Inhibition of Proinflammatory Cytokines in Cutibacterium acnes-Induced Inflammation in HaCaT Cells by Using Buddleja davidii Aqueous Extract

Acne is an inflammatory skin disorder; although some anti-inflammatory medicines for treating acne are available in a market, they have considerable side effects; therefore, new treatment options are needed. In the present study, among the 16 aqueous extracts of plants collected from Jeju Island in Korea which are used to test anti-inflammatory activity, B. davidii showed the strong decline of the proinflammatory cytokine expression against the inflammatory process caused by C. acnes in Human HaCaT keratinocyte cells. B. davidii downregulated the expression of 57% of COX-2, 41% of iNOS, and proinflammatory cytokines 29% of TNF-α, 32% of IL-1β, 21% of IL-6, and 35% of IL-8. Furthermore, B. davidii inhibited NF-κB and MAPK signaling cascades in keratinocytes that activated by toll-like receptor 2 (TLR-2) in response to C. acnes. Given those results, B. davidii is a potential agent to reduce the proinflammatory cytokine expression against C. acnes-induced inflammation and might provide an alternative to the current medications.


Introduction
Acne is a chronic inflammatory disorder of the sebaceous follicles on the chin, cheek, forehead, and back. Cysts and scarring can occur when the disease is severe. Although acne is a skin disease, it can lead to psychological issues that affect patient quality of life. In acne pathogenesis, C. acnes has been identified as one of the main causative agents. C. acnes, a commensal Gram-positive anaerobic, colonizes the duct of the sebaceous follicle, causing an innate immune response [1].
Inflammation is the body's initial complex response to infection. e processes of inflammation caused by C. acnes begin when peptidoglycan (PGN) and lipoteichoic acid (LTA) recognized by binding to toll-like receptors (TLRs) activate the TLR signaling pathway and trigger important intracellular signaling pathways including the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways.
is causes nuclear translocation of transcription factors, such as activator protein-1 (AP-1) and NF-κB, ultimately leading to production of a variety of proinflammatory cytokines including tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL-8, and IL-6 [2,3]. Adverse effects associated with anti-inflammatory drugs have become increasingly common and include increased risk of gastrointestinal ulcers, bleeding, heart attack, and kidney disease associated with nonsteroidal anti-inflammatory drugs (NSAIDs) [4]. Some anti-inflammatory drugs interfere with deactivated and hyperactivated cellular signaling networks or transcription factors [4]. erefore, to limit adverse effects, the next generation of anti-inflammatory drugs needs to target either enzymes or signaling pathways that are only operational in affected tissues or a small subset of inflammatory target genes.
Medicinal plants have long been used as sources of therapeutically active compounds, which is also a popular traditional medicine to treat inflammatory diseases. e use of traditional medicines for treatment of inflammation could provide safer alternatives compared with existing conventional treatments. Evidence suggests that herbal medicines have a lower rate of side effects compared with conventional medicines [5]. Furthermore, medicinal plants used for dermatological purposes with low or no side effects are gaining popularity in both traditional medicine and the cosmetic industry. Buddleja davidii is a perennial herbaceous plant that belongs to the Scrophulariaceae family with a pan tropical distribution across South Asia, Africa, and America. Its roots, leaves, and flowers are used in folk medicine for antioxidant, anti-inflammation, antiviral, antiplasmodium, and antifungal effects [6,7]. e purpose of the present study was to determine the anti-inflammatory activity of B. davidii against C. acnes infection and to elucidate its mechanism of action in human keratinocyte cell line.

Plant Material and Extraction.
Candidate plants, the herb medicine with anti-inflammation properties, were obtained from Jeju Island in South Korea. e plants were cleaned with distilled water and then dried using a freeze dryer (iLShin, Korea). After drying, 300 g of the dried herb was extracted three times in 3 liter of distilled water with refluxing at 80°C for 8 hours. 500 ml extract was concentrated under reduced pressure at 50°C using a rotary evaporator (EYELA, Japan) connected to a refrigerated bath circulator (Jeio Tech, Korea) [8]. e decoction was filtered using muslin cloth followed by Whatman (GE Healthcare, USA) grade 1 filter paper, lyophilized using a freeze dryer (iLShin, Korea), and stored at 4°C before further use [9]. To prepare the sample for experiments, the stock of extract powder (10 mg) was dissolved in 1 ml of dimethyl sulfoxide (DMSO).

Quantitative Real-Time PCR.
HaCaT cells (seeded at a density of 5 × 10 5 per well in a six-well plate) were incubated for 24 hours, pretreated with various concentrations (12.5, 25, 50 and 100 μg/ml) of aqueous extract of plants for 1 hour, and subsequently treated with heat-killed C. acnes (100 MOI, 5 × 10 7 CFU per well) for 24 hours. Total RNA was isolated from the HaCaT cells using TRIzol reagent (Life Technology, ermo Fisher Scientific, USA) according to the manufacturer's protocol. A reverse transcriptase (NanoHelix, Korea) reaction was prepared using 1 μg of RNA to obtain cDNA. e cDNA was used as the template for real-time PCR (qRT-PCR), which was carried out with QGreen 2X SybrGreen qPCR Master Mix (CellSafe, Korea). e primer sequences used to detect TLR-2, iNOS, COX-2, TNF-α, IL-8, IL-6, IL-1β, and GAPDH are listed in Table 1. GAPDH was used as an endogenous control. e delta-delta Cq formula is used to calculate the gene expression.

Statistical Analysis.
All data are presented as mean-± standard deviation (SD) of at least three independent experiments and were analyzed using one-way ANOVA. Student's t test was used when only two groups were compared. Differences were considered statistically significant at p < 0.05, p < 0.01.

Screening for Potential Anti-Inflammatory Activity of Aqueous Extract of Plants.
e effect of plant aqueous extract on the expression of genes is given in Table 2.
e inflammatory process increases the expression of COX-2 and NO synthase, which produce inflammatory mediators [12,17]. In addition, proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8 trigger and rapidly amplify the inflammatory response to limit the spreading of the infection [2,18]. In this study, 16 plants were extracted from Jeju Island (Korea) that were tested for their antiinflammatory activity against C. acnes-induced inflammation. e percentage of gene expression described in Table 2 was calculated based on the ratio of gene expression of C. acnes-infected HaCaT cells treated with aqueous plant extract over one without the plant aqueous extract as an increasing 100% compared with C. acnes-free HaCaT cells. Reduced values represent the inhibition of the expression of COX-2, NO, and proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8. Table 2 shows A. japonica A. Gray, B. davidii, I. verum, and P. fibrosa aqueous extract decreased the inflammation process caused by C. acnes. Among them, B. davidii aqueous extract showed the strongest inhibition toward the inflammatory process caused by C. acnes via reduced iNOS, COX-2, TNF-α, IL-1β, IL-6, and IL-8 expression (Table 2).

Suppression of C. acnes-Induced TLR-2 Expression in
HaCaT Cells by B. davidii Aqueous Extract. C. acnes contribute to the inflammation in acne through activation of toll-like receptors (TLRs), in particular, TLR-2 [2,19]. In the initial screening, B. davidii aqueous extracts displayed the strong decline of the proinflammatory cytokine expressions in C. acnes-treated HaCaT cells (Table 2). e effects of B. davidii aqueous extract on expression of the pattern recognition receptor TLR-2 showed C. acnes induced the expression of TLR-2 while treated with 50 μg/ml of B. davidii aqueous extracts and reduced the TLR-2 mRNA expression by 30% compared with C. acnes-treated HaCaT cells without B. davidii aqueous extract (Figure 1).

B. davidii Aqueous Extract
Reduced NF-kB Phosphorylation in C. acnes-Treated HaCaT Cells. TLR-2 signaling leads to the activation of the NF-κB pathway, and then NF-κB is released and translocates to the nucleus where it regulates expression of an array of genes, which in turn stimulates the release of proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α [2][3][4]. e NF-κB pathway was activated by C. acnes, indicated by increased expression of toll-like receptor 2 (TLR-2) ( Figure 1) compared with C. acnes-free. B. davidii aqueous extract dose-dependently reduced the amount of p-NF-κB and NF-κB protein which could be due to the decrease of the gene expression, as determined using western blotting ( Figure 2). ese results suggest that the anti-inflammatory activity of B. davidii aqueous extract in HaCaT cells with C. acnes-induced inflammation might be due to suppression of the NF-κB signaling pathway.

Inhibition of C. acnes-Induced p38 and JNK Expression and Phosphorylation by B. advidii Aqueous Extract.
e mitogen-activated protein kinase (MAPK) signaling pathways play an essential role in regulating the production of multiple inflammatory mediators [2,4,20]. e effects of B. davidii aqueous extract on C. acnes-induced MAPK pathway activity were examined using western blotting for p-JNK and p-p38 ( Figure 3). C. acnes-induced MAPK activity in HaCaT cells and treatment with B. davidii aqueous extract extracts dose-dependently suppressed this MAPK activity.
ese results indicate that B. davidii aqueous extract exerts its anti-inflammatory actions via inhibition of the MAPK signaling pathway.

Cytotoxicity of B. davidii Aqueous Extract in HaCaT C.
To determine the cytotoxic effects of B. davidii aqueous extracts on HaCaT cells, the MTT assay was used. e cells were treated with B. davidii aqueous extracts for 24 hours at a concentration ranging from 0.39 μg/ml to 100 μg/ml, and the B. davidii aqueous extracts had no significant cytotoxic effects on HaCaT cells ( Figure 5).

Discussion
Inflammation has been suggested as a key factor involved in the development and aggravation of acne vulgaris, although the exact pathogenesis of acne vulgaris has not been elucidated. It has been verified that C. acnes play a major role in the development of inflammatory acne lesions [1,4]. C. acnes promote the activation of TLR-2 in keratinocytes, aggravating the inflammation reactions [2,4]. In fact, the C. acnes infection begins to activate monocyte TLR-2, a major signaling pathway induced by activation of NF-κB or MAPKs signaling pathway, which in turn gives rise to expression of cytokines, chemokines, adhesion molecules, and   International Journal of Inflammation granulopoiesis factors [4,18,20]. On the basis of this information, B. davidii aqueous extract effectively prevents the inflammation caused by C. acnes through inhibition of the TLR-2 to NF-κB signaling cascade in keratinocytes (Figures 1 and 2).
Recently, it has been studied that the MAPK signaling pathway plays a critical role in regulation of the inflammatory response and coordination of the induction of many genes encoding inflammatory mediators [13,19,20]. B. davidii aqueous extract suppressed mitogen-activated protein kinase (MAPK) by inhibiting p-p38 and p-JNK in HaCaT cells treated with C. acnes (Figure 3). Activation of the TLR-2-mediated downstream MAPK signaling pathway is responsible for production of inflammatory cytokines. MAPK activation has been found in clinical acne lesions, indicating that inhibition of MAPK signaling is important for the pathogenesis of acne vulgaris in general [4,20]. Use of MAPK inhibitors is Relative protein expression of p-p38, p38, p-JNK, and JNK (relative to GAPDH) was determined using ImageJ. All data were expressed as mean ± SD. * p < 0.05, * * p < 0.01 compared with C. acnes-treated cells only.
emerging as an attractive alternative to anti-inflammatory drugs because they decrease synthesis of proinflammatory cytokines [20]. e results of this study suggest that B. davidii aqueous extract modulates the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) in the inflammation process through the inhibition of the NF-κB and MAPK kinase pathways (Figures 4 and 6). Hence, B. davidii aqueous extract might be a potential MAPK inhibitor to treat the inflammation caused by bacterial infections.
B. davidii is a well-known, traditional, herbal medicine and a promising candidate for the treatment of acne and other inflammatory diseases [6,7]. A better understanding of the mechanism of B. davidii's anti-inflammatory activity would not only offer insight into inflammation homeostasis but also provide opportunities for the discovery of new antiinflammatory compounds with fewer side effects in the future.
In conclusion, this study provides the first evidence that B. davidii has the anti-inflammation property through inhibiting two pathways that are NF-κB and MAPK kinase, leading to the reduced proinflammatory cytokine expression. ese findings suggest that B. davidii is a potential effective therapeutic agent for the treatment of acne vulgaris. However, further studies are needed for a full understanding of its mechanism of action as well as identification of the active components of B. davidii in anti-inflammatory activities such as luteolin, quercentin, hesperetin, and colchicine [6,24]. In addition, evaluation of the in vivo effects of B. davidii is required for use in clinical applications.

Data Availability
No data were used to support this study.

Conflicts of Interest
e authors declare that they have no conflicts of interest.