molecules Article
Effects of Linalyl Acetate on Thymic Stromal Lymphopoietin Production in Mast Cells Phil-Dong Moon 1,2,† , Na-Ra Han 1,† , Jin Soo Lee 1 , Hyung-Min Kim 1, * and Hyun-Ja Jeong 3, * 1 2 3
* †
Department of Pharmacology, College of Korean Medicine, Kyung Hee University, Seoul 02447, Korea;
[email protected] (P.-D.M.);
[email protected] (N.-R.H.);
[email protected] (J.S.L.) Center for Converging Humanities, Kyung Hee University, Seoul 02447, Korea Department of Food Science & Technology and Research Institute for Basic Science, Hoseo University, Chungnam 31499, Korea Correspondence:
[email protected] (H.-M.K.);
[email protected] (H.-J.J.); Tel.: +82-2-961-9448 (H.-M.K.); +82-41-540-9681(H.-J.J.) These authors contributed equally to this work.
Received: 11 June 2018; Accepted: 12 July 2018; Published: 13 July 2018
Abstract: Thymic stromal lymphopoietin (TSLP) is an important factor responsible for the pathogenesis of allergic diseases, such as atopic dermatitis and asthma. Because linalyl acetate (LA) possesses a wide range of pharmacological properties, being antispasmodic, anti-inflammatory, and anti-hyperpigmentation, we hypothesized that LA could inhibit TSLP. Therefore, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, caspase-1 assay, Western blot analysis, fluorescent analyses of the intracellular calcium levels, and the phorbol myristate acetate (PMA)-induced edema model were used to investigate how LA inhibits the production of TSLP in HMC-1 cells. LA reduced the production and mRNA expression of TSLP in HMC-1 cells. LA also inhibited the activation of nuclear factor-κB and degradation of IκBα. PMA plus A23187 stimulation up-regulated caspase-1 activity in HMC-1 cells; however, this up-regulated caspase-1 activity was down-regulated by LA. Finally, LA decreased intracellular calcium levels in HMC-1 cells as well as PMA-induced ear swelling responses in mice. Taken together, these results suggest that LA would be beneficial to treatment of atopic and inflammatory diseases by reducing TSLP. Keywords: thymic stromal lymphopoietin; linalyl acetate; nuclear factor-κB; caspase-1; intracellular calcium
1. Introduction Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by inflammation, pruritus, and eczematous lesions [1]. Approximately 25% of children and 1–3% of adults in industrialized countries are affected by AD [2]. Moreover, the prevalence of AD has grown greatly in the last ten years, notably in urban areas in developed countries [3]. This increase in prevalence has led to increased healthcare costs as well as decreased quality of life for patients with AD because of its symptoms, which include recurring skin lesions, itchiness, lack of sleep, and dietary limitations [4]. Thymic stromal lymphopoietin (TSLP) is an important factor responsible for the pathogenesis of allergic diseases, such as AD and asthma. Epicutaneous application of house dust mite resulted in the induction of TSLP gene expression in nonlesional skin of patients with AD [5]. Serum levels of TSLP were significantly elevated in AD patients compared with controls [6]. In atopic diseases, such as AD and asthma, mast cells play an important role in addition to keratinocytes and epithelial cells [7]. Indeed, many studies have reported that activation and the number of mast cells are up-regulated in AD models, suggesting the importance of mast cells in AD [8–12].
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Caspases are cysteine aspartic proteases with essential roles in programmed cell death; however, caspase-1 is a prototypic inflammatory caspase [13]. Although most caspases generally function in 2018, 23, x FOR PEER REVIEW 2 of 12 programed Molecules cell death, caspase-1 converts interleukin (IL)-1β from an inactive to an active form [14–16]. TSLP production was mediated caspase-1 and (NF)-κB pathway. addition, NF-κB Caspases are cysteine by aspartic proteases withnuclear essential factor roles in programmed cell death;In however, caspase-1 is by a prototypic inflammatory caspase [13].inhibitor, Although most caspasesthat generally function was down-regulated pretreatment with caspase-1 indicating caspase-1 is in an upstream programed cell death, caspase-1 converts interleukin (IL)-1β from an inactive to an active form [14– factor of NF-κB [17]. Molecules 2018, 23, x FOR PEER 2 of 12 16]. TSLP production wasREVIEW mediated by caspase-1 and nuclear factor (NF)-κB pathway. In addition, LinalylNF-κB acetate (LA, Figureby1)pretreatment is one ofwith main constituents of lavender [18]. is an Because LA was down-regulated caspase-1 inhibitor, indicating that caspase-1 Caspases are cysteine aspartic proteases with essential roles in programmed cell death; however, factor ofof NF-κB [17]. possesses aupstream wide range pharmacological properties, being antispasmodic, anti-inflammatory, caspase-1 is acetate a prototypic inflammatory Although caspases generally function in Linalyl (LA, [18–20], Figure 1) iswe onecaspase of main[13]. constituents of most lavender [18]. Because LA possesses and anti-hyperpigmentation hypothesized that LA could inhibit TSLP, which is an programed cell death, caspase-1 converts interleukin (IL)-1β from an inactive to an active form [14– a wide range of pharmacological properties, being antispasmodic, anti-inflammatory, and antiimportant 16]. factor in allergic diseases. Thus, we investigated how LA affects TSLP production in TSLP production[18–20], was mediated by caspase-1 factor (NF)-κB pathway. addition, hyperpigmentation we hypothesized thatand LAnuclear could inhibit TSLP, which is anInimportant NF-κB was down-regulated by pretreatment with caspase-1 inhibitor, indicating that caspase-1 is an HMC-1 cells. factor in allergic diseases. Thus, we investigated how LA affects TSLP production in HMC-1 cells. upstream factor of NF-κB [17]. Linalyl acetate (LA, Figure 1) is one of main constituents of lavender [18]. Because LA possesses a wide range of pharmacological properties, being antispasmodic, anti-inflammatory, and antihyperpigmentation [18–20], we hypothesized that LA could inhibit TSLP, which is an important factor in allergic diseases. Thus, we investigated how LA affects TSLP production in HMC-1 cells.
Figure 1. Chemical structure of linalyl acetate (LA).
Figure 1. Chemical structure of linalyl acetate (LA). 2. Results
2. Results
2.1. Effect of LA on TSLP Production in HMC-1 Cells Figure 1. Chemical structure of linalyl acetate (LA).
the effects of on TSLPCells production, we stimulated HMC-1 cells with PMA plus 2.1. Effect of LATo oninvestigate TSLP Production inLA HMC-1 A23187 for 7 h, after which ELISA was used to measure the levels of TSLP. Stimulation with PMA 2. Results
To investigate thestimulated effects of LAproduction on TSLP in production, stimulated HMC-1 cells with PMA plus plus A23187 TSLP HMC-1 cells; we however, this increase was significantly reduced to 0.104 ± 0.002, 0.096 ± 0.002, and 0.088 ± 0.002 by treatment with LA at 4 μg/mL 400 PMA plus A23187 for 2.1. 7 h,Effect afterof which ELISA was used to measure the levels of TSLP. Stimulationtowith LA on TSLP Production in HMC-1 Cells μg/mL, respectively (Figure 2A, p < 0.05). TSLP production levels in the control and blank groups A23187 stimulated TSLP production in HMC-1 cells; however, this increase was significantly reduced investigate the effects LA on TSLP production, we stimulated HMC-1the cells with PMA plus wereTo 0.121 ± 0.003 and 0.074 ±of 0.004, respectively. Four μg/mL of LA decreased TSLP production A23187 for 7±h, after which ELISA was used todid measure thethe levels of TSLP. with PMA to 0.104 ± 0.002, 0.096 ± 0.002, and 0.088 0.002 by with LA at 4when µg/mL to 400 µg/mL, up to 69.703 4.573%. Treatment with LA± alone not treatment affect production ofStimulation TSLP compared plus A23187 stimulated TSLP production in HMC-1 cells; however, thiscontrol increase was significantly with the blank (PBS-treated cells) group production (data not shown). When LAthe was pretreated at the doses ofgroups 4 respectively (Figure 2A, p < 0.05). TSLP levels in and blank were reduced to 0.104 ±cytotoxicity 0.002, 0.096was ± 0.002, and 0.088 ± 0.002 by treatment with LA2C). at 4 μg/mL to 400 to 400 μg/mL, the not shown by the treatment withdecreased LA (Figure 0.121 ± 0.003 and 0.074 ± 0.004, respectively. Four µg/mL of LA the TSLP production up to μg/mL, respectively (Figure 2A, p < 0.05). TSLP production levels in the control and blank groups 69.703 ± 4.573%. Treatment alone did not Four affect the production of the TSLP compared with were 0.121 ± 0.003 andwith 0.074LA ± 0.004, respectively. μg/mL of LA decreased TSLPwhen production up to 69.703 ± 4.573%. with LA alone did not affect the production of TSLP when at compared the blank (PBS-treated cells)Treatment group (data not shown). When LA was pretreated the doses of 4 to blank (PBS-treated cells) group (data not shown). When LA was pretreated at the doses of 4 400 µg/mL,with thethe cytotoxicity was not shown by the treatment with LA (Figure 2C). to 400 μg/mL, the cytotoxicity was not shown by the treatment with LA (Figure 2C).
Figure 2. Effects of LA on the production and mRNA expression of TSLP in HMC-1 cells. (A) HMC1 cells (3 × 105) were pretreated with LA at concentrations of 4 to 400 μg/mL for 1 h, and then stimulated by PMA plus A23187 for 7 h. The TSLP levels in the supernatant were assessed using ELISA method. (B) HMC-1 cells (1 × 106) were stimulated by PMA plus A23187 for 5 h. The levels of TSLP mRNA expression were measured using qPCR method. (C) HMC-1 cells (3 × 105) were Figure 2. Effects of LA ondoses the production mRNA TSLP in HMC-1 cells. (A)A23187 HMCpretreated with LA at the of 4 to 400 and μg/mL for 1expression h, and thenofstimulated PMA plus Figure 2. Effects of LA on the production and mRNA expression of TSLP inwith HMC-1 cells. (A) HMC-1 cells 5) were pretreated with LA at concentrations of 4 to 400 μg/mL for 1 h, and then 1forcells × 10 7 h.(3Cell viability was determined with an MTT assay. B, PBS-treated cells; C, PBS-treated, and 5 (3 × 10 ) were pretreated with LA at concentrations of 4 to 400 µg/mL for 1 h, and then stimulated by PMA stimulated PMA plus A23187 for 7cells. h. The TSLP levelsrepresents in the supernatant assessed then PMA by plus A23187-stimulated Each datum the meanwere ± S.E.M. of using three ) were stimulated by PMA plus A23187 5 h. The levels of (B) HMC-1 HMC-1 cellsin (1 the × 106supernatant plus A23187ELISA for 7method. h. The(B) TSLP levels were assessed usingfor ELISA method. 6 ) were were mRNA expressionbywere measured using qPCR (C) HMC-1 cells mRNA (3 × 105) expression cells (1 × 10TSLP stimulated PMA plus A23187 for 5 method. h. The levels of TSLP were pretreated with LA at the doses of 4 to 400 μg/mL for 1 h,5and then stimulated with PMA plus A23187 measured using qPCR method. (C) HMC-1 cells (3 × 10 ) were pretreated with LA at the doses of 4 to for 7 h. Cell viability was determined with an MTT assay. B, PBS-treated cells; C, PBS-treated, and 400 µg/mLthen for 1PMA h, and stimulated with plus A23187 forthe 7 h.mean Cell±viability plusthen A23187-stimulated cells. PMA Each datum represents S.E.M. ofwas threedetermined
with an MTT assay. B, PBS-treated cells; C, PBS-treated, and then PMA plus A23187-stimulated cells. Each datum represents the mean ± S.E.M. of three independent experiments. * p < 0.05; significantly different from the control (PBS-treated, and then PMA plus A23187-stimulated cells) value.
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independent experiments. * p < 0.05; significantly different from the control (PBS-treated, and then3 of 12 Molecules 2018, 23, 1711 PMA plus A23187-stimulated cells) value.
2.2. Effect of LA on TSLP mRNA Expression in HMC-1 Cells
assess the the effects effects of of LA LA on on the the TSLP TSLP mRNA mRNA expression, expression, HMC-1 cells were pretreated with To assess ofof LALA forfor 1 h1before PMA plusplus A23187 stimulation for 5for h. various concentrations concentrations (4 (4 to to400 400μg/mL) µg/mL) h before PMA A23187 stimulation qPCR analysis revealed that stimulation of HMC-1 cells with PMA plus A23187 up-regulated the 5 h. qPCR analysis revealed that stimulation of HMC-1 cells with PMA plus A23187 up-regulated TSLP mRNA expression; however, thethe up-regulated the TSLP mRNA expression; however, up-regulatedTSLP TSLPmRNA mRNAexpression expressionwas was reduced reduced by levels of of TSLP mRNA at doses of 4of to4400 pretreatment with with LA LA (Figure (Figure2B). 2B).The Therelative relativeexpression expression levels TSLP mRNA at doses to g/mL werewere 373.600 ± 9.868, 312.931 ± 9.220, 296.865 ± 6.640,±respectively, while those the control 400 g/mL 373.600 ± 9.868, 312.931 ± and 9.220, and 296.865 6.640, respectively, whileofthose of the and blank were 461.629 ± 17.307 1.933 0.580,±respectively. The inhibitory effect of 400 control andgroups blank groups were 461.629 ± and 17.307 and± 1.933 0.580, respectively. The inhibitory effect μg/mL of LA was greater than 4 than and 40 μg/mL; thus, we assessed the effects of 400of μg/mL of LA of in of 400 µg/mL of LA was greater 4 and 40 µg/mL; thus, we assessed the effects 400 µg/mL subsequent experiments. LA in subsequent experiments. 2.3. 2.3. Effect of LA on NF-κB Activation Activation and and IκBα IκBα Degradation Degradation To determine ifif the the inhibitory inhibitoryeffects effectsofofLA LAwere were mediated NF-κB IκBα signaling, mediated by by NF-κB andand IκBα signaling, we we analyzed activation NF-κB p65 and degradationofofIκBα IκBαby byWestern Westernblot blot analysis. analysis. Stimulation analyzed thethe activation of of NF-κB p65 and degradation of HMC-1 PMA plus A23187 increased the activation of NF-κB in the nuclear HMC-1cells cellswith with PMA plus A23187 increased the activation of NF-κB in the extract; nuclearhowever, extract; the increased activation responsein toresponse pretreatment with LA (Figure relative however, the NF-κB increased NF-κB decreased activation in decreased to pretreatment with3). LAThe (Figure 3). intensities of intensities NF-κB in the blank, control, and LA-pretreated groups were 0.226 ± 0.024, 0.021, The relative of NF-κB in the blank, control, and LA-pretreated groups were 0.401 0.226 ± ± 0.024, and ± 0.027, respectively. We examined thewhether inhibitory of LAeffect was of due its 0.4010.241 ± 0.021, and 0.241 ± 0.027, respectively. Wewhether examined the effect inhibitory LAtowas regulation of IκBα degradation because IκBα degradation results in NF-κB activation Stimulation due to its regulation of IκBα degradation because IκBα degradation results in NF-κB[21]. activation [21]. of HMC-1 cells with PMA plus A23187 increased the degradation of IκBα in the cytoplasmic extract; Stimulation of HMC-1 cells with PMA plus A23187 increased the degradation of IκBα in the however, theextract; increased IκBα degradation decreased in response to pretreatment with (Figure 3). cytoplasmic however, the increased IκBα degradation decreased in response toLA pretreatment The intensities IκBα inintensities the blank,ofcontrol, were 0.316 ±groups 0.016, withrelative LA (Figure 3). Theofrelative IκBα inand the LA-pretreated blank, control, groups and LA-pretreated 0.183 ± 0.012, and 0.273 ± 0.006, respectively. were 0.316 ± 0.016, 0.183 ± 0.012, and 0.273 ± 0.006, respectively.
Figure 3. of of NF-κB p65p65 andand degradation of IκBα in HMC-1 cells.cells. (A) Figure 3. Effects EffectsofofLA LAononthe theactivation activation NF-κB degradation of IκBα in HMC-1 6 6 pretreated with LALA at at the TheThe HMC-1 cellscells (5 ×(510 (A) HMC-1 × )10were ) were pretreated with thedose doseofof400 400μg/mL µg/mLfor for11 h, h, and then (B)(B) Each protein level waswas quantitated by densitometry. NE, stimulated by by PMA PMAplus plusA23187 A23187for for2 h. 2 h. Each protein level quantitated by densitometry. nuclear extract; CE, CE, cytoplasmic extract; B, B, PBS-treated cells; NE, nuclear extract; cytoplasmic extract; PBS-treated cells;C,C,PBS-treated, PBS-treated,and andthen then PMA PMA plus A23187-stimulated cells. cells. Each datum represents represents the the mean mean ± ± S.E.M. A23187-stimulated S.E.M.of ofthree threeindependent independent experiments. experiments. ** p < 0.05; significantly significantly different from the control (PBS-treated, and then PMA plus A23187-stimulated cells) value.
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2.4. 2.4. Effect Effect of of LA LA on on Caspase-1 Caspase-1 Activation Activation in in HMC-1 HMC-1 Cells Cells 2.4. Effect of LA on Caspase-1 Activation in HMC-1 Cells Caspase-1 the inhibitory inhibitory effect effect of of LA. LA. Caspase-1 activity activity was was measured measured to to investigate investigate whether whether it it mediated mediated the Caspase-1 activity cells was measured toplus investigate whether it the mediated the inhibitory effect of LA. Stimulation of HMC-1 with PMA A23187 increased activation of caspase-1; however, Stimulation of HMC-1 cells with PMA plus A23187 increased the activation of caspase-1; however, Stimulation of HMC-1 activation cells with PMAdown-regulated plus A23187 increased the activation of caspase-1; however, the the increased increasedcaspase-1 caspase-1 activationwas was down-regulatedby bytreatment treatmentwith withLA LA(Figure (Figure4).4).The Thecaspase-1 caspasethe increased caspase-1 activation was down-regulated by treatment with LA (Figure 4).0.342 The caspaseactivity values in the blank, control, and LA-pretreated groups were 0.290 ± 0.006, ± 0.003, 1 activity values in the blank, control, and LA-pretreated groups were 0.290 ± 0.006, 0.342 ± 0.003, and 1 activity values in respectively. the blank, control, and LA-pretreated groups were 0.290 ± 0.006, 0.342 ± 0.003, and and 0.295 ± 0.011, 0.295 ± 0.011, respectively. 0.295 ± 0.011, respectively.
Figure 4. Effect Effect of of LA LA on the the caspase-1activation activation inHMC-1 HMC-1 cells.HMC-1 HMC-1 cells(5(5×× 10 1066) were pretreated Figure were pretreated pretreated Figure 4. 4. Effect of LA on on the caspase-1 caspase-1 activationin in HMC-1cells. cells. HMC-1cells cells (5 × 106)) were with LA for 1 h, and then stimulated by PMA plus A23187 for 1 h. Each protein level was quantitated with Each protein protein level level was was quantitated quantitated with LA LA for for 11 h, h, and and then then stimulated stimulated by by PMA PMA plus plus A23187 A23187 for for 11 h. h. Each by densitometry. densitometry. B, B, PBS-treated PBS-treated cells; cells; C, C, PBS-treated, PBS-treated, and and then then PMA PMA plus plus A23187-stimulated A23187-stimulated cells. cells. by by densitometry. B, PBS-treated cells; C, PBS-treated, and then PMA plus A23187-stimulated cells. Each datum represents the mean ± S.E.M. of three independent experiments. * p < 0.05; significantly Each datum represents the mean ± S.E.M. of three independent experiments. * p < 0.05; significantly Each datum represents the mean ± S.E.M. of three independent experiments. * p < 0.05; significantly different from fromthe thecontrol control(PBS-treated, (PBS-treated,and andthen thenPMA PMAplus plusA23187-stimulated A23187-stimulatedcells) cells)value. value. different different from the control (PBS-treated, and then PMA plus A23187-stimulated cells) value.
2.5. Effect of LA on Intracellular Calcium Level in HMC-1 Cells 2.5. Effect of of LA LA on on Intracellular 2.5. Effect Intracellular Calcium Calcium Level Level in in HMC-1 HMC-1 Cells Cells Calcium is required for caspase-1 activation [22]; thus, we investigated the effects of LA on the Calcium is required [22]; thus, the effects effects of of LA Calcium is required for for caspase-1 caspase-1 activation activation [22]; thus, we we investigated investigated the LA on on the the level of intracellular calcium in HMC-1 cells. Stimulation of HMC-1 cells with PMA plus A23187 level of intracellular calcium in HMC-1 cells. Stimulation of HMC-1 cells with PMA plus A23187 level of intracellular calcium in HMC-1 cells. Stimulation of HMC-1 cells with PMA plus A23187 increased the level of intracellular calcium, but these increased intracellular calcium levels were increased increased the the level level of of intracellular intracellular calcium, calcium, but but these these increased increased intracellular intracellular calcium calcium levels levels were were reduced by pretreatment with LA (Figure 5). reduced reduced by by pretreatment pretreatment with with LA LA (Figure (Figure 5). 5).
Figure 5. Effect of LA on the level of intracellular calcium in HMC-1 cells. HMC-1 cells were Figure 5.5.Effect LALA on the of intracellular calcium in HMC-1 cells. HMC-1 were cells pretreated Effectofof on level the level of intracellular calcium in HMC-1 cells.cells HMC-1 were pretreated with LA for 20 min and then stimulated with PMA plus A23187. B, PBS-treated cells; C, with LA forwith 20 min then stimulated PMA plus B, PBS-treated C, PBS-treated, pretreated LA and for 20 min and thenwith stimulated withA23187. PMA plus A23187. B, cells; PBS-treated cells; C, PBS-treated, and then PMA plus A23187-stimulated cells. and then PMA plus A23187-stimulated cells. PBS-treated, and then PMA plus A23187-stimulated cells.
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2.6. Effect of LA on Pro-inflammatory Cytokine Levels in HMC-1 Cells To validate the effects of LA, we assessed the levels of pro-inflammatory cytokines, including IL-6 and TNF-α, in HMC-1 cells. Stimulation with PMA plus A23187 increased the production of IL-6 and TNF-α; however, these increased levels were down-regulated by treatment with LA (Figure 6). 2.7. Effect of LA in Animal Model HMC-1 cells do not express FcεRI, are immature, and have low expression of most mast cell markers with the exception of c-Kit and histamine. To overcome these limitations, we tested the effects of LA in an animal model. Topical application of PMA resulted in ear edema in a murine model [23]. In this study, ear thickness increased in response to topical application of PMA, but LA administration ameliorated the ear swelling response (Table 1). 3. Discussion When mast cells are activated by cross-linking of IgE receptors with antigen, protein kinase C (PKC) is activated and intracellular calcium levels are elevated [24,25]. PKC is involved in the activation of NF-κB and production of cytokines [24]. To reproduce this process, we used PMA to activate PKC and A23187 to elevate intracellular calcium levels in this study. Our previous study revealed that PMA plus A23187 stimulation increases cytokine TSLP production in HMC-1 cells [17], and skin lesions of patients with AD have been shown to express high levels of TSLP [26]. Moreover, Oyoshi et al. [27] reported that TSLP promotes skin inflammatory reactions in a mouse model of AD, while the well-known anti-inflammatory drug, dexamethasone, suppressed TSLP expression in an AD mouse model [28]. The results of the present study revealed that LA decreased the production and mRNA expression of TSLP in HMC-1 cells (Figure 2). Thus, we assume that LA might be beneficial in atopic and inflammatory diseases. As shown in Figure 2, the levels of mRNA and protein were quite different. Okayama et al. [29] reported that secreted TSLP was rapidly degraded by proteases secreted by mast cells. Similarly, the results of the present study also showed that the elevation in the amount of secreted TSLP protein was much lower than the increase in mRNA. Thus, we presuppose that a considerable amount of the secreted TSLP would be degraded by proteases that were secreted by mast cells. Dexamethasone is a well-known anti-inflammatory drug that has shown inhibitory effects against various inflammatory diseases, including pancreatitis and asthma, that occur through the down-regulation of NF-κB activation [30,31]. Lee and Ziegler [32] reported that TSLP gene regulation was controlled by NF-κB. Moreover, the production and mRNA expression of TSLP were found to be mediated by NF-κB in mast cells [17]. In the present study, LA reduced NF-κB activation and IκBα degradation (Figure 3). Furthermore, Shen et al. [33] reported that NF-κB is a key transcription factor in TSLP production. Thus, we assumed that LA could inhibit TSLP through down-regulation of NF-κB activation in HMC-1 cells and would be beneficial to treatment of various inflammatory diseases like dexamethasone. Typically, pro-inflammatory stimulation results in the activation of caspase-1 [34]. Several studies have shown that the activation of caspase-1 is induced by pro-inflammatory stimulation, such as PMA plus A23187 [35–37]. The results of the present study revealed that LA inhibited the activation of caspase-1 (Figure 4); therefore, it can be assumed that LA inhibits TSLP production and mRNA expression through blocking of caspase-1 activation in mast cells. Calcium is required for caspase-1 activation [22], and 1,2-bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid acetoxymethyl ester (BAPTA-AM) is an intracellular calcium chelator. Pretreatment with BAPTA-AM was shown to decrease the activation of caspase-1 in PMA plus A23187-stimulated cells [38], while the results of the present study showed that LA inhibits the level of intracellular calcium in HMC-1 cells (Figure 5). Taken together, these results provide evidence that LA inhibits TSLP through blockage of intracellular calcium/caspase-1/NF-κB signaling in HMC-1 cells (Figure 6).
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LA TSLP through down-regulation of the NF-κB/IκBα LA treatment treatment inhibited inhibited the the production production of of TSLP TSLP through throughdown-regulation down-regulation of ofthe theNF-κB/IκBα NF-κB/IκBα LA treatment inhibited the production of pathway in HMC-1 cells. Well-known cytokines including IL-6 and TNF-α have also been shown to pathway in HMC-1 cells. Well-known cytokines including IL-6 and TNF-α have also been shown to pathway in HMC-1 cells. Well-known cytokines including IL-6 and TNF-α have also been shown to be be regulated by the NF-κB/IκBα pathway [39,40]. As expected, LA treatment inhibited the production be regulated by the NF-κB/IκBα pathway [39,40]. expected, treatment inhibited production regulated by the NF-κB/IκBα pathway [39,40]. AsAs expected, LALA treatment inhibited thethe production of of IL-6 and TNF-α in HMC-1 cells (Figure 7). Thus, we validated our results through the inhibition of IL-6 and TNF-α in HMC-1 cells (Figure 7). Thus, we validated our results through the inhibition IL-6 and TNF-α in HMC-1 cells (Figure 7). Thus, we validated our results through the inhibition of of and TNF-α. ofIL-6 IL-6 TNF-α. IL-6 andand TNF-α.
5 were Figure pro-inflammatory cytokine levels in cells. HMC-1 cells (3 Figure 6. Effectof ofLA LAon on pro-inflammatory cytokine levels in HMC-1 cells. HMC-1 (3 105 ) Figure6. 6.Effect Effect of LA on pro-inflammatory cytokine levels inHMC-1 HMC-1 cells. HMC-1 cellscells (3××10 105)× ) were were pretreated with at concentration 400 µg/mL 1then h, and then stimulated PMA plus pretreated with at concentration of μg/mL for and stimulated by plus A23187 pretreated withLA LA atLA concentration of400 400of μg/mL for11h, h,for and then stimulated byPMA PMAby plus A23187 A23187 for 7 h. The levels of IL-6 and TNF-α in the supernatant were assessed using ELISA method. B, for 7 h. The levels of IL-6 and TNF-α in the supernatant were assessed using ELISA method. B, PBSfor 7 h. The levels of IL-6 and TNF-α in the supernatant were assessed using ELISA method. B, PBSPBS-treated cells; C, PBS-treated, and then PMA plus A23187-stimulated cells. Each datum represents treated cells; C, PBS-treated, and then PMA plus A23187-stimulated cells. Each datum represents the treated cells; C, PBS-treated, and then PMA plus A23187-stimulated cells. Each datum represents the the mean ± S.E.M. of three independent experiments.
