{'fraction_non_alphanumeric': 0.045245751033532386, 'fraction_numerical': 0.02549379880569591, 'mean_word_length': 4.755783212161269, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Enhancement of skin barrier and hydration-related molecules by protopanaxatriol in human keratinocytes

Background Protopanaxatriol (PPT) is a secondary intestinal metabolite of ginsenoside in ginseng. Although the effects of PPT have been reported in various diseases including cancer, diabetes and inflammatory diseases, the skin protective effects of PPT are poorly understood. Methods HaCaT cells were treated with PPT in a dose-dependent manner. mRNA and protein levels which related to skin barrier and hydration were detected compared with retinol. Luciferase assay was performed to explore the relative signaling pathway. Western blot was conducted to confirm these pathways and excavated further signals. Results PPT enhanced the expression of filaggrin (FLG), transglutaminase (TGM)-1, claudin, occludin and hyaluronic acid synthase (HAS) −1, −2 and −3. The mRNA expression levels of FLG, TGM-1, HAS-1 and HAS-2 were suppressed under NF-κB inhibition. PPT significantly augmented NF-κB-luc activity and upregulated Src/AKT/NF-κB signaling. In addition, PPT also increased phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 and upstream MAPK activators (MEK and MKK). Furthermore, transcriptional activity of AP-1 and CREB, which are downstream signaling targets of MAPK, was enhanced by PPT. Conclusion PPT improves skin barrier function and hydration through Src/AKT/NF-κB and MAPK signaling. Therefore, PPT may be a valuable component for cosmetics or treating skin disorders.


Introduction
Skin is a complex barrier consisting of tight junctions (TJs) and stratum corneum (SC) that protects our bodies from external invasion and water loss [1]. The TJs, intercellular contacts that form barriers in the stratum granulosum of the skin [2], mainly consist of occludin and claudin [3,4]. The SC is the outermost layer of the skin, and the cytoplasm of dead cells in the SC contains the keratin filament matrix [5]. The periphery of the cells in the SC, called the cornified cell envelope, is composed of eÀ(g-glutamyl) lysine crosslinked proteins, including involucrin, cornifins/small proline-rich proteins, keratin intermediate filaments, annexin I, and various other proteins [6e8]. Transglutaminases (TGMs) are membraneassociated enzymes that impart integrity to the SCs by catalyzing eÀ(g-glutamyl) lysine cross-linking reactions [9]. Filaggrin (FLG), a structural protein isolated from SC, also crosslinks with keratin in the SC layer to form a keratin matrix, a parallel structure that is densely packed with intermediate filaments (particularly keratin). The formed keratin matrix which acts as a scaffold to bind cornified-envelope proteins and lipids, thus plays a role in SC formation [10].
The skin maintains viscoelasticity through the moisture present in the SC; 10% or more moisture is essential to keep the elasticity of the SC [22,31]. Proper skin hydration gives the skin flexibility, protects it from damage and enables a process of desquamation by providing an environment in which hydrolytic enzymes can be activated [11e13]. In addition, maintaining the skin's moisture is necessary to optimize the barrier function of the SC. Furthermore, skin hydration deterioration with aging is closely related to wrinkle formation [14]. Key molecules that affect skin hydration include hyaluronic acid (HA) and aquaporins (AQPs) [15e17]. In the epidermis, HA is mainly located in the extracellular matrix (ECM) in the upper spinous-granular layers and inside the cells in the basal layer [18]. HA is a non-sulfated glycosaminoglycan (GAG) that captures large amounts of water, providing flexibility and moisture to the skin [19]. Most GAGs, which are proteoglycans, are produced in the Golgi apparatus that synthesizes the protein core; HA that is not covalently attached to the protein core is synthesized by hyaluronic acid synthase (HAS), a plasma membrane-bound enzyme [20]. There are three types of HAS (HAS-1, -2, and -3), and each of these has a different enzyme activity and enables synthesis of HA of different lengths [21]. AQPs are membrane proteins that act as channels for transporting water [22]. Among these proteins, AQP3, which is most abundant in the epidermis, is an aquaglyceroporin that is involved in hydration of mammalian skin. AQP3 can transport glycerol as well as water [16].
Ginseng is a medicinal plant that is widely used to treat cancer, diabetes, and heart disease in East Asian countries. The pharmacological activity of ginseng is mainly derived from ginsenosides, triterpenoid dammarane structures that belong to the saponin family. To date, over 30 ginsenosides have been isolated from ginseng, and these ginsenosides are classified into three types according to the type of carbohydrate backbone. The type I protopanaxadiol (PPD)-type ginsenosides consist of Rb1, Rb2, Rb3, Rc, Rd, F2, Rg3, and Rh2; the type II protopanaxatriol (PPT)-type ginsenosides are comprised of Re, Rg1, Rg2, Rg4, Rh1, and Rh4; and Ro is the type III oleanonic acid-type saponin [23]. PPT (shown in Fig. 1a) is a secondary metabolite of type II ginsenosides metabolized by the intestinal microflora and has been reported to alleviate steatosis [24], fatigue [25], inflammation [26], diabetes [27], and cancer [28] as well as to have photoprotective properties [29]. However, the effects of PPT on the skin environment are not well studied. Thus, we investigated the molecular mechanism of the pharmacological activity of PPT in the skin barrier and in skin hydration.

Materials
PPT was obtained from Chengdu Must Bio-Technology Co., Ltd.

Cell culture
HaCaT cells with 10 to 30 passage numbers are cultured in DMEM with 10% FBS and 1% antibiotics. HEK293Tcells are cultured in DMEM with 5% FBS and 1% antibiotics. These cell lines were incubated in a 5% humidified CO 2 incubator at the temperature of 37 C.

Cell viability assay
HaCaT cells were seeded in 96-well plates for 5 Â 10 5 cells/well overnight. PPT (3.125, 6.25, 12.5, and 25 mM) was treated in a dosedependent manner for 24 h. MTT assay was performed to check the cell viability [30]. MTT solution was injected at the dose of 10 mL/ well for 3-4 h. MTT stopping solution (10% sodium dodecyl sulfate in 1M HCl) was added to stopping the reaction. After incubating for 16-20 hours, solubilized formazan was measured at the absorbance of 570 nm by multi-plate reader (BioTek, Winooski, VT, USA).

Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis
HaCaT cells were seeded in 6-well plates. Injected PPT (12.5 or 25 mM) in the plates for 24 h. Semi-quantitative RT-PCR was performed as described before [31]. Primer sequences are illustrated in Table 1. performed by using the luciferase assay system.

Immunoblotting analysis
Treat PPT (12.5 or 25 mM), retinol (10 mg/mL), BAY 11-7082 (10 mM), U0126 (10 mM), SP600125 (20 mM) or SB203580 (20 mM) into HaCaT cells for 24 h. Cells were diattached with trypsin, washed with chilled 1x PBS and lysed with lysis buffer (containing 50 mM Tris-HCl, several protease and phosphorylase inhibitors) for 15min on ice. The lysates were used after being clarified by centrifugation at 12,000 rpm for 10 min at 4 C. Then, protein concentration was detected by Bradford assay [33]. The soluble fractions of the lysates were used to perform Western blot, and target proteins were detected as previously reported [34].

Cell morphology shooting
HaCaT cells were seeded into 6-well plate at the cell density of 1 Â 10 5 cells/mL. PPT treatment was at the dose of 25 mM. After 24 hours, 4X, 10X, and 20X microscope objective photos were taken (Olympus, Tokyo, Japan).

Statistical analysis
All the data we got are showed as mean ± standard deviation (SDs), and every experiment was consisted of six replications. Results were analyzed by the ManneWhitney U test to compare the statistical differences. A p value < 0.05 was considered as statistically significant. All the data were calculated by using SPSS software, version 25.0 (IBM Corp., Armonk, NY, USA).

The effect of PPT on skin barrier function and hydration in HaCaT cells
Before evaluating PPT activity, the cytotoxicity of PPT was assessed by MTT assay. The results confirmed that PPT did not induce cell death in HaCaT cells (Fig. 1B). In addition, PPT did not change the morphology but increased the growth rate of HaCaT cells at low density (Fig. 1C). Then, we examined mRNA expression of molecules associated with the skin barrier, FLG, TGM-1, claudin, and occludin, to verify the skin protective effect of PPT. We observed a significant increase in mRNA expression of FLG, TGM-1, claudin, and occludin in cells treated with 25 mM PPT (Fig. 1D).
Retinol was used as a positive control.
To examine the skin hydration efficacy of PPT, mRNA levels of HAS-1, HAS-2, HAS-3 and AQP-3 were investigated. For HAS-1 and HAS-2, the expression levels were significantly increased by treatment with 25 mM PPT (Fig. 1E). Consistent with the mRNA expression results, protein expression of HAS-2 was also elevated in PPT-treated HaCaT cells (Fig. 1F). PPT was found to rescue reduction of HAS-1 in UVB-irradiated HaCaT cells, whereas the mRNA level of HAS-2 was not recovered by this compound (Fig. 1G). These results suggest that PPT has a skin protective capacity through regulation of gene expression involved in skin barrier function and hydration.
3.2. The effect of PPT on the Src/AKT/NF-kB signaling pathway mRNA expression of FLG, TGM-1, HAS-1, and HAS-2 was investigated after treatment with PPT to elucidate the molecular mechanism of increased expression ( Fig. 2A). Expression of four genes was completely inhibited by treatment with Bay-11-7082 (Bay), an inhibitor of IKKa and IkBa, suggesting that the NF-kB signal is involved in the pharmaceutical mechanisms of skin barrier and skin hydration. Also, PPT may interact with the NF-kB pathway. Then, a luciferase assay was conducted to determine whether PPT could modulate NF-kB promoter activity. We observed that PPT increased NF-kB-mediated luciferase activity (Fig. 2B) and that PPTinduced NF-kB-luc activity was inhibited by Bay treatment (Fig. 2C). All of these results demonstrated that PPT can regulate the mechanisms of skin barrier and hydration through the NF-kB pathway. Furthermore, PPT augmented the phosphorylation of upstream molecules of NF-kB, including IkBa, IKKa/b, p50, and p65 (Fig. 2D). The tyrosine 416 phospho-Src was also enhanced while Src phosphorylated at tyrosine 527 was suppressed. This indicates that PPT can upregulate the protein-tyrosine enzyme activity by activating the kinase domain (Fig. 2E). Taken together, PPT enhances the expression of genes responsible for skin protection through upregulation of the Src/AKT/NF-kB signaling pathway.

The effect of PPT on MAPK-mediated AP-1 signaling
To validate the additional pharmacological activity of PPT, AP-1mediated luciferase activity was activated by PPT in a dosedependent manner (Fig. 3A). In addition, the involvement of MAP kinases was evaluated following PPT treatment. PPT-induced gene expression of FLG and HAS-1 was diminished by treatment with SP600126, an inhibitor of JNK (Fig. 3B). Similarly, upregulation of HAS-2 expression after PPT treatment was inhibited by U0126, an ERK inhibitor (Fig. 3B). TGM-1 expression was reduced by both U0126 and SB203580, ERK and p38 inhibitors, respectively, in PPTtreated cells (Fig. 3B). The phosphorylation of c-Jun and c-Fos was increased through PPT treatment (Fig. 3C). The levels of p-ERK, p-JNK, and p-p38 were also increased, with the total form of each MAPK unchanged, following treatment with 12.5 and 25 mM PPT (Fig. 3D). The phosphorylation of MEK1/2, MKK4, MKK7, and MKK3/6, all of which are upstream regulators of MAPK, was simultaneously upregulated after 12.5 and 25 mM PPT treatment (Fig. 3E).

The effect of PPT on MAPK-mediated CREB signaling
Previously, some papers reported that CREB is regulated by ERK [35,36], and HAS-2 expression is dependent on ERK and CREB [37]. Therefore, the effect of PPT on the transcriptional activity of CREB was studied. PPT gradually increased CREB-luc activity (Fig. 4A) and phosphorylation of CREB (Fig. 4B). However, the phosphorylation of PKA, upstream enzyme of CREB, was not reduced (Fig. 4C), while retinol suppressed the phosphorylation of PKA. Therefore, other upstream signaling mechanisms needed to be further detected. To this end, several MAPK inhibitors were treated singly or co-treated with PPT. The result demonstrated that SP600126 and SB203580 can dramatically downregulate p-CREB and that PPT can recover these depletions. However, U0126 had no significant effects (Fig. 4D). These data show that the phosphorylation of CREB is related to JNK and p38. Taken together, these results imply that PPT exhibits skin-protective activity through the regulation of MAPKmediated AP-1 activity and crosstalk with CREB signaling.

Discussion
In this study, we assessed the effects of PPT on the skin barrier including skin hydration in HaCaT cells. To this end, the effect of PPT on alteration of genes that encode representative skin barrier proteins such as FLG, TGM-1, claudin, and occludin was examined. In addition, we investigated whether PPT could regulate the expression of the HAS and AQP3 moisturizing factors. Additionally,   the action mechanism of PPT was dissected at a molecular level. In particular, since SC acts as the major barrier in the skin, we focused on understanding how PPT regulates the expression of four molecules present in the SC (FLG, TGM-1, HAS-1 and HAS-2).
Our results demonstrated that PPT strengthens the skin barrier by increasing the expression of the main elements of SC, FLG, and TGM-1, as well as claudin and occludin, important components of TJs. Also, PPT simultaneously enhances skin hydration by elevating the expression of HAS-1, -2, and -3. FLG is a constituent of the skin barrier. However, FLG monomers isolated from keratin by deamidation are degraded by proteases such as caspase 14 to form natural moisturizing factors (NMFs) [38,39]. Thus, increased expression of FLG may be associated with moisturizing. In addition, exogenous HA is rapidly degraded in the dermis, so HA synthesis or HA degradation should be targeted to improve skin hydration [40]. Thus, PPT, which can increase the expression of HAS-1, HAS-2, and FLG, is expected to be an effective skin moisturizer. Moreover, collapsed skin barriers and dry skin are known symptoms of various skinerelated diseases such as atopic dermatitis (AD), irritant contact dermatitis and psoriasis [13,41,42]. The expression of FLG is significantly reduced in AD patients [43]. Decreasing expression of occludin in flaky tail mice, an experimental animal model of AD, has also been reported [44]. Interestingly, our study showed that PPT treatment led to higher expression of FLG, TGM-1, and occludin than retinol treatment. Therefore, we expect that PPT can be utilized as a powerful active ingredient for the treatment or symptom relief of skin diseases such as atopic dermatitis [45].
Next, we sought to understand how PPT can regulate the expression of target genes. Gene expression of FLG, TGM-1, HAS-1, and HAS-2 was blocked by the inhibitor of IKKa and IkBa. In addition, PPT significantly enhanced NF-kB-luc activity and phosphorylation levels of IkBa, IKKa/b and AKT, indicating that the skin protective activity of PPT is dependent on the NF-kB signaling pathway. FLG, TGM-1, HAS-1, and HAS-2 expression levels were also blocked by ERK, JNK, and p38 inhibitors (U0126, SP600125, and SB203580, respectively). Therefore, the influence of PPT on AP-1, a downstream transcriptional factor of MAPK, was studied. PPT upregulated AP-1-mediated Luc activity as well as phosphorylation of ERK, JNK, and p38. Additionally, phosphorylation of MEK1/2, MKK4/7, and MKK3/6, the upstream regulators of ERK, JNK and p38, respectively, was increased by PPT. In contrast, retinol did not enhance the phosphorylation of MKK4/7, MKK3/6, and p38, implying that PPT has some different inhibitory characteristics compared to those of retinol in AP-1 signaling pathway. Furthermore, luc activity and phospho-CREB level, were upregulated by JNK and p38. Collectively, our results indicate that PPT increases gene expression of skin-barrier and moisturizing proteins via the regulation of NF-kB, MAPK/AP-1, and JNK/p38/CREB signal transduction. Interestingly, this study newly revealed that multifunctional transcription factors such as NF-kB and AP-1, which act as key regulators in inflammatory responses and cancer development [46e48], were also closely involved in the expression of FLG, TGM-1 and HAS. This suggests that substances with known antiinflammatory or anti-cancer effects may also function to improve the skin barrier. Indeed, PPT shows anti-inflammatory and antiallergic effects in macrophages and mast cells through the inhibition of NF-kB and AP-1 [49].
Taken together, our results demonstrate that PPT reinforces the skin barrier and skin hydration through the NF-kB, AP-1, and CREB pathways, as summarized in Fig. 5. Furthermore, we propose that PPT can be used as an active ingredient for skin moisturizing cosmetics as well as for the treatment of AD.

Declaration of competing interest
The authors have no conflicts of interest to declare.

Acknowledgments
This paper was supported by Konkuk University, South Korea, in 2020.