Rhein antagonizes P2X7 receptor in rat peritoneal macrophages

P2X7 receptor plays important roles in inflammation and immunity, and thereby it serves as a potential therapeutic target for inflammatory diseases. Rhein, an anthraquinone derivative, exhibits significant anti-inflammatory and immunosuppressive activities in therapy. However, the underlying mechanisms are largely unclear. Here, we aimed to investigate the effects of rhein on P2X7 receptor-mediated responses in vitro. In HEK293 cells expressing rat P2X7 receptor, we first found that rhein concentration-dependently blocked ATP-induced cytosolic calcium concentration ([Ca2+]c) elevation and pore formation of the plasma membrane, two hallmarks of the P2X7 receptor activation. These two inhibitory effects of rhein were also observed in rat peritoneal macrophages. Furthermore, rhein counteracted macrophage phagocytosis attenuation and suppressed reactive oxygen species (ROS) production triggered by ATP/BzATP. Meanwhile, rhein reduced ATP/BzATP-induced IL-1β release in lipopolysaccharide-activated macrophages. Prolonged application of ATP caused macrophage apoptosis, while the presence of rhein suppressed this cell cytotoxicity. Such ATP/BzATP-induced cellular reactions were also inhibited by a well-known rat P2X7 receptor antagonist, brilliant blue G, in a similar way to rhein. Together, our results demonstrate that rhein inhibit ATP/BzATP-induced [Ca2+]c increase, pore formation, ROS production, phagocytosis attenuation, IL-1β release and cell apoptosis by antagonizing the P2X7 receptor in rat peritoneal macrophages.


Effects of Rhein on ATP-induced [Ca
] c increase and pore formation in rP2X 7 -HEK293 cells. To examine whether rhein inhibits the P2X 7 receptor in vitro, we first tested the effects of rhein on ATP-induced [Ca 2+ ] c increase and pore formation, two hallmarks of P2X 7 receptor activation, in human embryonic kidney 293 cells stably expressing rat P2X 7 receptor (rP2X 7 -HEK293 cells). Firstly, application of ATP (5 mM) evoked a rapid increase in [Ca 2+ ] c followed by a sustained high plateau in rP2X 7 -HEK293 cells (Fig. 1a). Then, ATP-evoked [Ca 2+ ] c increases were significantly reduced in a dose dependent manner by pretreatment with rhein at 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μ M, respectively. Fitting the mean data with the Boltzmann equation yielded an IC 50 of 1.13 μ M (Fig. 1b). In addition, BBG, a potent rat P2X 7 receptor antagonist 40 , was used as a positive control to determine the potency and specificity of rhein. As shown in Fig. 1a (third line), the ATP-triggered [Ca 2+ ] c increase was also remarkably inhibited by BBG (Representative traces of 0.1, 1, 10 μ M were shown). The corresponding IC 50 of BBG is 0.80 μ M (Fig. 1b). Another salient functional property of P2X 7 receptor is that prolonged activation of the receptor causes formation of nonselective membrane pores which enables the cells to uptake cationic fluorescent dyes such as ethidium bromide (EB). Therefore, we subsequently examined the effect of rhein on membrane pore formation by detecting the ATP-induced uptake of EB in rP2X 7 -HEK293 cells. It showed that EB uptake triggered by ATP (2 mM) was concentration-dependently inhibited by rhein with an IC 50 of 1.31 μ M (Fig. 1c-e). In addition, ATP-triggered EB uptake was remarkably inhibited by BBG with an IC 50 of 0.84 μ M (Fig. 1c-e), indicating the involvement of P2X 7 receptor. Taken together, these data indicated that rhein had efficient inhibitory effects on ATP-induced [Ca 2+ ] c increase and pore formation by antagonizing P2X 7 receptor in rP2X 7 -HEK293 cells. 2+ ] c increase in rat peritoneal macrophages. Similarly, we monitored the effects of rhein on ATP/BzATP-induced [Ca 2+ ] c increase in rat peritoneal macrophages. It has been well established that P2X 7 receptor is highly expressed in macrophages 7 . In this study, we also confirmed the presence of P2X 7 receptors in rat peritoneal macrophages using RT-PCR (Fig. 2a). As expected, ATP (5 mM) elicited a rapid [Ca 2+ ] c increase in macrophages (Fig. 2b). Meanwhile, rhein robustly inhibited this [Ca 2+ ] c increase (Fig. 2b). The inhibition was dependent on the concentration of rhein with an IC 50 of 0.61 μ M (Fig. 2c). Furthermore, BzATP (500 μ M), a specific agonist of P2X 7 receptor, induced strong sustained elevation of [Ca 2+ ] c in macrophages as reported by many previous studies. Rhein also concentration-dependently inhibited BzATP-induced increase in [Ca 2+ ] c , with an IC 50 of 0.49 μ M (Fig. 2d). Besides, BBG exhibited similar inhibitory effects on ATP-and BzATP-induced increases in [Ca 2+ ] c with IC 50 of 1.08 μ M and 1.09 μ M, respectively (Fig. 2c,d). These results together suggested an efficient inhibitory effect of rhein on [Ca 2+ ] c increase mediated by P2X 7 receptor activation in macrophages.

Effects of Rhein on ATP/BzATP-induced [Ca
Scientific RepoRts | 5:14012 | DOi: 10.1038/srep14012 Effect of Rhein on ATP-induced pore formation in rat peritoneal macrophages. We further examined whether rhein inhibited pore formation by detecting ATP-induced uptake of EB in macrophages. As shown in Fig. 3, the EB uptake of macrophages induced by ATP was also concentration-dependently inhibited by rhein with an IC 50 of 1.04 μ M. The mean fluorescent intensity was significantly reduced to 94 ± 4% (ATP + 0.1 μ M rhein group), 83 ± 4% (ATP + 0.3 μ M rhein group), 51 ± 3% (ATP + 1 μ M rhein group), 25 ± 3% (ATP + 3 μ M rhein group) and 24 ± 3% (ATP + 10 μ M rhein group) as compared with the ATP alone group (Fig. 3c). ATP-induced EB uptake was also suppressed by BBG in a similar way to rhein, with an IC 50 of 0.97 μ M (Fig. 3c). These results suggested that rhein could significantly block the pore formation due to ATP-induced P2X 7 receptors activation in macrophages.  Effects of Rhein on ATP/BzATP-induced phagocytosis attenuation in rat peritoneal macrophages. Phagocytosis is a specific form of endocytosis which takes relatively large particles (> 250 nm) into vacuoles. ATP is reported to attenuate phagocytosis of macrophages 31 by acting on P2X 7 receptors. Therefore, the effect of rhein on ATP/BzATP-evoked phagocytosis attenuation of macrophages was examined. The phagocytic activity of macrophages was assessed by the uptake of 1.0 μ m diameter (a) P2X 7 mRNA was detected in rat peritoneal macrophages by RT-PCR. The four lanes in the gel were as follows: Marker (with a list of standardized DNA sequences from 100 bp to 1000 bp); P2X 7 + (experimental group with primers directed towards the P2X 7 mRNA); P2X 7 -(negative control group with nucleasefree water instead of DNA template); GAPDH (positive control group with primers directed towards the GAPDH mRNA). nile red fluorescent carboxylate-modified microspheres (Fig. 4). As shown in Fig. 4a, compared to control group, stimulation with ATP (2 mM) or BzATP (500 μ M) for 45 min evoked an obvious decrease in phagocytosis of microspheres, indicating that exposure to ATP or BzATP could lead to phagocytosis attenuation of macrophages. However, pretreatment with rhein (0.1, 0.3, 1, 3, 10 μ M) inhibited the decrease in microspheres uptake in a concentration-dependent manner (Fig. 4a,b). BBG (0.1, 0.3, 1, 3, 10 μ M) also counteracted this negative effect of ATP on the phagocytosis of macrophages, supporting that P2X 7 receptor contributed to the phagocytosis attenuation induced by ATP. Moreover, rhein inhibited BzATP-induced macrophage phagocytosis attenuation (Fig. 4a,b). The influences of rhein or BBG on phagocytic activity of macrophages were expressed as recovery rate, showed in Fig. 4c,d. It was found that pretreatment with 0.3, 1, 3 and 10 μ M rhein increased the recovery percentage of ATP to 27.5%, 51.3%, 72.5%, and 95.8%, respectively. Pretreatment with 0.3, 1, 3 and 10 μ M BBG increased the recovery rate of ATP to 10%, 41.2%, 80.8% and 85%, respectively. In addition, pretreatment with 0.3, 1, 3 and 10 μ M rhein increased the recovery percentage of BzATP to 15.9%, 43.2%, 80.3% and 92.5%, respectively. The IC 50 value of rhein and BBG on ATP-induced phagocytosis attenuation was 0.99 μ M and 1.24 μ M, respectively (Fig. 4c). The IC 50 value of rhein and BBG on BzATP-induced phagocytosis attenuation was 1.18 μ M and 0.65 μ M, respectively (Fig. 4d). These data together indicated that rhein could block the influence of ATP/BzATP on the phagocytic activity, which is exerted through activating the P2X 7 receptors.

Effects of Rhein on ATP/BzATP-evoked ROS production in rat peritoneal macrophages.
Next, the effect of rhein on ATP/BzATP-evoked intracellular ROS production was detected by fluorescent imaging with dihydroethidium (DHE). As shown in Fig. 5, compared with control (cells treated with vehicle solution), the stimulation with ATP (2 mM) or BzATP (0.5 mM) for 30 min induced remarkable generation of intracellular ROS. However, treatment with rhein (10 μ M) or BBG (10 μ M) alone had no obvious effect on ROS production (data not shown). Three hours' pretreatment with rhein (0.1, 0.3, 1, 3, 10 μ M) or BBG (0.1, 1, 10 μ M) effectively suppressed the ROS production evoked by ATP in concentration-dependent manners, respectively. The IC 50 of rhein was 1.56 μ M (Fig. 5c). Rhein also blocked BzATP-induced ROS generation (Fig. 5a,b). Taken together, these results suggested a suppressive effect of rhein on ATP/BzATP-induced ROS production, which was mediated by the activation of P2X 7 receptor.
Effects of Rhein on ATP/BzATP-induced IL-1β release in LPS-activated macrophages.
Furthermore, we examined ATP/BzATP-induced IL-1β release from LPS-activated rat peritoneal  macrophages, and further evaluated whether rhein inhibited the IL-1β secretion induced by ATP/BzATP. As previously reported 26 , the pro-IL-1β is accumulated in macrophages induced by bacterial LPS stimulation. Therefore, macrophages were pretreated with 5 μ g/ml LPS, LPS + rhein and LPS + BBG for 5 h, respectively. Then, the cells were stimulated with ATP (2 mM) or BzATP (500 μ M) for additional 2 h. As shown in Fig. 6a, both ATP and BzATP evoked massive secretion of IL-1β in LPS-primed macrophages (ATP group: 599.5 ± 62.4 pg/ml, 8 folds increase compared with the basal level 74.9 ± 4.5 pg/ml in control; BzATP group: 615.5 ± 65.0 pg/ml, 8.2 folds increase compared with control). Moreover, rhein reduced the IL-1β release triggered by ATP in a concentration-dependent way with an IC 50 of 1.48 μ M (Fig. 6b). Rhein also inhibited BzATP-induced IL-1β secretion. Similarly, BBG exhibited suppressive effects on IL-1β secretion. In addition, rhein alone had no effect on the basal IL-1β secretion (78.1 ± 5.1 pg/ml). ATP or LPS alone cannot cause massive extracellular accumulation of IL-1β (85.0 ± 5.0 pg/ml and 117.5 ± 11.1 pg/ml, respectively). Since ATP-induced IL-1β release from LPS-primed macrophages is directly associated with the activation of P2X 7 receptor 26,28 , these results indicated that rhein reduced ATP/BzATP-induced IL-1β release by inhibiting the activation of P2X 7 receptors.
Effect of Rhein on ATP-induced death of rat peritoneal macrophages. We tested whether rhein prevented macrophage death induced by high extracellular ATP. Stimulation with ATP (5 mM) evidently reduced macrophage viability as summarized in Fig. 7a. However, rhein reversed ATP-induced macrophage death in a concentration-dependent manner, with significant inhibition at concentrations of ≥ 0.1 μ M. Fitting the mean data with the Boltzmann equation derived an IC 50 of 0.4 μ M (Fig. 7b). In contrast, there was no cytolytic action with concentrations up to 10 μ M rhein (Fig. 7c). Besides, the ATP-induced cell death was suppressed by BBG in a similar fashion. These data suggested that rhein inhibited ATP-induced cell death through blocking P2X 7 receptors in rat peritoneal macrophages.

Discussion
In the present work, we aimed to investigate the effects of rhein on rat P2X 7 receptor by detecting the influences of rhein on various representative cellular responses or biochemical consequences mediated by P2X 7 receptor activation. It has been established that millimolar concentrations of ATP are needed for P2X 7 receptor activation 5,10 , and the optimal concentration of ATP chosen for each experiment in this study relies on previous reports 32, 44,45 and our preliminary experiments (Supplementary Fig. S1). Firstly, rP2X 7 -HEK293 cells, a cell line stably expressing the recombinant rat P2X 7 receptor, were used to determine the effects of rhein on the P2X 7 receptor through monitoring the change of [Ca 2+ ] c and pore formation, two hallmark properties directly associated with P2X 7 receptor activation. Except P2X 7 receptor, other P2X receptors do not exist in rP2X 7 -HEK293 cells. Thus, adoption of this cell line is a good approach to exclude the effects of other receptors and screen for specific antagonists for P2X 7 receptor. Our results showed that ATP (5 mM) evoked an increase in [Ca 2+ ] c via activation of P2X 7 receptor in rP2X 7 -HEK293 cells (Fig. 1a). Rhein had an efficient inhibition on this P2X 7 -mediated calcium response in a dose-dependent manner, which was similar to that of the specific P2X 7 receptor blocker BBG (Fig. 1a,b). Furthermore, we found that rhein also potently blocked ATP-triggered typical large transmembrane pores formation in rP2X 7 -HEK293 cells (Fig. 1c,d). These data presented initial evidence that rhein may be a potential antagonist for rat P2X 7 receptor. Based on the preliminary results acquired from rP2X 7 -HEK293 cells, the major focus of our study was to examine the effects of rhein on P2X 7 receptor-mediated inflammatory responses in a functional immune cell type, rat peritoneal macrophages. Our results showed that stimulation of macrophages with P2X 7 receptor agonists ATP/BzATP also induced rapid [Ca 2+ ] c increase (Fig. 2) and pore formation (Fig. 3), which was similar to those of rP2X 7 -HEK293 cells.
Subsequently, we investigated several other P2X 7 receptor-mediated processes in rat peritoneal macrophages. As mentioned in introduction, P2X 7 receptor is a key player in ATP-induced maturation and extracellular release of proinflammatory cytokine IL-1β 28 . Moreover, the activation of P2X 7 receptor is necessary for the generation of ROS and reduction of phagocytic activity triggered by millimolar ATP in macrophages 27,29,31 . Here we showed that both ATP/BzATP strongly enhanced IL-1β release in LPS-activated macrophages (Fig. 6). And the experiment with BBG further illustrated that these consequences were mediated by P2X 7 receptor activation (Fig. 6). Meanwhile, ATP/BzATP elicited remarkable intracellular ROS production (Fig. 5) and attenuated the phagocytic activity of macrophages (Fig. 4), which were also due to the activation of P2X 7 receptor. These results were in good accordance with previous literatures with regard to P2X 7 receptor expressing immune cells including macrophages 27,29,31 .
Moreover, the P2X 7 receptor has been demonstrated to be mainly responsible for ATP-induced macrophage death 20 . The cell death of macrophages occurred only upon exposure to high concentration of exogenous ATP (2-5 mM, as shown in Supplementary Fig. S1) . Such high agonist concentration will not only open cation channels, but also lead to the formation of large cytotoxic transmembrane pores 13,14 . The formation of permeable pores disrupts ionic gradients and induces an efflux of vital intracellular molecules of maximum 900 Da, which may lead to cell death [13][14][15] . Although how P2X 7 receptor activation causes cell death is not fully understood, some reports suggested that the caspases activation depending  on mitochondrial pathway was involved in the P2X 7 receptor-mediated apoptosis 16 . The present data indicated that stimulation with high extracellular ATP (5 mM) resulted in impairment of mitochondria, activation of caspase 3/7 (Figs 7 and 8), and eventually initiation of apoptotic cell death in rat peritoneal macrophages. Besides, our experiments with P2X 7 receptor selective antagonist BBG (Figs 2-8) and irreversible antagonist oxidized ATP (Supplementary Fig. S2) confirmed the contribution of P2X 7 receptors to these ATP-induced responses.
The most significant finding of present study was that we discovered and demonstrated that a natural anthraquinone derivative, rhein, could consistently inhibit all the P2X 7 receptor-mediated responses examined here. Firstly, it strongly suppressed ATP-elicited increases of [Ca 2+ ] c and formation of the EB dye permeable pore in both rP2X 7 -HEK293 cells (Fig. 1) and rat peritoneal macrophages (Figs 2  and 3), in a concentration dependent manner, respectively. Secondly, it effectively prevented ATP/ BzATP-evoked intracellular phagocytosis attenuation (Fig. 4), ROS production (Fig. 5), and massive secretion of IL-1β (Fig. 6) in rat peritoneal macrophages in a concentration dependent fashion. Rhein also reduced macrophage death caused by prolonged ATP stimulation (Fig. 7). Moreover, rhein potently reversed ATP-induced reduction in MMP and suppressed ATP-triggered caspases activation (Fig. 8). These results taken together provided compelling evidence to show the potent inhibitory effects of rhein on P2X 7 receptor-related consequences.
Actually, nowadays more and more attentions are being paid to the anti-inflammatory activity of rhein. Clinical applications have also demonstrated that rhein may be a potential cure for inflammatory diseases. For instance, rhein is an active metabolite for the drug of osteoarthritis diacetylrhein. Although the underlying mechanisms of the anti-inflammatory effect of rhein remain to be elucidated, studies so far have focused on the effects of rhein on the factors known to be critical in the pathogenesis of osteoarthritis 39 . It has been reported that rhein could significantly inhibit LPS-induced production of IL-1β and IL-1 downstream signaling events such as nuclear factor-κ B activation and nitric oxide generation 36,37 . Rhein could also block superoxide anion production, chemotaxis, phagocytosis and migration in neutrophils 46 and macrophages 38 . As mentioned above, many of these processes are now known to depend on the P2X 7 receptor activation. Thus, the P2X 7 receptor serves as a good candidate target of rhein.
Importantly, we obtained all the IC 50 values of rhein against rat P2X 7 receptor-mediated responses, which were ranged from 0.4 to 1.75 μ M. This is significantly lower than that of IC 50 values of rhein in other characterized cellular effects. For instance, rhein inhibited IKKβ in LPS-activated macrophages with an IC 50 of 11.79 μ M 38 . Rhein blocked IL-1β -induced activation of MEK/ERK pathway in cultured chondrocytes with hypoxia 36 , and protected endothelial cell from oxidative stress at ∼ 10 μ M magnitude 35 . Rhein-induced Hep-G2 cell death via mitochondrion permeability transition only occurred at concentrations higher than 100 μ M 34 . Such reports indicated that rhein was a more potent antagonist against rat P2X 7 receptors. Furthermore, the results from rP2X 7 -HEK293 cells suggested that rhein was highly specific and selective for P2X 7 receptor rather than other P2X receptors. Since rhein is a natural constituent of traditional Chinese herb, the discovery of rhein represents a significant advance in P2X 7 receptor pharmacology due to its significant anti-inflammatory activities. Besides, rhein showed nearly equivalent potency in blocking P2X 7 receptor-mediated calcium increase and pore formation in both rP2X7-HEK293 cells and macrophages, whereas the rat selective antagonist BBG and the human selective antagonist KN-62 are more potent (> 10-folds) in inhibiting the P2X 7 receptor-mediated pore formation than blocking the receptor-mediated calcium influx 40,41,47 . What's more, rhein also showed significant potency to block other P2X 7 receptor-mediated important inflammatory processes including ROS generation, phagocytic activity and cell death of macrophages, which have rarely been investigated in other reports on the antagonists of P2X 7 receptor.
In summary, our study clearly demonstrated that rhein could inhibit ATP/BzATP-induced various inflammatory responses including cytosolic calcium increase, membrane permeable pore formation, intracellular ROS production, IL-1β release, phagocytosis attenuation and cell apoptosis in rat peritoneal macrophages through antagonizing the P2X 7 receptor. Our finding provides a novel insight into the molecular mechanism or pathway underlying the anti-inflammatory effects of rhein. Furthermore, because of the important roles of P2X 7 receptor in immune response, inflammation and inflammatory diseases, the present investigation of rhein may facilitate development of therapeutic drugs targeting the P2X 7 receptors. (China). Fura-2/AM was from Biotium (USA). Rat IL-1β ELISA kits were purchased from NeoBioscience Technology Co.,Ltd. (China). RNAprep pure Cell/Bacteria Kit was from TIANGEN BIOTECH (China). Reverse Transcription System, GoTaq PCR Core system and Caspase-Glo assay kit were from Promega (USA). FluoSpheres carboxylate-modified, 1.0 μ m, nile red was purchased from Molecular Probes (USA). The rest of reagents, including ATP, BzATP, oxidized ATP, BBG, LPS, EB, DHE, rhodamine 123, dimethylsulfoxide (DMSO) and MTT were purchased from Sigma (USA).

Methods
Cell preparation and culture. Wistar rats were sacrificed according to institutional guidelines. Then, Hanks' balanced salt solution (HBSS) (NaCl 150 mM, KCl 5.4 mM, CaCl 2 2 mM, MgCl 2 1 mM, glucose 10 mM and HEPES 10 mM, pH = 7.4) was injected into the abdomen of each rat. Peritoneal cells were collected from the abdomen and isolated by centrifugation at 200 g for 10 min, which were subsequently cultured in RPMI1640 medium containing 10% FCS in a humidified incubator with 5% CO 2 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 previously described 44 . The rP2X 7 -HEK293 cells, a generous gift from Dr. Lin-Hua Jiang (Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK), were maintained in DMEM supplemented with 10% FCS and 2 mM L-glutamine in a humidified 5% CO 2 incubator at 37 °C.

Measurement of [Ca 2+ ] c in single cells.
[Ca 2+ ] c was measured as previously described 45 . In brief, macrophages or rP2X 7 -HEK293 cells were loaded with 2 μ M fura 2-AM in HBSS for 1 h, followed by extensive washing. Then, cells were bathed in fresh HBSS solution for monitoring the changes of [Ca 2+ ] c . [Ca 2+ ] c was measured with calcium imaging system built on an inverted fluorescence microscope (Olympus IX51). As a ratiometric fluorescent Ca 2+ indicator, fura-2 was alternately excited at 340 nm and 380 nm with a Lambda 10-2 Sutter (Sutter Instrument, USA). Fluorescence images (filtered at 515 nm ± 25 nm) were captured by a CCD camera (CoolSNAP fx-M, Roper Scientific, USA) and analyzed with MetaFluor software (Universal Imaging, PA, USA). [Ca 2+ ] 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.
EB Dye uptake assay. Macrophages or rP2X 7 -HEK293 cells were seeded in 6-well plates at ∼ 1 × 10 6 / well and incubated for 4 h. Then, cells were pretreated with rhein or BBG at indicated concentrations for 20 min. Subsequently, ATP was added for 10 min in the presence of 6 μ M EB. After extensive washing with HBSS solution, dye uptake positive cells were identified by illuminating with 488 nm light and detecting emission at 610 nm using an Olympus fluorescence microscope with a 20 × objective. The fluorescent intensity that indicates EB uptake in each cell was measured individually using MetaFluor software. The summation of intensity was divided by cell number to get the average intensity (F). The EB uptake in ATP alone group (F ATP -F control ) was taken as 100% (control group was treated with vehicle solution), and the percentages of EB uptake in other groups were normalized to the ATP group and calculated as: (F test -F control )/(F ATP -F control ) × 100%.
Phagocytosis of fluorescent microspheres assay. The phagocytic activity of macrophages was assessed by the uptake of 1.0 μ m diameter nile red fluorescent carboxylate-modified microspheres. After pretreatment with different concentrations of rhein or BBG for 3 h in serum-free DMEM medium at 37 °C, ATP (5 mM) or BzATP (0.5 mM) and microspheres (10 μ l) were applied to macrophages for 45 min, then washed thoroughly with ice cold PBS. Samples were quenched with PBS 1% BSA. The number of fluorescent particles taken up per macrophage was summarized to evaluate the phagocytic ability. The data were normalized to phagocytosis obtained on untreated macrophages. For imaging analysis, microphages were prepared in poly-L-lysine-coated glass coverslips at a density of 1 × 10 6 cells/ml. The images were captured by a Zeiss fluorescent microscope (Axio observer D1) with a 100 × oil immersion lens.
Measurement of 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 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). The concentration of IL-1β was determined by the standard curve.
Detection of intracellular ROS. DHE, a reduced form of ethidium bromide, was used to determine intracellular ROS. After treatment with indicated reagents, macrophages were incubated in HBSS with 5 μ M DHE for 30 min at 37 °C in dark. Then they were rinsed twice with HBSS and observed by a fluorescence microscope at the excitation wavelength of 488 nm and emission wavelength of 610 nm. The fluorescent intensity represents the intracellular ROS level.
Cell death assay. Macrophages were seeded in 96-well plates at ∼ 1 × 10 5 cells/well and cultured for 24 h. After pretreatment with rhein or BBG for 2.5 h, application of ATP (5 mM) to macrophages for 30 min. Then, cells were incubated in fresh culture medium for a further 24 h. Upon addition of 10 μ l MTT reagents (10 mg/ml in HBSS) into each well for 4 h, the culture medium was aspirated and replaced with 100 μ l lysis solution (50% DMSO and 50% ethanol). The absorbance at 570 nm (A 570 ) for each well was determined by an ELISA reader. The percentage of survival cells was derived as: A 570, test /A 570, control × 100%, and the percentage of dead cells was calculated as: (A 570, control -A 570, test )/A 570, control × 100%.
Detection of mitochondrial membrane potential. Rhodamine 123 was used to assess the depolarization of mitochondrial membrane potential (MMP). Macrophages were seeded into 96-well plate at ∼ 1 × 10 5 cells/well and incubated in HBSS with rhodamine 123 (10 μ g/ml) for 15 min at room temperature after treating with or without test agents. The rhodamine 123-loaded cells were washed and imaged with an inverted fluorescence microscope. The excitation wavelength and emission wavelength were 488 nm and 510 nm, respectively. A decrease in rhodamine 123 fluorescence intensity represents mitochondrial membrane depolarization.
Caspase assay. Caspase 3/7 activities in macrophages were measured to determine the apoptotic events, using a Caspase-Glo assay kit. Briefly, the luminogenic substrate containing the tetrapeptide sequence DEVD is cleaved by caspase 3/7. After caspase cleavage, a luciferase substrate is released, resulting in the luciferase reaction and the production of luminescent signal. Cell suspension (200 μ l; 1 × 10 5 cells/ml) was seeded into a 96-well plate and incubated with or without test reagents at 37 °C in DMEM with 5% FBS. After that, an equal volume of reagents was added to each well. Then, samples were incubated at room temperature for 2 h. Finally, the luminescence of each sample was measured by a luminometer (GloMax Multi Jr Detection System, Promega, USA). These fluorescent data from each group were proportional to the amount of caspase activity, which was assigned as the relative fluorescence unit (RFU). Data analysis. All data were presented as mean ± standard deviation (SD). The statistical comparison between two groups was carried out using Student's t-test (Origin 8.5), 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 concentrations producing half of the maximal inhibition (IC 50 ) were derived by fitting of the mean data to the Boltzmann equation: = + − + (( − ) / ) y A2 x A1 A2 1 exp IC50 dx , in which y is the inhibition ratio of increase in [Ca 2+ ] c , A1 is the asymptotic maximum, A2 is the asymptotic minimum, x is concentrations of rhein and dx is the time constant.