584 JOURNAL OF COSMETIC SCIENCE TSLP (id: qHsaCIP0030468), and GAPDH (id: qHsaCEP0041396) were obtained from Bio-Rad (Hercules, CA, USA). The cycling conditions were as follows: denaturation at 95°C for 2 min, followed by 40 cycles using a step program (95°C for 10 s and 60°C for 45 s), as described previously (33). The relative expression levels of FLG, IL31, TSLP, and POMC mRNA were normalized to GAPDH mRNA levels using the software program provided by the manufacturer. IMMUNOBLOT ANALYSIS Whole-cell lysates were prepared by lysing HaCaT cells in a buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 500 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM Na 3 VO 4 , 1 mM NaF, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 μm nitrocellulose membranes (Amersham Bioscience, Pittsburgh, PA, USA), as described previously (33). After blocking with 5% skimmed milk, the membrane was incubated with appropriate primary and secondary antibodies, and immunoreactive bands were detected using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). The intensities of the immunoreactive bands were measured using ImageJ (version 1.52a National Institutes of Health, Bethesda, MD, USA). Target protein levels were normalized and plotted as fold-changes relative to the untreated control value. ELISA HaCaT cells were serum-starved for 24 h and then treated with 20 ng/mL IL4. The culture medium was collected after 24 h of incubation and the concentrations of IL31 and β-endorphin were determined using commercially available human IL31 ELISA (BioLegend, Cat #445704) and human β-endorphin ELISA kits (Biovision, Cat #E4458) following the manufacturer’s instructions. Briefly After attachment of the capture antibody to a 96-well plate, standards and samples were incubated with the capture antibody overnight at 4°C. Unbound samples were removed by washing detection antibodies that bind to each sample protein were added and the plate was incubated for 1–2 h at 25°C. Then, avidin-conjugated horseradish peroxidase solution was added, and the plate was again incubated for 30 min at 25°C, followed by the addition of substrate solution and incubation for 15 min in the dark. The absorbance at 450 nm was measured on an EMax Endpoint ELISA Microplate Reader (Molecular Devices). DOUBLE IMMUNOFLUORESCENCE STAINING HaCaT cells cultured on coverslips were left untreated or treated with IL4 (for IL31 expression) or IL4 plus IL13 (IL4 + IL13) (for FLG expression). After 24 h, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 and 2% bovine serum albumin, and incubated with primary antibodies against FLG and IL31. For FLG staining, the cells were incubated with anti-FLG (1:100) antibodies for 2 h, followed by Alexa Fluor 488-conjugated secondary antibodies (green fluorescence) for 30 min. F-actin was counterstained with rhodamine-labeled phalloidin (1:200 red fluorescence).

585 SUPPRESSION OF ITCHING BY THREE HERBAL ETHANOLIC EXTRACTS For IL31 staining, the cells were incubated with anti-IL31 (1:100) and antitubulin (1:200, for counterstaining) antibodies for 2 h, followed by Alexa Fluor 555-conjugated (red fluorescence for anti-IL31 antibody) and Alexa Fluor 488-conjugated (green fluorescence for antitubulin antibody) secondary antibodies for 30 min. For staining nuclear DNA, the cells were incubated with Hoechst 333258 (blue fluorescence) for 10 min. Fluorescent images were captured using an EVOS fluorescence microscope (Advance Microscopy Group, Bothell, WA, USA). CLINICAL EVALUATION PREPARATION OF TOPICAL CREAM FOR CLINICAL TRIAL The A houstonianum, B falcatum, and S chinensis mixture cream for the clinical test was prepared by Hansol Bio Company (Seongnam, Republic of Korea) in a Cosmetic Good Manufacturing Practice facility. The cream was prepared by mixing 2% A houstonianum, B falcatum, and S chinensis with a basal cream containing distilled water, butylene glycol, hydrogenated polyisobutene, ethylhexyl palmitate, octyldodecanol, glycerin, cetyl alcohol, sodium polyacryloyldimethyl taurate, polysorbate 60, dipropylene glycol, xylityl glucoside, xylitol, anhydroxylitol, beta-glucan, glyceryl stearate, hydrogenated polydecene, sorbitan stearate, glucose, allantoin, Trideceth-10, dipotassium glycyrrhizate, PEG-100 stearate, arginine, disodium EDTA, hydroxyacetophenone, caprylyl glycol, 1,2-hexanediol, ethylhexylglycerin, eucalyptus leaf oil, and limonene. PARTICIPANTS AND STUDY PROTOCOL A total of 33 female volunteers ages 20–65 with itchy skin and impaired skin barrier function (transepidermal water loss (TEWL) 12 g/m2/h) were included. Dermatologists evaluated and recorded the degree of erythema, scaling, induration, and fissuring, scoring 0 to 3 points at each participant’s visit, with a total of four visits per participant. This resulted in a possible total of 0 to 12 points if a participant’s total exceeded 6 points, they were judged as a subject requiring medical treatment and excluded from the study. The participants did not receive antibiotics, steroids, immunosuppressants, antihistamines, retinoids, or phototherapy for at least for 1 week prior to the clinical study. The study procedures were clearly explained to all selected subjects before they signed the informed consent form. The test creams were randomly marked “left” or “right,” so control site (untreated site) and tested site were randomized. Participants were instructed to apply 1 g or more of the provided test cream to the area 3 cm below the center of the inner elbow twice a day in the morning and evening. Participants visited the institution on days 0, 14, and 28 after the trial to measure TEWL, moisture content, and itching intensity. Subjects were instructed not to use body care products, showering, or bathing 12 h prior to the test. At the institution visited, participants were first placed at rest for 30 min in a room with controlled temperature (22±2°C) and humidity (50±10%). In addition, the investigator obtained written consent from each subject not to use the test cream on the control area to prevent unauthorized use. Volunteer participants were recruited by the Korea Dermatology Research Institute (Seongnam, Republic of Korea). The study was conducted under the Korean Cosmetics Good Clinical Practice (GCP) guidelines (approved IRB number: KDRI-IRB-21540).