CD4+ T cells were separated from dLNs by magnetic-activated cell sorting (MACS) separation (Miltenyi Biotec, Bergisch Gladbach, Germany)

CD4+ T cells were separated from dLNs by magnetic-activated cell sorting (MACS) separation (Miltenyi Biotec, Bergisch Gladbach, Germany). 2.16. T cells that induce the expression IGLC1 of CD40L and CD25 (IL-2 receptor) for B cell helpers and proliferation [20,21]. The NFB pathway, including p65 translocation and MAPK pathway, are known to be involved in T cell activation. Understanding the process of T cell activation is critical for developing novel therapeutics of T cell-mediated diseases including atopic dermatitis (AD). AD is one of the multi-factorial diseases that is caused by environmental or genetic issues; hence, it is considered an incurable disease [22]. During recent decades, although many therapeutic approaches to conquer AD have been tried by understanding the mechanism of AD development, few trials have demonstrated the importance of T cells in AD. Once na?ve T cells are primed and activated by dendritic cells that load allergen peptides, they differentiate into effector T cells in lymph nodes to lead pathogenesis by producing effector cytokines, including IL-4, IL-5, IL-6, and IL-13 [23,24]. With Th2 cytokines milieu from effector T cells, AD is developed, and severe inflammatory response AGN 205327 is usually generated. As mentioned above, T cells play a critical role in AD progress, so that regulation of T cell activation is usually a promising strategy for improving AD symptoms [25,26]. However, it is still unknown whether treatment with liquiritigenin abrogates T cell activation in vitro and protects from atopic dermatitis in vivo. Here, we explored the effect of liquiritigenin isolated from on T cell activation with underlying mechanism and therapeutic potential of oral administration of liquiritigenin for AD pathogenesis. 2. Materials and Methods 2.1. Cell Culture Jurkat T cells were purchased from Korean Cell Line Lender (Seoul, Republic of Korea). The cells were cultured in RPMI medium (Welgene, Gyeongsan-si, Republic of Korea) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/mL), streptomycin (100 g/mL), and L-glutamine (2 AGN 205327 mM), and grown at 37 C in a humidified incubator made up of 5% CO2 and 95% air. 2.2. Mice Eight-week-old female BALB/c mice were obtained from Samtako and housed in specific pathogen-free (SPF) conditions. All experiments were approved by the Animal Care and Use Committee of the College of Pharmacy, Keimyung University (approval number: KM2019-005). 2.3. Herb Materials The dried of was purchased from the Yangnyeong herbal medicine market (Daegu, AGN 205327 Korea, in June 2019). A voucher specimen (KMU-2019-11-16) of the herb was deposited at the College of Pharmacy in Keimyung University. 2.4. Extraction and Isolation The dried stem of (10 kg) was refluxed with 100% ethanol for 3 h at boiling temperature. The dried EtOH (1.72 kg) extract was suspended with H2O, and the resulting H2O layer was partitioned three times with hexane (486 g), EtOAc (841 g), and H2O (393 g). The EtOAc-soluble fraction was loaded onto silica column (8 60 cm, silica-gel 70-230mesh), eluted in methanol in H2O (gradient from 0:100 to 100:0) to obtain seven fractions (Fr.1 to Fr.10). Among them, Fr.5 was subjected to Sephadex LH-20 column chromatography (35% MeOH to 100% MeOH) to obtain 8 fractions (Fr.5-1 to Fr.5-8). The Fr.5-8 was performed to C18 column chromatography followed by elution with a gradient solvent system of MeOH in H2O (45% MeOH to 100% MeOH) and purification with a semi-preparative high-performance liquid chromatography (HPLC) to giving liquiritigenin (274 mg). Isolated liquiritigenin was identified by comparing the values of spectroscopy data from previously published literature [27]. The isolated liquiritigenin was detected at 35.7 min with purity of 94% (Determine 1A, top), and liquiritigenin in EtOAc fraction of was also detected at 35.7min (Physique 1A, middle) but not in the hexane fraction (Physique 1A, bottom). The structure of liquiritigenin is usually shown in Physique 1B. Open in a separate window Physique 1 Liquiritigenin is usually isolated form EtOAc fraction of S. suberectus. (A) High-performance liquid chromatography (HPLC) chromatograms of isolated liquiritigenin (top), EtOAc fraction of (middle), and n-hexane fraction of at 280 nm. (B) Chemical structure of liquiritigenin. 2.5. Condition of High-Performance Liquid Chromatography (HPLC) Analysis Analyses were performed using a reversed-phase high-performance liquid chromatography.

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