Cnidium ocinale Makino Promotes Skin Health via Anti-Inammation Processes in Various Skin Cell Lines

Background Dermatitis is a worldwide health problem that is associated with quality of life. The skin continuously protects the body from the noxious environment. Cnidium ocinale Makino (CM) is an herb used in traditional medicine to treat skin diseases. Methods This study aimed to investigate whether CM exerted antioxidant and anti-inammatory effects and to describe the effect of CM on the moisturizing and whitening of human mast cells (HMC-1), keratinocytes and melanocyte cells. Antioxidant activity was measured by a DPPH free radical assay. The mRNA expression of hyaluronan synthases 1, 2, and 3, Filaggrin, Claudin-4 and Aquaporin3 was measured by RT-PCR. Microphthalmia-associated transcription factor (MITF), tyrosinase, TRP1, TRP2, AKT, Erk and NF-kB protein levels were evaluated by Western blotting analysis. Results We found that the levels of the DPPH free radical were decreased by CM treatments. CM exhibited anti-inammatory activities, including the suppression of inammation-associated molecules. We found that the levels of whitening-related proteins (MITF, tyrosinase, TRP1, and TRP2) were increased with CM treatment compared with α-MSH stimulation in B16F10 cells. CM induced the upregulation of hyaluronan synthases 1, 2, and 3, Filaggrin, Claudin-4 and Aquaporin3 mRNA expression in keratinocytes. Conclusions These ndings indicate that CM reduced several inammatory responses. CM exhibited antioxidant, skin-moisturizing and whitening activity, indicating that CM might be a useful drug for combating inammation and in skin care.


Background
Dermatitis is a worldwide health problem that is associated with quality of life. Millions of people worldwide suffer from in ammatory skin disorders [1,2]. The skin is the largest organ of the human body, and it is extensively exposed to the external environment. Additionally, it functions as the necessary interface between the internal and the external environment. The skin continuously protects the body from the noxious environment. The poor appearance of the skin resulting from dermatitis affects not only the body but also the mental condition of the patient. Therefore, general skin condition is an important indicator of health [3,4].
In ammation is a response to stimuli such as infections and tissue injury and leads to in ammatory cell migration, cytokine, prostaglandin, and leukotriene production and proin ammatory molecule release.
During in ammation, in ltrating neutrophils and cytokines are released [5][6][7]. As a result, uncontrolled or sustained in ammation induces several pathophysiological conditions, such as bacterial sepsis, rheumatoid arthritis, rhinitis, and skin in ammation [8][9][10]. Recently, moisturizers, antihistamines and corticosteroids have been used to treat skin in ammation, repair altered skin barrier function and reduce itching, but the use of steroids causes skin atrophy by reducing the amount of collagen [11]. A new therapy and intensive studies are needed. Previous studies have shown that various herbal medicines exert antioxidative, anti-in ammatory, and antimicrobial effects in animal models, thus increasing their use for therapeutic purposes [12,13]. Thus, herbal medicine is emerging as a novel alternative source of antioxidative and anti-in ammatory agents in food and cosmetics [14,15].
Mast cells are activated by IgE through the high-a nity IgE receptor, and activated mast cells secrete in ammatory mediators, histamine, leukotrienes, prostaglandin E2, cytokines and chemokines. As a result, mast cells mediate various immune responses and regulate allergic in ammation, including atopic dermatitis [16,17]. Additionally, keratinocytes play a pivotal role in the pathogenesis of in ammatory skin diseases, and activated keratinocytes induce skin in ammation by secreting Th2-related cytokines and chemokines [18,19]. These Th2-related cytokines and chemokines stimulate the in ltration of immune cells, including mast cells, into the site of in ammation on the skin and cause skin dermatitis [20,21]. Thus, inhibitors of these in ammatory mediators can be used for the treatment of in ammatory skin disease.
The root of Cnidium o cinale Makino (CM) is a perennial plant in the Umbelliferae family and is extensively cultivated in Korea, China and Japan. CM is traditional herbal medicine called ''Chunkung'' in Korea and has been used as a medicinal plant for a long time in Asia [22][23][24][25] Therefore, CM has a number of potential uses in various health-related elds, including the food processing, pharmaceutical, and cosmetics industries. However, the effect of CM on in ammation of the skin has not yet been elucidated. In the present study, we investigated the effects of the CM on skin in ammation.

Preparation of CM
CM was supplied by Han-poong Pharm Co., Ltd. (Jeonjoo, Republic of Korea). CM powder was dissolved in distilled water to a concentration of 20 mg/ml.

Cell Viability Assay
An MTS assay was performed to determine cell viability. In this assay, cells (HMC-1, HaCaT and B16F10 cells) were seeded in a 96-well plate at a density of 3 × 10 3 cells per well and treated 24 h later with varying concentrations of CM (5-1000 µg/mL) for an additional 24 h. HaCaT cells were treated with 1 µg/mL LPS, and B16F10 cells were treated with 100 ηM α-melanocyte-stimulating hormone (α-MSH) in the presence or absence of various concentrations of CM. Ten microliters of a solution of tetrazolium salt (WST) was added to each well of the plate, which was incubated in the dark at 37 °C for another 1 h. Optical density was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Versa Max; Molecular Devices LLC, Sunnyvale, CA, USA).

RT-PCR
RNA was isolated using an Easy-Blue RNA Extraction Kit (iNtRON Biotech, Republic of Korea). In brief, we harvested cells (HMC-1, HaCaT and B16F10 cells), and 1 mL of R&A-BLUE solution was added to each well. Next, 200 µL of chloroform was added to the lysate, and the mixture was vigorously vortexed for 10 seconds. Then, the lysate was centrifuged at 13,000 rpm for 10 min at 4 °C. We then transferred the appropriate volume of the aqueous phase into a clean tube, added 400 µL of isopropanol and thoroughly mixed the solution by inverting the tube 5 times. After centrifuging the tube at 13,000 rpm for 10 min, the supernatant was carefully removed without disturbing the pellet. Then, 1 mL of 75% ethanol was added, and the solution was thoroughly mixed by inverting the tube 4-5 times. The mixture was then centrifuged for 1 min at room temperature, and the supernatant was carefully discarded without disturbing the pellet. Finally, the remaining RNA pellet was dried and then dissolved in 20-50 µL of RNase-free water. The concentration of the isolated RNA was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, USA). We treated each sample with DNase. Two micrograms of total cellular RNA from each sample was reverse-transcribed using a cDNA synthesis kit (TaKaRa, Otsu, Shinga, Japan). PCR was conducted in a 20-µL reaction mixture composed of a DNA template, 10 pM of each gene-speci c primer, 10x Taq buffer, 2.5 mM of dNTP mixture, and 1 unit of Taq DNA polymerase (Takara, Otsu, Shinga, Japan). PCR was performed using the speci c primers listed in Table 1.

ELISA
The levels of IL-4 (BD 555194), IL-6 (BD 555220), IL-8 (BD 555244) and tumor necrosis factor (TNF) (BD 555212) were assessed using a Duoset ELISA system (BD Biosciences, USA) according to the manufacturer's instructions. In brief, to assess the levels of IL-4, IL-6, IL-8 and TNF in HMC-1 cells treated with CM, phorbol myristate acetate (PMA) and A23187, 96-well plates were coated with capture antibody in ELISA coating buffer and incubated overnight at 4 °C. The next day, the plates were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-T). Then, the plates were subsequently blocked with 10% FBS in PBS for 1 h at room temperature. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates, and the plates were incubated for 2 h at room temperature. After the plates were washed, biotin-conjugated anti-mouse IgE and streptavidin-conjugated horseradish peroxidase (SAv-HRP) were added to the plates, and the plates were incubated for 1 h at room temperature. Finally, the tetramethylbenzidine (TMB) substrate was added to the plates, and after 20 min of incubation in the dark, 50 µL of 2 N H2SO4 was added to stop the reaction.

Cell Migration
The human keratinocyte HaCaT cells were incubated at 3 × 10 5 cells/mL for 24 h in a cell culture incubator. Next, the cell monolayers were scratched with a 200-µL yellow tip and washed once with PBS.
Then, the cell monolayers were treated with different concentrations of GF and cultured in a CO 2 incubator for 24 h. Cell motility was assessed 24 h later, using a photomicroscope, and the scratched area was measured. Measurements were taken to determine the distance traveled in the 24 h period by measuring the scratched area by light microscopy (Olympus, Tokyo, Japan).

Melanin Content Assay
The melanin content assay was performed as previously described with some modi cations [33]. The mouse melanocyte B16F10 cells were treated with α-MSH (100 ηM) for 24 h and further treated with different concentrations of GF for another 24 h. After the treatments, the cells were detached by incubation with trypsin and subsequently centrifuged at 5000 xg for 5 min. Then, the cell pellets were solubilized in 1N NaOH at 60 °C for 60 min. The melanin content was assayed at 420 nm by an ELISA plate reader (Versa Max; Molecular Devices LLC, Sunnyvale, CA, USA).

Statistical Analysis
All experimental results are expressed as the mean ± SEM of at least three separate tests. Statistical signi cance was determined at < 0.05, < 0.01 and < 0.001 and is indicated with different symbols in the gures. Statistical analyses (ANOVA) were performed using PRISM software (GraphPad Software Inc., La Jolla, CA, USA).

CM Inhibited Agonist-Induced In ammatory Cytokine Production in HMC-1 Cells
To investigate whether CM affects cytokine expression in HMC-1 cells, we stimulated HMC-1 cells with A23187 and PMA before treatment with varying concentrations of CM. No signi cant effect on cell viability was observed in the HMC-1 cells treated with CM alone or in combination with A23187 and PMA ( Fig. 1A). Western blot analysis indicated that CM signi cantly reduced the agonist-stimulated protein expression of AKT, Erk, NF-κB and COX-2 in a dose-dependent manner (Fig. 1B). Moreover, RT-PCR analysis showed that CM dose-dependently suppressed the mRNA expression of IL-6, IL-8, IL-13, IL-17 and TNF-α that was induced by treatment with A23187 and PMA (Fig. 1C). We also demonstrated that CM inhibited the agonist-stimulated secretion of IL-6, IL-8, and TNF, as determined by ELISA (Fig. 1D).

Cm Suppressed Lps-induced In ammatory Responses In Hacat Cells
Furthermore, we evaluated the anti-in ammatory activities of CM in keratinocytes. Similar to HMC-1 cells, HaCaT cells showed no signi cant effect of toxicity was observed either when treated with CM alone or in combination with LPS ( Fig. 2A). Western blot analysis demonstrated that treatment with a high dose of CM reduced the levels of COX-2, p-AKT, and p-Erk and the activity of NF-KB in LPS-induced HaCaT cells (Fig. 2B). Finally, CM treatment decreased the mRNA levels of proin ammatory cytokines, including IL-6, IL-13, and TNF-α, in LPS-stimulated HaCaT cells (Fig. 2C).

Cm Promoted Dpph Radical Scavenging Activity
The antioxidant activity of CM was evaluated by measuring its ability to scavenge DPPH free radicals, and vitamin C (1-30 µg/mL) was used as a positive control. As shown in Fig. 3, CM demonstrated profound free radical scavenging activity with 65 and 90% inhibition at the concentrations of 500 and 1000 µg/ml, respectively. The free radical scavenging activities of vitamin C (10-30 µg/ml) and CM (250-1000 µg/ml) were similar.

Whitening Effect of CM via the Suppression of α-MSH-Induced Melanin Synthesis in B16F10 Cells
We investigated whether CM affects the whitening effect via the suppression of α-MSH-induced melanin synthesis in B16F10 cells. B16F10 cells were stimulated with α-MSH and then treated with varying concentrations of CM. No signi cant effect on cell viability was observed in B16F10 cells treated with CM alone or in combination with α-MSH (Fig. 4A). We next investigated the inhibitory effects of CM on α-MSH-induced melanin synthesis in B16F10 cells. To con rm the inhibitory effect of CM on α-MSHinduced melanin synthesis, we determined the melanin content in α-MSH-stimulated B16F10 cells in the absence or presence of CM. We demonstrated that CM suppresses the α-MSH-induced melanin accumulation in B16F10 cells (Fig. 4B). Because MITF is an essential transcription factor that regulates melanogenesis-associated gene expression through the α-MSH-PKA-CREB axis [34], we further investigated whether CM regulates these melanogenesis-associated signal transduction pathways.
Western blot analysis showed that treatment with a high dose of CM decreased TRP1, TRP2, MITF and tyrosinase levels in α-MSH-induced B16F10 cells (Fig. 4C).

CM Improves Skin Health via the Modulation of Gene Expression in HMC-1 and HaCaT Cells
We investigated whether CM affects skin health by modulating gene expression in HMC-1 and HaCaT cells. Newly activated T lymphocytes are able to produce IL-4, which is a major component of the in ammatory response in atopic dermatitis. We found that CM suppressed the levels of IL-4 mRNA and cytokines in HMC-1 cells in a dose-dependent manner (Fig. 5A, B). We next examined whether a high concentration of CM induces Aquaporin3, claudin-4, laggrin, hyaluronan synthase (HAS)-1, HAS-2, and HAS-3 expression in cultured skin keratinocytes. CM induced the mRNA expression of Aquaporin3, claudin-4, laggrin, HAS-1, HAS-2, and HAS-3 in keratinocytes (Fig. 5C). We investigated the effect of CM on keratinocyte migration in response to scratching. As shown in Fig. 5D, migration was increased in CMtreated keratinocytes compared to control keratinocytes in a dose-dependent manner.

Discussion
CM has been traditionally used as an anti-in ammatory agent for centuries. CM is considered an important source of various herbal medicines and is known to contain several major compounds, such as falcarindiol (FAD), 6-hydroxy-7-methoxy-dihydroligustilide, ligustilidiol, and senkyunolide H. [35][36][37]. Furthermore, FAD exhibited a potent inhibitory effect on the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in murine macrophages and macrophages from brain tissues [23,38]. In the present study, we investigated the effects of CM on skin in ammation.
TNF-α is a known in ammatory factor involved in a variety of in ammatory diseases [39][40][41]. The activation of TNF-α induces the autocrine and paracrine activation of macrophages. As a result, an increase in the generation of in ammatory cytokines, such as IL-6, IL-17 and COX-2, can lead to a chain reaction of in ammation [42][43][44]. TNF-α and IL-17 are important markers of skin in ammation, and the inhibition of in ammatory cytokines, such as TNF-α and IL-17, yields positive effects on the treatment of dermatitis [45][46][47][48]. Additionally, PI3K/mTOR/Akt inhibitors are known to act as therapies for in ammatory skin diseases, such as skin atrophy [49].
We investigated whether CM affects cytokine expression in HMC-1 and HaCaT cells. CM signi cantly reduced agonist-stimulated AKT, Erk, NF-κB and COX-2 protein expression in a dose-dependent manner in HMC-1 and HaCaT cells. CM dose-dependently suppressed IL-6, IL-8, IL-13, IL-17 and TNF-α mRNA expression in HMC-1 cells. Additionally, CM treatment reduced the mRNA levels of proin ammatory cytokines, including IL-6, IL-13, and TNF-α, in LPS-stimulated HaCaT cells. We also demonstrated that CM inhibited the agonist-stimulated secretion of IL-6, IL-8, and TNF, as determined by ELISA. We found that CM had an anti-in ammatory effect on HMC-1 and HaCaT cells.
In the DPPH radical scavenging method, DPPH free radicals were used to determine the antioxidant (scavenging) activity of various extracts. CM demonstrated profound free radical scavenging activity in a dose-dependent manner. Additionally, the free radical scavenging activities of vitamin C (10-30 µg/ml) and CM (250-1000 µg/ml) were similar.
α-MSH is known to be released from UV-exposed keratinocytes and can stimulate melanin biosynthesis. α-MSH leads to an increase in MITF. Moreover, MITF increases the gene expression of TRP1 and TRP2 in melanocytes [50]. We found that CM suppresses α-MSH-induced melanin accumulation in B16F10 cells. The levels of IL-4 mRNA and cytokines were decreased with CM treatment in HMC-1 cells in a dosedependent manner. Moreover, CM induced the mRNA expression of Aquaporin3, claudin-4, laggrin, HAS-1, HAS-2, and HAS-3 in keratinocytes. We found that CM impacts keratinocyte migration in response to scratching. Taken together, our results suggest that CM regulates proin ammatory cytokine production in mast cells and keratinocytes, thereby affecting skin health.

Conclusion
Our present study demonstrates that CM treatment suppresses the production of several in ammatory cytokines and the NF-kB and MAPK pathways in HMC-1 cells and HaCaT cells. In addition, our data indicate that CM treatment decreases melanin biosynthesis in UV-exposed cells, increases DPPH radical scavenging activity, and affects several markers of skin health. Taken together, our results propose that CM might be a potentially useful drug for skin care. Whole cell lysates were analyzed by Western blotting (B). IL-6, IL-8, IL-13, IL-17 and TNF-α mRNA expression was measured by RT-PCR (C). The culture medium of the cells was harvested, and TNF, IL-6 and IL-8 cytokine levels were measured by ELISA (D). The data are presented as the mean ± SEM of three independent experiments. #P < 0.05, ##P < 0.01 and ###P < 0.001 compared to the normal control group. * P < 0.05, * * P < 0.01 and * * * P < 0.001 compared to the A23187-and PMA-stimulated groups.

Supplementary Files
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