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Discussion

In this study, phenylethanoid glycosides obtained from Cistanche tubulosa did not affect cell viability on RBL-2H3 cells and KU812 cells at the 1.0-10.0 µg/mL treatments. But, acteoside was cytotoxic at the 100.0 µg/mL treatments (Fig. 2B), as has been reported by Saracoglu et al. [30] who have shown that acteoside isolated from Phlomic Armeniaca and Scutellaria salviifolia showed cytotoxic effects on dRLh-84, S-180, P-388/D1 cell lines with the IC₅₀ 30-221 µg/mL at different cell numbers. Our result suggested that the inhibition effect of acteoside on cell viability at 100 µg/mL treatment, may be affected by its apoptotic activity.

The immediate-type allergic reaction is involved in many allergic diseases such as asthma, allergic rhinitis, and sinusitis. Mast cells play a crucial role in inflammatory and immediate allergic responses. Our data shows that the acteoside has the highest inhibitory effect on the β-hexosaminidase release from IgE-sensitized, antigen-stimulated RBL-2H3 cells compared with other phenylethanoid glycosides at 1.0 μg/mL treatment (Fig. 3). The inhibition effect of acteoside on β-hexosaminidase release at 10.0 μg/mL treatment was lower than that at 1.0 μg/mL treatment. As Sugisawa et al. [31] demonstrated, this may be contributed to H₂O₂ induction at high concentrations of acteoside_, which might be regulated calcium signals and degranulation on the RBL-2H3 cells [32], influenced to β-hexosaminidase release at 10.0 μg/mL acteoside treatment.

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The degranulation of mast cells is closely related to [Ca²⁺]i. The inhibition of Ca²⁺ influx by anti-allergic drugs plays a crucial role in the suppression of degranulation in mast cells. [33-34]. Our results indicate that the intracellular [Ca²⁺]i level was lower in the acteoside-treated, DNP-BSA-stimulated RBL-2H3 cells (Fig. 4), which is consistent with other reports [34-35], and these results agree with those of Fig. 3. We consider from these observations that the decrease in intracellular [Ca²⁺]i is involved in the inhibitory effect of acteoside on β-hexosaminidase release. Nitric oxide (NO) and H₂O₂ are two major reactive oxygen species (ROS) known in the regulation of calcium signal and degranulation of mast cells. ROS are necessary for secretion of β-hexosaminidase and calcium influx, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is mainly responsible for ROS production in the IgE-mediated RBL-2H3 cells [29, 32]. The sustained elevation of cytosolic calcium through store-operated calcium entry was totally abolished when the ROS production was blocked. In addition, ROS has some relationship with ß-hexosaminidase and histamine release by protein kinase C (PKC) activation and IgE stimulation in RBL-2H3 cells [34]. Furthermore, Suzuki et al. [36] found that there was a significant correlation between inhibition effects on histamine release and 1, 1-diphenyl-2-piracy l-hydroxyl (DPPH) or superoxide anion radical scavenging activities of curcumin-related anti-oxidant compounds. On the other hand, acteoside has free radical scavenging properties on NO radical and DPPH radicals [9, 37]. Also, acteoside has shown scavenging activity in activated human leucocytes [38]. One of the possibilities is that the inhibition effect of acteoside on intracellular calcium release may be affected by its free radical scavenging activities of NADPH oxidase activities.

Acteoside dose-dependently inhibited histamine release on A23187 plus PMA-stimulated KU812 cell (Fig. 4A). Acteoside inhibited glutamate-induced intracellular Ca²⁺ influxes resulting in overproduction of NO and reduced formation of ROS [37]. Both PKC and Ca²⁺ signaling pathways are required for histamine and leukotrienes release from mast cells as well as rodent systems [39]. We hypothesized that acteoside may have an inhibitory effect on histamine release through Ca²⁺ influx. For this purpose, we used A21387 plus PMA to stimulate KU812 cells and demonstrated the histamine release inhibition effect with acteoside. This result supported our hypothesis that acteoside may decrease histamine release from KU812 cells by inhibition of Ca²⁺ influx or PKC activation.

It is believed without a doubt that stimulation of mast cells with compound 48/80 initiates the activation of a signal transduction pathway, which leads to histamine release. Senyshyn et al. [40] identified recombinant G subunit markedly synergized phospholipase D activation by compound 48/80 in permeabilized basophilic cells. Compound 48/80-induced secretion is associated with a transient increase in cytosolic Ca²⁺. This secretion was blocked by the calcium chelator and PKC inhibitor. In the present study, we observed that acteoside inhibited the compound 48/80-induced degranulation from basophilic cells. But, the inhibition effect of acteoside on histamine release at 0.1 μg/mL treatment was higher than that 1.0-10.0 μg/mL treatment (Fig. 4B). As Lau et al. [41] demonstrated, the inhibition effect of acteoside on compound 48/80 induced histamine release may be involved in the anti-inflammation effect of acteoside against vascular permeability-associated edema. Possibly, the acteoside may have several different ways of inhibiting chemical mediator release from basophilic cells, suggesting the complexity of its action. However, in order to reveal the mechanism of histamine release inhibition of this prescription, further studies should be performed.

Among cytokines produced by basophilic cells, TNF-α, IL-4, IL-13, and IL-5 are the key molecules. The reduction of pro-inflammatory cytokines from mast cells or basophilic cells is one of the key indicators of reduced allergic symptoms [42]. With regard to TNF-α, the production is mainly regulated by Ca²⁺ influx, but the release process is regulated by additional mechanisms possibly involving activation of PKC in KU812 cells. Inhibition of [Ca²⁺]i influx is involved in the expression of cytokines in mast cells and basophilic cells [34, 43]. Our result showed that 1.0 and 10.0 µg/mL acteoside reduced TNF-α and IL-4 production from A23187 plus PMA stimulated KU812 cells after treatment for 16 h. Acteoside might inhibit TNF-α and IL-4 production by decreasing the [Ca²⁺] I level in A23187 plus PMA stimulated KU812 cells. Moreover, our results suggested that the acteoside has an anti-allergic effect at the late phase. Confirmation of the effect of acteoside using FcεRI expressing mast cells or basophilic cells is necessary for the future.

In conclusion, we report for the first time that acteoside, echinacoside, and cistanoside A extracted from Cistanche tubulosa can inhibit the release of β-hexosaminidase from IgE-sensitized BSA-stimulated RBL-2H3 cells. Furthermore, acteoside can inhibit the histamine release, TNF-α, and IL-4 production in a dose-dependent manner on A23187 plus PMA stimulated KU812 cells. These results suggest that acteoside could be a good candidate for the therapeutic treatment of various allergic diseases. The detailed mechanism behind the anti-allergic effect of acteoside is the subject of a future study.

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