Dio 1: Protuupalni, antioksidativni, hidratantni i antimelanogenezni učinci kvercetin 3-O- -D-glukuronida u ljudskim keratinocitima i stanicama melanoma putem aktivacije puteva NF-κB i AP-1
Mar 21, 2022
Za više informacija kontaktirajtetina.xiang@wecistanche.com
Sažetak: kvercetin3-O- -D-glukuronid(Q-3-G), glukuronidni konjugat kvercetina, navodno imaprotuupalnosvojstva u makrofagima stimuliranim lipopolisaharidima, kao i antikancerogena i antioksidativna svojstva. Za razliku od kvercetina, za koji je opširno opisano da posjeduje širok raspon farmakoloških aktivnosti uključujući zaštitne učinke na kožu, farmakološke dobrobiti i mehanizme Q-3-G u koži tek treba razjasniti. Ova se studija usredotočila na karakterizaciju zaštitnih svojstava kože, uključujući protuupalna i antioksidativna svojstva, Q-3-protiv oksidativnog stresa izazvanog UVB-om ili H2O2-, učinke hidratacije iantimelanogenezaaktivnosti pomoću ljudskih keratinocita (HaCaT) i stanica melanoma (B16F10). Q-3-G smanjuje ekspresiju proupalnog gena i citokina kao što su ciklooksigenaza-2 (COX-2) i faktor nekroze tumora (TNF)- u HO, ili UVB- ozračene HaCaT stanice. Također smo pokazali da O-3-Gexhibits imaju antioksidativni učinak pomoću testova hvatanja slobodnih radikala, protočne citometrije i povećane ekspresije nuklearnog faktora eritroid 2- povezanog faktora 2 (Nrf2). Q-3-G smanjuje proizvodnju melanina u B16F10 stanicama izazvanim -melanocit-stimulirajućim hormonom (-MSH). Učinci hidratacije i mehanizmi Q-3-G ispitivani su procjenom gena povezanih s faktorom hidratacije, kao što su transglutaminaza-1(TGM-1), filagrin (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 fosforilaciju IkB, IkB kinaze(IKK)-, Akt i Src, uključenih u NF-kB put. Sve zajedno, pokazali smo da Q-3-G ispoljava protuupalna, antioksidativna, hidratantna i antimelanogenezna svojstva u ljudskim keratinocitima i stanicama melanoma putem puteva NF-kB i AP-1.
Ključne riječi: kvercetin 3-O- -D-glukuronid; UV zaštita; hidratantna; antimelanogeneza; AP-1; NF-kB
1. Uvod
Koža, najveći tjelesni organ, nudi barijeru koja igra važnu ulogu u zaštiti tijela od vanjskog okruženja i štetnih čimbenika kao što su ultraljubičasto zračenje, zarazni mikrobi i štetne kemikalije. Ova barijera također regulira tjelesnu ravnotežu elektrolita i temperaturu te sprječava gubitak vlage [1,2]. Ljudska koža sastoji se od tri sloja: epidermisa, dermisa s dodacima i potkožnog tkiva [3]. Epidermalni sloj ima važnu ulogu u održavanju tjelesne temperature.
Keratinociti, obilna komponenta epidermisa kože, usko sudjeluju s drugima putem dezmosoma i uskih spojeva koji omogućuju učinkovitu fizikalno-kemijsku 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 najcenzornijih čimbenika rizika [5]. UV svjetlo sastoji se od tri raspona valnih duljina: UVA (320-400 nm), UVB (280-320 nm) i UVC (200-280 nm). Osim vodikovog peroksida (H2O2), za koji je poznato da doprinosi oksidativnom stresu, UVB može dovesti do proizvodnje reaktivnih kisikovih vrsta (ROS) putem aktivacije 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 unutarstaničnih molekula koje potom dovode do raznih vrsta oštećenja kože i procesa povezanih sa starenjem kao što su stanična smrt, oksidativni stres, i upale [7,8]. Osim toga, poznato je da odgovor na oksidativni stres utječe na oštećenje različitih staničnih komponenti [9]. Oštećene stanice kože također mogu dovesti do upalnih reakcija, što rezultira kronično oštećenom kožom [10]. Jedan tip UVB ili H2O2-posredovanog oštećenja kože kroz biološke procese je ekspresija upalnih gena kao što su ciklooksigenaza-2 (COX-2) i proupalni citokini [11]. Stoga je razvoj sigurnijih, robusnijih i učinkovitijih zaštitnih učinaka kože ključan za prevenciju i blokiranje bolesti povezanih s oštećenjem kože posredovanim UVB ili H2O2.
Hidratacija kože također igra važnu ulogu u održavanju njezine normalne funkcije. Konkretno, stratum corneum (SC) ima zaštitni učinak protiv gubitka vode [12]. Sadržaj vode u stratum corneumu sudjeluje u deskvamaciji kože i pravilnom sazrijevanju, dok nedostatak vode u SC-u potiče nakupljanje i disfunkciju korneocita [12,13]. U završnim fazama diferencijacije keratinocita, njegova plazma membrana i stanične organele nestaju, a tada influks kalcija inducira transglutaminazu (TGM) za umrežavanje s drugim proteinima. Nedostatak ili gubitak TGM-1 dovodi do neispravnog umrežavanja stanične ovojnice. Lamelna ihtioza je primjer bolesti uzrokovane gubitkom TGM-1. Nadalje, filaggrin (FLG), formirani keratinski matriks, igra ulogu u stvaranju SC jer djeluje kao skela za vezanje proteina rožnate ovojnice i lipida [14] katalizirajući g-glutamil lizinske reakcije umrežavanja, ATGM i membranu -povezani enzimi, dajući cjelovitost SC-ima [15]. Osim toga, hijaluronska kiselina (HA) je vrsta prirodnog čimbenika vlažnosti (NMF) koji je uključen u zadržavanje vlage u koži i pokazuje poseban profil u intrinzičnom starenju kože, sredstvo je za isporuku lijekova i važna je komponenta izvanstaničnog matriksa [16,17]. HA igra ulogu u povećanju vlažnosti kože reguliranjem gena sintaze hijaluronske kiseline (HAS); osim toga, retinoična kiselina (vitamin A) može učinkovito regulirati HA u epidermisu [17]. TGM-1, FLG i HAS-12,3 geni su povezani s proizvodnjom NMF [18]. Štoviše, aktivacija transkripcijskih faktora kao što su protein aktivator-1(AP-1) i nuklearni faktor (NF)-kB uključeni su u proizvodnju proupalnih tvari koje optimiziraju kožnu barijeru i hidrataciju kože kroz AP-1 i NF-kB putove[19]. Uzvodni signalni enzimi u AP-1 putu uključuju mitogenom aktivirane proteinske kinaze (MAPK) kao što su c-Jun N-terminalna kinaza (JNK), izvanstanična kinaza regulirana signalom (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 pridonose određivanju boje kože i melanogenezi [21]. UVB zračenje i hormon koji stimulira melanocite (o-MSH) mogu stimulirati lučenje melanina, čime se pokreće melanogeneza [22. Tijekom melanogeneze, -MSH aktivira protein kinazu A (PKA), transkripcijski faktor povezan s proteinskom mikroftalmijom (MITF) i vezanje elementa odgovora cAMP (CREB)[23]. Jedna od funkcija melanina smatra se sprječavanje oštećenja kože UV zračenjem, uključujući i sunčevu svjetlost. Međutim, prekomjerna proizvodnja melanina ili nedostatak sadržaja melanina uzrokovan starenjem, hormonom koji stimulira melanocite i izloženošću UV zračenju potiče pjege, melazmu, senilne lentigine i druge poremećaje hiper/hipopigmentacije koji bi mogli imati negativan učinak na izgled i kožu. zdravlje [24]. Stoga je također poželjna kontrola melanogeneze kako bi se održao i zdravstveni i kozmetički izgled ljudskog tijela.
kvercetinje prevladavajući alocirani flavonoid tipa flavonola prisutan u povrću, voću, pićima i ljekovitom bilju [25]. Posljedično, 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 koji su koristili osjetljive i pouzdane metode pokazalo je da kvercetin nije otkriven u plazmi [30-33] i da se jedva nalazi u mozgu [34,35]. Kvercetin, koji se općenito pojavljuje kao oblik glikozida, dobro se apsorbira i većinom hidrolizira i metabolizira u tankom crijevu, debelom crijevu, jetri i bubrezima u konjugirane metabolite [30,36-38]. U cirkulirajućoj krvi otkrivaju se samo konjugirani metaboliti kao što su glukuronid ili sulfat (metilirani ili nemetilirani), a ne kvercetin aglikon ili njegovi glikozidi. 3/-metil-kvercetin-3-glukuronid, kvercetin-3-glukuronid i kvercetin-3'-sulfat identificirani su 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 faze II metabolizma [29,39,40]. Također je objavljeno da se konjugirani metaboliti kvercetina nakupljaju u ljudskoj plazmi u rasponu koncentracija od 10-' do 10- stupnjeva M nakon periodičnog uzimanja luka uz obroke tijekom 1 tjedna [41]. Poluvrijeme života metabolita kvercetina je dosta dugo, oko 11 do 28 sati [29]. Budući da su ovi spojevi promatrani kao glavni oblici konjugiranih metabolita kvercetina, studije usmjerene na biološku aktivnost flavonola pomoću ovih konjugiranih metabolita bile bi ključne, što nas je potaknulo na proučavanje biološke aktivnosti jednog od konjugiranih metabolita kvercetina. Jedan od najzastupljenijih metabolita glukuronida koji se uglavnom nalazi 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 generirati slične, jače ili slabije aktivnosti u usporedbi s izvornim spojevima u različitim modelima. Q-3-G se transportira do ciljnih tkiva putem plazme kako bi ispoljio svoju biološku aktivnost [39]. Također se pokazalo da je O-3-G učinkovit metabolit u plazmi štakora nakon oralne primjene kvercetina [43]. U drugim je studijama utvrđeno da se metabolit koji sadrži Q-3-G nakuplja u aorti nakon uzimanja 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 zeleni grah [46-49].Q-3-G opisano je kao protuupalna komponenta u uvjetima stimuliranim LPS-om i pokazalo je antioksidativna svojstva u krvnoj plazmi [50-52]. Međutim, u usporedbi s dugom poviješću primjene i širokim proučavanjem kvercetina, proučavanje Q-3-G tek treba istražiti. Stoga je Q-3-G odabran kao predstavnik metabolita kvercetina kao aktivnog in vivo igrača kvercetina. Osim toga, uloga Q-3-G na zaštitne učinke kože također još nije u potpunosti razjašnjena. U ovoj smo studiji, koristeći različite in vitro procjene, istraživali zaštitne učinke Q-3-G na kožu protiv UVBor H2O2-induciranog oksidativnog stresa i upale, hidrataciju kože u HaCaT (stanična linija ljudskih keratinocita) stanica, i aktivnost antimelanogeneze u stanicama B16F10 (stanična linija mišjeg melanoma).
2. Rezultat
2.1. Protuupalni i antioksidativni učinci Q-3-G protiv UVB ili H2O2-HaCaT stanica stimuliranih
Tkivo kože može biti oštećeno UV zračenjem. UV zračenje aktivira nekoliko štetnih čimbenika, uključujući oksidativni stres, apoptotičnu smrt stanica, upalu i starenje kože. Kako bismo odredili UV-zaštitne aktivnosti Q-3-G, prvo smo procijenili ima li Q-3-G ikakve učinke na vitalnost stanica ljudskih keratinocita. Koristeći test 3-(4-5-dimetiltiazol-2-il)-2,5-difenil-243 tetrazolij bromid (MTT), istražili smo citotoksične učinke O-3-G u HaCa stanicama. Kao što je prikazano na slici 2a, nije bilo značajne interferencije na vitalnost stanica pri koncentraciji od 2.5-20 uM po Q-3-G, što ukazuje da O-3-G nije pokazao citotoksičnost do 20 μM i nije oštetio keratinocite, površinski sloj stanica kože. Zatim smo procijenili zaštitne učinke Q-3-G na kožu u HaCaT stanicama izloženim UVB zračenju. Nakon UVB zračenja od 30 mJ/cm², Q-3-G je značajno zaštitio HaCaT stanice od smrti stanica izazvane UVB zračenjem na način ovisan o koncentraciji (Slika 2b). U skladu s MTT testom, upotrebom kamere spojene na mikroskop za snimanje morfologije HaCaT stanica, Q-3-G je zaštitio od stanične smrti zbog UVB-zračenih HaCaT stanica do koncentracije od 10 μM（Slika 2c） . Broj HaCaT stanica pod svakim uvjetom bio je 482 za netretiranu skupinu, 91 za skupinu s UVB 30 mJ/cm, 276 za skupinu s UVB 30 mJ/cm²plus 5 μM O-3-G i 320 za UVB 30 mJ/cm² plus 10 μM Q-3-G grupa (Slika 2d). Ovi rezultati potvrdili su da Q-3-G inhibira UVB-inducirano oštećenje HaCaT i spašava zaštitni učinak 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 aktiviraju makrofage i oslobađaju dio citokina. Upala je obilježje oksidativnog stresa. Istraživali smo protuupalni učinak Q-3-G protiv UVB ili HO2-inducirane upale. Dobro je poznato da UVB može potaknuti oštećenje DNK i upalu u stanicama kože 【10】. Ozračili smo HaCaT stanice s UVB 30mJ/cm² i inkubirali HaCaT stanice s H2O2 (500 uM). Upotrijebili smo PCR test u stvarnom vremenu kako bismo provjerili ekspresiju mRNA upalnog enzima COX-2 i proupalnog citokina TNF-. Kao što pokazuju rezultati na slici 2e,f, koncentracija od 10 μM O-3-G značajno je inhibirala ekspresiju mRNA COX-2 i TNF-. Posljedično, Q-3-G je potisnuo upalne odgovore izazvane u HaCaT stanicama 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 korištene modelne sustave za istraživanje aktivnosti čišćenja prirodnih spojeva in vitro—za procjenu antioksidativnih svojstava Q-3-G u različitim koncentracijama. Stanice HaCaT tretirane su s Q-3-G na način ovisan o koncentraciji od 2.5-40 μM u prisutnosti DPPH, a rezultat je pokazao značajnu aktivnost čišćenja pri više od 2,5 uM (Slika 2g) . ABTS test također je značajno pokazao učinke Q-3-G na uklanjanje slobodnih radikala na načine ovisne o koncentraciji s IC50 vrijednošću od 1,75 μM （Slika 2h）. Askorbinska kiselina （AA）, kao pozitivna kontrola, pokazala je najveće antioksidativne učinke u DPPH i ABTS testovima. Nuklearni faktor eritroid 2-srodan faktor 2 (Nrf2), jedan od gena ovisnih o elementu antioksidativnog odgovora (ARE), navodno regulira zaštitne oksidoreduktaze i njegove nukleofilne supstrate, potičući tako antioksidativne enzime za uklanjanje oštećenja i popravak sustavi [53]. Zatim smo ispitali razinu ekspresije Nrf2 u HaCaT stanicama ozračenim UVB zračenjem. Pojačavajući gornje nalaze, O-3-G je značajno povećao razinu ekspresije Nrf2 nakon UVB zračenja u usporedbi s kontrolom na način ovisan o koncentraciji, do 10 uM (Slika 2i), što implicira da je Q-3- G ima antioksidativni učinak i kemijski i biološki sa svojstvom hvatanja slobodnih radikala i povećava zaštitni učinak ARE signalizacije ovisne o Nrf2-. Nadalje, također smo proveli ROS protočnu citometriju koristeći diklorodihidrofluorescein diacetat (DCFDA) u UV uvjetima. Opće je poznato da antioksidans suzbija stanične stresore inhibiranjem proizvodnje ROS [54]. Rezultat je pokazao da je tretman s Q-3-G u koncentracijama od 5 i 10 μM smanjio razinu UVB-induciranog ROS（Slika 2j）. Uzeti zajedno, ovi podaci impliciraju da Q-3-G ima zaštitna svojstva kože kao što su protuupalni i antioksidativni učinci protiv H2O ili oksidativnog stresa izazvanog UVB zračenjem i upale u HaCaT stanicama.
![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. Učinak antimelanogeneze u B16F10 i hidratizirajući učinak u HaCaT stanicama Q-3-G
Kako bi se ispitali učinci O-3-G na melanogenezu, stanice melanoma tretirane su s -MSH tijekom 48 sati kako bi se lučio melanin u stanicama B16F10. Primjenom MTT testova prvo smo potvrdili da Q-3-G nije imao citotoksičnost u stanicama B16F10 pri koncentracijama do 20 μM tijekom 48 sati (Slika 3a). Ovaj je rezultat pokazao, za sljedeće procjene, da učinci O-3-G u B16F10 nisu bili posljedica stanične smrti. Zatim smo procijenili B16F10 s Q-3-Gand -MSH tijekom 48 sati kako bismo odredili učinke 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.
Molekula povezana s vlagom kože je hijaluronska kiselina (HA). Kao što je prethodno spomenuto, HA, raspoređen po epidermisu i dermisu, NMF je koji poboljšava hidrataciju kože [55]. Upotrijebili smo PCR (RT-PCR) test za ispitivanje razine transkripcije HAS-1 nakon 12 sati s koncentracijom Q-3-G do 30 uM. HAS potiče nakupljanje HA srednje veličine unutar keratinocita [12]. Kao što je prikazano na slici 3c, ekspresija HAS-1 povećana je tretmanom s Q-3-G do 10 μM. Osim faktora vlaženja, također smo ispitali ekspresiju FLG i TGM{{ 13}}. Koristili smo retinol kao pozitivnu kontrolnu skupinu. Razine transkripcije FLG i TGM-1 značajno su porasle nakon tretmana Q-3-G u koncentracijama do 10 μM (Slika 3d). Ovi nalazi sugeriraju da Q-3-G ima aktivnost vlaženja kože u HaCaT stanicama. Štoviše, koristeći SB203580 (inhibitor p38), SP600125 (inhibitor JNK), UO126 (inhibitor ERK) i Bay11-7082 (inhibitor NF-kB), ispitali smo uključenost signalnih putova MAPK i NF-kB u hidrataciji faktor Q-3-G-tretiranih HaCaT stanica. Na temelju ekspresije gena izazvane O-3-G, razine TGM-1 i FLG smanjene su tretmanom sa SP600125, inhibitorom JNK (Slika 3e), što sugerira da je JNK potrebna za povećanje ekspresije TGM-1 i FLG u smislu učinka O-3-G na vlaženje kože. Dodatno, ekspresiju FLG-a također je 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 putovi Q-3-G povezani s ovlaživanjem putem aktivacije AP-1 i NF-kB
Na temelju prethodnih rezultata mRNA, pretpostavili smo da se uključenost AP-1 i NF-kB signalizacije u učinak hidratacije Q-3-G može pojaviti i na razinama 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 stanica s Q-3-G tijekom 24 sata (Slika 4a). Razina p-INK također je povećana nakon tretmana Q-3-G u koncentracijama od 2,5,5 i 10 μM. Ukupni oblik JNK ostao je nepromijenjen (Slika 4b). Nadalje smo ispitali fosforilaciju MKK4 i TAK-1, koji su uzvodni regulatori JNK. Kao što je prikazano na slici 3c, nakon tretmana HaCaT stanica s O-3-G, razine proteina p-MKK4 i p-TAK1 također su bile regulirane prema gore.
Uz NF-kB signalizaciju, Q-3-G je povećao fosforilaciju p50 i p65, podjedinice NF-kB (Slika 4c). Štoviše, procijenili smo aktivaciju IKK i IkBa, koji su također bili povećani za Q-3-G do 10 uM u usporedbi s kontrolnom skupinom (Slika 4d). Na kraju smo utvrdili aktivaciju uzvodnih molekula uključenih u signalni put NF-kB. Fosforilacija Src i Akt (Ser473) pojačana je tretmanom O-3-G (Slika 4e). Sve u svemu, ovi podaci pokazuju da učinci Q-3-G zaštite kože ciljaju aktivaciju TAK1 i Src povećanjem razine proteina, čime se regulira aktivnost AP-1, odnosno NF-kB.
2.4. Učinci Q-3-G na transkripcijsku aktivaciju AP-1 i NF-kB
Istražili smo staničnu citotoksičnost Q-3-G u staničnoj liniji HEK293T stanica ljudskog embrija bubrega pomoću MTT testa (Slika 4f). Preživljavanje stanica HEK293T nije značajno promijenjeno nakon tretmana s Q-3-G（2.5-20 μM） u usporedbi s netretiranim stanicama.
Upotrijebili smo analizu luciferaze kako bismo odredili može li Q-3-G modulirati analize promotora NF-kB i AP-1. Potvrdili smo da O-3-G povećava aktivnost luciferaze posredovanu AP-1- (Slika 4g) i aktivnost luciferaze posredovanu NF-kB (Slika 4h) na način ovisan o koncentraciji. Ovi rezultati potvrđuju da su aktivacija AP-1 i aktivacija NF-KB važne farmakološke mete Q-3-G.
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