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Chicory extract-induced augmentation of lipogenesis in sz95 human sebaceous gland cells. SUMMARY This study was to explore the lipogenic effect by ethanol extracts of chicory and possible molecular mechanisms in sebocyte. When SZ95 sebocyte cell line was treated with the chicory extracts, lipid droplets were accumulated in the majority of cells. Chicory extracts increased expression of lipid droplets in the SZ95 cells. Chicory extracts augmented expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) and total cholesterol. These results suggest that chicory extracts induces lipogenesis in SZ95 cells through PPAR-γ activations. INTRODUCTION Mammalian skin is composed of two primary layers, the epidermis, which is derived from ectoderm, and the dermis, derived from mesoderm. The epidermis is composed, inter alia, of the outermost layers of the skin. It forms a protective barrier over the body's surface, responsible for keeping water in the body and preventing pathogens from entering, and is a stratified squamous epithelium, composed of proliferating basal and differentiated suprabasal keratinocytes. The epidermis also helps the skin regulate body temperature. In order to maintain healthy skin, the stratum corneum is a constant state of moisture and lipids, amino acids, organic acids, sebum and natural moisturizing factors have to be balanced. Dry skin, also called xerosis, is a very common skin condition that occurs at all ages. Usually, it doesn't represent a serious problem but sometimes it can be difficult to treat. It feels rough, scaly, sometimes painful, and itchy. Dry skin results when lipids are depleted and there is not enough water in the stratum corneum for it to function properly. Equally atopic dermatitis, a common dry skin condition in childhood, shows reduced lipids levels in the stratum corneum and, consequently, an important loss of water. Sebaceous glands secrete an oily substance called sebum that is made of fat and the debris of dead fat-producing cells in the skin of mammals. They are found in hair-covered areas where they are connected to hair follicles to deposit sebum on the hairs and bring it to the skin surface along the hair shaft. In the sebaceous glands, sebum is produced within specialized cells, which are called sebocytes, and is released as these cells burst. Sebaceous lipogenesis resulting in accumulation of lipid droplets and subsequent sebum secretion represents a major step in the terminal differentiation of sebocytes. Sebum excretion is associated with the functional maintenance of the surface of skin by controlling moisture balance and providing native immunity. Regarding sebaceous lipogenesis disorders, an excess secretion of sebum has been reported to cause the onset of acne vulgaris and seborrhoea, which are two of the most common skin diseases. In contrast, a decrease in sebum secretion has been associated with dry skin (xerosis), the development of which is related to ageing and environmental conditions. Therefore, pharmacological improvement of sebaceous gland functions is likely to result in the restoration of skin homeostasis. Chicory is one of the popular healthy vegetables because it has useful ingredients and abundant dietary fibers. It stimulates the immune system and alleviates intestinal diseases and harmful bacteria. Chicory contains Vitamin A, Vitamin B2, Vitamin C, potassium, iron, etc which is good for skin beauty and also helps digestion and blood circulation. The aim of this study was to address whether or not chicory extracts a biological activity to modulate sebaceous lipogenesis, which may be associated with the treatment of sebaceous disorders such as xerosis or childhood atopic dermatitis. MATERIAL AND METHODS Oil Red O staining The Oil Red O Method is a common staining technique used for demonstrating lipids in tissue. It is performed on fresh frozen sections, as alcohol and xylene dissolves the lipids that are in the tissue. Oil Red O has largely replaced many Sudan Dyes that were used for demonstrating lipids, such as Sudan III and Sudan IV, because Oil Red O provides a deeper red color that is easier to see. Total cholesterol assay Total cholesterol assay measure the total cholesterol within serum, plasma, cell lysate, or tissue samples. The assays will detect total cholesterol (cholesteryl esters plus free cholesterol) in the presence of cholesterol esterase or only free cholesterol in the absence of the esterase enzyme. Expression of Reverse transcription-polymerase chain reaction (PCR) Quantification was achieved by measuring light absorbency at 260 nm. In cases in which either total RNA quality or quantity was not sufficient to allow further analysis, the extraction procedure was repeated, using frozen tissue samples. The expression of PPAR-γ1 and PPAR-γ2 was assessed by quantitative RT-PCR. RESULTS 140 120 (% of control) Cell viability 100 80 60 40 20 0 % of control DMEM 100nM Control Linoleic Acid 100 94 25ug/ml 50ug/ml 100ug/ml 200ug/ml Chicory extract 111 118 117 120 Figure 1. Cell viability assay of Chicory extract on sz95 human sebocytes after 48h treatment determined by MTT assay. Positive control was sz95 treated with linoleic acid at the highest concentrations used in the tests. The bars indicate mean ±standard deviation of three independent experiments performed in triplicate. 160 120 (% of control) Absorbance of Oil Red O 140 100 80 60 40 20 0 % of control DMEM 100nM Control Linoleic acid 100 130 100ug/ml 200ug/ml Chicory extract 110 133 Figure 2. Effect of Chicory extract on lipid droplet enlargement in sz95 cells. Pictographic analysis after Oil red O staining. ⓐ The control group; ⓑ The linoleic acid treated group; ⓒ Low dose Chicory extract (100ug/ml) group; ⓓ High dose Chicory extract (200ug/ml) group. The contents of the lipid droplets were significantly higher in the different Chicory extract treatment groups than in the control group. The values are shown as the means ±standard deviation and are representative of three independent experiments. *p<0.1 compared to control; one-way ANOVA, followed by Newman-Keuls Multiple comparison test. 200 160 140 (% of control) PPARγ/β-Actin mRNA ratio 180 120 100 80 60 40 20 0 % of control DMEM 100nM Control Linoleic Acid 100 157 100ug/ml 200ug/ml Chicory extract 166 157 Figure 3. Effect of Chicory extract on sz95 human sebocytes mRNA expressions. Chicory extracts significantly up-regulated mRNA expression of PPAR-γ compared with control group. The 600 500 400 (% of control) Total cholesterol & Hyaluronan content expression of PPAR-γ was normalized against β-Actin mRNA expression. 300 200 100 0 DMEM 100nM 100ug/ml 200ug/ml Control Linoleic Acid Total cholesterol 100 442 324 333 Hyaluronan 100 123 149 300 Chicory extract Figure 4. Effect of Chicory extract on sz95 human sebocytes about total cholesterol and hyaluronan synthesis. Chicory extracts significantly increased total cholesterol and hyaluronan content compared with control group. CONCLUSION Human sebum is mainly composed of lipids including triglycerides, free fatty acids, and wax esters. The lipid film formed by sebum not only protects skin by providing a barrier against pathogens and ultraviolet light, but also protects the skin surface from dehydration as well as oxidative injuries. During the process of sebum formation, several transcription factors act as primary regulators in the induction of lipogenic gene expression. Peroxisome proliferator-activated receptors (PPARs) regulate lipid metabolism by binding with specific DNA sequences in the promoter region of lipogenic genes. PPAR-α and PPAR- γ are exuberantly expressed in the human sebaceous gland. In the present study, Chicory extracts treatment dramatically induced accumulation of lipid droplets in SZ95 sebocytes. Theses extracts significantly induced activation of PPAR- γ and Hyaluronic acid expression following a 48 h treatment. Through this study, we propose a pathologic function and molecular mechanisms for extracts-induced lipogenesis and provide valuable information for the deveolment in dry skin and childhood atopic dermatitis. REFERENCE 1) Epigallocatechin-3-Gallate Suppresses IGF-I-Induced Lipogenesis and Cytokine Expression in SZ95 Sebocytes. Journal of Investigative Dermatology (2012) 132, 2700–2708 2) Effect of Green Tea Extract on Lipid Synthesis in Human Sebocyte Cell Line. Korean J. Oriental Physiology & Pathology. 25(4):608 613, 2011 3) Differentiation and apoptosis in human immortalized sebocytes. J Invest Dermatol. 2003 Feb;120(2):175-81 4) Peroxisome proliferator-activated Dermatol. 2006 Sep;126(9):2002-9 receptors increase human sebum production. J Invest