Reactive oxygen species play a role in P2X7 receptor-mediated IL-6 production in spinal astrocytes

Abstract

Astrocytes mediate a remarkable variety of cellular functions, including gliotransmitter release. Under pathological conditions, high concentrations of the purinergic receptor agonist adenosine triphosphate (ATP) are released into the extracellular space leading to the activation of the purinergic P2X7 receptor, which in turn can initiate signaling cascades. It is well-established that reactive oxygen species (ROS) increase in macrophages and microglia following P2X7 receptor activation. However, direct evidence that activation of P2X7 receptor leads to ROS production in astrocytes is lacking to date. While it is known that P2X7R activation induces cytokine production, the mechanism involved in this process is unclear. In the present study, we demonstrated that P2X7 receptor activation induced ROS production in spinal astrocytes in a concentration-dependent manner. We also found that P2X7R-mediated ROS production is at least partially through NADPH oxidase. In addition, our ELISA data show that P2X7R-induced IL-6 release was dependent on NADPH oxidase-mediated production of ROS. Collectively, these results reveal that activation of the P2X7 receptor on spinal astrocytes increases ROS production through NADPH oxidase, subsequently leading to IL-6 release. Our results reveal a role of ROS in the P2X7 signaling pathway in mouse spinal cord astrocytes and may indicate a potential mechanism for the astrocytic P2X7 receptor in chronic pain.

Introduction

Astrocytes are a crucial component of the central nervous system (CNS), and key players in neurological diseases. Among many other roles, these specialized cells maintain homeostasis in the CNS, act as defensive cellular elements, and are responsible for numerous functions that support healthy neuronal networks as well as modulating neuronal pathology [1, 2]. However, reactive astrocytes also have harmful effects, such as producing proinflammatory cytokines and chemokines during injury or disease [3, 4]. Astrocytes mediate a remarkable variety of cellular functions, including gliotransmitter release and reactive oxygen species (ROS) production [5, 6].

Purinergic P2 receptors (P2Rs) are classified into two subfamilies: ionotropic P2X receptors and metabotropic P2Y receptors that are coupled to phospholipase C (PLC). Activation of these receptors by adenosine triphosphate (ATP) can facilitate the communication between cells in the CNS by regulating synaptic transmission and glial Ca²⁺ waves [7, 8]. Under pathological conditions, high concentrations of ATP are released into the extracellular space leading to the activation of the ionotropic nonselective cation channel, P2X7 receptor (P2X7R), which in turn initiates inflammatory signaling cascades or apoptosis/necrosis [9, 10]. Indeed, reports show that P2X7R plays a key role in pathophysiological conditions in the CNS such as neuroinflammation, pain, Alzheimer’s Disease, multiple sclerosis, and epilepsy [11–13]. However, the mechanism by which P2X7R mediates inflammation in disease remains unclear.

P2X7R activation can induce ROS production in neurons, microglia, erythroid cells, and macrophages [14–17]. These P2X7R-induced ROS play a vital role in various signaling pathways including transcription factor activation, pro-inflammatory cytokine release, activation of the MAPK pathway, and the regulation of cell death pathways in macrophage, monocytes and microglial cells [18–20]. Moreover, NADPH oxidase is known to play a role in P2X7R-mediated ROS production in cell types such as neurons, liver macrophages, and microglia [14, 21, 22]. Although P2X7R-mediated ROS production is well-established in many cell types, there is very little known about P2X7R-induced ROS production in astrocytes.

Downstream signaling of P2X7R that leads to inflammation has been extensively studied peripherally. Studies have shown that P2X7R is a key regulator of the inflammasome molecular complex, which ultimately leads to the subsequent release of proinflammatory cytokines [10]. Activation of P2X7R in macrophages can leads to the release of cytokines such as interleukin (IL)-1β, IL-6, and IL-18 [23]. Additionally, P2X7R knockout mice have altered cytokine production in response to lipopolysaccharide (LPS) injections due to their inability to release mature IL-1β [24]. P2X7R can also contribute to neuroinflammation by activating the inflammasome molecular complex [9, 25]. The inflammasome can then subsequently initiate cleavage of precursor interleukin (IL) molecules such as IL-1β and IL-18 prior to their release into the extracellular space [7]. Additionally, activation of P2X7R can induce the release of IL-6 from cultured mouse microglia, optic nerve head astrocytes, and retinal ganglion cells [26, 27]. Despite growing evidence that P2X7R activation leads to inflammation in the CNS, little is known regarding the mechanisms underlying cytokine release.

While expression of P2X7R in the CNS has been reported mainly in microglia, oligodendrocytes, and ependymal cells [7, 28], recent studies have demonstrated neurons and cerebellar astrocytes also express P2X7R [14, 29–31]. Nevertheless, P2X7R-mediated downstream events that may contribute to inflammation have yet to be explored in spinal astrocytes. In the current study, we investigate the downstream effects of P2X7R activation in primary spinal cord astrocytes, and its role in P2X7R-induced IL-6 release. Overall, we found that activation of P2X7R induces both ROS production and IL-6 release in spinal astrocytes, and that IL-6 release is dependent on NADPH oxidase-mediated ROS increases. Together, the data suggest that ROS play a critical role in the P2X7 signaling pathway and that astrocytic P2X7R may contribute to inflammation under pathological conditions. Our data elucidate that ROS increases contribute to P2X7R-mediated cytokine production.

Methods and materials

Cell culture

Spinal cord astrocyte cultures were prepared from neonatal (P3 – P5) mice as described previously [32]. Briefly, following hypothermic induction on ice, neonatal mice were decapitated and a laminectomy was performed. The spinal cord was removed and spinal cord strips were then incubated for 30 min at 37 °C in HBSS (Life Technologies, Carlsbad, CA) (in mM: 137 NaCl, 5.4 KCl, 0.4 KH₂PO₄, 1 CaCl₂, 0.5 MgCl₂, 0.4 MgSO₄, 4.2 NaHCO₃, 0.3 Na₂HPO₄, and 5.6 glucose) containing papain (15 U/ml; Worthington Biochemical, Lakewood, NJ). Spinal cord strips were then washed with HBSS, and placed in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific). Spinal cord strips were mechanically dissociated by gently triturating with a pipette and the resulting cells were plated onto a 75 cm² flask. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Culture medium was changed to fresh DMEM containing 10% FBS after 24 h and then allowed to grow for 7–8 days until 70% confluence. To detach cells sitting on top of the astrocyte monolayer such as microglia and other precursor cells, the 75 cm² flask was shaken in an orbital shaker (320 rpm) for 2 h [33, 34], which yielded astrocyte purity of 92% (GFAP positive cells) as described in our previous study [32]. Iba1 positive cells were less than 5% and NeuN positive cells were less than 1%. The adherent astrocytes were trypsinized and re-plated to 96-well plates (for enzyme-linked immunosorbent assay and MTT viability assays), 12-mm glass bottom dishes (for CellROX imaging) and 12-mm glass coverslips for calcium imaging) in DMEM culture medium.

Live-cell confocal imaging for oxidative stress

Astrocytes (1.5 × 10³) were cultured in 12-mm glass bottom 35 mm petri dishes for 24 h, and then loaded with 5 μM CellROX (Life Technologies, Carlsbad, CA) at 37 °C for 30 min in Tyrode’s solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, 5.6 glucose (pH 7.4) according to manufacturer’s instructions. Cells were washed twice with Tyrode’s solution. Time lapse images were captured at 2-min intervals using a 488-nm laser line for excitation, and emission through a 520-nm window in the Olympus FLUOVIEW FV1000 confocal microscope equipped with a 60x oil-immersion objective. ImageJ was used to quantify the increases in fluorescence by analyzing the mean intensity of individual cells. Values were normalized to intensity at their own time 0 prior to addition of any drugs or vehicles.

Live-cell calcium imaging

Calcium imaging was performed in cultured spinal cord astrocytes as we described previously [14, 32]. Briefly, spinal cord astrocytes were loaded with 4 μM Fura-2 AM (Life Technologies) for 30 min at room temperature in HBSS. Cells were then washed and incubated in Tyrode’s solution for an additional 20 min. Images were recorded at 3-s intervals using an Olympus inverted microscope equipped with a CCD camera (Hamamatsu ORCA-03G, Japan) at room temperature. The fluorescent images were captured and analyzed using the MetaFluor 7.79 software (Molecular Devices, LLC, and Downingtown, PA). Fluorescence ratio was determined by the fluorescence intensities excited at 340 and 380 nm with background subtraction. Only one recording was made from each coverslip.

Enzyme-linked Immunosorbent assay

Spinal cord astrocytes were cultured in 96-well plates (2 × 10⁴ cells/well). For measuring cytokine release, all astrocytes were maintained in DMEM containing 10% FBS for 6 h. For testing the effect of drugs on ATP- and benzoylbenzoyl-ATP (BzATP)-induced cytokine release, astrocytes were pre-treated with different concentrations of drugs for 30 min then co-treated with ATP or BzATP for 6 h. For the cytokine release in the presence of the specific endogenous catalase inhibitor, 3-amino-1,2,4-triazole (AT-3), astrocytes were pre-treated 2 h with the inhibitor then co-treated with BzATP for 6 h in DMEM culture medium. IL-6 in the supernatant was then measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Drugs

Adenosine 5′-triphosphate disodium salt hydrate (ATP), 2′(3′)-O-(4-Benzoylbenzoyl) adenosine-5′-triphosphate tri (triethylammonium) salt (BzATP), N-acetylcysteine (NAC), N-tert-butyl-α-phenylnitrone (PBN), Catalase (Cat), apocynin, and 3-amino-1,2,4-triazole (AT-3) were purchased from Sigma Aldrich (St. Louis, MO). A438079 was purchased from Tocris (Bristol, UK). Cat was reconstituted using 50 mM potassium phosphate buffer at a pH of 7.0. All other compounds were dissolved in Milli-Q water or dimethyl sulfoxide (DMSO) as stock solutions and further diluted to final concentrations in 0.1% DMSO.

Statistical analysis

Data are expressed as mean ± SEM. Treatment effects were statistically analyzed with a one-way analysis of variance (ANOVA). When ANOVA showed a significant difference, comparisons between means were performed by the post hoc Tukey’s test. Error probabilities of P < 0.05 were considered statistically significant. The statistical software GraphPad Prism 7 was used to perform all statistical analyzes.

Results

Purinoreceptor activation induces ROS production through P2X7R in spinal astrocytes

Previous studies have demonstrated that P2X7R activation can generate ROS in cell types such as macrophages and microglia [20, 35]. It has been shown that P2X7R induces ROS production in spinal cord slices [36]. However, the source of ROS is not clear. We recently found that P2X7R activation can increase ROS in spinal cord dorsal horn neurons [14]. To determine whether activation of P2 receptors by its endogenous ligand, ATP, could also result in ROS production in spinal astrocytes, cultured astrocytes were loaded with CellROX green and subjected to live-cell imaging for 20 min. ATP increased ROS production in a time- and concentration-dependent manner (Fig. 1a, b). To determine whether ATP-induced ROS production occurs through P2X7R, spinal astrocytes were pre-treated with A438079, a specific antagonist of P2X7R [37], for 10 min prior to co-treatment with ATP 1 mM. A438079 (10 μM) completely abolished ATP-mediated ROS increases (Fig. 1a, c), suggesting that ROS production occurs through P2X7R activation. To confirm that CellROX intensity was increasing due to ROS production, spinal astrocytes were also individually pre-treated with two different ROS scavengers, N-tert-butyl-α-phenylnitrone (PBN, 30 μM) and N-acetylcysteine (NAC, 10 mM), then co-treated with ATP 1 mM. Both ROS scavengers significantly decreased ATP-induced ROS increases at the commonly used effective concentration [38, 39] (Fig. 1a, c).

Fig. 1.

ATP induces ROS production in spinal astrocytes through P2X7R. (a) Representative of live-cell confocal images captured at time 0 min and after application of 1 mM ATP in the presence of vehicle, A438079 (A43), PBN, or NAC. (b) Time lapse of ROS production after administration of vehicle or ATP. (c) Summary of the effects of A438079, PBN or NAC on ATP-induced ROS production. Values represent mean ± SEM, n = 6–11 astrocytes. ***P < 0.001 compared to control, ^###P < 0.001 compared to ATP group by one-way ANOVA. Scale bar 20 μM

To further confirm that increases in ROS induced by ATP are occurring through P2X7R activation, a more specific P2X7R agonist BzATP for the receptor was used. Astrocytes treated with BzATP, showed a robust increase in ROS in both a time-dependent and concentration-dependent manner (Fig. 2a, b). Moreover, inhibiting P2X7R with its specific antagonist, A438079 (10 μM), completely abolished ROS increases induced by BzATP (300 μM) (Fig. 2a, c). Similar to ATP, individual pre-treatment with PBN and NAC reduced ROS induced by BzATP (Fig. 2a, c). Together, our data demonstrate that ATP and BzATP increase ROS in spinal astrocytes through P2X7R.

Fig. 2.

P2X7R agonist BzATP induces ROS production in spinal astrocytes. (a) Representative of live-cell confocal images captured before (0 min) and after application of BzATP in the presence of vehicle, PBN, A438079 (A43), or NAC. (b) Time lapse of ROS production after vehicle or BzATP administration. (c) Summary of the effects of A438079, PBN or NAC on BzATP-induced ROS production. Values represent mean ± SEM, n = 6–13 astrocytes. ***P < 0.001 compared to control, ^###P < 0.001 compared to vehicle by one-way ANOVA. Scale bar 20 μM

The ROS scavenger PBN does not interfere P2X7R-induced calcium entry

To determine whether inhibition of ROS by PBN occurs through blocking P2X7R function, we first confirmed whether P2X7R is functional in spinal astrocytes. Spinal astrocytes were loaded with Fura-2 AM in HBSS, and subjected to live-cell imaging. Following 2 min in 2 mM Ca²⁺ Tyrode’s solution, BzATP was applied to cells by a perfusion system. A robust intracellular Ca²⁺ increase was observed immediately after BzATP application (Fig. 3a). Pre-treatment for 10 min with a P2X7R antagonist, A438079, and prevented BzATP-induced Ca²⁺ increases (Fig. 3a). We then pre-treated spinal astrocytes for 30 min with the ROS scavenger PBN. As expected, BzATP-induced calcium entry was not altered by PBN pre-treatment (Fig. 3b). Thus, ROS scavenging by PBN occurs downstream of P2X7R activation.

Fig. 3.

ROS scavenging by PBN occurs downstream of P2X7R activation. (a) Left panel: Representative Ca²⁺ imaging recording of BzATP (100 μM)-induced intracellular Ca²⁺ increases with and without A438079 (A43). Right panel: Summary of A438079 effects on BzATP-induced Ca²⁺ response. (b) Left panel: Representative recording of BzATP-induced intracellular Ca²⁺ increases with and without PBN. Right panel: Summary of PBN effects on BzATP-induced Ca²⁺ response. Values represent mean ± SEM, n = 17–22 astrocytes. ***P < 0.001 compared to vehicle

NADPH oxidase contributes to P2X7R-mediated ROS production

P2X7R-mediated ROS production occurs through NADPH oxidase in microglia and spinal cord dorsal horn neurons [14, 21, 40]. NADPH oxidase is known expressed in mammalian astrocytes [41, 42]. To determine whether NADPH oxidase also contributes to BzATP-induced ROS increases in spinal cord astrocytes, we employed apocynin, a NADPH oxidase inhibitor [42, 43]. Pre-treatment with apocynin (125 or 250 μM, effective concentrations) for 30 min significantly attenuated BzATP (300 μM)-mediated ROS increases in a concentration-dependent manner (Fig. 4). Therefore, our data suggest that NADPH oxidase may contribute to P2X7R-mediated ROS production.

Fig. 4.

NADPH oxidase inhibition attenuates BzATP-mediated ROS production. (a) Representative of live-cell confocal images captured at time 0 min and after application of BzATP in the presence of vehicle or 250 µM apocynin (Apo). (b) Summary of the effects of apocynin on BzATP-induced ROS production. Values represent mean ± SEM, n = 3–10 astrocytes. ***P < 0.001 compared to control, ^###P < 0.001 compared to vehicle by one-way ANOVA. Scale bar 20 μM

P2X7R induces IL-6 release in spinal astrocytes

P2X7R activation induces IL-6 production in macrophages, microglia, and optic nerve head astrocytes [24, 26, 27, 44]. We therefore sought to determine whether activation of P2X7R in spinal astrocytes can induce IL-6 release. P2 purinoreceptor agonist ATP indeed induced IL-6 release in astrocytes after 6 h (Fig. 5a). To confirm that P2X7R solely contributes to ATP-induced IL-6 release in spinal astrocytes, we employed various inhibitors. U73122 (2 μM), a PLC inhibitor, was used to block downstream signaling from P2Y channels. Expression of the P2X4 receptor (P2X4R) has been reported in hippocampal astrocytes, pyramidal neurons, and spinal microglia [45–47]. Thus, 5-BDBD (5 μM), a potent P2X4R antagonist [48], was also used to determine whether P2X4R contributes to IL-6 release in spinal astrocytes. The P2X7R antagonist A438079 (10 μM) was used to determine whether P2X7R contributes to ATP-induced IL-6 release. Indeed, only A438079 significantly attenuated ATP-mediated IL-6 release (Fig. 5b), suggesting that P2X7R activation contributes to IL-6 release in spinal astrocytes.

Fig. 5.

P2X7R activation induces release of IL-6 in spinal astrocytes. (a) Effects of U73122, 5-BDBD, and A438079 (A43) on 1 mM ATP-induced IL-6 production. (b) Effect of A438079 on BzATP-induced IL-6 production. Values represent mean ± SEM, n = 6. ***P < 0.001 compared to control, ^###P < 0.001 compared to vehicle by one-way ANOVA

We then used BzATP to further confirm the role of P2X7R in IL-6 production of spinal astrocytes. BzATP (300 μM) significantly increased IL-6 release over a course of 6 h (Fig. 5b). Moreover, P2X7R antagonist A438079 (10 μM) largely attenuated BzATP-induced IL-6 release. The data thus confirm that P2X7R activation leads to IL-6 release in spinal astrocytes.

NADPH oxidase-dependent ROS production contributes to P2X7R-mediated IL-6 release

We also tested whether P2X7R-mediated IL-6 release is dependent on ROS production. We first tested effect of PBN on ATP-induced IL-6 production. Cultured astrocytes were pre-treated with 30 or 100 μM PBN for 30 min, and then co-treated with 1 mM ATP for 6 h. PBN decreased ATP-induced cytokine production in a concentration-dependent manner (Fig. 6a). Similarly, pre-treatment of astrocytes with ROS scavenger NAC (5 or 10 mM) for 30 min concentration-dependently reduced BzATP-induced IL-6 release (Fig. 6b). To confirm the role of ROS in Bz-ATP-induced IL-6 production, we tested whether inhibition of catalase (an endogenous ROS scavenger), could exacerbate Bz-ATP-induced IL-6 release from spinal astrocytes. As expected, pre-treatment with 3-amino-1,2,4-triazole (3-AT, 1 or 5 mM), the specific catalase inhibitor [49, 50], further increased IL-6 production in the presence of BzATP (300 μM) (Fig. 6c). Our results suggest that ROS play a significant role in P2X7R-mediated IL-6 release.

Fig. 6.

NADPH oxidase-dependent ROS production contributes to P2X7R-mediated IL-6 release. (a) Effect of ROS scavenger PBN (30 and 100 μM) on ATP-mediated IL-6 production. (b) Effect of ROS scavenger NAC (5 and 10 mM) on BzATP-mediated IL-6 production. (c) Effect of endogenous catalase inhibitor 3-AT on BzATP-mediated IL-6 production. (d) Effect of apocynin (Apo) on BzATP-mediated IL-6 production. Values represent mean ± SEM, n = 4–6. *P < 0.05, ***P < 0.001 compared to control, ^#P < 0.05, ^##P < 0.001 compared to vehicle by one-way ANOVA

Since we previously found NADPH oxidase contributes to ROS production in spinal astrocytes, we sought to determine whether NADPH oxidase inhibition can attenuate P2X7R-mediated IL-6 release. Interestingly, pre-treatment with NADPH oxidase inhibitor apocynin (125 or 250 μM) largely reduced IL-6 release induced by BzATP (300 μM) (Fig. 6d). Together, these data indicates that NADPH oxidase-mediated ROS production contributes to P2X7R-mediated IL-6 release.

Discussion

In this study, we demonstrated that P2X7R activation in spinal astrocytes leads to NADPH oxidase-mediated ROS increases that subsequently lead to IL-6 release (Fig. 7). Both endogenous ligand ATP and the more specific agonist, BzATP, increased ROS production, and P2X7R-mediated ROS increases were attenuated by inhibition of NADPH oxidase. Additionally, ATP and BzATP both induced IL-6 release from spinal astrocytes specifically through P2X7R, which was significantly reduced by inhibition of ROS and intensified by exacerbation of ROS. We also found ROS-mediated IL-6 release induced by P2X7R was partially dependent on NADPH oxidase. Taken together, our results establish a link between functional P2X7R in spinal astrocytes, NADPH oxidase-mediated ROS production, and ultimately IL-6 release that may contribute to inflammation under pathological conditions.

Fig. 7.

P2X7R activation leads to NADPH oxidase-mediated increases in IL-6 production

It is well-established that P2X7R activation leads to ROS increases in microglia and other cell types [15, 18]. Moreover, we previously found that P2X7R activation in spinal cord dorsal horn neurons increase ROS production [14]. It is known that P2X7R is expressed in astrocytes [27, 51, 52]. We therefore hypothesized that P2X7R activation in spinal astrocytes also led to ROS increases. Consistent with our hypothesis, live-cell imaging results show that activation of P2X7R with ATP and BzATP indeed increased ROS production in spinal astrocytes. Interestingly, in contrast to our previous study in spinal dorsal horn neurons [14], we found that ATP-induced ROS increases were completely inhibited by P2X7R antagonist A438079 in spinal astrocytes (Fig. 1). Our previous study suggested that ATP could induce ROS production in neurons through activation of other P2 purinoreceptors, such as P2Y receptors, which are expressed in neurons and astrocytes and have been reported to induce ROS increases following activation in mixed hippocampal cultures [53]. Although astrocytes express several P2XR and P2YR including P2X1/5, P2Y1/4 [54–56], our data indicates that P2X7R plays a prominent role in ATP-induced ROS production in spinal astrocytes.

Consistent with our previous results in spinal dorsal horn neurons [14], we found that BzATP-mediated ROS increases were dependent on NAPDH oxidase. Others have similarly reported on the role of NADPH oxidase in P2X7R-mediated ROS production in other CNS cell types such as microglia [21, 40]. Additionally, studies in Kupffer cells have also shown NADPH oxidase-mediated ROS production induced by P2X7R leads to downstream inflammatory liver injury [22]. Thus, NADPH oxidase is emerging as a key player in P2X7R signaling both peripherally and in the CNS.

P2X7R mediates a variety of signaling pathways that leads to inflammation [7]. While the majority of work elucidating the P2X7R inflammatory pathway has been carried out in peripheral cells such as macrophages, there is emerging evidence of the importance of P2X7R in CNS inflammation [57, 58]. Previous studies have found that P2X7R activation induced increases in IL-6 release in cultured mouse microglia [26, 59]. Another recent report has shown that P2X7R activation leads to IL-6 release in retinal ganglion cell neurons and optic nerve head astrocytes [27]. Collectively, these studies suggest that P2X7R activation leads to cytokine release in the CNS that could subsequently contribute to inflammation.

While P2X7R-mediated ROS production has been shown to contribute to microglia activation and inflammation [40], no studies have been reported its role in cytokine production in astrocytes. Interestingly, we found that BzATP-mediated IL-6 release was partially attenuated by ROS scavenger NAC. We also found that inhibition of endogenous ROS abrogation exacerbated BzATP-mediated IL-6 release. Thus, P2X7R-induced ROS production may, at least partially, contribute to cytokine production. Other pathways may indeed be involved in IL-6 release mediated by P2X7R activation. For example, a recent report states that high levels of free fatty acids can induce P2X7R-mediated IL-6 release via activation of p38, a member of the MAPK pathway [60]. We are therefore currently investigating the role of the MAPK pathway in P2X7R-mediated IL-6 release in astrocytes. Since we found ROS production occurs through NADPH oxidase, we also tested whether its inhibitor, apocynin, could attenuate IL-6 release. Indeed, we found a significant reduction on IL-6 release with pre-treatment of apocynin. Therefore, we conclude that NADPH oxidase may contribute to ROS-mediated IL-6 release induced by P2X7R activation. However, the exact mechanisms by which NADPH oxidase-mediated ROS production can contribute to cytokine release remain to be explored.

Spinal astrocytes have been recognized as active participants in chronic pain conditions [61, 62]. They respond to peripheral inflammation or injury by releasing cytokines, which are thought to be involved in central mechanisms underlying the maintenance and exaggeration of chronic pain [63, 64]. Spinal P2X7R has been reported to be involved in acute and chronic pain [14, 65–67]. Previous studies have also demonstrated that ROS play a role in neuropathic pain [68–70]. Our findings reveal that activation of P2X7 increases ROS formation, which leads to IL-6 production. Our study establishes a novel link between P2X7 receptor-induced oxidative stress and proinflammatory cytokine production in spinal astrocytes and unveils a potential underlying mechanism for astrocytic P2X7R in chronic pain.

Compliance with ethical standards

Funding information

This work was supported by NIH Grants R21NS077330 and R01NS087033 to H.H.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All experiments were performed in accordance with the guidelines of the National Institutes of Health (NIH) and with the guidelines of the Committee for Research and Ethical Issues of IASP and were approved by the Animal Care and Use Committee of Rutgers New Jersey Medical School.