mob tau yees duab.

Kev tshuaj xyuas los ntawm lub sijhawm tiag tiag kom muaj nuj nqis thim rov qab polymerase saw cov tshuaj tiv thaiv

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) tsom xam tau ua raws li tau piav qhia yav dhau los.11,12 Tag Nrho RNA tau muab rho tawm los ntawm NHDF cell qauv siv cov khoom siv RNeasy (Qiagen NV, Venlo, Netherlands). Ntxiv DNA tau tsim los ntawm tag nrho RNA nrog SuperScript First-Strand Synthesis System (Thermo Fisher Scientific) thiab random hexamer primers. Kev ntsuas PCR tiag tiag ntawm ib tus neeg ntxiv DNAs tau ua tiav siv SYBR ntsuab dye los ntsuas kev tsim cov DNA duplex nrog Thermo Fisher Scientific system (StepOne Plus) thiab normalized rau kev qhia ntawm glyceraldehyde 3-phosphate dehydrogenase. Cov primers thiab probes siv nyob rau hauv real-time qRT-PCR tau piav nyob rau hauv Table 1.

Western blot tsom xam

Western blot tsom xam tau ua raws li tau piav qhia dhau los. 13-15 NHDF hlwb raug kho nrog Cistanche Extract extract rau 24 teev, ntxuav ob zaug hauv dej khov-txias phosphate-buffered saline, thiab homogenized ntawm 4 degree hauv lysis tsis muaj 50 mM Tris–HCl, pH 7.5, 1% Triton X{{10}}, 300 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium vanadate, 20 mM -glycerol phosphate, 2 mM dithiothreitol, 1 mM thiab leupeptin 10 mM p-nitrophenyl phosphate. Homogenates tau centrifuged ntawm 15, 000 × g rau 10 feeb thiab supernatants tau siv rau Western blot tsom xam. Cov protein concentration ntawm lysates tau ntsuas los ntawm bovine serum albumin protein assay, thiab 50 ug ntawm cov cell lysates tau fractionated los ntawm 10 feem pua ​​sodium dodecyl sulfate-polyacrylamide gel thiab blotted mus rau polyvinylidene difluoride membrane. Tom qab thaiv nrog 5 feem pua ​​​​skim mis nyuj Tris-buffered saline muaj ing 0.02 feem pua ​​​​Tween 20, daim nyias nyias tau soj ntsuam nrog cov tshuaj tiv thaiv uas cuam tshuam thiab pom los ntawm kev txhim kho chemiluminescence, raws li cov chaw tsim khoom cov lus qhia (GE Healthcare, Pittsburgh, PA, USA). Actin tau siv los tswj kev thauj khoom.

Table 1 Primer teem rau lub sijhawm tiag tiag PCR

ROS tsom xam

NHDF hlwb raug plated thiab incubated ntawm ib tug ceev ntawm 1.0 × 106 hlwb/zoo nyob rau hauv rau-zoo kab lis kev cai daim hlau rau 24 teev. Hnub tom qab, cov hlwb raug UV irradiated ntawm 40 J rau 180 vib nas this nrog UV Crosslinker (Vilber Loirmat, Eberhardzell, Lub teb chaws Yelemees), tsis kho, thiab kho nrog Cistanche Extract extracts rau 24 teev. Cells raug ntxuav thiab stained nrog 5 mM CM-H2 DCFDA (Thermo Fisher Scientific) hauv Hank cov kua ntsev sib npaug, incubated ntawm 37 degree rau 30 feeb. FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) tau siv los ntsuas H2 O2 kev ua ub no, siv lub emission lim ntawm 532 nm. Tsawg kawg yog 10,{17}} cov hlwb raug tshuaj xyuas hauv txhua ntawm peb qhov kev sim ywj pheej.

Kev kho mob-kho nrog nas

C57Bl/6 txiv neej nas (7 lub lis piam-hnub, n=3) los ntawm Jungang Animal Company (Seoul, Kauslim) tau ua thawj zaug tshuaj loog, thiab thaj chaw raug mob raug shaved. Plaub 0.25 cm2 (0.5 ×0.5 cm) excisional qhov txhab raug tsim rau ntawm daim tawv nqaij dorsal (kev tswj tsis zoo thiab 1, 10, 50 µg/mL Cistanche Extract Extract ). Kev kho mob ntawm daim tawv nqaij raug mob nrog Cistanche Extract extracts tau ua tiav nrog paj rwb swab rau 4 hnub (ib zaug ib hnub). Cov nas tau nyob hauv tus qauv nas tawb nrog 12- teev lub teeb / tsaus ntuj. Cov tsiaj raws tu qauv hauv peb txoj kev tshawb fawb tau pom zoo los ntawm pawg saib xyuas tsiaj thiab kev coj ncaj ncees ntawm Chungnam National University. Tag nrho cov txheej txheem tuav cov tsiaj txhu tau ua raws li Phau Ntawv Qhia rau Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Laboratory ntawm Kaus Lim Kauslim Lub Tsev Haujlwm Saib Xyuas Kev Noj Qab Haus Huv thiab ua raws li cov lus qhia ntawm Txoj Cai Kev Nyab Xeeb Tsiaj.

Kev txheeb xyuas txheeb cais Quantification ntawm Western blot tsom xam tau ua tiav siv ImageJ program (NIH, Bethesda, MD, USA). Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± SD. Cov txiaj ntsig tau raug tshuaj xyuas los ntawm Stu dent's unpaired t-test (SPSS version 12.0 software, SPSS Inc., Chicago, IL, USA). *P,0.

Cov txiaj ntsig

Cell viability thiab qhov txhab-kho los ntawm Cistanche Extract Extract hauv NHDF hlwb

Cistanche Extract tau muab rho tawm los ntawm nplooj ntawm Kammaru (Daim duab 1A). NHDF hlwb tau kho nrog Cistanche Extract ntawm koob tshuaj 1, 10, thiab 50 µg/mL rau 24 teev. Cells kho nrog Cistanche Extract extract koob tshuaj ntawm 0, 10, thiab 50 µg / mL pom tsis muaj kev cuam tshuam ntawm cell viability piv rau cov tsis kho cov hlwb (Daim duab 1B). Hauv kev kuaj mob ntawm tes-kho zoo, cov cell monolayers tau raug mob los ntawm kev khawb khawb thiab tom qab ntawd kho nrog Cistanche Extract extracts ntawm qhov ntau ntawm 1 txog 50 µg / mL rau 24 teev. Cistanche Extract-kho cov hlwb tau pom kev kho kom zoo ntawm thaj chaw khawb hauv ib qho kev noj tshuaj (Daim duab 1C thiab D).

Enhanced extracellular matrix noob qhia theem hauv Cistanche Extract-kho NHDF hlwb

Collagen yog cov txheej txheem loj ntawm cov protein uas muaj nyob rau hauv qhov chaw extracellular ntawm ntau cov ntaub so ntswg hauv cov tsiaj.16 Elastin yog ib qho elastic connective cov ntaub so ntswg protein uas pab tswj cov ntaub so ntswg zoo tom qab ncab los yog cog lus los ntawm kev xyuas kom meej tias cov ntaub so ntswg rov qab mus rau lawv tus qub txoj hauj lwm. Hyaluronan los yog hyaluronic acid (HA) yog ib feem ntawm lub extracellular matrix (ECM) koom nyob rau hauv lubricating pob qij txha thiab sau qhov chaw los ntawm cov hlwb yuav migrate. Thaum kho qhov txhab thiab kho cov ntaub so ntswg, HA muab lub hauv paus rau fibroblasts. HAS-2 yog hyaluronan synthase (HAS). Tom qab kev kho mob nrog ntau koob tshuaj ntawm Cistanche Extract extract rau 24 teev, ECM cov noob xws li collagen, elastin, thiab HAS-2 qhia qib tau nce ntau npaum li kev noj tshuaj. MMP-1 encoded los ntawm MMP1 noob nyob rau hauv tib neeg yog lub npe hu ua interstitial thiab fibroblast collagenase.16–18 MMPs koom nrog hauv kev tawg ntawm ECM hauv cov txheej txheem physiological thiab kho cov ntaub so ntswg, thiab MMP-1 tshwj xeeb tawg down lub interstitial collagen hom I, II, thiab III. Peb pom tau hais tias tus tub txib RNA qib ntawm MMP-1 tau txo qis hauv Cistanche Extract-kho cov hlwb nyob rau hauv koob tshuaj (Daim duab 2).

Daim duab 1 Cell viability thiab qhov txhab kho qhov txhab. Lus Cim: Cistanche Extract nplooj thiab qia duab. (A) Cistanche Extract extract tau thov ntawm 1 thiab 10 ug/mL pom tias tsis muaj cytotoxicity hauv EXCyto assay siv NHDF hlwb. (B) Kev kuaj mob ntawm tes-kho kho mob tau teeb tsa 24 teev tom qab khawb lub cell monolayer thiab kho nrog Cistanche Extract extract ntawm cov koob tshuaj qhia. (C) Thaum cov hlwb mus txog qhov sib txuam, ib lub qhov txhab tau ua nyob rau hauv nruab nrab ntawm lub monolayer siv 10 ml pipette taub, thiab cov hlwb raug kho nrog qhov qhia tau hais tias cov ntsiab lus ntawm Cistanche Extract extracts. Tom qab 24- teev incubation, ib daim duab tau coj ntawm × 10 magnification nyob rau hauv lub tshuab ntsuas. (D) Cov duab thaij duab los ntawm (C) hauv koob tshuaj tau tshuaj xyuas rau thaj tsam sib txawv. Cov txiaj ntsig tau nthuav tawm raws li qhov nruab nrab ± tus qauv sib txawv ntawm peb qhov kev sim ywj pheej. ** P, 0.01. Cov ntawv luv: Conc, concentration; NHDF, tib neeg dermal fibroblast; Cistanche Extract, Piper cambodianum P. Plaub.

Txhim kho ECM protein qhia hauv Cistanche Extract-kho NHDF hlwb

Protein extracts ntawm untreated los yog Cistanche Extract-kho hlwb raug soj ntsuam los ntawm Western blot tsom. Cistanche Extract-kho cov hlwb pom tau tias muaj collagen thiab elastin protein ntau ntxiv piv nrog cov kev tswj tsis tau kho (Daim duab 3). ERK signaling pathway regulates type I collagen gene expression in human fibroblasts.17 Peb cov ntaub ntawv qhia tau hais tias cov protein ntau ntawm ERK tau txhim kho qhov tseem ceeb, thiab interstitial collagenase enzyme MMP-3 yog downregulated ho los ntawm Cistanche Extract extract nyob rau hauv ib koob tshuaj. yam (Daim duab 3). Cov neeg xa xov RNA qib ntawm collagen thiab elastin tau cuam tshuam nrog kev hloov pauv hauv lawv cov qib protein. Cov txiaj ntsig no tau qhia tias qhov kev tiv thaiv kev laus ntawm Cistanche Extract ntawm NHDF hlwb muaj feem cuam tshuam nrog kev txhais lus thiab kev hloov pauv ntawm cov molecules koom nrog ECM.

Daim duab 2 ECM gene qhia theem los ntawm q-PCR. Lus Cim: RNAs los ntawm cov tshuaj tsis kho lossis Cistanche Extract-kho cov hlwb raug tshuaj xyuas los ntawm lub sijhawm tiag tiag q-PCR nrog (A) collagen-, (B) elastin-, (C) hyaluronan synthase (HAS)-2, thiab (D ) matrix metalloproteinase (MMP)-1-cov primers tshwj xeeb. Cov txiaj ntsig tau nthuav tawm raws li qhov nruab nrab ± SD ntawm peb qhov kev sim ywj pheej. *P,0.05, **P,0.01. Cov ntawv luv: Conc, concentration; ECM, extracellular matrix; HAS, hyaluronan synthase; MMP, matrix metalloproteinase; mRNA, tub txib RNA; NHDF, tib neeg dermal fibroblast; Cistanche Extract, Piper cambodianum P. Plaub.; q-PCR, quantitative polymerase saw cov tshuaj tiv thaiv; RNA, ribonucleic acid; SD, tus qauv sib txawv.