Ntu 1: Anti-Inflammatory, Antioxidant, Moisturizing, Thiab Antimelanogenesis Los Ntawm Quercetin 3-O- -D-Glucuronide nyob rau hauv tib neeg Keratinocytes Thiab Melanoma Cells Ntawm Kev Ua Kom NF-κB Thiab AP -1 Txoj Kev
Mar 21, 2022
Yog xav paub ntxiv, hu rautina.xiang@wecistanche.com
Abstract: Quercetin3-O- -D-glucuronide(Q-3-G), glucuronide conjugate ntawm quercetin, tau tshaj tawm tias muajanti-inflammatoryCov khoom hauv lipopolysaccharide-stimulated macrophages, nrog rau cov tshuaj tiv thaiv kab mob thiab antioxidant zog. Tsis zoo li quercetin, uas tau piav qhia dav dav kom muaj ntau yam tshuaj ua haujlwm nrog rau kev tiv thaiv tawv nqaij, cov txiaj ntsig tshuaj thiab cov txheej txheem ntawm Q-3-G hauv daim tawv nqaij tseem yuav tsum tau piav qhia. Txoj kev tshawb no tau tsom mus rau tus yam ntxwv ntawm kev tiv thaiv ntawm daim tawv nqaij, suav nrog cov tshuaj tiv thaiv kab mob thiab antioxidant, ntawm Q-3- Tawm tsam UVB-induced los yog H2O2-induced oxidative stress, hydration los, thiabantimelanogenesisCov dej num siv tib neeg keratinocytes (HaCaT) thiab melanoma (B16F10) hlwb. Q-3-G down-regulated cov kev qhia ntawm pro-inflammatory gene thiab cytokine xws li cyclooxygenase-2 (COX-2) thiab qog necrosis factor (TNF)- hauv HO, los yog UVB- irradiated HaCaT hlwb. Peb kuj tau qhia tias O-3-Gexhibits muaj cov nyhuv antioxidant siv cov tshuaj dawb radical scavenging, flow cytometry, thiab kev nthuav qhia ntxiv ntawm nuclear factor erythroid 2- ntsig txog yam 2 (Nrf2). Q-3-G txo melanin ntau lawm hauv -melanocyte-stimulating hormone (-MSH)-induced B16F10 hlwb. Cov teebmeem hydration thiab cov txheej txheem ntawm Q-3-G tau tshuaj xyuas los ntawm kev soj ntsuam cov moisturizing factor-related genes, xws li transglutaminase-1(TGM-1), filaggrin(FLG), thiab hyaluronic acid synthase (HAS)-1. Ntxiv rau, Q-3-G nce phosphorylation ntawm c-Jun, Jun N-terminal kinase (JNK), Mitogen-activated protein kinase (MAPK) kinase 4(MKK4), thiab TAK1, koom nrog hauv MAPKs/AP -1 txoj kev, thiab cov phosphorylation ntawm IkB , IkB kinase (IKK)- , Akt, thiab Src, koom nrog txoj hauv kev NF-kB. Ua ke, peb tau ua pov thawj tias Q-3-G tawm dag zog tiv thaiv kab mob, antioxidant, moisturizing, thiab antimelanogenesis zog hauv tib neeg keratinocytes thiab melanoma hlwb los ntawm NF-kB thiab AP-1 txoj hauv kev.
Ntsiab lus: quercetin 3-O- -D-glucuronide; tiv thaiv UV; moisturizing; antimelanogenesis; AP-1; NF-kB
1. Taw qhia
Cov tawv nqaij, lub cev loj tshaj plaws ntawm lub cev, muaj qhov cuam tshuam uas ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv lub cev los ntawm ib puag ncig sab nraud thiab ua rau muaj kev puas tsuaj xws li hluav taws xob ultraviolet, kab mob microbes, thiab tshuaj lom neeg. Qhov teeb meem no tseem tswj hwm lub cev electrolyte tshuav thiab kub thiab tiv thaiv kom tsis txhob ya raws [1,2]. Tib neeg daim tawv nqaij yog tsim los ntawm peb txheej: epidermis, dermis nrog appendages, thiab cov ntaub so ntswg subcutaneous [3]. Cov txheej txheej epidermal plays lub luag haujlwm tseem ceeb hauv kev tswj lub cev kub.
Keratinocytes, muaj ntau yam nyob rau hauv daim tawv nqaij epidermis, nruj nreem cuam tshuam nrog lwm tus los ntawm desmosomes thiab nruj junctions uas tso cai rau ib tug zoo physicochemical barrier [4]. Txawm hais tias daim tawv nqaij ua haujlwm raws li kev tiv thaiv kev tiv thaiv, ultraviolet (UV) hluav taws xob muaj qhov cuam tshuam loj rau feem ntau cov hlwb, suav nrog keratinocytes, txheej txheej ntawm daim tawv nqaij. UV hluav taws xob tuaj yeem ua rau tawv nqaij laus, tawv nqaij puas, wrinkles, thiab hyperpigmentation, uas yog suav tias yog ib qho ntawm cov feem ntau censorious yam xwm txheej [5].UV lub teeb muaj peb wavelength ranges: UVA({2}} nm), UVB (280-320 nm), thiab UVC (200-280 nm). Be-sides hydrogen peroxide (H2O2), uas tau paub tias yuav pab txhawb oxidative kev nyuaj siab, UVB tuaj yeem ua rau kev tsim cov pa oxygen reactive (ROS) los ntawm kev ua kom ROS-generating enzymes [6]. Kev tswj tsis tau ntawm ROS hauv cov hlwb suav nrog keratinocytes tau piav qhia los ua oxidative hloov pauv rau lipids, proteins, nucleic acids, thiab lwm yam intracellular molecules uas tom qab ua rau ntau hom tawv nqaij puas thiab cov txheej txheem kev laus xws li kev tuag ntawm tes, oxidative stress, thiab mob [7,8]. Tsis tas li ntawd, cov lus teb oxidative kev nyuaj siab yog paub los cuam tshuam kev puas tsuaj ntawm ntau yam cellular Cheebtsam [9]. Cov kab mob ntawm daim tawv nqaij puas tuaj yeem ua rau cov lus teb inflammatory, uas ua rau cov tawv nqaij puas lawm [10]. Ib hom UVB lossis H2O2- kho cov tawv nqaij puas tsuaj los ntawm cov txheej txheem lom neeg yog qhov qhia txog cov kab mob inflammatory xws li cyclooxygenase-2 (COX-2) thiab pro-inflammatory cytokines [11]. Yog li ntawd, kev txhim kho kev nyab xeeb, muaj zog dua, thiab muaj txiaj ntsig zoo ntawm cov tawv nqaij tiv thaiv yog qhov tseem ceeb rau kev tiv thaiv thiab thaiv cov kab mob cuam tshuam nrog UVB lossis H2O2, - kho cov tawv nqaij puas.
Daim tawv nqaij hydration kuj ua lub luag haujlwm tseem ceeb hauv kev tswj nws txoj haujlwm ib txwm muaj. Tshwj xeeb, stratum corneum (SC) muaj kev tiv thaiv kev tiv thaiv dej [12]. Cov dej ntsiab lus ntawm stratum corneum koom nrog rau hauv daim tawv nqaij desquamation thiab kom loj hlob zoo, whereas dej tsis txaus nyob rau hauv SC txhawb lub tsub zuj zuj thiab dysfunction ntawm corneocytes [12,13]. Nyob rau theem kawg ntawm kev sib txawv ntawm keratinocyte, nws cov plasma membrane thiab cellular organelles ploj mus, thiab tom qab ntawd calcium influx induces transglutaminase (TGM) rau crosslinking nrog lwm cov proteins. Ib qho tsis txaus lossis poob ntawm TGM-1 ua rau muaj kev cuam tshuam ntawm kev sib txuas ntawm cov cell envelopment. Lamella ichthyosis yog ib qho piv txwv ntawm tus kab mob tshwm sim los ntawm kev poob ntawm TGM-1. Tsis tas li ntawd, filaggrin (FLG), tsim keratin matrix, plays lub luag hauj lwm hauv SC tsim vim nws ua raws li ib tug scaffold los khi cornified-lub hnab ntawv cov protein thiab lipids [14] los ntawm catalyzing g-glutamyl lysine cross-linking tshua, ATGMs, thiab daim nyias nyias. -kev koom tes enzymes, imparting kev ncaj ncees rau SCs [15]. Tsis tas li ntawd, hyaluronic acid (HA) yog ib hom ntawm ntuj ya raws qhov tseem ceeb (NMF) koom nrog hauv kev khaws cov dej noo hauv daim tawv nqaij thiab nthuav tawm cov ntaub ntawv sib txawv hauv cov tawv nqaij laus, yog cov tshuaj xa tshuaj, thiab yog ib qho tseem ceeb ntawm cov kab mob extracellular matrix. [16, 17]. HA plays lub luag haujlwm hauv kev ua kom cov tawv nqaij noo los ntawm kev tswj cov hyaluronic acid synthase (HAS) noob; Tsis tas li ntawd, retinoic acid (vitamin A) tuaj yeem tswj hwm HA hauv epidermis [17]. TGM-1, FLG, thiab HAS{15}},3 yog cov noob muaj feem xyuam nrog NMF ntau lawm [18]. Tsis tas li ntawd, kev ua kom cov khoom siv hloov pauv xws li activator protein-1(AP-1) thiab nuclear factor (NF)-kB tau koom nrog hauv kev tsim cov tshuaj tiv thaiv kab mob uas ua kom zoo rau daim tawv nqaij thiab daim tawv nqaij hydration. los ntawm AP -1 thiab NF-kB txoj hauv kev[19]. Txoj hauv kev tshaj tawm cov enzymes hauv AP-1 txoj hauv kev suav nrog mitogen-activated protein kinases (MAPKs) xws li c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), thiab p38, thaum Src, Akt, IkB -kinase (IKKo/ ), thiab KBO (IkBa) belongs rau txoj kev NF-KB [20. Cov tawv nqaij kuj tsim melanin nyob rau hauv lub epidermal txheej, tshwj xeeb tshaj yog nyob rau hauv lub melanocytes - lub loj qhov chaw ntawm melanin - uas pab mus txiav txim ntawm daim tawv nqaij xim thiab melanogenesis [21]. UVB irradiation thiab -melanocyte-stimulating hormone (o-MSH) tuaj yeem ua rau melanin secretion, yog li ua rau melanogenesis [22. Thaum lub sij hawm melanogenesis, -MSH activates protein kinase A (PKA), protein microphthalmia-associated transcription factor (MITF), thiab cAMP teb cov ntsiab lus-binding (CREB) [23]. Ib qho ntawm cov haujlwm ntawm melanin yog suav tias yog kev tiv thaiv ntawm daim tawv nqaij puas tsuaj los ntawm UV, suav nrog hauv tshav ntuj. Txawm li cas los xij, ntau dhau ntawm melanin lossis tsis muaj cov ntsiab lus melanin tshwm sim los ntawm kev laus, melanocyte-stimulating hormone, thiab UV raug txhawb nqa freckles, melasma, senile lentigines, thiab lwm yam hyper / hypogmentation mob uas tuaj yeem cuam tshuam tsis zoo rau cov tsos thiab daim tawv nqaij. noj qab haus huv [24]. Yog li, tswj melanogenesis kuj xav kom tswj tau ob qho tib si kev noj qab haus huv thiab kev zoo nkauj ntawm tib neeg lub cev.
Quercetinyog ib qho tseem ceeb faib flavonol-hom flavonoid tam sim no hauv zaub, txiv hmab txiv ntoo, dej haus, thiab cov nroj tsuag tshuaj [25]. Yog li ntawd, quercetin tau kawm ntau ntxiv kom muaj cov txiaj ntsig pharmacological suav nrog kev tiv thaiv kab mob, tiv thaiv atherosclerotic,antioxidant, anticancer, thiab tiv thaiv daim tawv nqaij, thiab muaj feem xyuam rau melanogenesis [24, 26-29]. Txawm li cas los xij, ntau txoj haujlwm uas siv cov txheej txheem rhiab thiab txhim khu kev qha tau pom tias quercetin tsis tau kuaj pom hauv plasma [30-33] thiab tsis tshua pom hauv lub hlwb [34,35]. Quercetin, uas feem ntau nthuav tawm raws li daim ntawv glycoside, yog absorbed zoo thiab feem ntau hydrolyzed thiab metabolized nyob rau hauv cov hnyuv me, hnyuv, siab, thiab raum rau hauv conjugated metabolites [30,36-38]. Tsuas yog cov conjugated metabolites xws li glucuronide los yog sulfate (methylated los yog unmethylated, feem), es tsis yog quercetin aglycone los yog nws glycosides, raug kuaj pom nyob rau hauv cov ntshav ncig. 3/-methyl-quercetin{10}}glucuronide, quercetin-3-glucuronide, thiab quercetin-3'-sulfate tau raug txheeb xyuas tias yog cov metabolites loj hauv plasma tom qab 1.5 teev los ntawm kev noj cov dos kib[30, 32] ib. Cov quercetin metabolites yog tsim nyob rau hauv cov hnyuv me thiab daim siab los ntawm biotransformation enzymes nrog rau UDP-glucuronyltransferases raws li theem II metabolism [29,39,40]. Nws kuj tau tshaj tawm tias conjugated metabolites ntawm quercetin accumulates hauv tib neeg plasma nyob rau hauv qhov concentration ntau ntawm 10-'rau 10- degree M tom qab noj cov dos ib ntus nrog zaub mov rau 1 lub lis piam[41]. Ib nrab-lub neej ntawm quercetin metabolites yog siab heev, ntawm 11 txog 28 h [29]. Txij li cov tebchaw no tau pom tias yog cov qauv tseem ceeb ntawm conjugated quercetin metabolites, cov kev tshawb fawb tsom mus rau kev ua haujlwm lom neeg ntawm flavonols siv cov conjugated metabolites yuav yog qhov tseem ceeb, uas ua rau peb kawm txog kev ua haujlwm lom neeg ntawm ib qho ntawm conjugated quercetin metabolites. Ib qho ntawm ntau tshaj glucuronide metabolites pom nyob rau hauv plasma thiab cov ntaub so ntswg nrog rau daim siab, raum, thiab lub hlwb yog quercetin 3-O- -D-glucuronide(Q-3-G)(Daim duab 1) [31,35,40,42,43], uas tej zaum yuav tsim kom muaj kev ua haujlwm zoo sib xws, muaj zog, lossis tsis muaj zog piv nrog cov niam txiv sib txuas hauv cov qauv sib txawv. Q-3-G raug thauj mus rau lub hom phiaj cov ntaub so ntswg ntawm cov ntshav plasma kom ua rau nws lub cev muaj zog [39]. O-3-G kuj tau pom tias yog cov metabolite muaj txiaj ntsig zoo hauv cov ntshav plasma tom qab kev tswj qhov ncauj ntawm quercetin [43]. Hauv lwm cov kev tshawb fawb, Q-3-G-muaj cov metabolite tau pom muaj nyob rau hauv lub aorta tom qab kev tswj hwm ntawm quercetin-nplua nuj cov zaub mov thiab tau raug txheeb xyuas raws li lub hauv paus ntsiab lus tseem ceeb hauv tib neeg cov ntshav xws li 0.{{48} } μM 【44】. Tsis tas li ntawd, Q-3-G tuaj yeem pom hauv cawv [45] thiab hauv cov nroj tsuag tshuaj xws li Hypericum hirsutum, Nelumbo nucifera, Oenothera biennis, thiab taum ntsuab [46-49].Q-3-G tau piav raws li cov tshuaj tiv thaiv kab mob nyob rau hauv LPS-stimulated tej yam kev mob thiab pom muaj antioxidant zog nyob rau hauv cov ntshav plasma [50-52]. Txawm li cas los xij, piv nrog cov keeb kwm ntev ntawm daim ntawv thov thiab kev kawm dav ntawm quercetin, txoj kev kawm ntawm Q-3-G tseem yuav tau tshawb nrhiav. Yog li ntawd, Q-3-G tau raug xaiv los ua tus neeg sawv cev ntawm quercetin metabolites raws li kev ua haujlwm hauv vivo player ntawm quercetin. Tsis tas li ntawd, lub luag haujlwm ntawm Q-3-G ntawm kev tiv thaiv ntawm daim tawv nqaij kuj tseem tsis tau qhia meej meej. Hauv txoj kev tshawb no, siv ntau yam kev ntsuam xyuas hauv vitro, peb tshawb xyuas cov kev tiv thaiv ntawm daim tawv nqaij ntawm Q-3-G tiv thaiv UVBor H2O2-induced oxidative stress thiab o, daim tawv nqaij hydration hauv HaCaT (ib tug tib neeg keratinocyte cell kab) hlwb, thiab kev ua haujlwm antimelanogenesis hauv B16F10 (ib murine melanoma cell line) hlwb.
2. Qhov tshwm sim
2.1.Anti-Inflammatory and Antioxidant Effects of Q-3-G tiv thaiv UVB lossis H2O2-Timulated HaCaT Cells
Cov tawv nqaij tuaj yeem raug puas los ntawm UV hluav taws xob. UV hluav taws xob ua kom muaj ntau yam teeb meem, suav nrog oxidative kev nyuaj siab, apoptotic cell tuag, o, thiab tawv nqaij laus. Txhawm rau txiav txim siab txog UV tiv thaiv kev ua ub no ntawm Q-3-G, thawj zaug, peb tau soj ntsuam seb Q-3-G puas muaj kev cuam tshuam rau lub cell viability ntawm tib neeg keratinocytes. Siv ib qho 3-(4-5-Dimethylthiazol-2-yl)-2, 5-diphenyl-243 tetrazolium bromide (MTT) assay, peb tshawb xyuas cov teebmeem cytotoxic ntawm O-3-G hauv HaCa hlwb. Raws li pom nyob rau hauv daim duab 2a, tsis muaj kev cuam tshuam tseem ceeb ntawm cell viability ntawm qhov concentration ntawm 2.5-20 uM los ntawm Q-3-G, uas tau qhia tias O-3-G tsis pom muaj cytotoxicity txog li. 20 μM thiab tsis ua mob rau keratinocytes, txheej txheej ntawm daim tawv nqaij. Tom ntej no, peb tau soj ntsuam cov kev tiv thaiv ntawm daim tawv nqaij ntawm Q-3-G hauv HaCaT hlwb raug UVB. Tom qab UVB irradiation ntawm 30 mJ/cm², Q-3-G tiv thaiv HaCaTcells los ntawm UVB-induced cell tuag nyob rau hauv ib tug concentration-dependent yam (Daim duab 2b). Raws li kev pom zoo nrog MTT qhov kev soj ntsuam, siv lub koob yees duab txuas nrog lub tshuab ntsuas kom ntes cov morphology ntawm HaCaT hlwb, Q-3-G tiv thaiv kev tuag ntawm tes vim UVB-irradiated HaCaT hlwb mus txog qhov concentration ntawm 10 μM (Daim duab 2c) . Tus naj npawb ntawm HaCaT hlwb nyob rau hauv txhua tus mob yog 482 rau pab pawg tsis kho, 91 rau UVB 30 mJ / cm-group, 276 rau UVB 30 mJ / cm² ntxiv rau 5μM O-3-G pawg, thiab 320 rau UVB 30 mJ/cm² plus 10μM Q-3-G Group (Daim duab 2d). Cov txiaj ntsig no tau lees paub tias Q-3-G inhibits UVB-induced HaCaT kev puas tsuaj thiab cawm cov tawv nqaij tiv thaiv los ntawm UVB irradiation.
![i-inflammatory and antioxidant effects of Q-3-G. (a) Treatment of HaCaT cells with various concentrations of Q-3-G (0–20 μM) for 24 h, using a conventional MTT assay to determine cell viability. (b) HaCaT cells irradiated with UVB (30 mJ/cm3) for 10 s in the absence or presence of Q-3-G (0–20 μM), followed by incubation for 24 h. A conventional MTT assay was used to determine cell viability. (c,d) HaCaT cells were treated with Q-3-G (5, 10 μM) and UVB irradiation (30 mJ/cm3) for 24 h. The representative cell morphology of Q-3-G against UVB was observed via microscopy, and the number of cells was then plotted. (e,f) Anti-inflammatory effect of Q-3-G in HaCaT cells. The HaCaT cells were irradiated with UVB (30 mJ/cm3) for 10 s or incubated with H2O2 (500 μM) with or without Q-3-G (5–10 μM) and further incubated for 12 h. The mRNA expression levels of COX-2 (e) and TNF-α (f) were determined by quantitative real-time PCR. GAPDH was used as a housekeeping gene. (g–j) Antioxidant effect of Q-3-G in HaCaT cells. Q-3-G (0–40 μM) was reacted with 2,2-diphenylpicrylhydrazyl (DPPH) in the dark at 37°C for 30 min. The absorbance at 517 nm was measured spectrophotometrically (g). Q-3-G was reacted with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) at different concentrations in the dark at 37°C for 30 min. The absorbance at 730 nm was measured spectrophotometrically (h). Ascorbic acid was used as a control substance. (i) The HaCaT cells were irradiated with UVB (30 mJ/cm3) for 10 s with or without Q-3-G (5– 10 μM) and after incubating for 12 h, the mRNA level of Nrf2 was determined by quantitative realtime PCR. GAPDH was used as a housekeeping gene. (j) The representative result of intracellular ROS production was measured by flow cytometry using DCFDA staining in HaCaT cells with or without UVB irradiation (30 mJ/cm3) and Q-3-G (0-10 μM) were treated for 24 h. Results (a,b,d–i) are expressed as mean ± SD. # p < 0.05 and ## p < 0.01 compared to normal group (no treatment), and * p < 0.05 and ** p < 0.01 compared to inducer alone (UVB, H2O2, DPPH, or ABTS). 2.2. Antimelanogenesis Effect in B16F10 and Moisturizing Effect in HaCaT Cells of Q-3-G To examine the effects of Q-3-G on melanogenesis, the melanoma cells were treated with α-MSH for 48 h to secrete melanin in B16F10 cells. By employing MTT assays, we first confirmed that Q-3-G had no cytotoxicity in B16F10 cells at concentrations up to 20 μM for 48 h (Figure 3a). This result showed, for the next assessments that the effects of Q- 3-G in B16F10 were not due to cell death. Next, we evaluated B16F10 with Q-3-G and α- MSH for 48 h to determine the effects of Q-3-G on melanogenesis. As shown in Figure 3b, Q-3-G significantly inhibited melanin secretion. The positive control arbutin also significantly decreased melanin secretion. The skin moisture-related molecule is hyaluronic acid (HA). As previously mentioned, HA, distributed throughout the epidermis and dermis, is an NMF that enhances skin hydration [55]. We used a reverse transcription PCR (RT-PCR) assay to examine the transcription level of HAS-1 after 12 h with a concentration of Q-3-G of up to 30 μM. HAS promotes the accumulation of intermediate-sized HA within keratinocytes [12]. As shown in Figure 3c, the expression of HAS-1 was increased by treatment with Q-3-G up to 10 μM. In addition to the moisturizing factor, we also examined the expression of FLG and TGM- 1. We used retinol as a positive control group. The transcription levels of FLG and TGM-1 increased notably after treatment with Q-3-G at concentrations up to 10 μM (Figure 3d). Figure 2. Anti-inflammatory and antioxidant effects of Q-3-G. (a) Treatment of HaCaT cells with various concentrations of Q-3-G (0–20 µM) for 24 h, using a conventional MTT assay to determine cell viability. (b) HaCaT cells irradiated with UVB (30 mJ/cm3 ) for 10 s in the absence or presence of Q-3-G (0–20 µM), followed by incubation for 24 h. A conventional MTT assay was used to determine cell viability. (c,d) HaCaT cells were treated with Q-3-G (5, 10 µM) and UVB irradiation (30 mJ/cm3 ) for 24 h. The representative cell morphology of Q-3-G against UVB was observed via microscopy, and the number of cells was then plotted.](/Content/uploads/2022842169/2022032111072748381b7599e14e8e8a252699414e284f.png "i-inflammatory and antioxidant effects of Q-3-G. (a) Treatment of HaCaT cells with various concentrations of Q-3-G (0–20 μM) for 24 h, using a conventional MTT assay to determine cell viability. (b) HaCaT cells irradiated with UVB (30 mJ/cm3) for 10 s in the absence or presence of Q-3-G (0–20 μM), followed by incubation for 24 h. A conventional MTT assay was used to determine cell viability. (c,d) HaCaT cells were treated with Q-3-G (5, 10 μM) and UVB irradiation (30 mJ/cm3) for 24 h. The representative cell morphology of Q-3-G against UVB was observed via microscopy, and the number of cells was then plotted. (e,f) Anti-inflammatory effect of Q-3-G in HaCaT cells. The HaCaT cells were irradiated with UVB (30 mJ/cm3) for 10 s or incubated with H2O2 (500 μM) with or without Q-3-G (5–10 μM) and further incubated for 12 h. The mRNA expression levels of COX-2 (e) and TNF-α (f) were determined by quantitative real-time PCR. GAPDH was used as a housekeeping gene. (g–j) Antioxidant effect of Q-3-G in HaCaT cells. Q-3-G (0–40 μM) was reacted with 2,2-diphenylpicrylhydrazyl (DPPH) in the dark at 37°C for 30 min. The absorbance at 517 nm was measured spectrophotometrically (g). Q-3-G was reacted with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) at different concentrations in the dark at 37°C for 30 min. The absorbance at 730 nm was measured spectrophotometrically (h). Ascorbic acid was used as a control substance. (i) The HaCaT cells were irradiated with UVB (30 mJ/cm3) for 10 s with or without Q-3-G (5– 10 μM) and after incubating for 12 h, the mRNA level of Nrf2 was determined by quantitative realtime PCR. GAPDH was used as a housekeeping gene. (j) The representative result of intracellular ROS production was measured by flow cytometry using DCFDA staining in HaCaT cells with or without UVB irradiation (30 mJ/cm3) and Q-3-G (0-10 μM) were treated for 24 h. Results (a,b,d–i) are expressed as mean ± SD. # p < 0.05 and ## p < 0.01 compared to normal group (no treatment), and * p < 0.05 and ** p < 0.01 compared to inducer alone (UVB, H2O2, DPPH, or ABTS). 2.2. Antimelanogenesis Effect in B16F10 and Moisturizing Effect in HaCaT Cells of Q-3-G To examine the effects of Q-3-G on melanogenesis, the melanoma cells were treated with α-MSH for 48 h to secrete melanin in B16F10 cells. By employing MTT assays, we first confirmed that Q-3-G had no cytotoxicity in B16F10 cells at concentrations up to 20 μM for 48 h (Figure 3a). This result showed, for the next assessments that the effects of Q- 3-G in B16F10 were not due to cell death. Next, we evaluated B16F10 with Q-3-G and α- MSH for 48 h to determine the effects of Q-3-G on melanogenesis. As shown in Figure 3b, Q-3-G significantly inhibited melanin secretion. The positive control arbutin also significantly decreased melanin secretion. The skin moisture-related molecule is hyaluronic acid (HA). As previously mentioned, HA, distributed throughout the epidermis and dermis, is an NMF that enhances skin hydration [55]. We used a reverse transcription PCR (RT-PCR) assay to examine the transcription level of HAS-1 after 12 h with a concentration of Q-3-G of up to 30 μM. HAS promotes the accumulation of intermediate-sized HA within keratinocytes [12]. As shown in Figure 3c, the expression of HAS-1 was increased by treatment with Q-3-G up to 10 μM. In addition to the moisturizing factor, we also examined the expression of FLG and TGM- 1. We used retinol as a positive control group. The transcription levels of FLG and TGM-1 increased notably after treatment with Q-3-G at concentrations up to 10 μM (Figure 3d). Figure 2. Anti-inflammatory and antioxidant effects of Q-3-G. (a) Treatment of HaCaT cells with various concentrations of Q-3-G (0–20 µM) for 24 h, using a conventional MTT assay to determine cell viability. (b) HaCaT cells irradiated with UVB (30 mJ/cm3 ) for 10 s in the absence or presence of Q-3-G (0–20 µM), followed by incubation for 24 h. A conventional MTT assay was used to determine cell viability. (c,d) HaCaT cells were treated with Q-3-G (5, 10 µM) and UVB irradiation (30 mJ/cm3 ) for 24 h. The representative cell morphology of Q-3-G against UVB was observed via microscopy, and the number of cells was then plotted.")
Qhov tshwm sim thiab exacerbation ntawm o nyob rau hauv lub cev qee zaus qhib macrophages thiab tso qee cov cytokines. Inflammation yog ib qho cim ntawm oxidative kev nyuaj siab. Peb tau tshawb xyuas cov tshuaj tiv thaiv kab mob Q-3-G tiv thaiv UVB lossis HO2- ua rau mob. Nws paub zoo tias UVB tuaj yeem txhawb DNA kev puas tsuaj thiab o ntawm daim tawv nqaij 【10】. Peb irradiated HaCaT hlwb nrog UVB 30mJ / cm² thiab incubated lub HaCaT hlwb nrog H2O2 (500 uM). Peb tau ua haujlwm PCR lub sijhawm tiag tiag los kuaj xyuas mRNA qhia ntawm inflammatory enzyme COX-2 thiab pro-inflammatory cytokine TNF- . Raws li pom los ntawm cov txiaj ntsig hauv daim duab 2e,f, ib qho 10 μM concentration ntawm O-3-G cuam tshuam rau mRNA qhia ntawm COX-2 thiab TNF- . Yog li ntawd, Q-3-G tshem tawm cov lus teb inflammatory induced hauv HaCaT hlwb tom qab raug UVB irradiation los yog H2O2.
Peb tau siv 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) thiab 2,2-diphenyl-lpicrylhydrazyl(DPPH) radical scavenging assays—-cov qauv siv dav dav los soj ntsuam cov kev ua ub no hauv vitro ntawm natural compounds - los soj ntsuam cov khoom antioxidant ntawm Q-3-G ntawm ntau qhov sib txawv. HaCaT hlwb raug kho nrog Q-3-G nyob rau hauv ib tug concentration-dependent yam ntawm 2.5-40 μM nyob rau hauv lub xub ntiag ntawm DPPH, thiab qhov tshwm sim pom ib tug tseem ceeb scavenging kev ua si ntawm ntau tshaj 2.5 uM (Daim duab 2g) . Qhov kev ntsuam xyuas ABTS kuj tseem pom tau tias muaj txiaj ntsig zoo ntawm Q-3-G ntawm cov dawb radical scavenging nyob rau hauv concentration-dependent yam ntxwv nrog IC50 tus nqi ntawm 1.75 μM (Daim duab 2h). Ascorbic acid (AA), raws li kev tswj hwm zoo, tau pom cov tshuaj tiv thaiv antioxidant zoo tshaj plaws hauv DPPH thiab ABTS kev soj ntsuam. Nuclear factor erythroid 2-related factor 2 (Nrf2), ib qho ntawm cov tshuaj tiv thaiv antioxidant (ARE)-nyob ntawm cov noob, tau raug tshaj tawm los tswj cov kev tiv thaiv oxidoreductases thiab nws cov nucleophilic substrates, yog li txhawb cov antioxidant enzymes kom tshem tawm kev puas tsuaj thiab kho. systems [53]. Tom ntej no, peb tshuaj xyuas cov theem qhia ntawm Nrf2 hauv UVB-irradiated HaCaT hlwb. Ntxiv dag zog rau cov kev tshawb pom saum toj no, O-3-G tau txhim kho qhov kev qhia theem ntawm Nrf2 raws li UVB irradiation piv rau kev tswj nyob rau hauv ib tug concentration-dependent yam, mus txog 10 uM(Figure2i), uas txhais tau tias Q-3- G nthuav tawm cov nyhuv antioxidant ob qho tib si tshuaj lom neeg thiab lom nrog cov khoom siv dawb radical scavenging thiab ua rau kom muaj kev tiv thaiv ntawm Nrf2-raws li kev taw qhia, raws li. Tsis tas li ntawd, peb kuj tau ua ROS flow cytometry siv dichlorodihydrofluorescein diacetate (DCFDA) hauv UV mob. Cov tshuaj antioxidant yog dav paub los tiv thaiv cellular stressors los ntawm inhibiting ROS ntau lawm [54]. Cov txiaj ntsig tau pom tias kev kho mob nrog Q-3-G ntawm qhov siab ntawm 5 thiab 10 μM txo qis qib ntawm UVB-induced ROS (Daim duab 2j). Ua ke, cov ntaub ntawv no qhia tau hais tias Q-3-Gexerts cov khoom tiv thaiv tawv nqaij xws li tshuaj tiv thaiv kab mob thiab antioxidant tiv thaiv H2O, lossis UVB-induced oxidative stress thiab o hauv HaCaT hlwb.
![A level of Nrf2 was determined by quantitative realtime PCR. GAPDH was used as a housekeeping gene. (j) The representative result of intracellular ROS production was measured by flow cytometry using DCFDA staining in HaCaT cells with or without UVB irradiation (30 mJ/cm3) and Q-3-G (0-10 μM) were treated for 24 h. Results (a,b,d–i) are expressed as mean ± SD. # p < 0.05 and ## p < 0.01 compared to normal group (no treatment), and * p < 0.05 and ** p < 0.01 compared to inducer alone (UVB, H2O2, DPPH, or ABTS). 2.2. Antimelanogenesis Effect in B16F10 and Moisturizing Effect in HaCaT Cells of Q-3-G To examine the effects of Q-3-G on melanogenesis, the melanoma cells were treated with α-MSH for 48 h to secrete melanin in B16F10 cells. By employing MTT assays, we first confirmed that Q-3-G had no cytotoxicity in B16F10 cells at concentrations up to 20 μM for 48 h (Figure 3a). This result showed, for the next assessments that the effects of Q- 3-G in B16F10 were not due to cell death. Next, we evaluated B16F10 with Q-3-G and α- MSH for 48 h to determine the effects of Q-3-G on melanogenesis. As shown in Figure 3b, Q-3-G significantly inhibited melanin secretion. The positive control arbutin also significantly decreased melanin secretion. The skin moisture-related molecule is hyaluronic acid (HA). As previously mentioned, HA, distributed throughout the epidermis and dermis, is an NMF that enhances skin hydration [55]. We used a reverse transcription PCR (RT-PCR) assay to examine the transcription level of HAS-1 after 12 h with a concentration of Q-3-G of up to 30 μM. HAS promotes the accumulation of intermediate-sized HA within keratinocytes [12]. As shown in Figure 3c, the expression of HAS-1 was increased by treatment with Q-3-G up to 10 μM. In addition to the moisturizing factor, we also examined the expression of FLG and TGM- 1. We used retinol as a positive control group. The transcription levels of FLG and TGM-1 increased notably after treatment with Q-3-G at concentrations up to 10 μM (Figure 3d). Figure 2. Anti-inflammatory and antioxidant effects of Q-3-G. (a) Treatment of HaCaT cells with various concentrations of Q-3-G (0–20 µM) for 24 h, using a conventional MTT assay to determine cell viability. (b) HaCaT cells irradiated with UVB (30 mJ/cm3 ) for 10 s in the absence or presence of Q-3-G (0–20 µM), followed by incubation for 24 h. A conventional MTT assay was used to determine cell viability. (c,d) HaCaT cells were treated with Q-3-G (5, 10 µM) and UVB irradiation (30 mJ/cm3 ) for 24 h. The representative cell morphology of Q-3-G against UVB was observed via microscopy, and the number of cells was then plotted. (e,f) Anti-inflammatory effect of Q-3-G in HaCaT cells. The HaCaT cells were irradiated with UVB (30 mJ/cm3 ) for 10 s or incubated with H2O2 (500 µM) with or without Q-3-G (5–10 µM) and further incubated for 12 h. The mRNA expression levels of COX-2 (e) and TNF-α (f) were determined by quantitative real-time PCR. GAPDH was used as a housekeeping gene. (g–j) Antioxidant effect of Q-3-G in HaCaT cells. Q-3-G (0–40 µM) was reacted with 2,2- diphenylpicrylhydrazyl (DPPH) in the dark at 37 ◦C for 30 min. The absorbance at 517 nm was measured spectrophotometrically (g). Q-3-G was reacted with 2,2-azinobis-(3-ethylbenzothiazoline-6- sulfonic acid (ABTS) at different concentrations in the dark at 37 ◦C for 30 min. The absorbance at 730 nm was measured spectrophotometrically (h). Ascorbic acid was used as a control substance. (i) The HaCaT cells were irradiated with UVB (30 mJ/cm3 ) for 10 s with or without Q-3-G (5–10 µM) and after incubating for 12 h, the mRNA level of Nrf2 was determined by quantitative real-time PCR. GAPDH was used as a housekeeping gene. (j) The representative result of intracellular ROS production was measured by flow cytometry using DCFDA staining in HaCaT cells with or without UVB irradiation (30 mJ/cm3 ) and Q-3-G (0-10 µM) were treated for 24 h. Results (a,b,d–i) are expressed as mean ± SD. # p < 0.05 and ## p < 0.01 compared to normal group (no treatment), and * p < 0.05 and ** p < 0.01 compared to inducer alone (UVB, H2O2 , DPPH, or ABTS)](/Content/uploads/2022842169/202203211108455bff4d0223ef483dbe3172e2d3349d71.png "A level of Nrf2 was determined by quantitative realtime PCR. GAPDH was used as a housekeeping gene. (j) The representative result of intracellular ROS production was measured by flow cytometry using DCFDA staining in HaCaT cells with or without UVB irradiation (30 mJ/cm3) and Q-3-G (0-10 μM) were treated for 24 h. Results (a,b,d–i) are expressed as mean ± SD. # p < 0.05 and ## p < 0.01 compared to normal group (no treatment), and * p < 0.05 and ** p < 0.01 compared to inducer alone (UVB, H2O2, DPPH, or ABTS). 2.2. Antimelanogenesis Effect in B16F10 and Moisturizing Effect in HaCaT Cells of Q-3-G To examine the effects of Q-3-G on melanogenesis, the melanoma cells were treated with α-MSH for 48 h to secrete melanin in B16F10 cells. By employing MTT assays, we first confirmed that Q-3-G had no cytotoxicity in B16F10 cells at concentrations up to 20 μM for 48 h (Figure 3a). This result showed, for the next assessments that the effects of Q- 3-G in B16F10 were not due to cell death. Next, we evaluated B16F10 with Q-3-G and α- MSH for 48 h to determine the effects of Q-3-G on melanogenesis. As shown in Figure 3b, Q-3-G significantly inhibited melanin secretion. The positive control arbutin also significantly decreased melanin secretion. The skin moisture-related molecule is hyaluronic acid (HA). As previously mentioned, HA, distributed throughout the epidermis and dermis, is an NMF that enhances skin hydration [55]. We used a reverse transcription PCR (RT-PCR) assay to examine the transcription level of HAS-1 after 12 h with a concentration of Q-3-G of up to 30 μM. HAS promotes the accumulation of intermediate-sized HA within keratinocytes [12]. As shown in Figure 3c, the expression of HAS-1 was increased by treatment with Q-3-G up to 10 μM. In addition to the moisturizing factor, we also examined the expression of FLG and TGM- 1. We used retinol as a positive control group. The transcription levels of FLG and TGM-1 increased notably after treatment with Q-3-G at concentrations up to 10 μM (Figure 3d). Figure 2. Anti-inflammatory and antioxidant effects of Q-3-G. (a) Treatment of HaCaT cells with various concentrations of Q-3-G (0–20 µM) for 24 h, using a conventional MTT assay to determine cell viability. (b) HaCaT cells irradiated with UVB (30 mJ/cm3 ) for 10 s in the absence or presence of Q-3-G (0–20 µM), followed by incubation for 24 h. A conventional MTT assay was used to determine cell viability. (c,d) HaCaT cells were treated with Q-3-G (5, 10 µM) and UVB irradiation (30 mJ/cm3 ) for 24 h. The representative cell morphology of Q-3-G against UVB was observed via microscopy, and the number of cells was then plotted. (e,f) Anti-inflammatory effect of Q-3-G in HaCaT cells. The HaCaT cells were irradiated with UVB (30 mJ/cm3 ) for 10 s or incubated with H2O2 (500 µM) with or without Q-3-G (5–10 µM) and further incubated for 12 h. The mRNA expression levels of COX-2 (e) and TNF-α (f) were determined by quantitative real-time PCR. GAPDH was used as a housekeeping gene. (g–j) Antioxidant effect of Q-3-G in HaCaT cells. Q-3-G (0–40 µM) was reacted with 2,2- diphenylpicrylhydrazyl (DPPH) in the dark at 37 ◦C for 30 min. The absorbance at 517 nm was measured spectrophotometrically (g). Q-3-G was reacted with 2,2-azinobis-(3-ethylbenzothiazoline-6- sulfonic acid (ABTS) at different concentrations in the dark at 37 ◦C for 30 min. The absorbance at 730 nm was measured spectrophotometrically (h). Ascorbic acid was used as a control substance. (i) The HaCaT cells were irradiated with UVB (30 mJ/cm3 ) for 10 s with or without Q-3-G (5–10 µM) and after incubating for 12 h, the mRNA level of Nrf2 was determined by quantitative real-time PCR. GAPDH was used as a housekeeping gene. (j) The representative result of intracellular ROS production was measured by flow cytometry using DCFDA staining in HaCaT cells with or without UVB irradiation (30 mJ/cm3 ) and Q-3-G (0-10 µM) were treated for 24 h. Results (a,b,d–i) are expressed as mean ± SD. # p < 0.05 and ## p < 0.01 compared to normal group (no treatment), and * p < 0.05 and ** p < 0.01 compared to inducer alone (UVB, H2O2 , DPPH, or ABTS)")
2.2.Antimelanogenesis Effect in B16F10 thiab Moisturizing Effect in HaCaT Cells of Q-3-G
Txhawm rau tshuaj xyuas qhov cuam tshuam ntawm O-3-G ntawm melanogenesis, cov hlwb melanoma tau kho nrog -MSH rau 48 teev kom tso melanin hauv B16F10 hlwb. Los ntawm kev ua haujlwm MTT kev soj ntsuam, peb thawj zaug tau lees paub tias Q-3-G tsis muaj cytotoxicity hauv B16F10 hlwb ntawm qhov siab txog 20 μM rau 48 h (Daim duab 3a). Cov txiaj ntsig no tau pom, rau kev ntsuam xyuas tom ntej tias qhov cuam tshuam ntawm O-3-G hauv B16F10 tsis yog vim kev tuag ntawm tes. Tom ntej no, peb ntsuas B16F10 nrog Q-3-Gand -MSH rau 48 teev los txiav txim qhov cuam tshuam ntawm Q-3-G ntawm melanogenesis. Raws li pom hauv daim duab 3b, Q-3-G inhibited melanin secretion heev. Qhov zoo tswj arbutin kuj txo qis melanin secretion.
Cov molecules ntawm daim tawv nqaij molecules yog hyaluronic acid (HA). Raws li tau hais dhau los, HA, faib thoob plaws hauv daim tawv nqaij thiab cov tawv nqaij, yog NMF uas txhim kho cov tawv nqaij hydration [55]. Peb tau siv qhov kev hloov pauv rov qab PCR (RT-PCR) kev ntsuas los tshuaj xyuas qib kev hloov pauv ntawm HAS-1 tom qab 12 teev nrog qhov siab ntawm Q-3-G txog li 30 uM. HAS txhawb kev sib sau ntawm nruab nrab-loj HA hauv keratinocytes [12]. Raws li pom hauv daim duab 3c, qhov kev qhia ntawm HAS{10}} tau nce los ntawm kev kho nrog Q-3-G mus txog 10 μM.Ntxiv rau qhov moisturizing yam, peb kuj tau tshuaj xyuas cov lus qhia ntawm FLG thiab TGM{{ 13}} ib. Peb tau siv retinol los ua pawg tswj hwm zoo. Cov qib kev hloov pauv ntawm FLG thiab TGM{14}} tau nce ntxiv tshwj xeeb tom qab kho nrog Q-3-G ntawm qhov siab txog li 10 μM (Daim duab 3d). Cov kev tshawb pom no qhia tias Q-3-G muaj cov dej noo ntawm daim tawv nqaij hauv HaCaT hlwb. Ntxiv mus, siv SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), UO126 (ERK inhibitor), thiab Bay11-7082 (NF-kB inhibitor), peb tau tshuaj xyuas qhov kev koom tes ntawm MAPK thiab NF-kB cov cim qhia txoj hauv kev moisturizing. qhov tseem ceeb ntawm Q-3-G-kho HaCaT hlwb. Raws li O-3-G-induced gene qhia, ob qho tib si theem ntawm TGM-1 thiab FLG raug txo los ntawm kev kho mob nrog SP600125, JNK inhibitor (Daim duab 3e), qhia tias JNK yuav tsum tau nce qhov kev qhia. ntawm TGM-1 thiab FLG raws li daim tawv nqaij moisturizing nyhuv ntawm O-3-G. Tsis tas li ntawd, FLG kev qhia kuj tau txo qis los ntawm Bay11-7082, ib qho inhibitor ntawm IKK thiab IkB , qhia tias cov tshuaj siv tshuaj ntawm Q-3-G kuj tuaj yeem koom nrog NF-kB kev taw qhia txoj hauv kev.
2.3.Moisturizing-Related Signaling Pathways of Q-3-G ntawm Kev Ua Haujlwm ntawm AP-1 thiab NF-kB
Raws li cov txiaj ntsig mRNA yav dhau los, peb xav tias qhov kev koom tes ntawm AP-1 thiab NF-kB teeb liab ntawm qhov dej ntawm Q-3-G tuaj yeem tshwm sim hauv cov qib protein. Siv Western blot assay, peb tshuaj xyuas cov phosphorylation ntawm cov dej ntws ntawm cov molecules ntawm AP-1 thiab NF-kB. Ua ntej, hauv AP-1 txoj hauv kev, phosphorylation ntawm c-Jun tau nce tom qab incubation ntawm HaCaT hlwb nrog Q-3-G rau 24 h (Daim duab 4a). Qib ntawm p-INK kuj tau nce ntxiv tom qab kev kho mob nrog Q-3-G ntawm qhov siab ntawm 2.5,5 thiab 10 μM. Tag nrho daim ntawv ntawm JNK tseem tsis hloov pauv (Daim duab 4b). Peb tau tshuaj xyuas ntxiv txog phosphorylation ntawm MKK4 thiab TAK{18}}, uas yog cov tswj hwm ntawm JNK. Raws li pom nyob rau hauv daim duab 3c, tom qab kev kho mob ntawm HaCaT hlwb nrog O-3-G, cov protein ntau ntawm p-MKK4 thiab p-TAK1 kuj tau kho.
Ntxiv nrog rau NF-kB signaling, Q-3-G tau nce phosphorylation ntawm p50 thiab p65, ib subunit ntawm NF-kB (Daim duab 4c). Ntxiv mus, peb tau ntsuas qhov ua kom IKK thiab IkBa, uas kuj tau nce los ntawm Q-3-G txog 10 uM piv rau pawg tswj hwm (Daim duab 4d). Thaum kawg, peb tau txiav txim siab ua kom cov dej ntws ntws mus rau hauv lub NF-kB teeb liab txoj hauv kev. Lub phosphorylation ntawm Src thiab Akt (Ser473) tau txhim kho los ntawm O-3-G kev kho (Daim duab 4e). Zuag qhia tag nrho, cov ntaub ntawv no qhia tias cov kev tiv thaiv ntawm daim tawv nqaij ntawm Q-3-G lub hom phiaj TAK1 thiab Src ua kom los ntawm kev nce qib protein, yog li txhawb kev ua haujlwm ntawm AP-1 thiab NF-kB, raws li.
2.4.Effects ntawm Q-3-G ntawm Transcriptional Activation ntawm AP-1 thiab NF-kB
Peb tau tshawb xyuas cov cell cytotoxicity ntawm Q-3-G hauv tib neeg lub raum kab mob raum kab mob HEK293T hlwb los ntawm kev soj ntsuam MTT (Daim duab 4f). Kev muaj peev xwm ntawm HEK293T hlwb tsis cuam tshuam loj heev tom qab kho nrog Q-3-G(2.5-20 μM) piv rau cov hlwb tsis kho.
Peb ua haujlwm ib qho kev ntsuam xyuas luciferase los txiav txim seb Q-3-G tuaj yeem hloov kho NF-kB thiab AP-1 kev soj ntsuam tus neeg txhawb nqa. Peb tau lees paub tias O-3-G nce AP-1-kev ua haujlwm nruab nrab ntawm luciferase (Daim duab 4g) thiab NF-kB-kB-mediated luciferase kev ua (Daim duab 4h) nyob rau hauv ib qho concentration-dependent yam. Cov txiaj ntsig no tau lees paub tias AP-1 ua kom thiab NF-KB ua kom muaj txiaj ntsig tseem ceeb ntawm lub hom phiaj tshuaj ntawm Q-3-G.
Nyem qhov txuas kom tau txais cov ntaub ntawv ntxiv:https://www.xjcistanche.com/news/part-2-anti-inflammatory-antioxidant-moistu-55118278.html
