keratinocytes (PromoCell GmbH, Heidelberg, Germany) were maintained in Keratinocyte Growth Medium-2 in the presence of supplements and 0.06mM CaCl2 (PromoCell). Additional CaCl2 was added to induce differentiation at the point of treatment. Keratinocytes were treated for 48 hours in the presence of 1.2 mM CaCl2 with colloidal oatmeal extract or vehicle (DMSO).
RNA was extracted from primary human keratinocytes or epidermal human skin equivalents using Qiagen RNeasy Plus Mini kit with DNase I digestion (Qiagen, Valencia, CA). Reverse transcription was performed using High Capacity cDNA kit (Life Technologies, Grand Island, NY). 40 to 60ng of cDNA samples were used in a qPCR reaction to measure CLDN4, CLDN7, TGM1, ELOVL4, UGCG, HMGCR, and PPARβ/δ using ABI 7500 fast amplifier. All gene expression data were normalized by reference gene, polymerase (RNA) II polypeptide A (POLR2A), and verified using GAPDH. Statistical significance (P< 0.05) was determined by one-way ANOVA. Gene expression is reported relative to untreated samples or vehicle control.
Epidermal skin equivalents were homogenized on ice in RIPA buffer (Alfa Aesar, Ward Hill, MA) in the presence of protease inhibitors (Sigma, St Louis, MO). Homogenates were centrifuged at 14,000 × g at 4°C for 15 min. Total protein in was measured in supernatants by bicinchoninic acid protein assay (BCA) (Pierce Biotechnology, Rockford, IL) and ANGPTL4 immunoassay was carried out using Milliplex Map Human Liver Protein Magnetic Bead Panel hANGPTL4-MAG (EMD Millipore, Billerica, MA) according to manufacturer’s instructions on a Luminex xMAP platform (Luminex Corporation, Austin, TX). ANGPTL4 concentrations were normalized per mg protein. Involucrin protein level was assessed in keratinocytes whole cell extracts using Milliplex Map Human Skin Magnetic Bead INVOL-MAG (EMD Millipore, Billerica, MA).
Extracts prepared for buffering capacity determination were in the form of water insoluble solids. The solids were dispersed in water to enable pH measurements and titration with hydrochloric acid (HCl). The pH of the dispersed samples was measured before and after the titration. Hydrochloric acid solutions (0.001 to 0.01N) were used to drop the pH by one unit. The initial pH of the samples ranged between pH 5 to 9. Buffering capacity was determined and expressed as the micro equivalent of hydrogen ions required to change the pH of the sample equivalent by one pH unit using previously published procedures (28). The given formula was used for calculation of the buffering capacity (BC) value: BC (μeq H+ ions) = (Na*Va)/W, wherein: Na = Normality of acid (mol/L); Va = Volume of acid used (L); W = weight of material used (g).
Measurement of Transepithelial Electrical Resistance (TEER)
For TEER immature epidermal skin equivalents (EPI-20, MatTek Corporation, Ashland, MA) were transferred to medium containing 100ng/ml IL-4, 100 ng/ml IL-13, 50ng/ml IL-31 and 30ng/ml TNFα (R&D Systems, Minneapolis, MN). Colloidal oatmeal skin protectant lotion was applied topically twice every 24 hours after 4 hours of pretreatment with cytokines. TEER was measured at 0, 24, and 48 hours by using Millicell ERS-2 Epithelial Volt-Ohm Meter (Milipore). Tissues were placed in six-well tissue culture plates containing 5ml of culture medium and overlaid with 400 ml of PBS for the time required to measure electrical resistance The percentage change in TEER between time 0 (100%) and time 24 hours was expressed as follows: (TEER24hours/TEER0hours)X100.
Clinical Study Design
A five-week investigator-blinded randomized clinical study was conducted to demonstrate the effectiveness of an oatmeal skin protectant lotion in improving the moisture and barrier function of moderate to severe dry skin and to measure the residual skin effects after treatment is stopped. A standard Kligman Regression model was utilized. The protocol was approved by an IRB and informed consent was obtained from all subjects. Following a conditioning period, subjects used the oatmeal skin protectant lotion on their lower leg twice a day for a period of three weeks (Days 1-21). For the following 2 weeks (Days 22-34), subjects did not use the test product or any other lotions on their legs. Dry skin was evaluated by an expert graded and instrumental analysis (trans-epidermal water loss and Skicon) measured barrier function and skin moisture. Statistical analysis of data was performed to determine efficacy.
50 subjects completed the study. Subjects were healthy females, between the ages of 18-65 years old, with moderate to severe dry skin on both lower legs at the time of enrollment. Subjects washed with a standardized soap for five days prior to the baseline visit.