Dio 1: Anti-inflamatorni, antioksidativni, hidratantni i antimelanogenezni efekti kvercetina 3-O- -D-glukuronida u ljudskim keratinocitima i ćelijama melanoma putem aktivacije NF-κB i AP-1 puteva
Mar 21, 2022
Za više informacija kontaktirajtetina.xiang@wecistanche.com
Abstract: kvercetin3-O- -D-glukuronid(Q-3-G), glukuronidni konjugat kvercetina, prijavljeno je da imaprotuupalnosvojstva u makrofagima stimuliranim lipopolisaharidom, kao i antikancerogena i antioksidativna svojstva. Za razliku od kvercetina, za koji je opširno opisano da posjeduje širok spektar farmakoloških aktivnosti, uključujući zaštitne efekte kože, farmakološke prednosti i mehanizmi Q-3-G u koži ostaju da se razjasne. Ova studija se fokusirala na karakterizaciju zaštitnih svojstava kože, uključujući antiinflamatorna i antioksidativna svojstva, Q-3-protiv UVB-indukovanog ili H2O{6}}indukovanog oksidativnog stresa, efekata hidratacije iantimelanogenezaaktivnosti pomoću ljudskih keratinocita (HaCaT) i ćelija melanoma (B16F10). Q-3-G je smanjio ekspresiju proinflamatornog gena i citokina kao što je ciklooksigenaza-2 (COX-2) i faktor nekroze tumora (TNF)- u HO, ili UVB- ozračene HaCaT ćelije. Također smo pokazali da O-3-Gexhibiti imaju antioksidativni efekat koristeći testove uklanjanja slobodnih radikala, protočnu citometriju i povećanu ekspresiju faktora 2 vezanog za nuklearni faktor eritroid 2- (Nrf2). Q-3-G je smanjio proizvodnju melanina u B16F10 ćelijama izazvanim -melanocit-stimulirajućim hormonom (-MSH). Efekti hidratacije i mehanizmi Q-3-G ispitani su procjenom gena vezanih za hidratantni faktor, kao što su transglutaminaza-1(TGM-1), filaggrin (FLG) i sintaza hijaluronske kiseline (IMA)-1. Osim toga, Q-3-G je povećao fosforilaciju c-Jun, Jun N-terminalne kinaze (JNK), mitogen-aktivirane protein kinaze (MAPK) kinaze 4 (MKK4) i TAK1, uključenih u MAPKs/AP -1 put, i fosforilacija IkB, IkB kinaze(IKK)-, Akt i Src, uključenih u NF-kB put. Uzeti zajedno, pokazali smo da Q-3-G ispoljava antiinflamatorna, antioksidativna, hidratantna i antimelanogeneza svojstva u ljudskim keratinocitima i ćelijama melanoma putem NF-kB i AP-1 puteva.
Ključne riječi: kvercetin 3-O- -D-glukuronid; UV zaštita; hidratantna; antimelanogeneza; AP-1; NF-kB
1. Uvod
Koža, najveći organ u tijelu, nudi barijeru koja igra važnu ulogu u zaštiti tijela od vanjskog okruženja i štetnih faktora kao što su ultraljubičasto zračenje, infektivni mikrobi i štetne kemikalije. Ova barijera također reguliše ravnotežu elektrolita i temperaturu tijela i sprječava gubitak vlage [1,2]. Ljudska koža se sastoji od tri sloja: epidermisa, dermisa sa dodacima i potkožnog tkiva [3]. Epidermalni sloj igra važnu ulogu u održavanju tjelesne temperature.
Keratinociti, sastavni dio epiderme kože, usko stupaju u interakciju s drugim putem dezmozoma i čvrstih spojeva koji omogućavaju efikasnu fizičko-hemijsku barijeru[4]. Iako koža funkcionira kao zaštitna barijera, ultraljubičasto (UV) zračenje ima značajan učinak na većinu stanica, uključujući keratinocite, vanjski sloj kože. UV zračenje može uzrokovati starenje kože, oštećenje kože, bore i hiperpigmentaciju, što se smatra jednim od najcenzurnijih faktora rizika [5].UV svjetlo se sastoji od tri opsega talasnih dužina: UVA(320-400 nm), UVB (280-320 nm) i UVC (200-280 nm). Pored vodikovog peroksida (H2O2), za koji je poznato da doprinosi oksidativnom stresu, UVB može dovesti do proizvodnje reaktivnih vrsta kisika (ROS) kroz aktivaciju enzima koji stvaraju ROS [6]. Opisano je da nekontrolirano nakupljanje ROS-a u stanicama uključujući keratinocite uzrokuje oksidativne modifikacije lipida, proteina, nukleinskih kiselina i drugih intracelularnih molekula koje potom dovode do različitih vrsta oštećenja kože i procesa starenja kao što su smrt stanica, oksidativni stres, i upala [7,8]. Osim toga, poznato je da odgovor na oksidativni stres utječe na oštećenje različitih ćelijskih komponenti [9]. Oštećene stanice kože također mogu dovesti do upalnih odgovora, što rezultira kronično oštećenom kožom [10]. Jedna vrsta oštećenja kože posredovana UVB ili H2O2-biološkim procesima je ekspresija upalnih gena kao što su ciklooksigenaza-2 (COX-2) i proinflamatorni citokini [11]. Stoga je razvoj sigurnijih, robusnijih i efikasnijih zaštitnih efekata kože neophodan za prevenciju i blokiranje bolesti povezanih sa oštećenjem kože posredovanim UVB ili H2O2.
Hidratacija kože također igra važnu ulogu u održavanju njene normalne funkcije. Posebno, stratum corneum (SC) ima zaštitni učinak protiv gubitka vode [12]. Sadržaj vode u stratum corneum-u učestvuje u deskvamaciji kože i pravilnom sazrevanju, dok nedovoljna količina vode u SC potiče akumulaciju i disfunkciju korneocita [12,13]. U završnim fazama diferencijacije keratinocita, njegova plazma membrana i ćelijske organele nestaju, a zatim dotok kalcija inducira transglutaminazu (TGM) za umrežavanje s drugim proteinima. Nedostatak ili gubitak TGM-1 dovodi do neispravnog umrežavanja ćelijskog omotača. Lamela ihtioza je primjer bolesti uzrokovane gubitkom TGM-1. Nadalje, filaggrin (FLG), formirani keratinski matriks, igra ulogu u formiranju SC jer djeluje kao skela za vezanje proteina rožnate ovojnice i lipida[14] kataliziranjem reakcija umrežavanja g-glutamil lizina, ATGM-ova i membrane -povezani enzimi, koji daju integritet SC-ima [15]. Osim toga, hijaluronska kiselina (HA) je vrsta prirodnog faktora vlage (NMF) uključenog u zadržavanje vlage u koži i pokazuje poseban profil u unutrašnjem starenju kože, sredstvo je za isporuku lijekova i važna je komponenta ekstracelularnog matriksa. [16,17]. HA ima ulogu u povećanju vlažnosti kože regulacijom gena sintaze hijaluronske kiseline (HAS); osim toga, retinoična kiselina (vitamin A) može efikasno regulisati HA u epidermisu [17]. TGM-1, FLG i HAS-12,3 su geni povezani sa proizvodnjom NMF [18]. Štaviše, aktivacija faktora transkripcije kao što su protein aktivator-1(AP-1) i nuklearni faktor (NF)-kB uključeni su u proizvodnju proupalnih supstanci koje optimiziraju kožnu barijeru i hidrataciju kože kroz AP-1 i NF-kB puteve[19]. Uzvodni signalni enzimi u AP-1 putu uključuju protein kinaze aktivirane mitogenom (MAPK) kao što su c-Jun N-terminalna kinaza (JNK), ekstracelularna signalno regulirana kinaza (ERK) i p38, dok Src, Akt, IkB -kinaza (IKKo/) i KBO(IkBa) pripadaju NF-KB putu [20. Koža također proizvodi melanin u epidermalnom sloju, posebno u melanocitima – glavnom izvoru melanina – koji doprinose određivanju boje kože i melanogenezi [21]. UVB zračenje i hormon koji stimulira melanocite (o-MSH) mogu stimulirati lučenje melanina, čime pokreću melanogenezu [22. Tokom melanogeneze, -MSH aktivira protein kinazu A (PKA), transkripcijski faktor povezan sa proteinskom mikroftalmijom (MITF) i cAMP element za vezivanje (CREB)[23]. Smatra se da je jedna od funkcija melanina sprečavanje oštećenja kože od UV zračenja, uključujući i sunčevu svjetlost. Međutim, prekomjerna proizvodnja melanina ili odsustvo sadržaja melanina uzrokovano starenjem, hormonom koji stimulira melanocite i izlaganjem UV zračenju izaziva pjege, melazmu, senilne lentigine i druge hiper/hipopigmentacijske poremećaje koji mogu negativno utjecati na izgled i kožu. zdravlje [24]. Stoga je kontrola melanogeneze poželjna i za održavanje zdravlja i kozmetičkog izgleda ljudskog tijela.
kvercetinje dominantni flavonoid tipa flavonola prisutan u povrću, voću, pićima i ljekovitom bilju [25]. Shodno tome, kvercetin je opsežno proučavan da posjeduje farmakološke prednosti, uključujući protuupalno, antiaterosklerotsko,antioksidans, antikancerogena i zaštitna svojstva kože, a povezana je s melanogenezom [24,26-29]. Međutim, nekoliko radova korištenjem osjetljivih i pouzdanih metoda pokazalo je da kvercetin nije otkriven u plazmi [30-33] i da se jedva nalazi u mozgu [34,35]. Kvercetin, koji se općenito predstavlja u obliku glikozida, dobro se apsorbira i uglavnom hidrolizira i metabolizira u tankom crijevu, debelom crijevu, jetri i bubrezima u konjugirane metabolite [30,36-38]. Samo konjugirani metaboliti kao što su glukuronid ili sulfat (metilirani ili nemetilirani), umjesto kvercetin aglikona ili njegovih glikozida, otkrivaju se u cirkulirajućoj krvi. 3/-metil-kvercetin-3-glukuronid, kvercetin-3-glukuronid i kvercetin{12}}'-sulfat su identifikovani kao glavni metaboliti u plazmi nakon 1,5 h od konzumiranja prženog luka[30, 32]. Ovi metaboliti kvercetina nastaju u tankom crijevu i jetri biotransformacijskim enzimima uključujući UDP-glukuroniltransferaze kao rezultat metabolizma faze II [29,39,40]. Također je objavljeno da se konjugirani metaboliti kvercetina akumuliraju u ljudskoj plazmi u rasponu koncentracija od 10-'do 10- stepena M nakon periodičnog uzimanja crnog luka uz obroke tokom 1 sedmice[41]. Poluživot metabolita kvercetina je prilično visok, oko 11 do 28 sati [29]. Budući da su ovi spojevi opaženi kao glavni oblici konjugiranih metabolita kvercetina, studije koje se fokusiraju na biološku aktivnost flavonola koristeći ove konjugirane metabolite bile bi ključne, što nas je potaknulo da proučavamo biološku aktivnost jednog od konjugiranih metabolita kvercetina. Jedan od najzastupljenijih metabolita glukuronida koji se nalazi uglavnom u plazmi i tkivima uključujući jetru, bubrege i mozak je kvercetin 3-O- -D-glukuronid(Q-3-G) (Slika 1) [31,35,40,42,43], koji mogu generisati slične, jače ili slabije aktivnosti u poređenju sa matičnim jedinjenjima u različitim modelima. Q-3-G se transportuje do ciljnih tkiva putem plazme da bi pokazao svoju biološku aktivnost [39]. O-3-G se takođe pokazao kao efikasan metabolit u plazmi štakora nakon oralne primjene kvercetina [43]. U drugim studijama, otkriveno je da se metabolit koji sadrži Q-3-akumulira u aorti nakon davanja hrane bogate kvercetinom i identificiran je kao aktivni princip otkriven u ljudskoj krvi u rasponu od 0.{{48} } μM【44】. Osim toga, Q-3-G se može naći u vinu [45] i u ljekovitim biljkama kao što su Hypericum hirsutum, Nelumbo nucifera, Oenothera biennis i mahune [46-49].Q-3-G je opisan kao antiinflamatorna komponenta u uslovima stimulisanim LPS-om i pokazao je antioksidativna svojstva u krvnoj plazmi [50-52]. Međutim, u poređenju sa dugom istorijom primjene i širokim proučavanjem kvercetina, studija Q-3-G ostaje da se istraži. Stoga je Q-3-G izabran kao predstavnik metabolita kvercetina kao aktivni in vivo igrač kvercetina. Osim toga, uloga Q-3-G u zaštitnim efektima kože također još nije u potpunosti razjašnjena. U ovoj studiji, koristeći različite in vitro procjene, istražili smo zaštitne efekte Q-3-G protiv UVBor H2O2-indukovanog oksidativnog stresa i upale, hidrataciju kože u HaCaT (ćelijska linija ljudskih keratinocita) ćelije, i aktivnost antimelanogeneze u ćelijama B16F10 (ćelijska linija mišjeg melanoma).
2. Rezultat
2.1.Protuupalni i antioksidativni efekti Q-3-G protiv UVB ili H2O2-stimuliranih HaCaT ćelija
Tkivo kože može biti oštećeno UV zračenjem. UV zračenje aktivira nekoliko štetnih faktora, uključujući oksidativni stres, apoptotičku smrt ćelija, upalu i starenje kože. Kako bismo odredili UV-zaštitne aktivnosti Q-3-G, na početku smo procijenili da li Q-3-G ima bilo kakve efekte na vitalnost ćelija ljudskih keratinocita. Koristeći 3-(4-5-dimetiltiazol-2-il)-2,5-difenil-243 tetrazolijum bromid (MTT) test, istražili smo citotoksične efekte od O-3-G u HaCa ćelijama. Kao što je prikazano na slici 2a, nije bilo značajnih smetnji na vitalnost ćelije pri koncentraciji od 2.5-20 uM od strane Q-3-G, što ukazuje da O-3-G nije pokazao citotoksičnost do 20 μM i nije oštetio keratinocite, površinski sloj ćelija kože. Zatim smo procijenili zaštitne efekte Q-3-G u HaCaT ćelijama izloženim UVB-u. Nakon UVB zračenja pri 30 mJ/cm², Q-3-G je značajno zaštitio HaCaT ćelije od UVB-indukovane ćelijske smrti na način ovisan o koncentraciji (slika 2b). U dogovoru s MTT testom, korištenjem kamere spojene na mikroskop za snimanje morfologije HaCaT ćelija, Q-3-G je zaštitio od ćelijske smrti zbog HaCaT ćelija ozračenih UVB-om do koncentracije od 10 μM (slika 2c) . Broj HaCaT ćelija pod svakim uslovom bio je 482 za netretiranu grupu, 91 za UVB 30 mJ/cm-grupu, 276 za UVB 30 mJ/cm² plus 5μM O-3-G grupu i 320 za UVB 30 mJ/cm² plus 10μM Q-3-G grupa (slika 2d). Ovi rezultati su potvrdili da Q-3-G inhibira oštećenje HaCaT izazvano UVB-om i spašava učinak zaštite kože od UVB zračenja.
![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.")
Pojava i pogoršanje upale u tijelu povremeno aktivira makrofage i oslobađa neke citokine. Upala je obilježje oksidativnog stresa. Istraživali smo antiinflamatorni efekat Q-3-G protiv UVB ili HO{2}}indukovane upale. Dobro je poznato da UVB može potaknuti oštećenje DNK i upalu u stanicama kože 【10】. Ozračili smo HaCaT ćelije sa UVB 30mJ/cm² i inkubirali HaCaT ćelije sa H2O2 (500 uM). Koristili smo PCR test u realnom vremenu da provjerimo ekspresiju mRNA upalnog enzima COX-2 i proinflamatornog citokina TNF-. Kao što pokazuju rezultati na slici 2e,f, koncentracija O-3-G od 10 μM značajno je inhibirala ekspresiju mRNA COX-2 i TNF-. Posljedično, Q-3-G je potisnuo inflamatorne odgovore inducirane u HaCaT ćelijama nakon izlaganja UVB zračenju ili H2O2.
Koristili smo 2,2'-azinobis-(3-etilbenzotiazolin-6-sulfonsku kiselinu)(ABTS) i 2,2-difenil-lpikrilhidrazil(DPPH) testove uklanjanja radikala – široko korišteni modelni sistemi istražiti aktivnosti čišćenja in vitro prirodnih jedinjenja – procijeniti antioksidativna svojstva Q-3-G u različitim koncentracijama. HaCaT ćelije su tretirane sa Q-3-G na način ovisan o koncentraciji od 2.5-40 μM u prisustvu DPPH, a rezultat je pokazao značajnu aktivnost čišćenja na više od 2,5 uM (Slika 2g) . ABTS test je također značajno pokazao efekte Q-3-G na uklanjanje slobodnih radikala na načine zavisne od koncentracije sa IC50 vrijednošću od 1,75 μM (Slika 2h). Askorbinska kiselina （AA）, kao pozitivna kontrola, pokazala je najveće antioksidativne efekte u DPPH i ABTS testovima. Faktor 2 (Nrf2), jedan od gena zavisnih od elementa antioksidativnog odgovora (ARE), reguliše zaštitne oksidoreduktaze i njene nukleofilne supstrate, promovišući antioksidativne enzime za uklanjanje oštećenja i popravku. sistemi [53]. Zatim smo ispitali nivo ekspresije Nrf2 u UVB ozračenim HaCaT ćelijama. Pojačavajući gore navedene nalaze, O-3-G je značajno povećao nivo ekspresije Nrf2 nakon UVB zračenja u poređenju sa kontrolom na način ovisan o koncentraciji, do 10 uM (Slika 2i), što implicira da je Q-3- G ima antioksidativni efekat i hemijski i biološki sa svojstvom uklanjanja slobodnih radikala i povećava zaštitni efekat ARE signalizacije zavisne od Nrf2-. Nadalje, izveli smo i ROS protočnu citometriju korištenjem dihlorodihidrofluorescein diacetata (DCFDA) u UV uvjetima. Poznato je da antioksidans potiskuje ćelijske stresore inhibiranjem proizvodnje ROS-a [54]. Rezultat je pokazao da tretman sa Q-3-G u koncentracijama od 5 i 10 μM smanjuje nivo UVB-indukovanog ROS-a (slika 2j). Uzeti zajedno, ovi podaci impliciraju da Q-3-Geksira zaštitna svojstva kože, kao što su antiinflamatorni i antioksidativni efekti protiv H2O, ili UVB izazvanog oksidativnog stresa i upale u HaCaT ćelijama.
![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.Efekat antimelanogeneze u B16F10 i hidratantni efekat u HaCaT ćelijama Q-3-G
Da bi se ispitali efekti O-3-G na melanogenezu, ćelije melanoma su tretirane sa -MSH tokom 48 sati kako bi se lučili melanin u B16F10 ćelijama. Koristeći MTT testove, prvo smo potvrdili da Q-3-G nema citotoksičnost u B16F10 ćelijama pri koncentracijama do 20 μM tokom 48 h (Slika 3a). Ovaj rezultat je za naredne procjene pokazao da efekti O-3-G u B16F10 nisu posljedica smrti ćelije. Zatim smo procijenili B16F10 sa Q-3-Gand-MSH tokom 48 h da bismo odredili efekte Q-3-G na melanogenezu. Kao što je prikazano na slici 3b, Q-3-G je značajno inhibirao lučenje melanina. Arbutin pozitivne kontrole također je značajno smanjio lučenje melanina.
Molekul vezan za vlagu kože je hijaluronska kiselina (HA). Kao što je ranije spomenuto, HA, raspoređena po epidermisu i dermisu, je NMF koji poboljšava hidrataciju kože [55]. Koristili smo PCR test reverzne transkripcije (RT-PCR) da ispitamo nivo transkripcije HAS-1 nakon 12 h sa koncentracijom Q-3-G do 30 uM. HAS podstiče akumulaciju HA srednje veličine unutar keratinocita [12]. Kao što je prikazano na slici 3c, ekspresija HAS-1 je povećana tretmanom sa Q-3-G do 10 μM. Pored hidratantnog faktora, ispitali smo i ekspresiju FLG i TGM{{ 13}}. Koristili smo retinol kao pozitivnu kontrolnu grupu. Nivoi transkripcije FLG i TGM-1 su se značajno povećali nakon tretmana sa Q-3-G u koncentracijama do 10 μM (Slika 3d). Ovi nalazi ukazuju na to da Q-3-G ima aktivnost hidratacije kože u HaCaT ćelijama. Štaviše, koristeći SB203580 (p38 inhibitor), SP600125 (inhibitor JNK), UO126 (ERK inhibitor) i Bay11-7082 (inhibitor NF-kB), ispitali smo učešće MAPK i NF-kB signalnih puteva u hidrataciji faktor Q-3-G-tretiranih HaCaT ćelija. Na osnovu ekspresije gena izazvane O-3-G, i nivoi TGM-1 i FLG su smanjeni tretmanom sa SP600125, JNK inhibitorom (slika 3e), što sugerira da je JNK potreban za povećanje ekspresije TGM-1 i FLG u smislu hidratantnog efekta O-3-G. Pored toga, ekspresiju FLG je također smanjio Bay11-7082, inhibitor IKK i IkB, što sugerira da bi farmaceutski mehanizam Q-3-G također mogao pripadati NF-kB signalnom putu.
2.3. Signalni putevi Q-3-G vezani za hidrataciju putem aktivacije AP-1 i NF-kB
Na osnovu prethodnih rezultata mRNK, pretpostavili smo da se uključenost AP-1 i NF-kB signalizacije na efekat hidratacije Q-3-G također može pojaviti u nivoima proteina. Koristeći Western blot test, ispitali smo fosforilaciju uzvodnih molekula AP-1 i NF-kB. Prvo, u AP-1 putu, fosforilacija c-Jun je povećana nakon inkubacije HaCaT ćelija sa Q-3-G tokom 24 h (slika 4a). Nivo p-INK-a je također povećan nakon tretmana sa Q-3-G u koncentracijama od 2,5,5 i 10 μM. Ukupni oblik JNK ostao je nepromijenjen (slika 4b). Dalje smo ispitali fosforilaciju MKK4 i TAK-1, koji su uzvodni regulatori JNK. Kao što je prikazano na slici 3c, nakon tretmana HaCaT ćelija sa O-3-G, nivoi proteina p-MKK4 i p-TAK1 su takođe povećani.
Pored NF-kB signalizacije, Q-3-G je povećao fosforilaciju p50 i p65, podjedinice NF-kB (slika 4c). Štaviše, procijenili smo aktivaciju IKK i IkBa, koji su također povećani za Q-3-G do 10 uM u poređenju sa kontrolnom grupom (slika 4d). Na kraju smo utvrdili aktivaciju uzvodnih molekula uključenih u NF-kB signalni put. Fosforilacija Src i Akt (Ser473) je poboljšana O-3-G tretmanom (Slika 4e). Sve u svemu, ovi podaci ukazuju da efekti zaštite kože Q-3-G ciljaju aktivaciju TAK1 i Src povećanjem nivoa proteina, čime se povećava aktivnost AP-1 i NF-kB, respektivno.
2.4.Efekti Q-3-G na transkripcijsku aktivaciju AP-1 i NF-kB
Ispitivali smo ćelijsku citotoksičnost Q-3-G u ćelijama ljudske embrionalne ćelijske linije bubrega HEK293T MTT testom (slika 4f). Vijabilnost ćelija HEK293T nije bila značajno pogođena nakon tretmana sa Q-3-G（2.5-20 μM) u poređenju sa netretiranim ćelijama.
Koristili smo test luciferaze da odredimo da li Q-3-G može modulirati testove promotora NF-kB i AP-1. Potvrdili smo da je O-3-G povećao aktivnost luciferaze posredovanu AP{{4} (slika 4g) i aktivnost luciferaze posredovanu NF-kB (slika 4h) na način ovisan o koncentraciji. Ovi rezultati potvrđuju da su AP{10}} aktivacija i aktivacija NF-KB važne farmakološke mete Q-3-G.
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