Nepetin, a Flavonoid Derived From Saussureae Involucratae, Suppresses The LPS-Induced Inammatory Response in Cultured Human Keratinocytes

Background: Saussureae Involucratae Herba, known as “snow lotus” in Uyghur and/or Chinese medicines, is the dried aerial part of Saussurea involucrata (Kar. et Kir.) Sch.-Bip. (Asteraceae). One of the known pharmacological applications of this herb is to suppress chronic inammation. Nepetin is considered as a bioactive avonoid of Saussureae Involucratae Herba. Here, we are probing the ecacy of nepetin in modulating lipopolysaccharide (LPS)-stimulated inammatory responses in cultured human keratinocytes. Results: In cultured keratinocytes, applied nepetin prevented the LPS-induced cell death. In parallel, the productions of inammatory mediators, i.e. iNOS, COX-2 (cid:0) PGES2 and NO, were declined after nepetin challenge in LPS-treated keratinocytes. Besides, the treatment of nepetin in LPS-induced cultures suppressed the expressions of cytokines, i.e. IL-1β, IL-6 and TNF-α, in a dose-dependent manner. The productions of inammatory modulators and/or cytokines could be accounted by nepetin-mediated NF-kB translocation from cytosol to nucleus within the inammatory activated keratinocytes. In accord to this notion, the formation of ROS, as induced by LPS, could be reduced by nepetin challenge. Conclusions: The aforementioned results suggested that nepetin could account the anti-inammatory properties of Saussureae Involucratae Herba, at least during the skin inammation, e.g. atopic dermatitis.


Introduction
In ammation is commonly associated with many chronic diseases. The in ammatory process is triggered by recruitment of activated immune cells, e.g. mast cell, monocyte, macrophage and lymphocyte, to the site of lesion [1]. Skin in ammatory disorder is classi ed as typical dermatologic disease, categorized as acute and chronic conditions [2]. Acute disorder is commonly triggered by UV light, allergen, and physical/chemical irritants. The symptoms of acute in ammation are itching, rash and skin redness, and which are appearing and disappearing quickly [3]. On the other hand, chronic in ammation is di cult to deal with, and which could be further divided into atopic dermatitis, seborrheic dermatitis, psoriasis, and rosacea [4,5]. In fact, atopic dermatitis is the most common recurrent dermatological problem. Atopic dermatitis, known as atopic eczema, is caused by genetic and/or environmental factors [3], which is leading to long-term in ammation of skin, resulted in itchy, red, swollen and cracked skin. The incidence of atopic dermatitis is increasing, estimated about 230 million people suffering from this medical problem globally in 2010 [6]. Unfortunately, there is no complete cure for atopic dermatitis.
Reactive oxygen species (ROS) has been reported to play as a second messenger, and which activates immune modulatory responses [7]. Oxidative stress is the starting point, and which initiates nuclear factor kappa B (NF-κB) translocation and cytokine release [8,9]. High levels of cytokines, e.g. interleukin 1-beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α), as well as over expressions of in ammatory mediators, e.g. inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E synthase 2 (PGES2) and nitric oxide (NO), aggravate the skin barrier at the injury site [10]. Corticosteroid is the standard treatment for atopic dermatitis; the usage is effective in attenuating dermatitis for shortterm application [11]. However, longer period administration of steroid may induce skin macular atrophy, striae, telangiectasia, and other side effects [12]. Therefore, the treatment of atopic dermatitis requires better and safer therapy. Several lines of evidence have suggested the possible application of avonoidenriched herbal product in treating dermatitis [5,6,13,14].
Saussureae Involucratae Herba, known as "snow lotus" in Uyghur and/or Chinese medicines, is the dried aerial part of Saussurea involucrata (Kar. et Kir.) Sch.-Bip. (Asteraceae). This herb has known e cacy in treating in ammatory-related diseases [!5, 16]. Nepetin is a avonoid found in Saussureae Involucratae Herba: this avonoid has been proposed to account for anti-cancer and anti-oxidative stress properties of the herb [17,18]. In human retinal pigment epithelial cells, application of nepetin decreased the phosphorylations of ERK1/2, JNK and MAPK: the IC 50 of NO release was at < 7 µM of nepetin [17,19,20].
Furthermore, nepetin inhibited asthma, induced by coal y dust [21]. However, the potential of nepetin in treating atopic dermatitis has not been fully reported. Here, we are probing the e cacy of nepetin in modulating the lipopolysaccharide (LPS)-stimulated in ammatory responses in cultured human keratinocytes by: (i) rate of cell apoptosis; (ii) production of in ammatory mediators; (iii) release of cytokines; (iv) activation of NF-кB; and (v) formation of ROS.

Keratinocyte culture
Human keratinocyte cell line, named HaCaT cell, was purchased and shipped from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modi ed Eagles medium enriched with 100 IU/mL penicillin, 100 µg/mL of streptomycin and 10% fetal bovine serum. Cultured human keratinocyte were growth and incubated at 37 °C in a water-saturated 5 % CO 2 incubator. Cells were pre-incubated with LPS (1 µg/mL) for 24 hours before any medical treatment. Dexamethasone (Dex, 10 µM) was used as a positive control in the experiments. All the chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

MTT assay
The cell viability was measured by MTT assay (Sigma-Aldrich). In brief, cells were seeded and growth in 96-well plate. After drug treatment for 48 hours, MTT solution was added into the cultures at the nal concentration of 0.5 mg/mL. After incubating for 2 hours, the production of purple crystal was dissolved by DMSO. The absorbance was set at 570 nm. Cell viability was calculated as the percentage of absorbance of blank group and value was set at 1.

PI-Annix staining assay
Cells were seeded in 35-mm culture plates and allowed to grow for 24 hours in medium before drug treatment. The Annexin V-FITC/PI Apoptosis Detection kit was employed using ow cytometry (BD Biosciences, Franklin Lakes, NJ). Brie y, cells were washed by phosphate-buffered saline (PBS) twice and then harvested in PBS. Cells were incubated in 100 µL of binding buffer, containing annexin-V/FITC and propidium iodide (PI), for 15 min at room temperature in dark. The samples were automatically acquired using the loader with acquisition criteria of 10,000 events for each tube, and the quadrants were set according to the population of viable. The results were analyzed using the FlowJo v10.6 software.

NO production
NO Fluorometric measurement methods were performed using an Olympus Fluoview FV1000 laser

Cytosolic and nuclear protein separation
After drug presence for 48 hours in cultured cells, 1X PBS was used to wash the cultures, and then using Qproteome Nuclear Protein Kit (Qiagen, Francisco, CA) to separate nuclear and cytosol NF-κB protein.
The band intensities were calculated and analyzed by Image J.

NF-kB translocation assay
The translocation of p65 was revealed with Olympus Fluoview FV1000 confocal system (Olympus America) mounted on an inverted Olympus microscope (63X objective). The antibodies for p65 and βactin were diluted in cold PBS containing 2.5 % fetal bovine serum and 0.1% Triton X-100 (Sigma-Aldrich) after cell xed, using 4% methanol-free paraformaldehyde treatment for 10 min. Samples were kept in cold room for overnight, and then 1:1,000 dilution of FITC-labeled anti-rabbit antibody (Jackson Laboratories, West Grove, PA) was used as secondary antibody, incubated in dart at room temperature for 3 hours. After incubating, the cells were washed for 3 times and. DAPI (1: 5,000 dilution) was used to stain nucleus before performing assay.

ROS formation
Fluorometric measurements of ROS were utilized CellROX Deep Red Fluorescence Flow Cytomer detection kit (BD Biosciences) under manufacturer's suggested protocol. Brie y, cells at were incubated in 100 µL of binding buffer for 15 min at room temperature. Samples were automatically acquired using the loader with acquisition criteria of 10,000 events for each tube, and the quadrants were set according to population of viable. The cytometer results were analyzed with Flowjo v7.6 software.

Chromatography
The chromatography of methanol extract of Saussureae Involucratae Herba was described by Gong et al. (2020b). Agilent 1200 series system (Agilent, Waldbronn, Germany), supplied with degasser, binary pump, auto-sampler and thermo-stated column compartment was utilized. Chromatographic run was carried out on an Agilent, Eclipse Plus, C18 column (4.6 x 250 mm, 5 µm) with acetonitrile (as solvent A) and 0.01% formic acid (as solvent B) in the mobile phase at a ow rate of 1.0 mL/min. Ten µL sample or nepetin marker (Sigma-Aldrich) were injected.

Statistical analyses and other assays
Protein concentrations were calculated measured by Bradford protein assay dye from Bio-Rad (Bio-Rad Laboratories, Hercules, CA). The protein sample was collected from cell lysis. Then, the aliquot of sample or different doses of BSA standard was mixed with assay dye by gently shaking for 1 min followed by measured at 595 nm using Multiskan™ FC Microplate Photometer. The protein concentration was calculated according to the standard curve. Each result is represented as the Mean ± SEM. Comparisons of the means for untreated control cells and treated cells were analyzed using one-way ANOVA (Bonferroni's post-test). Signi cant values were indicated by * p < 0.05, * * p < 0.01 and ***p < 0.001 as compared to the blank group. Signi cant values were indicated by ^p < 0.05, ^^p < 0.01 and ^^^p < 0.001 as compared to the LPS group.

Nepetin decreases LPS-induced responses
Prior conducting biological analysis, the purity of nepetin, determined by HPLC, and the maximal working concentration of nepetin in cultured cells, determined by MTT analysis, were calibrated. Nepetin below 10 µM did not show toxicity to cultured keratinocytes ( Supplementary Fig. 1), and therefore safety concentration of nepetin was used here. From the results, the purity of nepetin was higher than 98%, and which could be utilized for the following assays ( Supplementary Fig. 1). The inhibition of cell apoptosis was analyzed in LPS-induced cultured HaCaT cells in the presence or absence of nepetin. Annexin V-FITC-and PI-labeled keratinocytes were subjected to ow cytometry analysis. HaCaT cell viability was dramatically altered in the presence of LPS at 1 µg/mL (Fig. 1A&B). The dose of LPS was in accord to the published reports [8,9,17]. The LPS at 1 µg/mL induced apoptotic cell to ~ 12-fold increase, as compared to the control group, i.e. inducing cell death to ~ 60% of total cell population (Fig. 1B). Therefore, LPS at 1 µg/mL was applied for the following experiments. Dexamethasone at 10 µM prevented the LPS-induced cell death signi cantly to ~ 20% of total (Fig. 1A&B). Nepetin at different doses decreased the LPSinduced HaCaT cell apoptosis in a dose-dependent manner (Fig. 1A&B). In the presence of 3 µM nepetin in LPS-induced cells, the apoptotic rate was deceased to ~ 50%, as compared to the LPS-treated group (Fig. 1B).
Skin keratinocyte is the rst line of defensive barrier against external stimulus, and which triggers immune responses of skin by producing in ammatory mediators and cytokines. In cultured HaCaT cells, the treatment of LPS stimulated the expressions of iNOS, COX-2 and PGES2, as compared to the blank control ( Fig. 2A). LPS triggered the levels of in ammatory mediators to ~ 2.5-fold for iNOS, ~ 5-fold for COX-2 and ~ 4.5-fold for PGES2. To examine the potential anti-in ammatory properties of nepetin on LPS-induced dermatitis in cultured HaCaT cells, nepetin was applied onto the culture for 48 hours. The expression levels of iNOS, PGES2 and COX-2 were declined after nepetin treatment in the LPS-treated cultures: this suppression was in a dose-dependent manner ( Fig. 2A). Nepetin at 3 µM showed the highest suppression on the protein expressions. Moreover, the increased production of NO, induced by LPS, in cultured keratinocytes was abolished by nepetin in a dose-dependent manner, as revealed by DAF-FM uorescence (Fig. 2B). Nepetin at 3 µM restricted the LPS-induced NO production to ~ 60%. Dexamethasone was a positive control, and which suppressed the LPS-mediated in ammatory responses back to the blank control (Fig. 2A&B).
The amounts of cytokines, i.e. IL-1β, IL-6 and TNF-α, in cultured keratinocytes after exposure of LPS were measured. LPS induced the production of cytokines signi cantly, at least over 10-folds, in dosedependent manners (Fig. 3). Flow cytometer data showed that the production of IL-1β was signi cantly down-regulated by nepetin in LPS-treated keratinocyte, as compared with LPS model group (Fig. 3A). Three µM of nepetin reduced the IL-1β production to ~ 30% of the LPS-treated group. Similar scenarios were observed for the releases of IL-6 and TNF-α in exposing to nepetin for 48 hours in the LPSstimulated keratinocytes (Fig. 3B&C).

Nepetin mitigates NF-κB translocation and ROS formation
The induction of ROS formation and NF-κB signaling have been reported to be involved in the pathogenesis of atopic dermatitis. Agents having the activity of modulating ROS formation and maintaining NF-κB transduction have been considered to use in treating in ammatory skin diseases [7,22]. NF-κB is an ubiquitous transcription factor, and the primary role is responsible to mediate in ammatory stimuli. The inactivated NF-κB, located in cytosol, is being complexed with an inhibitory protein IκBα. Once being stimulated, IκBα undergoes degradation and release of NF-κB migrating to nucleus [8,16]. The translocation of NF-κB was noted in cultures with LPS (Fig. 4). Immunostaining results indicated that the present of LPS could induce NF-κB p65 translocation from cytosol to nucleus (Fig. 4). Dexamethasone suppressed the translocation of p65 in LPS-induced keratinocytes. The exposure of nepetin in LPS-treated cultures, dose-dependently, modi ed the translocation of p65 (Fig. 4). To further con rm the p65 activity, the amounts of p65 within separated cytosolic and nucleus fractions were determined by western blotting. The expression of p65 in nucleus was increased by applied LPS in cultured keratinocytes (Fig. 5A). Application of nepetin was able to suppress the p65 translocation, induced by LPS, in the nucleus: this effect was in a dose-dependent manner. As expected, the positive control dexamethasone showed similar suppression. In parallel, the level of NF-kB in cytosolic fraction was measured. Contrasting to nucleus, LPS reduced the level of NF-kB in cytosol, and this reduction was partially restored by application of nepetin in cultured keratinocytes, as in a dose-dependent manner (Fig. 5B). Moreover, the protein expression level of IκBα in cytosol was determined, and which was reduced by LPS application. Nepetin, or dexamethasone, could restore the reduction, at least partially (Fig. 5B).
The inhibition of ROS generation has been reported to be important in preventing dermal tissue damage during skin barrier infection and in ammation [10,11]. Having this notion, the ROS formation in the LPSstimulated keratinocytes, with or without nepetin, was measured. Application of LPS markedly induced the formation of ROS, at least over 12-fold of activation (Fig. 6). The treatment of nepetin, dosedependently, suppressed the LPS-induced ROS formation in a concentration-dependent manner (Fig. 6A&B). The ROS formation under dexamethasone showed neutralization effect in the LPS-treated keratinocytes.

Discussion
Flavonoids have a benzo-γ-pyrone basic structure and consist of myriad polyphenolic compounds. Flavonoids work as secondary metabolites by encouraging plant to adapt to environment, producing pigmentation and protecting the plant against pathogens [23]. Flavonoid is a multi-functional phytochemical presenting substantial characteristics that can be exploited for therapeutic agents targeting on various diseases, pharmaceutically. Flavonoids are believed as pivotal components for daily supplement [23]. Various avonoid-enriched functional foods have been reported to have an antiin ammatory function, e.g. Astragali Radix, green tea, Ginkgo Folium [24]. Studies have demonstrated that theses functional foods perform anti-in ammatory properties by blocking the in ammatory mediators, e.g. NO, COX-2, TNF-α, IL-1β, IL-6, IL-15 and interferon-γ [25]. Therefore, these functional foods are likely to modulate TLRs, NF-κB, AP-1, and IRFs in ammatory signaling pathways, as well as to suppress intracellular in ammatory signaling molecules [25].
In ammation is critical in responding to infection or pathogen by activating expression of cytokine and/or in ammatory mediator, aiming to recruit T-cells and myeloid cells [5]. The in ammatory mediators, i.e. NO, has been con rmed to be monitored by inducible isoforms of iNOS and COX-2 during in ammation [27]. Abnormal production of in ammatory mediators has been noticed after external stimulus: this abnormality is associated with various immune-related diseases, as well as tumorigenesis in different cell types [9]. Several lines of evidence have reported that atopic dermatitis is closely associated with in ammation [28,29]. LPS, a well-studied external stimulus, leads to accelerate apoptotic rate of human keratocytes, as well as to trigger production of in ammatory mediators [30]. Here, we have shown that an application of nepetin in cultured keratinocytes could reduce LPS-induced cell death.
Moreover, nepetin inhibited LPS-induced expression/production of iNOS, COX-2, PGES2 and NO. Thitilertdecha et al. (2019) found that nepetin exhibited suppressive function on early T-cell activation by altering cell death rate [31]. Makino et al. (2008) reported that the herbal formulae mitigated atopic dermatitis-like symptoms in skin, and the herbal treatment decreased the expressions of IL-4 and IFN-γ mRNAs [32]. Interestingly, these herbal formulae all contained nepetin as major chemical. Taken together, the aforementioned results suggest that nepetin, or its parental herb Saussureae Involucratae Herba, has the potential pharmaceutical value in treating skin atopic dermatitis.
Chinese herbal medicines have been well-recognized clinically and widely recommended for treatment of atopic dermatitis [33,34]. However, there is no scienti c evidence, or proposed action mechanism, to support the utilization of herbal medicine, and therefore patients and/or consumers are still concerned about the e cacy and safety [35]. Bioactive agents identi ed and isolated from natural resources have drawn great attention with precise mechanism and lower side effects, as compared to the synthesized medicine. Therefore, herbal medicine is playing critical function in drug discovery. Having an increase usage of herbal products in clinics for atopic dermatitis, active chemicals have been isolated for such treatment, e.g. methoxsalen isolated from Ammi majus and capsaicin isolated from chill pepper [36]. Despite nepetin has shown positive function in responding to in ammatory progression in vitro, animal and more controlled clinical data are required to further con rm the e cacy and safety of this plantisolated chemical in dermatology. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.