Emodin inhibits ATP-induced IL-1β secretion, ROS production and phagocytosis attenuation in rat peritoneal macrophages via antagonizing P2X₇ receptor

Animals and reagents

Healthy male Wistar rats with weight of 200 ± 50 g were obtained from the Institute of Health and Environmental Medicine, Academy of Military Medical Sciences (Tianjin, China, Certification Number: SCXK 2010-0002). DMEM and fetal calf serum (FCS) were purchased from Gibco (Grand Island, NY) and HyClone (Logan, UT), respectively. Emodin was obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Fura-2/AM was from Biotium (Hayward, CA). IL-1β ELISA kits were purchased from NeoBioscience Technology Co., Ltd. (China). Neutral red was from Damao Chemical reagent Factory (Tianjin, China). SOD (superoxide dismutase) was purchased from Beyotime Institute of Biotechnology (Haimen, China). The rest of reagents, including ATP, LPS (lipopolysaccharide), DHE (dihydroethidium), BBG (brilliant blue G), DPI (diphenylene iodonium) and DMSO (dimethylsulfoxide), were from Sigma (St. Louis, MO).

Millimolar concentrations of ATP are required for P2X₇R activation (Surprenant et al., Citation1996), and the optimal concentration of ATP chosen for each experiment was based on previous reports (Fang et al., Citation2009; Zhou et al., Citation2006). The concentrations of emodin (0.1, 0.3, 1, 3, 10 μM) were selected based on our preliminary experiments and the effectual dose applied in previous studies (Kaneshiro et al., Citation2006; Tang et al., Citation2007). The specific antagonist of P2X₇R BBG was set as positive control, and the doses that ranged from 0.1 to 10 μM were also chosen according to previous work (Jiang et al., Citation2000; Liu et al., Citation2010).

Macrophage isolation and culture

Rat peritoneal macrophages were isolated and cultured as previously described (Zhou et al., Citation2006). Briefly, Wistar rats were sacrificed according to institutional guidelines. Hanks’ balanced salt solution (HBSS) (NaCl 150 mM, KCl 5.4 mM, CaCl₂ 2 mM, MgCl₂ 1 mM, glucose 10 mM and HEPES 10 mM, pH 7.4) was injected into the abdomen of each rat. Peritoneal cells were isolated from the abdomen, collected by centrifugation at 200 g for 10 min and then cultured in DMEM containing 10% FCS in a humidified incubator with 5% CO₂ at 37 °C before analysis. The adherent cells were used in our experiment. Nonspecific esterase staining showed that approximately 95% of them were macrophages as characterized previously.

Measurement of cytosolic Ca²⁺ concentrations ([Ca²⁺]_c)

[Ca²⁺]_c was measured as previously described (Hu et al., Citation2012). After treatment, macrophages were loaded with 2 μM fura-2/AM in HBSS for 1 h at room temperature in dark. After a gentle washing step, cells were bathed in fresh HBSS solution for monitoring the changes in [Ca²⁺]_c triggered by ATP. [Ca²⁺]_c was measured with a calcium imaging system built on an inverted fluorescence microscope (Olympus IX51). The ratiometric fluorescent Ca²⁺ indicator fura-2 was alternately excited at 340 nm and 380 nm with a Lambda 10-2 Sutter (Sutter Instrument, Novato, CA). Fluorescence images (filtered at 515 nm ± 25 nm) were acquired by a CCD camera (CoolSNAP fx-M, Roper Scientific Inc., Tucson, AZ) and analyzed with MetaFluor (Universal Imaging Corporation, Downingtown, PA). [Ca²⁺]_c was represented by the ratio of fluorescence intensity at 340 nm/fluorescence intensity at 380 nm (F340/F380). At least three independent experiments were done for each condition. One curve of calcium changes was plotted as the representation of other similar traces.

Measurement of interleukin-1 beta (IL-1β) release

Extracellular IL-1β was detected by the rat IL-1β ELISA kit. Briefly, after treatment with indicated reagents, the supernatant of macrophages in culture in each group was transferred into an ELISA plate seeded with the IL-1β antibody, and incubated for 90 min. Then the plate was washed with the washing buffer for five times. After that, the secondary antibody was applied for 60 min and then washed extensively. Subsequently, the HRP-conjugate reagent was introduced to each well of the plate to promote the formation of antibody–antigen–enzyme–antibody compounds for 30 min. After repeated washing steps, the chromogen solution and stop solution was sequentially added into each well for an incubation step of 15 min and 5 min, respectively. Finally, the absorbance at 450 nm representing the relative level of IL-1β in each well was measured by an ELISA reader (Bio-Rad Imark Microplate Reader, Hercules, CA). The concentration of IL-1β in each group was determined by the standard curve.

Detection of intracellular reactive oxygen species (ROS)

Dihydroethidium (DHE), a reduced form of ethidium bromide, was used to detect intracellular ROS. After treatment with indicated reagents, macrophages were incubated in HBSS with 5 μM DHE for 30 min at 37 °C in dark. Then they were rinsed twice with HBSS and observed with a fluorescence microscope at the excitation wavelength of 488 nm and emission wavelength of 610 nm. The fluorescent intensity represents the intracellular ROS level.

Measurement of phagocytic activity of macrophages

The phagocytic activity of macrophages was analyzed by the uptake assay of neutral red (Man et al., Citation2012). Briefly, after the pre-incubation with emodin or BBG for 3 h, macrophages were treated simultaneously with ATP (1 mM) and neutral red (0.1%) for 30 min. After the two washing steps with HBSS, the cells were treated with lysis buffer (50% acetic acid and 50% ethanol) shaking for 15 min. The absorbance at 550 nm (A₅₅₀) for each well was determined by an ELISA reader (Bio-Rad Imark, Hercules, CA). The relative phagocytic activity in each group was represented by A_{550, sample}. The inhibition of phagocytic activity in ATP alone group was taken as 100% (control group was treated with vehicle solution), and the inhibitory rates in other groups were normalized to the ATP group and calculated as: . Thus, the recovery rates of phagocytic activity can be derived as: .

Statistical analysis

All data are presented as mean ± standard deviation (SD) from three independent experiments. The statistical comparison between two groups was carried out using Student’s t-test (Origin 8.1), and the analysis for multiple groups was using Dunnett's test (SPSS 18.0, one-way ANOVA). p < 0.05 was considered to be statistically significant. The values of half maximal inhibitory concentration (IC₅₀) were calculated according to the dose–response curve fitting with the Boltzmann equation: , in which y is the inhibition ratio of increase in [Ca²⁺]_c, A1 is the asymptotic maximum, A2 is the asymptotic minimum, x is concentrations of emodin and dx is the time constant.

Emodin inhibited ATP-triggered increase in cytosolic Ca²⁺ concentrations in macrophages

The influence of emodin on ATP-induced [Ca²⁺]_c increase was measured by Ca²⁺ imaging. A rise in F340/F380 ratio indicated an increase in [Ca²⁺]_c. To activate P2X₇R completely, a high concentration of ATP (5 mM) was chosen. Representative [Ca²⁺]_c profiles are shown in Figure 1A. The application of ATP (indicated by arrows) triggered a rapid increase in [Ca²⁺]_c, and this [Ca²⁺]_c increase was significantly inhibited by pretreatment with different concentrations of emodin (0.1, 0.3, 1, 3, 10 µM) for 20 min. The inhibition was dependent on the dose of emodin. The peak values of [Ca²⁺]_c increase were plotted against the concentration of emodin. As shown in Figure 1B, the peak values of ATP-induced [Ca²⁺]_c increase were reduced by 26.9, 40.3, 69.8, 90.0 and 98.6% in the presence of 0.1, 0.3, 1, 3 and 10 μM emodin, respectively. The dose-dependent curve was fitted, with an IC₅₀ of 0.5 μM. We also tested the effect of BBG, a potent P2X₇R antagonist in parallel experiments as a positive control (Bulanova et al., Citation2005; Jiang et al., Citation2000). It can be seen from Figure 1C that the ATP-triggered increase in [Ca²⁺]_c was remarkably reduced by different concentrations of BBG (0.1, 1, 10 µM). This inhibition of BBG indicated the involvement of P2X₇R in [Ca²⁺]_c increase triggered by ATP. In addition, emodin alone had no effect on [Ca²⁺]_c at the same experimental conditions (data not shown). These results together indicated the efficient inhibitory effect of emodin on ATP-induced increase in [Ca²⁺]_c, and this effect was dependent on antagonizing P2X₇R.

Figure 1. Inhibition of emodin on ATP-evoked increase in [Ca²⁺]_c in macrophages. (A) Representative [Ca²⁺]_c traces for stimulating macrophages with ATP (5 mM) in the absence (control) and in the presence of emodin (0.1, 0.3, 1, 3, 10 µM). Emodin was added 20 min before the application of ATP. Arrows indicated the application of ATP. (B) The statistic peak values of increase in F340/F380 ratio after ATP application from experiments shown in A (n = 15 for each case). *p < 0.05 by one-way ANOVA, compared with control group. The smooth curve represented the fitting to the equation of with an IC₅₀ value of 0.5 μM. (C) Representative [Ca²⁺]_c traces for stimulating macrophages with ATP (5 mM) in the absence (control) and in the presence of BBG (0.1, 1, 10 µM). BBG was added 20 min before the stimulating with ATP.

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Emodin reduced ATP-induced IL-1β release from LPS-activated macrophages

The following experiment examined ATP-induced IL-1β release from LPS-activated macrophages, and further evaluated whether emodin can inhibit the ATP-induced IL-1β secretion. As previously suggested (Sanz & Virgilio, Citation2000), macrophages were pretreated with 5 µg/ml LPS or LPS plus different doses of emodin (0.1, 0.3, 1, 3, 10 µM)/BBG (0.1, 1, 10 µM) for 5 h. Then, the cells were stimulated by 2 mM ATP for additional 2 h in the presence of LPS and emodin/BBG. As shown in Figure 2, ATP evoked a massive secretion of IL-1β in LPS-primed macrophages (490.4 ±62.3 pg/ml, 6.4-fold increase compared with the basal level 76.5 ± 4.5 pg/ml in control). Moreover, emodin reduced the IL-1β release triggered by ATP in a concentration-dependent way with an IC₅₀ of 1 μM. Similarly, BBG exhibited suppressive effects on IL-1β secretion. In addition, emodin alone had no effect on IL-1β secretion (78.1 ± 5.1 pg/ml). ATP or LPS alone cannot cause massive extracellular accumulation of IL-1β (85.6 ± 5.7 pg/ml and 124.0 ±11.1 pg/ml, respectively). Because ATP-induced IL-1β release from LPS-primed macrophages is directly associated with the activation of P2X₇R (Ferrari et al., Citation2006; Kahlenberg & Dubyak, Citation2004), the data above suggested that emodin reduced ATP-induced IL-1β release by inhibiting the activation of P2X₇ receptors.

Figure 2. Inhibition of emodin on IL-1β release induced by ATP in LPS-primed macrophages. Macrophages were pretreated with LPS (5 µg/ml) and emodin (0.1, 0.3, 1, 3, 10 µM) or BBG (0.1, 1, 10 µM) for 5 h simultaneously, then stimulated with ATP (2 mM) for an additional 2 h. IL-1β production in supernatants was measured by ELISA kits. The concentration of IL-1β (pg/ml) in each group was determined by the standard curve (n = 5 for each group). *p < 0.05 by Student’s t-test, compared with control group (cells treated with vehicle solution); #p < 0.05 by one-way ANOVA, compared with ATP plus LPS group.

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Emodin suppressed ATP-evoked ROS production in macrophages

Next, the influence of emodin on ATP-evoked intracellular ROS production was detected with DHE fluorescent live imaging. As shown in Figure 3A and B, compared with control (cells treated with vehicle solution), the stimulation with ATP (1 mM) for 30 min induced a remarkable intracellular ROS generation, whereas treatment with emodin (10 µM) or BBG (10 µM) alone had no obvious effect (data not shown). Three hour pretreatment with emodin (0.1, 0.3, 1, 3, 10 µM) or BBG (0.1, 1, 10 µM) suppressed the ROS production evoked by ATP in a dose-dependent manner, respectively. The IC₅₀ of emodin was 1.6 μM at this condition. Besides, ROS generation was inhibited by pretreatment for 3 h with two typical ROS scavengers, DPI (0.1, 1, 10 µM, a NAD(P)H oxidase inhibitor), and SOD (10, 100 U/ml), as positive control. Taken together, these results suggested the suppressive effect of emodin on ATP-induced ROS production, which was related to the activation of P2X₇R.

Figure 3. Inhibition of emodin on ATP-induced ROS production in macrophages. (A) Dihydroethidium (DHE) fluorescence images, captured using a 40× objective, are shown. The fluorescence intensity represents the ROS concentration. The cells were pretreated with indicated concentration of BBG (0.1, 1, 10 µM), emodin (0.1, 0.3, 1, 3, 10 µM), DPI (0.1, 1, 10 µM) and SOD (10, 100 U/ml) for 3 h, and stimulated with ATP (1 mM) for additional 30 min. (B) Statistic data of the fluorescence intensity (n = 30). *p < 0.05 by Student’s t-test, compared with control group (cells treated with vehicle solution); #p < 0.05 by one-way ANOVA, compared with ATP alone group.

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Emodin inhibited ATP-induced phagocytosis attenuation of macrophages

Finally, the effects of emodin on ATP-evoked phagocytotic attenuation of macrophages were examined. The phagocytic ability of macrophages was represented by the uptake assay of neutral red. As shown in Figure 4A, compared with the control group (cells treated with vehicle solution), stimulation with ATP (1 mM) for 30 min evoked an obvious decrease in neutral red uptake, indicating phagocytotic attenuation of macrophages caused by ATP exposure. Three hour pretreatment with emodin (0.1, 0.3, 1, 3, 10 µM) counteracted this negative effect of ATP on the phagocytosis of macrophages in a concentration-dependent manner. The inhibition of BBG (0.1, 1, 10 µM) on decrease in neutral red uptake is shown in Figure 4B, supporting that P2X₇R contributed to the phagocytosis attenuation induced by ATP. The influences of emodin or BBG on phagocytic activity of macrophages expressed as recovery rates are shown in Figure 4C and D, respectively. Pretreatment with 0.3, 1, 3 and 10 μM emodin increased the recovery to 37.4, 75.4, 94.8 and 96.7%, respectively (Figure 4C), and pretreatment with 0.1, 1 and 10 μM BBG increased the recovery to 19.9, 57.4 and 90.0%, respectively (Figure 4D). The IC₅₀ value of emodin was 0.7 μM. These data indicated that emodin inhibited ATP-induced phagocytosis attenuation of macrophages, in which P2X₇ receptors played an essential role.

Figure 4. Inhibition of emodin on ATP-induced phagocytosis decrease in macrophages. The phagocytic activity of macrophages was tested by a neutral red uptake assay. (A and B) Macrophages were pretreated with emodin (0.1, 0.3, 1, 3, 10 µM) or BBG (0.1, 1, 10 µM) for 3 h, and then stimulated simultaneously with ATP (1 mM) and neutral red (0.1%) for additional 30 min. The relative phagocytic activity in each group was represented by A₅₅₀ (n = 5). *p < 0.05 by Student’s t-test, compared with control (cells treated with vehicle solution); #p < 0.05 by one-way ANOVA, compared with ATP alone group. (C and D) Statistic data of the recovery rates of phagocytic activity, which were derived from the calculation of . The recovery rates in C and D were calculated by the values of A₅₅₀ given in A and B, respectively (n = 5). *p < 0.05 by Student’s t-test, compared with control; #p < 0.05 by one-way ANOVA, compared with ATP alone group.

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