Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium

The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton’s tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca2+ levels ([Ca2+]i), whereas PP2 prolonged the time required for [Ca2+]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca2+.

lytic enzymes can also damage healthy surrounding tissue, resulting in deleterious inflammatory diseases, such as acute lung injury, chronic obstructive pulmonary disease, and asthma [19][20][21][22] . In order to ameliorate these conditions, many studies have investigated the pharmacological modulation of activated human neutrophils by natural products and their mechanisms of action.
This present study investigated the modulatory effects of a P. frutescens var. crispa extract (PFE) in activated human neutrophils. We found that a non-toxic level of PFE reduced superoxide anion (O 2˙-) production, elastase release, ROS formation, CD11b expression, and chemotactic migration in N-formyl-Met-Leu-Phe (fMLF)-induced human neutrophils. Neutrophils express the formyl-peptide receptor (FPR) that sense invading pathogen and tissue damage. Diverse intracellular signaling pathways, including G-proteins, calcium (Ca 2+ ) mobilization , tyrosine protein kinases, adapter proteins, and cytoskeletal rearrangement are triggered by FPR and are responsible for neutrophil activation 23 . Many of the observations made in this study demonstrated that the anti-inflammatory effects of PFE were mediated through two pathways: blockade of Src family kinases (SFKs) and reducing intracellular Ca 2+ mobilization.

Results
PFE inhibited O 2˙− production, elastase release, and ROS formation in fMLF-activated human neutrophils. In order to evaluate whether PFE affected neutrophil function and inflammatory responses, we first investigated the effects of PFE on O 2˙− production, elastase release, and ROS formation in fMLF-activated human neutrophils. Our experiments revealed that O 2˙-and elastase, which were detected by ferricytochrome c and elastase substrate, respectively, were reduced by PFE (1, 3, and 10 μ g/ml) in a concentration-dependent manner, with IC 50 values of 3.18 ± 0.32 and 3.82 ± 0.27 μ g/ml, respectively (Fig. 1A,B). ROS formation in activated and elastase release were detected spectrophotometrically using cytochrome c reduction and elastase substrate, respectively. (C) The fluorescence intensity of dihydrorhodamine 123 (DHR123) was used to detect the intracellular ROS. The ROS formation in fMLF-activated neutrophils pretreated with PFE (red line) was decreased, as compared with those without PFE (black line). The dashed line indicated neutrophils that were not treated with fMLF (basal group). (D) The mean values of fluorescence intensity from panel C. Data are expressed as the mean ± standard error of the mean, n = 7, *p < 0.05, ***p < 0.001, as compared to the fMLF group.

PFE inhibited CD11b expression and cell migration in fMLF-activated human neutrophils.
CD11b/CD18 is involved in cellular adhesion between activated neutrophils and endothelial cells. When neutrophils are stimulated, they rapidly immobilize through activation of integrin CD11b/CD18, and subsequent modulation of this attachment allows migration. Our results demonstrated that fMLF stimulation of human neutrophils resulted in significant up-regulation of CD11b expression, while PFE (1, 3, and 10 μ g/ml) inhibited CD11b expression in fMLF-activated human neutrophils with an IC 50 value of 4.49 ± 1.39 μ g/ml ( Fig. 2A,B). Furthermore, fMLF-induced neutrophil migration was reduced in the presence of PFE (3 and 10 μ g/ml; Fig. 2C). The IC 50 for this effect was 5.36 ± 1.06 μ g/ml.
PFE inhibited the activation of SFKs. SFKs are protein-tyrosine kinases that play important roles in neutrophil activation triggered by the chemotactic peptide, fMLF 24 . Our immunoblotting analyses demonstrated that fMLF stimulated the phosphorylation of SFKs (Tyr416), Src (Tyr416), and Lyn (Tyr 396) in human neutrophils; PFE attenuated phosphorylation of these proteins (Fig. 3A). PP2, a selective inhibitor of SFKs, also inhibited phosphorylation of these SFKs (Fig. 3B). In addition, we investigated whether PFE inhibited the enzymatic activities of Src and Lyn. The results of this analysis indicated that PFE inhibited Src and Lyn tyrosine kinase activities in a concentration-dependent manner (IC 50 = 5.21 ± 0.36 μ g/ml and 2.51 ± 0.29, respectively) ( Fig. 4A,B). PP2 was used as a positive control. (C) Neutrophils (5 × 10 6 cells/ml) were pre-incubated with DMSO or PFE (1, 3, and 10 μ g/ml) for 5 min in the top chamber. Migrated neutrophils were counted after 60 min. Data are expressed as the mean ± standard error of the mean, n = 3-4, *p < 0.05, **p < 0.01, ***p < 0.001, as compared to the fMLF group.
Scientific RepoRts | 5:18204 | DOI: 10.1038/srep18204 PFE attenuated the phosphorylation of Burton's tyrosine kinases (Btk). Btk belongs to the Tec family of tyrosine kinases, which includes Itk, Tec, Txk, and Bmx [25][26][27] . Btk is involved in G-protein coupled receptor (GPCR) signal transduction [27][28][29] . Immunoblotting for phospho-Btk demonstrated that both PFE (3 and 10 μ g/ml) and PP2 (1 μ M) attenuated the phosphorylation of Btk Tyr223 (Fig. 5A,B). Furthermore, we used a Btk inhibitor, LFM-A13, to explore the role of Btk in the regulation of neutrophil activation. Figure   After transferring the blots onto nitrocellulose membranes, we immediately cropped the targeted blots according to referenced indicating markers, and then targeted proteins were immunoblotted with its specific monoclonal antibody. (A,B) Representative images from one of four independent experiments of Western blotting using anti-phospho antibodies directed against SFKs, Src, and Lyn were shown. Bands on the blots were analyzed using a densitometer, and the quantitative ratios for all samples were normalized to the corresponding total protein or to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (3 ng/ml) was incubated with dimethylsulfoxide (DMSO, as control), PFE (3, 10, and 30 μ g/ml), or PP2 (3 μ M) for 10 min, and then ATP/substrate was added to the reaction mixture for 60 min prior to measurement of Src or Lyn activity, as described in the Methods section. Data are expressed as the mean ± standard error of the mean, n = 3, ***p < 0.001, as compared to the control group.

Discussion
P. frutescens is a popular vegetable and food condiment that also commonly utilized as an herbal medicine in many Asian countries. Previous studies demonstrated that P. frutescens has important anti-inflammatory effects 5,6 . Nevertheless, little is known about the pharmacological mechanisms underlying these effects in human neutrophils. In the present study, we found that PFE inhibited the respiratory burst, degranulation, and chemotactic migration of fMLF-activated human neutrophils through inhibiting SFKs (Src and Lyn) pathway and intracellular Ca 2+ mobilization.
Many studies have revealed that compounds found in P. frutescens have anti-oxidative effects 7,8 . In this study, we found that PFE reduced O 2˙-production and ROS formation in fMLF-induced human neutrophils. We also used a cell-free xanthine/xanthine oxidase system and found that higher concentration of PFE (10 μ g/ml and 30 μ g/ml) had direct O 2˙-scavenging activity (IC 50 = 23.43 ± 0.34 μ g/ml; data not shown); this finding was consistent with a previous report 7 . These findings suggested that PFE reduced ROS levels, either by modulating cellular signaling or by direct free radical scavenging activity.
SFKs are non-receptor tyrosine kinases that regulate cell growth, differentiation and activation via various intracellular signaling pathways. Several SFKs such as Lyn, Hck and Fgr are expressed in human neutrophils 34,35 . These kinases are involved in fMLF-activated signal transduction processes 24,[36][37][38] . In our studies, a selective inhibitor of SFKs (PP2) significantly reduced O 2˙-production and elastase release in neutrophils stimulated with fMLF. CD11b expression and chemotactic migration of fMLF-induced neutrophils were also reduced in the presence of PP2 (data not shown). Other studies 24 and our data support the role of SFKs in signal transduction triggered by fMLF receptors and neutrophil activation. Furthermore, we demonstrated that PFE inhibited the phosphorylation of the SFKs, Src, and Lyn in fMLF-activated neutrophils. Furthermore, we found that PFE directly inhibited the enzymatic activities of Src and Lyn. These data suggestedthat PFE influenced the activities of SFKs, which plays an important role in the functional responses to fMLF.
Btk belongs to the Tec family of tyrosine kinases. Previous reports have revealed a tight relationship between SFKs and the Tec family 27,29,39 . The phosphorylation of Btk at Tyr551and subsequent autophosphorylation at Tyr223, which is necessary for its full activation, are closely correlated with the activity of SFKs 40,41 . In the present study, we found that an inhibitor of Btk (LFM-A13) reduced O 2˙− production and elastase release in fMLF-activated human neutrophils, which was consistent with a previous study 29 describing the role of Btk in neutrophil activation. Moreover, we observed that the phosphorylation of Btk was significantly inhibited by PP2, suggesting that Btk was downstream of SFKs. In addition, Vav acts as a GEF for Rac, which is a subfamily of the RHO family, leading  24 . Our immunoblotting data showed that both PP2 and LFM-A13 inhibited the phosphorylation of Vav (Fig. 5C), suggesting that SFKs and Btk modulated Vav activation. In agreement with this finding, PFE significantly inhibited the fMLF-induced phosphorylation of Btk and Vav in human neutrophils. Taken together, the results of this study provided evidence for the role of SFKs/ Btk/Vav in human neutrophil activation triggered by fMLF, and indicated that PFE regulated human neutrophil activation by inhibiting this SFKs/Btk/Vav signaling pathway.  Ca 2+ is a vital intracellular second messenger that contributes to neutrophil activation 42,43 . The binding of fMLF to GPCR triggers a rapid and transient increase in [Ca 2+ ] i , resulting in neutrophil activation. Some specific inhibitors of Ca 2+ signaling have been investigated as anti-inflammatory drugs because of their suppressive effects on neutrophil functions 44 . Our experiments using a Ca 2+ chelator (BAPTA-AM) proved that increased [Ca 2+ ] i was required for the fMLF-induced respiratory burst and elastase release. Furthermore, our study showed that PFE reduced the fMLF-induced amplification of [Ca 2+ ] i . However, PP2 prolonged the time required for [Ca 2+ ] i to resume its original equilibrium concentration. These findings inferred that the Ca 2+ mobilization inhibited by PFE was independent of SFKs. Intracellular Ca 2+ transients and SFKs are both important signal transduction pathways in fMLF-induced neutrophils 23 . Obviously, additional work is required to define the signal events linking SFKs Ca 2+ mobilization in human neutrophils.
Based on these findings, we conclude that PFE significantly inhibited fMLF-induced human neutrophil activation, including O 2˙-production, elastase release, ROS formation, CD11b expression, and chemotactic migration. PFE inhibited activation of SFKs and Ca 2+ mobilization, which represent two signaling pathways involved in fMLF-induced neutrophil activation (Fig. 9). Because the PFE used in the present study was a crude extract of P. frutescens, any of the chemical components of PFE may be involved in the effects and mechanisms described above. Further studies aimed at clarifying whether these two signaling pathways are regulated by one or more of the different chemical constituents of PFE should contribute to the development of more effective therapeutic options.

Methods
Preparation of P. frutescens extract. PFE was prepared by our co-author, Dr. Leu. P. frutescens (L.) Britt leaf powder was purchased from the Sun-Ten Pharmaceutical Co., Ltd. (Taipei, Taiwan). This contained the P. frutescens extract (67%) and corn starch (33%). The powder (1 g) was suspended in 10 ml ethanol at 37 °C for 4 h and then centrifuged at 5000 g for 20 min. The supernatant was filtered and lyophilized. The resulting PFE was suspended in dimethylsulfoxide (DMSO) at a concentration of 10 mg/ml and stored at − 20 °C until use. The voucher specimen (CGU-NP-PFE-327) was secured at the Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan .

Isolation of human neutrophils. This study protocol was investigated and approved by the Institutional
Review Board at Chang Gung Memorial Hospital, and written informed consent was obtained from every volunteer. The methods were carried out in accordance with the approved guidelines. Blood was drawn from healthy volunteers (aged 20-30 years) who had no congenital or systemic disease and did not take any medicine during the week prior to sample collection. Human neutrophils were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll-Hypaque gradient and hypotonic lysis of red blood cells 45 . The granulocyte layer was harvested and resuspended in Ca 2+ -free Hank's balanced salt solution (HBSS) at pH 7.4, and maintained at 4 °C until use. Greater than 98% cell viability was confirmed by trypan blue exclusion. The data presented for each specific experiment were derived from 4 to 7 samples.
Assessment of elastase release. Human neutrophils (6 × 10 5 cells/ml) were equilibrated with an elastase substrate (MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide, 100 μ M) at 37 °C for 2 min and then incubated with DMSO, PFE, PP2, LFM-A13, or BAPTA/AM for 5 min. Cells were activated by fMLF (0.1 μ M)/CB (0.5 μ g/ml ) for a further 10 min. Elastase release was determined by measuring the changes in absorbance at 405 nm using a spectrophotometer (U-3010; Hitachi, Tokyo, Japan). The results were expressed as a percentage of the elastase release in the fMLF/CB-activated, drug-free, control group 48 .
Determination of ROS formation. Cell-permeable DHR123, which is not fluorescent until oxidized, was used to detect intracellular ROS. Human neutrophils (1 × 10 6 cells/ml) were incubated in HBSS containing DHR123 Evaluation of LDH release. LDH release was used as an indicator of cell membrane integrity and served as a general means to assess cytotoxicity. We used commercially available reagents (Promega) to determine the LDH level. Human neutrophils were treated with PFE (10 μ M) for 60 min. LDH assay reagents was then added to the supernatant. LDH release was expressed as a percentage of the amount of enzyme liberated following incubation of human neutrophils with 0.1% Triton X-100 for 30 min at 37 °C.

Measurement of CD11b expression.
CD11b/CD18 is a heterodimeric glycoprotein that is expressed on the plasma membrane of neutrophils. Neutrophils (5 × 10 6 cells/ml) were preincubated with PFE, PP2, or LMF-A13 for 5 min and then activated by fMLF (0.1 μ M)/CB (0.5 μ g/ml ) for a further 5 min. The reaction was stopped by placing the cells on ice prior to centrifugation at 4 °C. The supernatant was discarded and the cells were resuspended in 0.5% bovine serum albumin for staining using a FITC-labeled-anti-CD11b antibody (1 μ g) for 90 min at 4 °C. The fluorescence intensity of FITC-labeled anti-CD11b was then monitored using flow cytometry.
Chemotactic migration assay. Human neutrophil chemotactic migration was assessed using a microchemotaxis chamber with a 3-μ m filters (Millipore) 49 . Neutrophils (5 × 10 6 cells/ml) were incubated with DMSO or PFE (3 and 10 μ g/ml) for 5 min at 37 °C and then placed into the top chamber. HBSS containing fMLF (0.1 μ M) was added to the bottom chamber. After incubation in a 5% CO 2 incubator for 60 min, the number of cells that migrated was determined by a MoxiZ automatic cell counter (ORFLO).
Src and Lyn kinase activity. Src and Lyn kinase activity were determined by the ADP-Glo TM kinase assay kit (Promega) according to the manufacturer's instructions. Briefly, Src or Lyn kinase reactions were performed using a buffer containing Src or Lyn kinase, Src or Lyn kinase substrate, ATP (50 μ M), and PFE (3, 10, and 30 μ g/ ml) or PP2 (3 μ M) for 60 min. ADP-GloTM reagent was added to terminate the kinase reaction and deplete the remaining ATP. The luciferase/luciferin luminescence was recorded with a microplate reader (Infinite 200 Pro; Tecan, Männedorf, Switzerland). Statistical analysis. Results were expressed as mean ± standard error of the mean (SEM). Computation of the 50% inhibitory concentration (IC 50 ) was computer-assisted (PHARM/PCS v.4.2). Statistical comparisons were made between groups using Student's t-test. Values of p less than 0.05 were considered to be statistically significant.