Anti-Inflammatory Activity of 1,4-Naphthoquinones Blocking P2X7 Purinergic Receptors in RAW 264.7 Macrophage Cells

Abstract

P2X7 receptors are ligand-gated ion channels activated by ATP and play a significant role in cellular immunity. These receptors are considered as a potential therapeutic target for the treatment of multiple inflammatory diseases. In the present work, using spectrofluorimetry, spectrophotometry, Western blotting and ELISA approaches, the ability of 1,4-naphthoquinone thioglucoside derivatives, compounds U-286 and U-548, to inhibit inflammation induced by ATP/LPS in RAW 264.7 cells via P2X7 receptors was demonstrated. It has been established that the selected compounds were able to inhibit ATP-induced calcium influx and the production of reactive oxygen species, and they also exhibited pronounced antioxidant activity in mouse brain homogenate. In addition, compounds U-286 and U-548 decreased the LPS-induced activity of the COX-2 enzyme, the release of pro-inflammatory cytokines TNF-α and IL-1β in RAW 264.7 cells, and significantly protected macrophage cells against the toxic effects of ATP and LPS. This study highlights the use of 1,4-naphthoquinones as promising purinergic P2X7 receptor antagonists with anti-inflammatory activity. Based on the data obtained, studied synthetic 1,4-NQs can be considered as potential scaffolds for the development of new anti-inflammatory and analgesic drugs.

Tetracyclic 1,4-Naphthoquinone Thioglucoside Conjugate U-556 Blocks the Purinergic P2X7 Receptor in Macrophages and Exhibits Anti-Inflammatory Activity In Vivo

“…In this study, we did not find a pronounced dose-effect relationship for compound U-556. The same lack of clear dose dependence has been previously described for other 1,4-NQs, which is likely due to the non-linear mode of P2X7R inhibition [12,13].…”

“…In our experiments, acute inflammation induced by carrageenan showed significant mitigation by U-556 in the second phase after 4 and especially 24 h. Apparently, this is a consequence of the conjugate U-556 impact on P2X7R followed by inhibition of pro-inflammatory cytokine release and ROS generation responsible for paws edema. COX-2 inhibition by the studied compound described for some 1,4-NQs in our previous investigation [13] may also contribute to diminishing the edema rate in the second phase.…”

“…Some of the natural and synthetic compounds were found to inhibit histamine, IL-1β, IL-6, and TNF-α release, cyclooxygenase-2 (COX-2) activity, and reduce edema in the carrageenan model of inflammation [6]. Some synthetic 1,4-NQs were observed to effectively block the P2X7R functions in mouse neuronal and macrophage cells [7][8][9][10][11][12][13]. In this study, we proved that synthetic tetracyclic naphthoquinone-thioglucoside conjugate U-556, is able to effectively block the functioning of P2X7R in mouse macrophages.…”

“…Acetylated thioglucoside 3 was characterized as red solid with R f 0.43 (hexane-benzene-acetone, 2:1:1 v/v, silufol plates), yield 81%, mp 169-171 • C. 1 H NMR (700 MHz, CDCl 3 ): δ 1.95 (s, 3H), 2.01 (s, 3H), 2.02 (s, 3H), 2.08 (s, 3H), 2.26 (s, 3H), 2.27 (s, 3H), 3.70 (ddd, 1H, J 2.4, 5.1, 10.0 Hz), 4.06 (dd, 1H, J 2.4, 12.3 Hz), 4.15 (m, 1H, J 5.1, 12.3 Hz), 4.21 (s, 3H), 5.09 (dd, 1H, J 9.3, 10.0 Hz), 5.11 (dd, 1H, J 9.3 10.0 Hz), 5.26 (dd, 1H, J 9.3, 9.4 Hz), 5.50 (d, 1H, J 10.0 Hz), 12.06 (s, 1H), 13.41 (s, 1H). 13 Tetracyclic conjugate 4 (U-556) was prepared according to [12] by treatment 3 with MeONa solution in dry MeOH. The red precipitate of 4 was filtered off, dried, and yielded (2R,3S,4S,4aR,12aS)-3,4,7,10-tetrahydroxy-2-hydroxymethyl-3,4,4a,12a-tetrahydro- [1,4]oxathiine-6,11-dione (4); R f 0.47 (benzene-ethylacetatemethanol, 7:4:2 v/v, silufol plates), yield 80%, mp 314-316 • C [21].…”

“…This work continues a series of studies of the biological activity of a number of 1,4-naphthoquinones synthesized by us and their ability to influence the functioning of purinergic P2X7 receptors in various cell types. We have already evaluated the effect of these compounds on Neuro-2a neuronal cells [12] and the effect of some of them on the functioning of P2X7R in macrophage cells [13]. In this study, we focused on the extended investigation of the synthetic tetracyclic thioglucoside conjugate, U-556, impact on RAW 264.7 macrophage P2X7R functioning, data for which have not been presented before.…”