Multiple Components Rapidly Screened from Perilla Leaves Attenuate Asthma Airway Inflammation by Synergistic Targeting on Syk

Abstract

Background: Perilla frutescens (L.) Britt., a classic medicinal plant, has been demonstrated to have anti-inflammatory and anti-allergic effects in asthma. Perilla leaves extract (PLE) exerted significant therapeutic effect against allergic asthma inflammation through Syk inhibition. But the active chemical ingredients from PLE are complex and unclear, it is difficult to fully elucidate its pharmacological mechanisms.

Methods: A method was established for rapid screening and characterization of active ingredients from PLE that targeted Syk, with which three potential active ingredients were identified. By using OVA-induced allergic asthma mouse model in vivo, OVA-induced human PBMCs inflammation model and DNP-IgE/BSA-induced RBL-2H3 cells model in vitro, the effects and mechanisms of PLE and its active components were evaluated.

Results: Using Syk-affinity screening method, roseoside (RosS), vicenin-2 (Vic-2) and rosmarinic acid (RosA) were identified from PLE. In vitro, PLE and its ingredients showed significant inhibitory activities against Syk, with their mixture (Mix, prepared by RosS, Vic-2 and RosA in accordance with their ratio in Syk-conjugated beads bound fraction) showing a stronger inhibitory activity. RosS, Vic-2 and RosA also showed significant effects on allergic asthma, and a synergistic effect of Mix was observed. Moreover, treatment with PLE, RosS, Vic-2, RosA, and Mix significantly inhibited the expression and phosphorylation of Syk, PKC, NF-κB p65, and cPLA₂ in allergic mice lung tissue and in RBL-2H3 cells.

Conclusion: PLE may alleviate allergic airway inflammation partly through the multiple components synergistic targeting on Syk and its downstream inflammatory pathway.

Keywords: PLE; Syk; allergic airway inflammation; multi-component; synergistic effect.

Figure 1

Extraction and identification of Syk affinitive components from PLE and their inhibited activity on Syk in vitro. (A) HPLC chromatograms: 1 – PLE, 2 – Eluent, 3 – Blank, and 4 – RosS, 5 – Vic-2, and 6 – RosA Standard reference substances. (B) Ion chromatograms (EIC) of LC-ESI-IT-TOF-MS extracted analysis of the sample peak at 18.750 min. (C) EIC of LC-ESI-IT-TOF-MS extracted analysis of the sample peak at 23.017 min. (D) EIC of LC-ESI-IT-TOF-MS extracted analysis of the sample peak at 33.300 min. (E) The relative abundance of Syk affinitive compounds as calculated by mass concentration (mg/mL) and molar concentration (mol/L). (F) In vitro Syk inhibitory activities of PLE, RosS, Vic-2, RosA, and Mix (n = 3).

Figure 2

Effects of Syk affinitive compounds from PLE on allergic airway inflammation in vivo. (A) Pathological changes in lung tissue were determined by H&E staining (magnified ×100). (B) The scoring of inflammatory cells infiltration in H&E stained lung tissues (n =20 was carried out using independent slices from 4 mice in each group). (C) Total and differential leukocyte counts in BALF (n = 8). (D–H) The levels of IL-6, TNF-α, IL-4, IFN-γ and the ratio of IL-4/IFN-γ in BALF (n = 8). (I–L) The levels of IgE, IgG1, IgG2a and IgG2b in serum (n = 8). Data were shown as mean ± SEM; ^##P < 0.01 vs control group; *P < 0.05 and **P < 0.01 vs model group.

Figure 3

Effects of Syk affinitive compounds from PLE on allergic airway inflammation in vitro. (A, B) The levels of IL-6 and IL-8 in OVA-induced human PBMCs (n = 42). (C) The level of TNF-α in DNP-IgE/BSA-induced RBL-2H3 cells (n = 3 independent experiments). (D) The release rate of β-Hex (%) in DNP-IgE/BSA-induced RBL-2H3 cells (n = 3 independent experiments). (E) (a) Images of DNP-IgE/BSA-induced RBL-2H3 cells stained by toluidine blue staining (magnified ×400) with (b) its degranulation rate (%) (n = 3). Data were shown as mean ± SEM; ^##P < 0.01 vs control group; *P < 0.05 and **P < 0.01 vs model group.

Figure 4

Syk affinitive components from PLE decreased the expression and phosphorylation of Syk and its downstream signaling proteins in vivo. (A–H) The expression of Syk, p-Syk, PKC, p-PKC, p65, p-p65, cPLA₂, and p-cPLA₂ in lung tissues of OVA-induced asthmatic mice were determined by WB (n = 5). Data were shown as mean ± SEM. ^#P < 0.05 and ^##P < 0.01 vs control group, *P < 0.05 and **P < 0.01 vs model group.

Figure 5

Syk affinitive components from PLE decreased the expression and phosphorylation of Syk and its downstream signaling proteins in vitro. (A) The expression of Syk were determined by immunofluorescence staining, (a) representative photomicrographs (×200) of Syk in RBL-2H3 cells and (b) their relative fluorescence intensities (n = 3). (B) The expression of p-Syk were determined by immunofluorescence staining, (a) representative photomicrographs (×200) of Syk in RBL-2H3 cells and (b) their relative fluorescence intensities (n = 3). (C–J) The expression of Syk, p-Syk, PKC, p-PKC, p65, p-p65, cPLA₂, and p-cPLA₂ in DNP-IgE/BSA-induced RBL-2H3 cells as determined by WB (n = 5). Data were shown as mean ± SEM. ^##P < 0.01 vs control group, *P < 0.05 and **P < 0.01 vs model group.

Figure 6

Verification that Syk, PKC, NF-κB and cPLA₂ were associated with Mix attenuated Inflammation in DNP-IgE/BSA-induced RBL-2H3 cells. (A) (a) Images of DNP-IgE/BSA-induced RBL-2H3 cells stained by toluidine blue (magnified ×400), (b) the percent of degranulation (%) calculated (n = 3), (c) the release rate of β-Hex (%) and (d) the level of TNF-α in cells co-treated Syk affinitive compounds with Syk inhibitor (n = 3 independent experiments). (B) The release rate of β-Hex and (C) production of TNF-α in cells co-treated Syk affinitive compounds with PKC, NF-κB or cPLA₂ inhibitors (n = 3 independent experiments). Data were shown as mean ± SEM. ^##P < 0.01 vs control group; **P < 0.01 vs model group.

Figure 7

Syk affinitive compounds in PLE attenuated allergic inflammation by synergistically targeting on Syk in DNP-IgE/BSA-induced RBL-2H3 cell. (A) (a) Representative photomicrographs (×200) of Syk expression in RBL-2H3 cells and (b) their relative fluorescence intensities as determined by immunofluorescence staining (n = 3). (B) The expression of Syk in DNP-IgE/BSA-induced RBL-2H3 cells with Mix of Syk affinitive compounds co-treated with Syk inhibitor detected by WB (n = 5). (C) (a) Representative photomicrographs of (×200) p-Syk in RBL-2H3 cells and (b) their relative fluorescence intensities as determined by immunofluorescence staining (n = 3). (D) The expression of p-Syk in DNP-IgE/BSA-induced RBL-2H3 cells with Mix of Syk affinitive compounds co-treated with Syk inhibitor detected by WB (n = 5). (E–J) The expression of PKC, p-PKC, p65, p-p65, cPLA₂, and p-cPLA₂ in DNP-IgE/BSA-induced RBL-2H3 cells with Mix of Syk affinitive compounds treatment with the Syk inhibitor detected by WB (n = 5). (K–P) The expression of Syk and p-Syk in DNP-IgE/BSA-induced RBL-2H3 cells with Mix of Syk affinitive compounds co-treated with PKC, NF-κB and cPLA₂ inhibitors detected by WB (n = 5). Data were shown as mean ± SEM. ^##P < 0.01 vs control group; *P < 0.05 and **P < 0.01 vs model group.