Hyaluronic Acid Decreases Lipid Synthesis in Sebaceous Glands Yu Ra Jung, Chul Hwang, Jeong-Min Ha, Dae-Kyoung Choi, Kyung-Cheol Sohn, Young Lee, Young-Joon Seo, Young-Ho Lee, Chang- Deok Kim, Jeung-Hoon Lee, Myung Im  Journal of Investigative Dermatology  Volume 137, Issue 6, Pages 1215-1222 (June 2017) DOI: 10.1016/j.jid.2017.01.017 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Effects of HA on hamster sebaceous glands. Different groups of six hamster auricles were injected with 1 mg/ml HA or PBS as a control. (a) Hematoxylin-eosin and (b) Oil Red O staining were performed on frozen tissue sections of the ear 7 days after injection. (c) After hematoxylin-eosin staining, the sebaceous gland size was determined in 30 consecutive sebaceous glands. (d) Changes in the mean sebum output level (μg/cm2) after HA injection into the auricles of hamsters. Scale bars = 20 mm. Data represent mean ± SEM (n = 6). Data were analyzed using Student’s t test (**P < 0.01). HA, hyaluronic acid; PBS, phosphate buffered saline; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 1215-1222DOI: (10.1016/j.jid.2017.01.017) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Expression of CD44 in human sebaceous glands and sebocytes. (a) Human sebaceous glands were evaluated for CD44 by immunohistochemistry. (b) Immunofluorescence labeling of CD44 (red) in human sebocytes. Nuclei were counterstained with DAPI (blue). (c, d) Western blotting and RT-PCR of sebocyte lysates. Human keratinocytes and fibroblasts overexpressing CD44 were used as positive controls and HepG2 cells as negative control. Scale bars = 50 μm. FB, fibroblasts; GAPFH, glyceraldehyde-3-phosphate dehydrogenase; KC, keratinocytes; NC, negative control using isotype control antibody; RT-PCR, reverse transcription-polymerase chain reaction; Sebo, sebocytes. Journal of Investigative Dermatology 2017 137, 1215-1222DOI: (10.1016/j.jid.2017.01.017) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Effects of HA on cell proliferation in human sebocytes. (a) The effects of hyaluronic acid on sebocyte viability were examined by LDH release assay. (b) Rate of [3H]-thymidine incorporation into sebocytes treated with various doses of HA, calculated as a percentage of the value in untreated cells. (c) Dose-dependent effects of HA in sebocyte proliferation examined using microscopy from low cell seeding assays. Scale bars = 400 μm. Data represent mean ± SEM (n = 6). Data were analyzed using Student’s t test (*P < 0.05). HA, hyaluronic acid; LDH, lactate dehydrogenase; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 1215-1222DOI: (10.1016/j.jid.2017.01.017) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Effects of HA on lipid production in human sebocytes. (a) Lipid levels in sebocytes treated with various doses of HA, calculated as percentages of the value of untreated cells. (b) Detection of intracellular lipids in sebocytes treated with HA using microscopy after Oil Red O and Nile red staining. Scale bars = 20 μm. (c) Relative abundance of major lipid classes determined by thin-layer chromatography. Human sebocytes grown in the presence of [14C]-acetate after treatment with HA, and changes in specific lipid components such as triglyceride, cholesterol, wax ester, and squalene were analyzed. Data represent mean ± SEM (n = 10). Data were analyzed using Student’s t test (*P < 0.05, **P < 0.01). HA, hyaluronic acid; Pre, preconfluence; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 1215-1222DOI: (10.1016/j.jid.2017.01.017) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Inhibition of lipid synthesis by HA in human sebocytes via CD44/RhoA signaling. (a) After sebocytes were treated with HA, whole-cell lysates were prepared and analyzed by western blotting. Blots were incubated with specific antibodies. Sebocytes were transfected with control or CD44-targeting siRNA and intracellular lipid levels were measured by (b) Nile red staining and (c) thin-layer chromatography. Scale bars = 20 μm. Data represent mean ± SEM (n = 6). Data were analyzed by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). HA, hyaluronic acid; Post, postconfluence; Pre, preconfluence; siRNA, small-interfering RNA; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 1215-1222DOI: (10.1016/j.jid.2017.01.017) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Effects of HA on facial sebum production. (a) Photographs showing clinical improvement of hyperseborrhea. In each set of images, one photograph was taken before and the other was taken 4 weeks after HA injection. (b) Changes in the mean sebum output level (μg/cm2) after HA injection in facial skin. Data are presented as mean ± SEM (n = 20). Data were analyzed by Student’s t test (*P < 0.05 vs. before treatment). HA, hyaluronic acid; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 1215-1222DOI: (10.1016/j.jid.2017.01.017) Copyright © 2017 The Authors Terms and Conditions