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• W2084558224 abstract "Acne vulgaris is a nearly universal cutaneous disease characterized by multifactorial pathogenic processes. Because current acne medications have various side effects, investigating new pharmacologically active molecules is important for treating acne. As natural products generally provide various classes of relatively safe compounds with medicinal potentials, we performed activity-guided purification after a series of screenings from the extracts of five medicinal plants to explore alternative acne medications. Lupeol, a pentacyclic triterpene, from the hexane extract of Solanum melongena L. (SM) was identified after instrumental analysis. Lupeol targeted most of the major pathogenic features of acne with desired physicochemical traits. It strongly suppressed lipogenesis by modulating the IGF-1R/phosphatidylinositide 3 kinase (PI3K)/Akt/sterol response element–binding protein-1 (SREBP-1) signaling pathway in SEB-1 sebocytes, and reduced inflammation by suppressing the NF-κB pathway in SEB-1 sebocytes and HaCaT keratinocytes. Lupeol exhibited a marginal effect on cell viability and may have modulated dyskeratosis of the epidermis. Subsequently, histopathological analysis of human patients’ acne tissues after applying lupeol for 4 weeks demonstrated that lupeol markedly attenuated the levels of both the number of infiltrated cells and major pathogenic proteins examined in vitro around comedones or sebaceous glands, providing solid evidence for suggested therapeutic mechanisms. These results demonstrate the clinical feasibility of applying lupeol for the treatment of acne. Acne vulgaris is a nearly universal cutaneous disease characterized by multifactorial pathogenic processes. Because current acne medications have various side effects, investigating new pharmacologically active molecules is important for treating acne. As natural products generally provide various classes of relatively safe compounds with medicinal potentials, we performed activity-guided purification after a series of screenings from the extracts of five medicinal plants to explore alternative acne medications. Lupeol, a pentacyclic triterpene, from the hexane extract of Solanum melongena L. (SM) was identified after instrumental analysis. Lupeol targeted most of the major pathogenic features of acne with desired physicochemical traits. It strongly suppressed lipogenesis by modulating the IGF-1R/phosphatidylinositide 3 kinase (PI3K)/Akt/sterol response element–binding protein-1 (SREBP-1) signaling pathway in SEB-1 sebocytes, and reduced inflammation by suppressing the NF-κB pathway in SEB-1 sebocytes and HaCaT keratinocytes. Lupeol exhibited a marginal effect on cell viability and may have modulated dyskeratosis of the epidermis. Subsequently, histopathological analysis of human patients’ acne tissues after applying lupeol for 4 weeks demonstrated that lupeol markedly attenuated the levels of both the number of infiltrated cells and major pathogenic proteins examined in vitro around comedones or sebaceous glands, providing solid evidence for suggested therapeutic mechanisms. These results demonstrate the clinical feasibility of applying lupeol for the treatment of acne. free fatty acid phosphatidylinositide 3 kinase Solanum Melongena L. sterol response element–binding protein-1 Toll-like receptor-2 Acne vulgaris affects nearly 90% of adolescents worldwide and can leave permanent scarring if it is not properly treated (James, 2005James W.D. Clinical practice. Acne.N Eng J Med. 2005; 352: 1463-1472Crossref PubMed Scopus (234) Google Scholar; Williams et al., 2012Williams H.C. Dellavalle R.P. Garner S. Acne vulgaris.Lancet. 2012; 379: 361-372Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). Although current medications are moderately effective in treating acne, they may be associated with various side effects (Strauss et al., 2007Strauss J.S. Krowchuk D.P. Leyden J.J. et al.Guidelines of care for acne vulgaris management.J Am Acad Dermatol. 2007; 56: 651-663Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar; Thiboutot et al., 2009Thiboutot D. Gollnick H. Bettoli V. et al.New insights into the management of acne: an update from the Global Alliance to Improve Outcomes in Acne group.J Am Acad Dermatol. 2009; 60: S1-50Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). For example, oral isotretinoin, one of the most effective treatments, has potentially serious side effects including teratogenicity and dyslipidemia. Topical retinoids, as well as topical and systemic antibiotics, may cause a burning sensation and antibiotic resistance. Therefore, the need to investigate new antiacne ingredients has been growing, and natural products provide important clues for identifying novel drugs with relative safety that, so far, research has neglected (Ji et al., 2009Ji H.F. Li X.J. Zhang H.Y. Natural products and drug discovery. Can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight against cancer and dementia?.EMBO Rep. 2009; 10: 194-200Crossref PubMed Scopus (456) Google Scholar). They are a matchless source of pharmacologically active drugs even compared with high-throughput combinatorial screening, although only limited numbers have been tested for the acne model. Because of the innate complexity of acne pathogenesis, a variety of candidate molecules should be screened simultaneously for multiple pathogenic processes involving seborrhea, inflammation, Propionibacterium acnes (P. acnes), and follicular dyskeratosis (Zouboulis et al., 2005Zouboulis C.C. Eady A. Philpott M. et al.What is the pathogenesis of acne?.Exp Dermatol. 2005; 14: 143-152Crossref PubMed Google Scholar). Therefore, stepwise fractionation and screening of natural products would be a more efficient and systematic approach compared with the hypothesis-driven study for a single molecule. Lupeol (Lup-20(29)-en-3b-ol), a pentacyclic lupane-type triterpene, is present in several species of the plant kingdom and is abundant in medicinal plants (Saleem, 2009Saleem M. Lupeol, a novel anti-inflammatory and anti-cancer dietary triterpene.Cancer Lett. 2009; 285: 109-115Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar; Siddique and Saleem, 2011Siddique H.R. Saleem M. Beneficial health effects of lupeol triterpene: a review of preclinical studies.Life Sci. 2011; 88: 285-293Crossref PubMed Scopus (221) Google Scholar). The compound possesses wide-spectrum pharmacological activities, including lipid-lowering, anti-inflammatory, and anticancerous effects (Lee et al., 2007Lee T.K. Poon R.T. Wo J.Y. et al.Lupeol suppresses cisplatin-induced nuclear factor-kappaB activation in head and neck squamous cell carcinoma and inhibits local invasion and nodal metastasis in an orthotopic nude mouse model.Cancer Res. 2007; 67: 8800-8809Crossref PubMed Scopus (113) Google Scholar; Liu et al., 2013Liu F. He Y. Liang Y. et al.PI3-kinase inhibition synergistically promoted the anti-tumor effect of lupeol in hepatocellular carcinoma.Cancer Cell Int. 2013; 13: 108Crossref PubMed Scopus (34) Google Scholar). In addition, a few preclinical studies have suggested its potential as an anti-inflammatory or chemopreventive agent (Saleem et al., 2001Saleem M. Alam A. Arifin S. et al.Lupeol, a triterpene, inhibits early responses of tumor promotion induced by benzoyl peroxide in murine skin.Pharmacol Res. 2001; 43: 127-134Crossref PubMed Scopus (74) Google Scholar; Tarapore et al., 2013Tarapore R.S. Siddiqui I.A. Adhami V.M. et al.The dietary terpene lupeol targets colorectal cancer cells with constitutively active Wnt/beta-catenin signaling.Mol Nutr Food Res. 2013; 57: 1950-1958Crossref PubMed Scopus (34) Google Scholar). These observations, along with the fact that acne is mainly related to lipogenesis and inflammation, suggest that it may improve acne. In this research, we conducted activity-guided purification after a series of screenings from five medicinal plants. Lupeol purified from the Solanum melongena L. (SM) extract has potent antiacne effects. These include sebosuppressive and anti-inflammatory effects on human SEB-1 sebocytes and HaCaT keratinocytes, as well as beneficial effects on follicular dyskeratosis. Further biochemical and cellular studies indicate that the modulation of IGF-1R/phosphatidylinositide 3 kinase (PI3K)/Akt and NF-κB signaling pathways mediate lupeol’s sebosuppressive and anti-inflammatory effects, respectively. We subsequently confirmed these experimental results in vivo after analyzing histopathological changes of human patients’ acne tissues after applying lupeol for 4 weeks. To explore antiacne ingredients from natural compounds, we screened five candidate medicinal plants (Agrimonia pilosa, Aleriana fauriei, Lycopodium clavatum, Solanum melongena L., and Curcuma longa) that are known to be effective for acne from the literature or complementary medicine. Methanol extracts of each plant were separated on the basis of polarity and acidity (Figure 1a). Each fraction of these plant extracts was tested for biologic activities such as toxicity, antilipogenesis, anti-inflammation, and antimicrobial activities. Among them, the acidic hexane fraction of SM showed most desired results, including antiproliferative effects on SEB-1 sebocytes with no direct cytotoxicity on the HaCaT keratinocytes and 3T3-L1 adipocytes, and suppression of intracellular lipid contents per equal cell counts (Figure 1b and c). The fraction also demonstrated anti-inflammatory effects by attenuating cytokine gene expressions induced by P. acnes–stimulated SEB-1 sebocytes (Figure 1d). Antimicrobial activity against P. acnes was also observed at a relatively high concentration (400 μg ml−1) of the hexane fraction. On the basis of these initial screening tests, we isolated and identified specific antiacne chemical components from the SM extract based on activity-guided purification procedures. After separating SM extracts by open-column chromatography and following preparatory HPLC steps, each subfraction was evaluated by serial antiacne screening steps including toxicity, antilipogenic effects, anti-inflammatory effects, and antimicrobial activities (Figure 2a). Subfractions showing the most desired effects were further purified. The results are summarized in Supplementary Table S1 online. With these procedures, a molecular structure of a single final fraction that showed the best antiacne traits was characterized after analyzing data from gas chromatography–mass spectrometry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy (Figure 2b–d). Lup-20(29)-en-3β (lupeol), a pentacyclic triterpene, was identified (Figure 2e). Download .pdf (.46 MB) Help with pdf files Supplementary Material Although hyperseborrhea is critical in the pathogenesis of acne, few topical drugs that are currently used can reduce sebum secretion effectively. To examine whether lupeol modulates lipid synthesis in human SEB-1 sebocytes, intracellular neutral lipid content was analyzed by Nile Red staining after treating the cells with different concentrations of lupeol. Lupeol significantly decreased lipid by 58% in SEB-1 sebocytes treated with 20 μM lupeol (Figure 3a and b). Changes in specific free fatty acid (FFA) components, which have important roles in the innate immunity of acne inflammation, were further analyzed using quantitative fatty acid methyl ester analysis with gas chromatography–mass spectrometry (Figure 3c). Overall, fatty acid content was reduced as lupeol concentration increased, with significant decreases in major FFA components (palmitic acid [C16:0], stearic acid [C18:0], and oleic acid [C18:1]). Addition of lupeol robustly decreased 14C acetate incorporation into fatty acids, cholesterol, and squalene, which are major components of human sebum, dose dependently after cell count normalization, further confirming the lupeol-induced reduction of intracellular lipid synthesis (Figure 3d). We tested the hypothesis of whether sterol regulatory element–binding proteins (SREBPs), major transcriptional factors responsible for the regulation of cholesterol/fatty acid metabolism, were involved in the antilipogenic effects of lupeol. Lupeol significantly decreased both precursor and mature forms of SREBP-1 proteins after 24 hours (Figure 3e). Quantitative real-time PCR showed that lupeol also decreased mRNA levels of SREBP-1a, SREBP-1c, and SREBP-2, as well as key downstream targets of all SREBPs such as fatty acid synthase, acetyl-CoA carboxylase, HMG-CoA reductase, and HMG-CoA synthase. These suggest that lupeol inhibited the expression of lipogenic molecules at the transcriptional level (Figure 3f). To identify upstream regulators that are responsible for the lupeol-mediated suppression of the SREBP pathway, we focused on the IGF-1R/PI3K/Akt pathway for the following reasons: (1) previous studies show that lupeol mitigates the PI3K/Akt pathway in a CD-1 mouse model and in AsPC-1 cell lines (Saleem et al., 2004Saleem M. Afaq F. Adhami V.M. et al.Lupeol modulates NF-kappaB and PI3K/Akt pathways and inhibits skin cancer in CD-1 mice.Oncogene. 2004; 23: 5203-5214Crossref PubMed Scopus (230) Google Scholar, Saleem et al., 2005Saleem M. Kaur S. Kweon M.H. et al.Lupeol, a fruit and vegetable based triterpene, induces apoptotic death of human pancreatic adenocarcinoma cells via inhibition of Ras signaling pathway.Carcinogenesis. 2005; 26: 1956-1964Crossref PubMed Scopus (119) Google Scholar), and (2) IGF-1 treatment induces lipogenesis via activation of the IGF-1R/PI3K/Akt pathway in SEB-1 sebocytes (Smith et al., 2008Smith T.M. Gilliland K. Clawson G.A. et al.IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway.J Invest Dermatol. 2008; 128: 1286-1293Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Lupeol decreased the IGF-1R/IRS-1/PI3K/Akt pathway in a dose-dependent manner after 3 hours and subsequently downregulated SREBP-1 protein expressions (Figure 3e). Co-treatment with lupeol and the PI3K/Akt inhibitor LY 294002 potently blocked the expression of phosphorylated Akt. Importantly, this inhibition did not synergistically decrease protein content of precursor and mature SREBP-1 or intracellular lipid content (Figure 3g and h), suggesting that lupeol mainly suppressed lipogenesis through the PI3K/Akt pathway. Together, these data suggest that lupeol suppressed sebum mainly through inhibition of the IGF-1R/PI3K/Akt/SREBP-1 signaling pathway in human SEB-1 sebocytes. In acne, P. acnes–induced inflammatory response around pilosebaceous gland, mainly through the secretion of various proinflammatory cytokines, represents a key pathogenic factor leading to disease initiation and aggravation (Kang et al., 2005Kang S. Cho S. Chung J.H. et al.Inflammation and extracellular matrix degradation mediated by activated transcription factors nuclear factor-kappaB and activator protein-1 in inflammatory acne lesions in vivo.Am J Pathol. 2005; 166: 1691-1699Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar; Nagy et al., 2006Nagy I. Pivarcsi A. Kis K. et al.Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes.Microbes Infect. 2006; 8: 2195-2205Crossref PubMed Scopus (288) Google Scholar; Agak et al., 2014Agak G.W. Qin M. Nobe J. et al.Propionibacterium acnes induces an IL-17 response in acne vulgaris that is regulated by Vitamin A and Vitamin D.J Invest Dermatol. 2014; 134: 366-373Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We investigated anti-inflammatory effects of lupeol in SEB-1 sebocytes (Figure 4a–d) and HaCaT keratinocytes (Figure 4e–h), two major cutaneous parenchymal cells associated with acne. Heat-inactivated P. acnes induces several proinflammatory cytokines including IL-8 and IL-6 in SEB-1 sebocytes and tumor necrosis factor-α and IL-6 in HaCaT keratinocytes, respectively (Figure 4a, b, d, e, and f). To further examine underlying molecular mechanisms, we measured the activation of associated proteins related with the NF-κB pathway. We found that protein expressions of NF-κB p65 and phospho-IκB were significantly increased after P. acnes treatment, strongly suggesting that the activation of NF-κB may induce proinflammatory cytokine expressions (Figure 4c and g). Then, we tested the anti-inflammatory effect of lupeol in this P. acnes-induced inflammatory model. Lupeol significantly decreased protein expression of cytokines in a dose-dependent manner for both SEB-1 sebocytes and HaCaT keratinocytes, strongly supporting its anti-inflammatory effect. Messenger RNA expressions of proinflammatory cytokines in HaCaT keratinocytes showed consistent patterns (Figure 4h). Furthermore, protein expressions of phospho-IκB and NF-κB were reduced with lupeol, confirming that lupeol inhibited innate immunity of two major cutaneous cells associated with inflammatory acne by mitigating the NF-κB pathway induced by heat-inactivated P. acnes (Figure 4a–h). To test the possible toxicity of lupeol, we investigated the effect of lupeol treatment on the viability of SEB-1 sebocytes and HaCaT keratinocytes. Using both MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell counting kit-8 assays, we demonstrated that lupeol treatment was not cytotoxic in conditions identical to other cellular experiments (concentration range 0–20 μM for 24 hour of incubation) in either cell type. Only a marginal effect on cell viability was observed at 20 μM after 48 hours in SEB-1 sebocytes, and no toxicity was observed in HaCaT keratinocytes (Figure 5a and b). Because follicular epidermal dyskeratosis is another major triggering factor of acne pathogenesis, we investigated the possible beneficial effects of lupeol on the abnormal differentiation of epidermal keratinocytes. Lupeol significantly suppressed protein and mRNA expressions of IL-1α, a strong inducer of hypercornification of the infundibulm, and Toll-like receptor-2 (TLR-2), a key molecule in IL-1α release during comedogenesis, in HaCaT keratinocytes stimulated by heat-inactivated P. acnes (Figure 5c and d). In accord with these findings, lupeol also downregulated keratin 16 expression, a marker of abnormal differentiation and epidermal proliferation, in inflamed HaCaT keratinocytes (Figure 5c and e). Then, we investigated the antibacterial effects of lupeol against P. acnes, playing critical roles in innate immunity of acne. To examine this hypothesis, the minimal concentration of lupeol needed to inhibit P. acnes growth in culture medium was compared with vehicle control used for dissolution. Only lupeol-containing solution effectively inhibited the growth of P. acnes at rather higher concentrations, suggesting that it may also partly contribute to suppress abnormal colonization of P. acnes for acne development in vivo (Figure 5f and g). A schematic diagram of lupeol’s therapeutic mechanisms based on whole experimental results is illustrated in Figure 5h. To determine the consistency with our in vitro findings, we further examined changing patterns of inflammatory skin pathologies and several target protein expressions associated with acne pathogenesis in the human tissue after lupeol application. Skin specimens including typical acne lesions were acquired from patients before and after applying 2% lupeol twice daily for 4 weeks. The major findings of hematoxylin and eosin staining demonstrated that lupeol attenuated the manifestation of infiltrated inflammatory cells around comedones or sebaceous glands (Figure 6a). This small molecule also significantly decreased expressions of IGF-1R, SREBP-1, NF-κB p65, and IL-8 in the human acne lesion, consistent with results from our cellular assays (Figures 6b–e). As a real control of the baseline state of skin, the expression of these four major pathogenic proteins in nonacne skin was significantly lower than in the skin with acne lesions at both time points (IGF-1R: 0.8, SREBP-1: 0.5, NF-κB p65: 0.8, IL-8: 0.8). These hematoxylin and eosin staining and immunohistochemical analyses confirmed that lupeol decreased lipogenesis and inflammation, two critical steps in acne pathogenesis, from human acne lesions in vivo. We also found that expression levels of IL-1α and TLR-2 were significantly decreased after lupeol application for 4 weeks (Figure 6f and g). In addition, the expression of keratin 16 was significantly downregulated around the whole epidermis (Figure 6h). The expression of these three proteins was also significantly lower in nonacne skin than in skin containing acne lesions at both time points (IL-1α: 0.5, TLR-2: 0.5, keratin 16: 0.3). These results suggest that lupeol may have preventive roles in the progression of pathogenic cornification in the early phase of acne pathogenesis. Despite the near-universal prevalence of acne and corresponding demands for treatment, the development of effective antiacne medications has been hampered by the complexity of acne pathogenesis. In our previous study, we discovered that epigallocatechin-3-gallate improves acne by modulating multiple pathogenic factors, including hyperseborrhea, inflammation, and P. acnes overgrowth (Yoon et al., 2013Yoon J.Y. Kwon H.H. Min S.U. et al.Epigallocatechin-3-gallate improves acne in humans by modulating intracellular molecular targets and inhibiting P. acnes.J Invest Dermatol. 2013; 133: 429-440Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In spite of great efficacy in properly controlled condition, relative molecular instability of this antioxidant could be affected by various external factors including solvent, pH, temperature, ionic strength, and oxidative stress (Proniuk et al., 2002Proniuk S. Liederer B.M. Blanchard J. Preformulation study of epigallocatechin gallate, a promising antioxidant for topical skin cancer prevention.J Pharm Sci. 2002; 91: 111-116Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Possible molecular degradation during drug formulation or delivery processes may lead to significant decreases of therapeutic potentials. To resolve this issue by exploring new class of small molecules, we performed activity-guided purification after a series of screening tests from extracts of various medicinal plants, and finally isolated lupeol showing desirable therapeutic effects against all major targets of acne. This systematic methodology would have implications for future research. Lupeol is quite lipophilic, has no ionizable moiety in the physiologic pH, and it is chemically stable (Laszczyk, 2009Laszczyk M.N. Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy.Planta Med. 2009; 75: 1549-1560Crossref PubMed Scopus (406) Google Scholar). It is also useful in maintaining skin texture and integrity in animal studies (Harish et al., 2008Harish B.G. Krishna V. Santosh Kumar H.S. et al.Wound healing activity and docking of glycogen-synthase-kinase-3-beta-protein with isolated triterpenoid lupeol in rats.Phytomedicine. 2008; 15: 763-767Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These physicochemical and biological properties would enhance the absorption rate through the solid skin barrier and accumulation around the pilosebaceous unit during a long period of time without notable side effects (Mitragotri, 2003Mitragotri S. Modeling skin permeability to hydrophilic and hydrophobic solutes based on four permeation pathways.J Control Release. 2003; 86: 69-92Crossref PubMed Scopus (249) Google Scholar). Excessive sebum secretion is crucial in acne pathogenesis, and only oral isotretinoin and hormonal therapy have been shown to reduce seborrhea (Nelson et al., 2008Nelson A.M. Zhao W. Gilliland K.L. et al.Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid-induced apoptosis of human sebaceous gland cells.J Clin Invest. 2008; 118: 1468-1478Crossref PubMed Scopus (106) Google Scholar). In this study, lupeol had sebosuppressive effects mainly through the downregulation of IGF-1R/PI3K/Akt/SREBP-1 signaling pathway in SEB-1 sebocytes with a marginal effect on cell viability. Significant decreases in typical FFAs, including palmitic acid and oleic acid, were remarkable because these FFAs enhance innate immune system by inducing antimicrobial peptides (Nakatsuji et al., 2010Nakatsuji T. Kao M.C. Zhang L. et al.Sebum free fatty acids enhance the innate immune defense of human sebocytes by upregulating beta-defensin-2 expression.J Invest Dermatol. 2010; 130: 985-994Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar; Makrantonaki et al., 2011Makrantonaki E. Ganceviciene R. Zouboulis C. An update on the role of the sebaceous gland in the pathogenesis of acne.Dermatoendocrinol. 2011; 3: 41-49Crossref PubMed Scopus (155) Google Scholar). However, the concentration of linoleic acid, a polyunsaturated FFA that attenuates related inflammation by decreasing NF-κB-mediated host immune response (Zhao et al., 2005Zhao G. Etherton T.D. Martin K.R. et al.Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells.Biochem Biophys Res Commun. 2005; 336: 909-917Crossref PubMed Scopus (256) Google Scholar), did not change with lupeol treatment. Therefore, changing patterns of these fatty acid components may subsequently alleviate inflammation. We confirmed this antilipogenic effect from other lipid-laden cell lines by showing that lupeol also decreased intracellular lipid accumulation in adipocytes without notable cytotoxicity (Supplementary Figure S1 online). Although lupeol normalizes the serum lipid profiles in animals fed a high-fat and cholesterol diet, associated cellular mechanisms have never been investigated extensively (Sudhahar et al., 2006Sudhahar V. Kumar S.A. Varalakshmi P. Role of lupeol and lupeol linoleate on lipemic-oxidative stress in experimental hypercholesterolemia.Life Sci. 2006; 78: 1329-1335Crossref PubMed Scopus (78) Google Scholar; Ardiansyah and Shirakawa, 2012Ardiansyah YamaguchiE Shirakawa H. et al.Lupeol supplementation improves blood pressure and lipid metabolism parameters in stroke-prone spontaneously hypertensive rats.Biosci Biotechnol Biotech. 2012; 76: 183-185Crossref PubMed Scopus (18) Google Scholar). Taken together, lupeol may provide another therapeutic strategy to target lipogenesis in acne or seborrhea itself. Many cutaneous diseases are associated with chronic inflammation that also aggravates acne and frequently leaves remnant scars (Harper and Thiboutot, 2003Harper J.C. Thiboutot D.M. Pathogenesis of acne: recent research advances.Adv Dermatol. 2003; 19: 1-10PubMed Google Scholar; Yamasaki et al., 2007Yamasaki K. Di Nardo A. Bardan A. et al.Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.Nat Med. 2007; 13: 975-980Crossref PubMed Scopus (612) Google Scholar). We found that lupeol alleviated the inflammatory response in well-established in vitro models of inflammatory acne by inhibiting the NF-κB pathway (Lee et al., 2014Lee W.R. Kim K.H. An H.J. et al.The protective effects of Melittin on Propionibacterium acnes-induced inflammatory responses in vitro and in vivo.J Invest Dermatol. 2014; 134: 1922-1930Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Although the exact mechanism by which lupeol mitigates NF-κB remains elusive, the decrease of phospho-IκB that we observed strongly suggests that lupeol acts upstream of IκB degradation (Pikarsky et al., 2004Pikarsky E. Porat R.M. Stein I. et al.NF-kappaB functions as a tumour promoter in inflammation-associated cancer.Nature. 2004; 431: 461-466Crossref PubMed Scopus (2144) Google Scholar). The uniqueness of acne inflammation is not in the nature of the signaling cascade but in the localization of the process (specialized sebaceous follicles) (Kang et al., 2005Kang S. Cho S. Chung J.H. et al.Inflammation and extracellular matrix degradation mediated by activated transcription factors nuclear factor-kappaB and activator protein-1 in inflammatory acne lesions in vivo.Am J Pathol. 2005; 166: 1691-1699Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Downregulation of the NF-κB pathway, a representative indicator of inflammation, may provide insight into the molecular mechanism underlying lupeol’s effects on the significant improvement of inflammatory skin pathologies. Consistent with our results, downregulation of NF-κB is the major mechanism underlying the anticancer properties of lupeol against various cancer cell lines (Saleem et al., 2004Saleem M. Afaq F. Adhami V.M. et al.Lupeol modulates NF-kappaB and PI3K/Akt pathways and inhibits skin cancer in CD-1 mice.Oncogene. 2004; 23: 5203-5214Crossref PubMed Scopus (230) Google Scholar; Lee et al., 2007Lee T.K. Poon R.T. Wo J.Y. et al.Lupeol suppresses cisplatin-induced nuclear factor-kappaB activation in head and neck squamous cell carcinoma and inhibits local invasion and nodal metastasis in an orthotopic nude mouse model.Cancer Res. 2007; 67: 8800-8809Crossref PubMed Scopus (113) Google Scholar). As NF-κB and Akt pathways are functionally interconnected (Guo et al., 2009Guo C. Gasparian A.V. Zhuang Z. et al.9-Aminoacridine-based anticancer drugs target the PI3K/AKT/mTOR, NF-kappaB and p53 pathways.Oncogene. 2009; 28: 1151-1161Crossref PubMed Scopus (100) Google Scholar), lupeol may target a protein commonly shared by these two pathways. We found elevated levels of the proinflammatory cytokine IL-1α in acne lesions in vivo. Previous works have shown that exposing isolated infundibula to IL-1α in vitro induces epidermal dyskeratosis (Guy et al., 1996Guy R. Green M.R. Kealey T. Modeling acne in vitro.J Invest Dermatol. 1996; 106: 176-182Abstract Full Text PDF PubMed Scopus (172) Google Scholar; Graham et al., 2004Graham G.M. Farrar M.D. Cruse-Sawyer J.E. et al.Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL.Br J Dermatol. 2004; 150: 421-428Crossref PubMed Scopus (184) Google Scholar; Kim, 2005Kim J. Review of the innate immune response in acne vulgaris: activation of Toll-like receptor 2 in acne triggers inflammatory cytokine responses.Dermatology. 2005; 211: 193-198Crossref PubMed Scopus (228) Google Scholar), and a recent report suggests that activation of TLR-2 in basal and infundibular keratinocytes provokes the release of IL-1α, thereby initiating comedogenesis (Selway et al., 2013Selway J.L. Kurczab T. Kealey T. et al.Toll-like receptor 2 activation and comedogenesis: implications for the pathogenesis of acne.BMC Dermatol. 2013; 13: 10Crossref PubMed Scopus (63) Google Scholar). Remarkably, lupeol decreased both IL-1α and TLR-2 from inflamed HaCaT keratinocytes in our study. Differentiation-inducing activities of lupeol have been reported in other cell lines (Hata et al., 2005Hata K. Hori K. Murata J. et al.Remodeling of actin cytoskeleton in lupeol-induced B16 2F2 cell differentiation.J Biochem. 2005; 138: 467-472Crossref PubMed Scopus (32) Google Scholar). Therefore, these results suggest that lupeol modulates a comedogenic process in the early phase of acne development. We also discovered that P. acnes was vulnerable to lupeol. Although minimal inhibitory concentration was rather higher compared with the concentration range shown in other experiments, the antibiotic effect of lupeol, which has been reported in several studies (Tanaka et al., 2004Tanaka T. Ikeda T. Kaku M. et al.A new lignan glycoside and phenylethanoid glycosides from Strobilanthes cusia BREMEK.Chem Pharm Bull. 2004; 52: 1242-1245Crossref PubMed Scopus (55) Google Scholar; Ahmed et al., 2010Ahmed Y. Sohrab M.H. Al-Reza S.M. et al.Antimicrobial and cytotoxic constituents from leaves of Sapium baccatum.Food Chem Toxicol. 2010; 48: 549-552Crossref PubMed Scopus (47) Google Scholar), should be further examined in vivo around acne lesions for the following reasons: First, sebocytes may demonstrate less cytotoxicity in vivo. Second, P. acnes related to acne inflammation located above the sebaceous glands (e.g., the gland duct) may be exposed to a much higher concentration of lupeol than sebocytes within the gland. On the basis of these in vitro results, we further investigated histopathological changes of typical acne lesions of human patients after applying 2% lupeol for 4 weeks. The results confirmed our proposed therapeutic mechanisms in human acne tissue. Levels of both infiltrated inflammatory cell numbers around comedones or sebaceous glands and six major pathogenic proteins involved with lipogenesis, inflammation, and follicular dyskeratosis were significantly decreased. Keratin 16 is the type I keratin partner of keratin 6 in the intermediate filament heterodimer formation and is upregulated in all abnormally differentiating and hyperproliferative suprabasal keratinocytes (Aldana et al., 1998Aldana O.L. Holland D.B. Cunliffe W.J. Variation in pilosebaceous duct keratinocyte proliferation in acne patients.Dermatology. 1998; 196: 98-99Crossref PubMed Scopus (33) Google Scholar; Ramot et al., 2013Ramot Y. Sugawara K. Zakany N. et al.A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytes in vitro and in situ.Peer J. 2013; 1: e40Crossref PubMed Scopus (45) Google Scholar). Therefore, decreases of typical inducers and a marker of abnormal differentiation in vivo further support that lupeol not only has potential therapeutic effects against active acne as described, but also exerts a prophylactic activity in the prevention of comedone formation. Follow-up studies are required. In summary, lupeol modulated the key pathological factors contributing to acne, including hyperseborrhea, inflammation, follicular dyskeratosis, and P. acnes overgrowth. These results strongly suggest the potential clinical feasibility of lupeol in acne treatment. The SEB-1-immortalized human sebocyte cell line was generated by transfection of secondary sebocytes with SV40 large T antigen, as previously described (Thiboutot et al., 2003Thiboutot D. Jabara S. McAllister J.M. et al.Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).J Invest Dermatol. 2003; 120: 905-914Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). SEB-1 cells were cultured and maintained in standard culture medium containing DMEM (Invitrogen, Carlsbad, CA), 5.5 mM glucose/Ham’s F-12 3:1 (Invitrogen), fetal bovine serum 2.5% (HyClone, Logan, UT), adenine 1.8 × 10−4M (Sigma, St Louis, MO), hydrocortisone 0.4 mg ml−1 (Sigma), insulin 10 ng ml−1 (Sigma), epidermal growth factor 3 ng ml−1 (Austral Biologicals, San Ramon, CA), and cholera toxin 1.2 × 10−10M (Sigma) at 37 °C in a 5% CO2 incubator. The human keratinocyte cell line HaCaT was maintained in DMEM (Invitrogen) supplemented with 5% fetal bovine serum, 20 mM L-glutamine, 1 mM sodium pyruvate, and antibiotic/antimycotic solution (10 U ml−1 penicillin, 10 μg ml−1 streptomycin, and 0.25 μg ml−1 amphotericin; Invitrogen) at 37 °C in a 5% CO2 incubator. Protein was extracted using cell lysis buffer (Cell Signaling Technology, Beverly, MA). Protein contents in lysates were determined using the BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of protein were run on 10% SDS-PAGE gels and then transferred to a polyvinylidene difluoride membrane. The blots were primarily probed with Akt rabbit antibody, Phospho-Akt (Thr308) rabbit antibody, Phospho-IRS-1 (Ser 307) rabbit antibody, IκBα rabbit antibody, Phospho-IκBα (Ser32/36) mouse antibody, Phospho-PI3K p85(Tyr 458)/p55 (Tyr199) rabbit antibody (Cell Signaling Technology), β-actin mouse antibody, NF-κB p65 mouse antibody, SREBP-1 rabbit antibody, keratin 16 mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA), Phospho-IGF1 receptor rabbit antibody, TLR-2 rabbit antibody (Abcam, Cambridge, UK), and IL-1α mouse antibody (R&D Systems, Minneapolis, MN). Secondary anti-rabbit IgG and anti-mouse IgG antibody (Cell Signaling Technology) were used to detect primary antibodies. Blots were developed with WESTSAVE Up (LabFrontier, Seoul, South Korea) and exposed to the film. Films of blots were analyzed and quantified using a densitometric program (TINA; Raytest Isotopenmebgerate, Straubenhardt, Germany). SEB-1 cells were grown to 80% confluence in 60 mm dishes and treated with control or lupeol (5, 10, and 20 μM) for 24 hours. In all experiments, cells were counted to normalize the data. The remaining cells were suspended in a DMEM solution containing 2 μCi 14C-acetate and incubated for 2 hours at 37 °C with agitation, and extracted twice with ethyl ether and nonradioactive carrier lipids. Samples were dissolved in a small volume of ethyl acetate and spotted on 20-cm silica gel thin-layer chromatography plates (Merck, Darmstadt, Germany), which were run until the solvent front reached 19.5 cm in hexane, followed by 19.5 cm in benzene, and finally to 11 cm in hexane/ethyl ether/glacial acetic acid (69.5:30:1.5). Lipid spots were visualized, excised, and radioactivity in each spot was quantified in a liquid scintillation counter. The radio-TLC was also checked using TLC aluminum sheets 20 × 20cm silica gel (Merck) as a stationary phase. To analyze changes of specific fatty acid components after lupeol treatments, SEB-1 sebocytes treated with control or different concentrations of lupeol were grounded after freeze drying. Then, they were placed in tubes with Teflon caps. Pentadecanoic acid (15:0) is used as an internal standard. Methylation and extraction steps were performed as previously described (Garces and Mancha, 1993Garces R. Mancha M. One-step lipid extraction and fatty acid methyl esters preparation from fresh plant tissues.Anal Biochem. 1993; 211: 139-143Crossref PubMed Scopus (502) Google Scholar). Gas chromatography (7890 A, Agilent, Santa Clara, CA) with flame ionization detector was used for detection. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid" @default.
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• W2084558224 title "Activity-Guided Purification Identifies Lupeol, a Pentacyclic Triterpene, As a Therapeutic Agent Multiple Pathogenic Factors of Acne" @default.
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