Dihydrotanshinone, a Natural Diterpenoid, Preserves Blood-Retinal Barrier Integrity via P2X7 Receptor.

Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1ß, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.

ATP induces PAD4 in renal proximal tubule cells via P2X7 receptor activation to exacerbate ischemic AKI.

We previously demonstrated that renal tubular peptidylarginine deiminase-4 (PAD4) is induced after ischemia-reperfusion (IR) injury and this induction of PAD4 exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation and neutrophil infiltration. However, the mechanisms of renal tubular PAD4 induction after IR remain unknown. Here, we tested the hypothesis that ATP, a proinflammatory danger-associated molecular pattern (DAMP) ligand released from necrotic cells after IR injury, induces renal tubular PAD4 and exacerbates ischemic AKI via P2 purinergic receptor activation. ATP as well as ATPγS (a nonmetabolizable ATP analog) induced PAD4 mRNA, protein, and activity in human and mouse renal proximal tubule cells. Supporting the hypothesis that ATP induces renal tubular PAD4 via P2X7 receptor activation, A804598 (a selective P2X7 receptor antagonist) blocked the ATP-mediated induction of renal tubular PAD4 whereas BzATP (a selective P2X7 receptor agonist) mimicked the effects of ATP by inducing renal tubular PAD4 expression and activity. Moreover, ATP-mediated calcium influx in renal proximal tubule cells was blocked by A804598 and was mimicked by BzATP. P2X7 activation by BzATP also induced PAD4 expression and activity in mouse kidney in vivo. Finally, supporting a critical role for PAD4 in P2X7-mediated exacerbation of renal injury, BzATP exacerbated ischemic AKI in PAD4 wild-type mice but not in PAD4-deficient mice. Taken together, our studies show that ATP induces renal tubular PAD4 via P2X7 receptor activation to exacerbate renal tubular inflammation and injury after IR.

The role of microglial P2X7: modulation of cell death and cytokine release.

BACKGROUND: ATP-gated P2X7 is a non-selective cation channel, which participates in a wide range of cellular functions as well as pathophysiological processes including neuropathic pain, immune response, and neuroinflammation. Despite its abundant expression in microglia, the role of P2X7 in neuroinflammation still remains unclear. METHODS: Primary microglia were isolated from cortices of P0-2 C57BL/6 wild-type or P2X7 knockout (P2X7-/-) mouse pups. Lipopolysaccharide, lipopolysaccharide plus IFNγ, or IL4 plus IL13 were used to polarize microglia to pro-inflammatory or anti-inflammatory states. P2rx7 expression level in resting or activated mouse and human microglia was measured by RNA-sequencing and quantitative real-time PCR. Microglial cell death was measured by cell counting kit-8 and immunocytochemistry, and microglial secretion in wild-type or P2X7-/- microglia was examined by Luminex multiplex assay or ELISA using P2X7 agonist BzATP or P2X7 antagonist A-804598. P2X7 signaling was analyzed by Western blot. RESULTS: First, we confirmed that P2rx7 is constitutively expressed in mouse and human primary microglia. Moreover, P2rx7 mRNA level was downregulated in mouse microglia under both pro- and anti-inflammatory conditions. Second, P2X7 agonist BzATP caused cell death of mouse microglia, while this effect was suppressed either by P2X7 knockout or by A-804598 under both basal and pro-inflammatory conditions, which suggests the mediating role of P2X7 in BzATP-induced microglial cell death. Third, BzATP-induced release of IL1 family cytokines including IL1α, IL1ß, and IL18 was blocked in P2X7-/- microglia or by A-804598 in pro-inflammatory microglia, while the release of other cytokines/chemokines was independent of P2X7 activation. These findings support the specific role of P2X7 in IL1 family cytokine release. Finally, P2X7 activation was discovered to be linked to AKT and ERK pathways, which may be the underlying mechanism of P2X7 functions in microglia. CONCLUSIONS: These results reveal that P2X7 mediates BzATP-induced microglial cell death and specific release of IL1 family cytokines, indicating the important role of P2X7 in neuroinflammation and implying the potential of targeting P2X7 for the treatment of neuroinflammatory disorders.

Muscle hyperalgesia induced by peripheral P2X3 receptors is modulated by inflammatory mediators.

ATP, via activation of P2X3 receptors, has been highlighted as a key target in inflammatory hyperalgesia. Therefore, the aim of this study was to confirm whether the activation of P2X3 receptors in the gastrocnemius muscle of rats induces mechanical muscle hyperalgesia and, if so, to analyze the involvement of the classical inflammatory mediators (bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines and neutrophil migration) in this response. Intramuscular administration of the non-selective P2X3 receptor agonist α,ß-meATP in the gastrocnemius muscle of rats induced mechanical muscle hyperalgesia, which, in turn, was prevented by the selective P2X3 and P2X2/3 receptors antagonist A-317491, the selective bradykinin B1-receptor antagonist Des-Arg9-[Leu8]-BK (DALBK), the cyclooxygenase inhibitor indomethacin, the ß1- or ß2-adrenoceptor antagonist atenolol and ICI 118,551, respectively. Also, the nonspecific selectin inhibitor fucoidan. α,ß-meATP induced increases in the local concentration of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß), which were reduced by bradykinin antagonist. Finally, α,ß-meATP also induced neutrophil migration. Together, these findings suggest that α,ß-meATP induced mechanical hyperalgesia in the gastrocnemius muscle of rats via activation of peripheral P2X3 receptors, which involves bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines release and neutrophil migration. It is also indicated that bradykinin is the key modulator of the mechanical muscle hyperalgesia induced by P2X3 receptors. Therefore, we suggest that P2X3 receptors are important targets to control muscle inflammatory pain.

Protection of differentiated neuronal NG108-15 cells from P2X7 receptor-mediated toxicity by taurine.

BACKGROUND: Strong P2X7 receptor (P2X7R) activation causes Ca(2+) overload and consequent cell death. We previously showed that depletion of Ca(2+) stores and endoplasmic reticulum (ER) stress in differentiated NG108-15 neuronal cells contributed to P2X7R-mediated cytotoxicity. In this work, we assessed whether taurine (2-aminoethanesulfonic acid) could prevent this P2X7R-mediated cytotoxicity in this neuronal cell line. METHODS: Cytotoxicity markers were assessed by MTT assay and Western blotting. Cytosolic Ca(2+) and mitochondrial Ca(2+) concentrations were measured microfluorimetrically using fura-2 and rhod-2, respectively. Intracellular reactive oxygen species (ROS) production was assayed by the indicator 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: Selective P2X7R agonist BzATP treatment causes neuronal cell death by causing cytosolic Ca(2+) overload, depletion of Ca(2+) stores, endoplasmic reticulum (ER) stress, and caspase-3 activation (cleaved caspase 3). Remarkably, taurine (10mM) pretreatment could prevent P2X7R-mediated neuronal cell death by blocking BzATP-mediated ER stress as determined by phosphorylated eukaryotic translation initiation factor 2α (peIF2α) and C/EBP-homologous protein (CHOP). However, taurine did not block BzATP-induced Ca(2+) overload and depletion of ER Ca(2+) stores. Interestingly, P2X7R activation did not result in mitochondrial Ca(2+) overload, nor did it affect mitochondrial membrane potential. BzATP-induced generation of intracellular reactive oxygen species (ROS) was prevented by taurine. CONCLUSIONS: The neuroprotective effect by taurine is attributed to the suppression of P2X7R-mediated ER stress and ROS formation.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier integrity through a mechanism involving P2X7 receptors.

The recreational drug 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') produces a neuro-inflammatory response in rats characterized by an increase in microglial activation and IL-1ß levels. The integrity of the blood-brain barrier (BBB) is important in preserving the homeostasis of the brain and has been shown to be affected by neuro-inflammatory processes. We aimed to study the effect of a single dose of MDMA on the activity of metalloproteinases (MMPs), expression of extracellular matrix proteins, BBB leakage and the role of the ionotropic purinergic receptor P2X7 (P2X7R) in the changes induced by the drug. Adult male Dark Agouti rats were treated with MDMA (10 mg/kg, i.p.) and killed at several time-points in order to evaluate MMP-9 and MMP-3 activity in the hippocampus and laminin and collagen-IV expression and IgG extravasation in the dentate gyrus. Microglial activation, P2X7R expression and localization were also determined in the dentate gyrus. Separate groups were treated with MDMA and the P2X7R antagonists Brilliant Blue G (BBG; 50 mg/kg, i.p.) or A-438079 (30 mg/kg, i.p.). MDMA increased MMP-3 and MMP-9 activity, reduced laminin and collagen-IV expression and increased IgG immunoreactivity. In addition, MDMA increased microglial activation and P2X7R immunoreactivity in these cells. BBG suppressed the increase in MMP-9 and MMP-3 activity, prevented basal lamina degradation and IgG extravasation into the brain parenchyma. A-438079 also prevented the MDMA-induced reduction in laminin and collagen-IV immunoreactivity. These results indicate that MDMA alters BBB permeability through an early P2X7R-mediated event, which in turn leads to enhancement of MMP-9 and MMP-3 activity and degradation of extracellular matrix.