Effects of Resveratrol on Thymic Stromal Lymphopoietin Expression in Mast Cells

Background and objectives: Cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathogenesis of atopic diseases such as atopic dermatitis, allergic rhinitis, and asthma. Resveratrol (RSV) exerts various pharmacological effects such as antioxidant, anti-inflammatory, neuroprotective, and anticancer. Although, it has been verified the beneficial effects of RSV on various subjects, the effect of RSV on thymic stromal lymphopoietin (TSLP) regulation has not been elucidated. Materials and Methods: Here, we examined how RSV regulates TSLP in HMC-1 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, Western blotting, and calcium assay were performed to evaluate the effect of RSV. Results: TSLP production and mRNA expression were reduced by RSV. RSV down-regulated nuclear factor-κB activation, IκBα phosphorylation as well as activation of receptor-interacting protein2 and caspase-1 in HMC-1 cells. In addition, RSV treatment decreased the up-regulation of intracellular calcium in HMC-1 cells. Conclusions: These results suggest that RSV might be useful for the treatment of atopic diseases through blocking of TSLP.


Introduction
Atopic dermatitis (AD) is a common chronic skin disease worldwide [1][2][3]. The prevalence of AD is 2-10% of adults and 5-20% of children throughout the world [4]. The lifetime prevalence of AD has elevated over the past decades, particularly in industrialized countries [5]. AD induced a poor quality of life. Want of sleep, school or work absenteeism, and psychological stress arose from AD. Medical costs were elevated compared with those of patients without AD [6].
Cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathogenesis of atopic diseases such as atopic dermatitis, allergic rhinitis, and asthma. Intradermal injection of recombinant TSLP up-regulated scratching frequency in a murine AD model [7]. TSLP knockout mice showed decreased AD-like skin lesions [8]. Staphylococcal enterotoxin B plus 2,4-dinitrochlorobenzene induced significant TSLP elevation in AD-like skin lesions [9]. Epithelial cells and keratinocytes play important roles in AD; however, mast cells also contribute to the induction of AD [10]. Numerous reports suggest that mast cell population and mast cell activation were elevated in AD, indicating the significance of mast cells in AD [11][12][13].
Resveratrol (RSV, Figure 1) is a dietary polyphenol found in various plants such as grapes, pines, apples, knotweed, blueberries, plums, and peanuts [19,20]. RSV exerts various pharmacological effects such as antioxidant, anti-inflammatory, neuroprotective, and anticancer [21][22][23]. Recently, Cheng and colleagues [24] reported that RSV ameliorates renal damage in obese mice via protection of inflammation. However, the beneficial effects of RSV on TSLP expression in mast cells have not been fully understood. In the present study, we studied whether RSV can regulate the TSLP expression in HMC-1 human mast cell line.

MTT Assay
MTT assay was used to analyze cytotoxicity as described previously [25]. Various concentrations of RSV (0.03, 0.3, and 3 µM) or PBS were pretreated in HMC-1 cells (3 × 10 5 /mL) for 1 h and incubated with 5 mg/mL of MTT for 4 h. MTT formazan was dissolved by 1 mL of dimethyl sulfoxide and transferred 200 µL of supernatant into a 96-well microplate. The plate was measured at 540 nm.

Enzyme-Linked Immunosorbent Assay (ELISA)
Various concentrations of RSV (0.03, 0.3, and 3 µM) or PBS were pretreated in HMC-1 cells (3 × 10 5 /mL) for 1 h and incubated with PMA plus calcium ionophore (PMACI) for 7 h considering our previous reports [18,26]. An ELISA was utilized for assessment of TSLP levels from culture supernatants as described previously [27]. In short, anti-TSLP capture antibody was incubated on 96-well microplates overnight. After washing, TSLP standard solution and culture supernatants were added. After washing, anti-TSLP detection antibody was added. After washing again, avidin-peroxidase was incubated at 37 • C. Finally, substrate solution was added and the microplates were analyzed at 450 nm.

Real-Time Polymerase Chain Reaction (PCR)
Various concentrations of RSV (0.03, 0.3, and 3 µM) or PBS were pretreated in HMC-1 cells (1 × 10 6 /mL) for 1 h and incubated with PMACI for 5 h considering our previous reports [18]. Real-time PCR was performed by using a Power SYBR Green PCR master mix. ABI StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA) was used as described previously [28].

Extraction of Nuclear and Cytoplasmic Proteins
HMC-1 cells (5 × 10 6 /mL) were pretreated with RSV for 1 h before PMACI stimulation. Proteins were extracted by using hypotonic buffer and cold saline buffer as described previously [29,30].

Western Blotting
After incubation at 95 • C for 5 min, samples were cooled on ice. Each protein was separated by 15% SDS-polyacrylamide gel and transferred to nitrocellulose paper. Primary antibodies were treated overnight and then secondary antibodies were treated for 1 h. Protein bands were visualized using enhanced chemiluminescent reagent (Amersham Co., Newark, NJ, USA) as described previously [31].

Calcium Assay
For fluorescence measurement, fura-2/AM was added in HMC-1 cells (1 × 10 5 /mL) for 30 min. The cells then were harvested to perform the calcium assay as described previously [32].

Statistics
All results were analyzed by IBM SPSS software (version 23, SPSS Inc., Chicago, IL, USA). Statistical analysis was conducted by using independent t-test and ANOVA with a Tukey post hoc test. The results were considered significant at values of p < 0.05 and expressed as the mean ± standard error of the mean (SEM).

Effect of RSV on TSLP Production
PMA plus calcium ionophore (PMACI) were used to induce mast cell activation.

Effect of RSV on Calcium Levels
Calcium is essential for the activation of RIP2 and caspase-1 [35,36]. To determine if RSV influences the intracellular calcium in mast cells, HMC-1 cells were seeded in 96-well plates and pretreated with RSV (3 µM) for 20 min. PMACI were able to induce elevation of intracellular calcium, which could be reduced by RSV (3 µM, Figure 5).

Discussion
In this study, RSV reduced the TSLP production and mRNA expression. Additionally, RSV attenuated the NF-κB activation, IκBα phosphorylation as well as RIP2/caspase-1 activation. Lastly, RSV inhibited intracellular calcium levels.
In general, mast cell activation results from the binding of IgE receptors with multivalent antigens. Activation of protein kinase C (PKC), as well as increment of intracellular calcium are shown in activated mast cells [37]. To make a similar condition, PMA was used to activate PKC, and calcium ionophore was used to increase intracellular calcium in this experiment. TSLP production and mRNA expression were elevated by incubation with PMACI in HMC-1 cells [18]. Serum TSLP levels increased in patients with AD compared with those of healthy subjects [38]. Diesel exhaust particles increased mRNA and production of TSLP from bronchial asthmatic epithelial cells that isolated from bronchus of patients with asthma [39]. The results of this study also presented that the production and mRNA expression of TSLP are reduced by RSV treatment in HMC-1 cells (Figure 2). It will be possible to suggest that RSV may contribute to the treatment for atopic and asthmatic diseases.
A study suggested that TSLP was regulated by NF-κB in epithelial cells [40]. Additionally, TSLP production and mRNA expression were regulated by NF-κB in mast cells [18]. Moreover, it has been suggested that NF-κB plays a critical role in the production of TSLP [41]. Our results presented that NF-κB activation and IκBα phosphorylation were down-regulated by RSV (Figure 3), suggesting that inhibition of TSLP by RSV is regulated by NF-κB/IκBα in HMC-1 cells. External treatment of NF-κB inhibitor (IMD-0354) reduced AD symptoms [42]. Jiang et al. [43] suggested that topical treatment of specific NF-κB inhibitor (dehydroxymethylepoxyquinomicin) improves 2,4-dinitrochlorobenzene and oxazolone-induced AD-like skin lesions. In recent research, AD symptoms were ameliorated by topical treatment of specific NF-κB inhibitor (dehydroxymethylepoxyquinomicin) in chemical irritants plus horny layer removing-induced AD murine model [44]. Thus, we presume that RSV may be helpful for us to improve AD symptoms via blocking of NF-κB.
When cells are exposed to pro-inflammatory stimuli, RIP2 binds and activates caspase-1 [17]. Numerous studies demonstrated that activation of RIP2 and caspase-1 resulted from pro-inflammatory stimuli including PMACI or ovalbumin [14,45,46]. Our findings presented that the activation of RIP2 and caspase-1 was reduced by RSV ( Figure 4). The results of the present study implied that RSV might be active in reducing TSLP through blocking RIP2 and caspase-1 in HMC-1 cells.
Increment of intracellular calcium induced activation of RIP2 and caspase-1 [35,36]. Han et al. [35] reported that calcium chelator BAPTA-AM decreases the protein levels of RIP2 and caspase-1. Our results presented that intracellular calcium was reduced by RSV ( Figure 5). Hence, the results implied that RSV may reduce TSLP through down-regulating calcium/RIP2/caspase-1/NF-κB signals in HMC-1 cells (Figure 6). Figure 6. A schematic diagram of TSLP reduction by RSV. When mast cells are exposed to proinflammatory stimuli, intracellular calcium levels were elevated, the elevated intracellular calcium resulted in activation of RIP2 and caspase-1. NF-κB activation resulted from the activation of RIP2 and caspase-1. Lastly, the NF-κB activation resulted in TSLP production. In the present study, RSV decreased the TSLP production through blocking of calcium/RIP2/caspase-1/NF-κB signals in mast cells.
Finally, daily administration of RSV (300 mg/kg) for 4 weeks did not show any toxicity in rats [47]. In the present study, we treated 3 µM of RSV (approximately 0.684 mg/kg). Thus, we presuppose that 3 µM of RSV would not be toxic to humans.

Conclusions
Collectively, the present study showed that RSV reduced TSLP production and mRNA expression. RSV inhibited NF-κB activation, IκBα phosphorylation, as well as activation of RIP2 and caspase-1 in HMC-1 cells. Furthermore, RSV reduced intracellular calcium levels. Therefore, these findings suggest that RSV might have important implications for screening promising drugs in atopic diseases.