Effects of Honokiol on Activation of Transient Receptor Potential Channel V1 and Secretion of Thymic Stromal Lymphopoietin in HaCaT Keratinocytes

2020-04-03BoXieShaShaSongYongFangWangJianBingWuXinYuLi

国际皮肤性病学杂志 2020年1期

Bo Xie, Sha-Sha Song, Yong-Fang Wang, Jian-Bing Wu, Xin-Yu Li,∗

1Department of Pharmacology, 2Department of Dermatology, Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, Jiangsu 210042, China.

Abstract Objective: This study was performed to investigate the effects of honokiol on the activation of transient receptor potential channel V1(TRPV1)and the secretion of thymic stromal lymphopoietin(TSLP)in a human benign epidermal keratinocyte line (HaCaT).Methods: HaCaT keratinocytes were cultivated and divided into six groups: capsaicin-induced model control group,capsazepine control group,solvent control group,and three honokiol treatment groups(7.81,15.63,and 31.25 mg/L of honokiol).The effect of honokiol on calcium(Ca2+)influx was measured by a Ca2+fluorescence imaging system.The fluorescence intensity(F)of cells was measured.The rate of change in F(ΔF/F0)was calculated,and the ΔF/F0-time curve was constructed.HaCaT keratinocytes were stimulated with polyinosinic:polycytidylic acid,recombinant human tumor necrosis factor α,and recombinant human interleukin 4.Different concentrations of honokiol(15.63,7.81,and 3.91mg/L) were added to the cells in the respective honokiol groups; 20mg/L of dexamethasone or 0.5% dimethyl sulfoxide was added to the cells in the positive control group or solvent control group.The TSLP concentration in the HaCaT keratinocytes of each group was detected by enzyme-linked immunosorbent assay. Statistical analysis was performed by one-way analysis of variance and Dunnett t test.Results:The capsaicin-induced Ca2+fluorescence intensity in HaCaT keratinocytes was significantly inhibited in the 31.25mg/L honokiol group;ΔF/F0 at 45second was 0.76 in the model control group and 0 in the 31.25mg/L honokiol group.The TSLP level in the 15.63 and 7.81mg/L honokiol groups was lower than that in the solvent control group(t=7.382,P=0.003,and t=2.766,P=0.023,respectively),while the TSLP level in the 3.91mg/L honokiol group was not significantly different from that in the solvent control group (t=1.872, P=0.124).Conclusions: Honokiol inhibited the Ca2+ influx induced by capsaicin (TRPV1 agonist) in HaCaT keratinocytes.Honokiol has an inhibitory effect on TSLP secretion in HaCaT keratinocytes.

Keywords: honokiol, keratinocytes, transient receptor potential channel V1, thymic stromal lymphopoietin

Introduction

Itch is a common symptom associated with many conditions, such as urticaria, pruritus, atopic dermatitis(AD), psoriasis, Sézary syndrome, prurigo nodularis,jaundice, uremia, and diabetes.1-2The exact mechanism underlying the development of itch is not fully understood.Itch can be divided into histaminergic and non-histaminergic itch according to its stimulus. Many inflammatory mediators play key roles in the development of nonhistaminergic pruritus, such as thymic stromal lymphopoietin(TSLP),interleukin(IL)-4,IL-13,IL-31,and others;these inflammatory mediators are particularly closely related to chronic itch.3TSLP was initially identified in the thymus and is expressed primarily by epithelial cells at barrier surfaces such as the skin (i.e., keratinocytes in the epidermis), gut, and lung. TSLP production may be activated by a variety of signals,including cytokines such as IL-4, tumor necrosis factor α (TNF-α), and infection agents and their products such as viruses, bacterial peptidoglycans, lipoteichoic acid, and double-stranded RNA during response to stimulating signals.4TSLP exerts its function by activating toll-like receptor 3, proteaseactivated receptor 2, and the transient receptor potential channel vanilloid type 1 (TRPV1). TSLP can directly activate cutaneous sensory neurons to promote the sensation of itch.5Increased expression of TSLP has been found in patients with AD, and this mediator is recognized as a pivotal pathogenetic factor in the development of AD.6

In addition to inflammatory mediators, many nociceptive receptors take part in the mechanism of itch.TRPV1 is a cation channel [mainly calcium (Ca2+)] that belongs to the transient receptor potential (TRP) family. Many stimuli can activate TRPV1, such as pungent extracts(e.g., capsaicin), protons (pH ＜5.9), vanilloids, and inflammatory mediators. When TRPV1 is activated, Ca2+ flows into neurons and produces an itch-related action potential on nerve fibers. This itch signal travels to the nerve center and produces the sensation of itch.7Interestingly, although TRPV1 is highly expressed in nociceptor neurons of the dorsal root ganglia, it is also found on nonsensory tissues such as human epidermal keratinocytes and airways, as well as on fibroblasts,monocytes,dendritic cells,mast cells,vascular endothelial cells, sweat glands, pneumocytes, smooth muscle, hair follicles, thymocytes, urothelium, and gut epithelium.8In recent years, many studies have shown that the TRPV1 expressed on these cells is closely related to many inflammatory factors. For instance, activation of TRPV1 can cause the secretion of prostaglandin E2 and IL-8 in HaCaT, a human benign epidermal keratinocyte line.9

Treatment of chronic itch is more difficult because of the complexity of the underlying mechanisms.5Some traditional Chinese medicine prescriptions can reportedly improve itch symptoms. Among these prescriptions,Magnolia officinalis is a commonly used Chinese herb.M.officinalis is a plant in the family Magnoliaceae.10The main active constituents of M. officinalis are several phenolic compounds. Honokiol is one of these phenolic compounds.Research has shown that honokiol can inhibit the increased intracellular Ca2+influx into bovine adrenal chromaffin cells stimulated by acetylcholine (ACh)11and the activation of inositol 1,4,5-trisphosphate receptors(IP3 receptors) 1 of the distal ileum in diarrhea mouse model through Ca2+channel blockade.12Honokiol can also reduce the allergic effects caused by immunoglobulin E complexes and the number of scratches induced by compound 48/80 in mice.8In a previous study,we found that honokiol had an antipruritic effect in two animal models and an inhibitory effect on Ca2+influx in the dorsal root ganglion cells of the rat spine.13However, whether honokiol affects TSLP secretion and activation of TRPV1 in keratinocytes remains unknown. In this study, we observed the effect of honokiol on Ca2+influx induced by a TRPV1 agonist and TSLP secretion induced by exogenous stimuli in HaCaT keratinocytes by fluorescence imaging and enzyme-linked immunosorbent assay(ELISA),respectively.

Materials and methods

Cell line

HaCaT keratinocytes were purchased from Biotechnology Co., Ltd. Shanghai Enzyme Research, China. The cells were cultured at 37°C in 5% carbon dioxide (CO2) and subcultured in a conventional manner.

Compound, main reagents, and instruments

Honokiol (analytical standard, ≥98% purity by highperformance liquid chromatography, HPLC) was from Nanjing SenBeiJia Biotechnology Co., Ltd. (Nanjing,China).The honokiol was dissolved in dimethyl sulfoxide(DMSO) and then diluted with either Dulbecco Modified Eagle Medium(DMEM)+10%fetal bovine serum(FBS)or Hank balanced salt solution (HBSS) to a definite concentration. Capsaicin and dexamethasone (analytical standard, ≥98% purity by HPLC) were from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai,China) and dissolved in DMSO and then diluted with HBSS and DMEM+10%FBS,respectively.Capsazepine(≥98% purity by HPLC) was from Toronto Research Chemicals Inc. (Toronto, ON, Canada); it was first dissolved in DMSO and then diluted with HBSS.Recombinant human TNF-α(rhTNF-α)and recombinant human IL-4 (rhIL-4) were got from PeproTech, Inc.(Rocky Hill, NJ). Polyinosinic:polycytidylic acid [poly(I:C)]was got from InvivoGen,Inc.(San Diego,CA).DMEM+ 10% FBS and DMEM without FBS were got from KeyGen Biotech Co.,Ltd.(Nanjing,China).Fluo-3 AM,a Ca2+fluorescence indicator,was got from Sigma-Aldrich,Inc. (St. Louis, MO). Pluronic F-127 (a nonionic surfactant) and HBSS with Ca2+were from Beyotime Biotechnology (Shanghai, China). The Human TSLP ELISA kit was from Beijing 4A Biotech Co.,Ltd.(Beijing,China). The microplate reader and CO2cell incubator were from Thermo Fisher Scientific (Waltham, MA). The confocal laser scanning microscope was from Olympus Corporation (Tokyo, Japan).

Ca2+ fluorescence imaging

HaCaT keratinocytes (5×104cells/mL) were seeded with DMEM + 10% FBS in a 35-mm-diameter glass-bottom dish at 37°C in 5% CO2. At 80% confluence, the cells were divided into six groups: the capsaicin (500μmol/L,TRPV1 agonist)-induced model control group, capsazepine (500μmol/L, TRPV1 antagonist) group, solvent control group, and three honokiol treatment groups(7.81, 15.63, and 31.25mg/L of honokiol). After the medium was discarded, the cells in each group were washed twice with HBSS. Next, 300μL of the Ca2+fluorescence indicator Fluo-3AM/Pluronic F-127 was added to the cells in 5%CO2at 37°C incubation for 30 minutes. Then, 65μL of HBSS was added to the cells in the model control group, 65μL of 1,000μmol/L capsazepine was added to the cells in the capsazepine group, 65μL of different concentrations of honokiol(15.63,31.25,and 62.5mg/L,respectively)were added to the cells in honokiol treatment groups,and 65μL of 0.1%DMSO was added to the cells in the solvent control group. The cells were observed under a laser confocal scanning microscope, and the fluorescence intensity (F)was monitored continuously for up to 300seconds;65μL of 1,000μmol/L capsaicin was added to the culture of each group between 15 and 30seconds. Images at different times were recorded by taking photographs every 15seconds.6,14The fluorescence intensity at different time points was analyzed using Olympus FV1000 software. The percent increase in the fluorescence intensity (ΔF/F0×100%) was calculated to construct a relative fluorescence intensity-time curve(ΔF=Fmax-F0,where Fmax is the maximum fluorescence intensity at a definite time and F0is the initial value of fluorescence intensity).14

MTT assay

To test the effect of honokiol on cell viability, HaCaT keratinocytes(3×105cells/mL)were seeded with DMEM+ 10% FBS in a 96-well plate at 37°C in 5% CO2for 24hours. Different concentrations of honokiol (final concentrations of 3.91, 7.81, 15.63, 31.25, 62.5, and 125mg/L,respectively)were added to the cells,which were then cultivated for another 24hours.Next,20μL of MTT(0.5mg/mL)was added to each well.The 96-well plate was incubated for 4hours in the absence of light. Finally, the precipitates were suspended in 150μL of DMSO after the medium was discarded. The optical density (OD) at a wavelength of 550nm was measured with a microplate reader. The cell survival rate was calculated as follows:(OD in experimental group/OD in normal control group)×100%.

ELISA for assessment of TSLP secretion in HaCaT keratinocytes

HaCaT keratinocytes were cultured (3×105cells/mL)with DMEM + 10% FBS in 96-well plates (100μL/well)at 37°C in 5% CO2for 7hours. The cells were then cultured continually for 17hours with DMEM without FBS. The HaCaT keratinocytes were randomly divided into seven groups: normal control group, model control group, solvent control group, positive control group(dexamethasone), and three honokiol treatment groups(final concentrations of 15.63, 7.81, and 3.91mg/L,respectively). The cells in all groups (except the normal control group) were treated with poly(I:C) (100mg/L),rhTNF-α (100mg/L), and rhIL-4 (100ng/mL) for 48 hours,in which different concentrations of honokiol were added to the cells of the honokiol treatment groups at 24 hours of the above-mentioned stimulation. Similarly,dexamethasone (final concentration of 20mg/L) was added to the cells of the positive control group(dexamethasone), and DMSO (final concentration of 0.5%) was added to the cells of the solvent control group. The concentrations of TSLP secreted by HaCaT keratinocytes in supernatant were measured by ELISA according to the manufacturer’s protocol.

Statistical analysis

The effect of honokiol on the viability of HaCaT keratinocytes and TSLP concentrations is represented as mean±standard deviation. Statistical significance was analyzed using SPSS 20.0 (IBM Corp., Armonk, NY,USA). One-way analysis of variance with Dunnett t test was used.A P value of ＜0.05 was considered statistically significant.

Results

Effects of honokiol on capsaicin-induced TRPV1 activation in HaCaT keratinocytes

After the preparation and culture of HaCaT keratinocytes in the 35mm glass-bottom dish(5×104cells/mL),Fluo-3 AM/Pluronic F-127 was used to treat the cells. Capsaicin was used to activate TRPV1,and the fluorescence intensity of each group was detected. Figure 1 shows that at 45 second after addition of capsaicin in the model control group, the fluorescence intensity dramatically increased and the ΔF/F0value approached 0.8, indicating that the TRPV1 on the HaCaT cells was open and that extracellular Ca2+was flowing into the cells. As time passed (about 60seconds later), the fluorescence intensity gradually weakened, and the ΔF/F0value decreased to about 0.4 at 300seconds. After pretreating the cells with different concentrations of honokiol, the capsaicinstimulated relative fluorescence intensity appeared to be weakened at each point in time. In particular, the ΔF/F0value decreased to 0 at 45second in the 31.25mg/L honokiol treatment group,indicating that the Ca2+influx was significantly inhibited. In the 15.63 and 7.81mg/L honokiol groups, the fluorescence intensity peaked at nearly 90 and 75seconds, but the peaks were obviously delayed and the ΔF/F0values of the peaks were relatively lower than those in the model control group. These findings indicated that honokiol at these two concentrations could partially inhibit the Ca2+influx.The peak in the solvent group was slightly lower than that in the model control group at around 45second.Capsazepine,a TRPV1 inhibitor, significantly inhibited Ca2+influx,and no fluorescence intensity peak was observed. The fluorescence image in each group at 15 and 45seconds is shown in Fig. 2.

Effects of honokiol on secretion of TSLP in HaCaT keratinocytes

We used 15.63mg/L as the maximum test concentration of honokiol according to the trials of viability of HaCaT keratinocytes (Table 1). After stimulation of the HaCaT cells with 100mg/L of poly(I:C)combined with 100mg/L of rhTNF-α and 100ng/mL of rhIL-4 for 48hours, the TSLP concentration in the model control group was significantly higher than that in the normal control group(t=17.323,P=0.000).There was no significant difference between the model control group and the solvent control group (t=0.198, P=0.976). The TSLP concentrations in the 15.63 and 7.81mg/L honokiol groups were significantly lower than those in the solvent control group (t=7.382, P=0.003, and t=2.766, P=0.023, respectively),indicating that honokiol at these two concentrations inhibited TSLP secretion in HaCaT keratinocytes. The TSLP concentration was not significantly different between the 3.91mg/L honokiol group and solvent control group(t=1.872,P=0.124).The TSLP concentration was significantly lower in the dexamethasone group than in the solvent control group(t=6.104,P=0.004).All results are shown in Figure 3.

Discussion

Itch is an unpleasant feeling on the skin or mucous membranes that causes the desire to scratch. A crosssectional study of 19,000 adults showed that 8%-9%had acute itch and that 13.5%had chronic itch.15Pruritus can severely affect people’s physical and mental health. It is a common clinical manifestation of many diseases. For example, 87%-100% patients with AD have chronic itch.2However, treatment of chronic itch is often minimally effective.5M. officinalis, a traditional Chinese medicine drug, originates from the bark of Magnolia.Honokiol is an active constituent of M. officinalis.In our previous study,we demonstrated the antipruritic effect of honokiol in both a histamine-induced itch model and an acetone, ether, and water itch model.13TSLP is an inflammatory cytokine secreted by keratinocytes.Research has shown that an elevated TSLP level can directly stimulate TSLP receptors on skin nerve endings in patients with AD, further activating itch-related receptors to produce an itch sensation and transmit it to the center of the nerve.3Drugs acting on itch-related inflammatory mediators such as TSLP, IL-4, IL-13, and IL-31 are thought to have good treatment prospects.The monoclonal antibodies of these itch-related cytokines are being clinically researched.3,16The present study showed that honokiol significantly inhibited the secretion of TSLP in HaCaT keratinocytes at concentrations of 15.63 and 7.81 mg/L. These results suggest that honokiol can weaken pruritus by reducing the production of the itch-related inflammatory mediator TSLP.TRPV1 is a nociceptor that is expressed in primary sensory afferent neurons and plays an important role in the itch sensation. Interestingly,TRPV1 was recently found to be expressed not only in neurons but also in other cells such as keratinocytes,mast cells,dendritic cells,and fibroblasts.3In a previous study,we found that honokiol can significantly inhibit capsaicin(TRPV1 agonist)-induced Ca2+influx into rat dorsal root ganglion cells,suggesting that its antipruritic efficacy may be related to its effect on TRPV1.13The results of the present study indicate that honokiol at a concentration of 31.25mg/L has a significant inhibitory effect on Ca2+influx into HaCaT keratinocytes activated by capsaicin to open TRPV1. Activation of TRPV1 in HaCaT keratinocytes can reportedly induce the secretion of many inflammatory mediators.9The combination of our previous and present results suggests that the antipruritic effect of honokiol may not only be related to the inhibition of TRPV1 activation on dorsal root ganglion cells but also to the inhibition of the open TRPV1 on HaCaT keratinocytes,thereby further inhibiting the secretion of downstream itch-related inflammatory cytokines.To further elucidate the clinical value of honokiol against pruritus, the molecular relationship between its inhibition of TSLP secretion and the inhibition of TRPV1 channel activation in HaCaT keratinocytes needs to be explored in detail.

Figure 1. Effects of different concentrations of honokiol on Ca2+influx in the capsaicin-induced model of TRPV1 in HaCaT keratinocytes.MC:model control group;SC:solvent control group;PC,positive control group,capsazepine;Hon1,7.81mg/L honokiol treatment group;Hon2,15.63mg/L honokiol treatment group; Hon3, 31.25mg/L honokiol treatment group; ΔF/F0, rate of change in fluorescence intensity; ΔF,variation in fluorescence intensity(calculated as Fmax-F0,where Fmax is the maximum fluorescence intensity and F0 is the initial fluorescence intensity).

Figure 2. Fluorescence photographs of each group of HaCaT keratinocytes at different time points after capsaicin induction.(A1)The 15th second in the model control group.(A2)The 45th second in the model control group.(B1)The 15th second in the solvent control group.(B2)The 45th second in the solvent control group.(C1)The 15th second in the positive control,capsazepine group.(C2)The 45th second in the positive control,capsazepine group.(D1)The15thsecondinthe7.81mg/Lhonokiolgroup.(D2)The45thsecondinthe7.81mg/Lhonokiolgroup.(E1)The15thsecondinthe15.63 mg/L honokiol group.(E2)The 45th second in the 15.63mg/L honokiol group).(F1)The 15th second in the 31.25mg/L honokiol group.(F2)The 45th second in the 31.25mg/L honokiol group.