Synoviocytes play a major role in gout because MSU induce inflammatory, and OS associated with phagocytosis [11]. The working doses of the anti-inflammatory agent PGAL were established from viability curves at 24 h in synoviocytes stimulated with 1-500 µg/ml. In this range of concentrations, no morphological changes suggestive of cell damage or death or decreased synovial viability were identified; therefore, the doses selected were 100 and 200 µg/ml. In addition, in a recent study by our group, it was demonstrated that PGAL at these concentrations was capable of modulating IL-6, IL-8 and TNF-α in THP-1 monocytes activated with phorbol myristate acetate [18].
In the present model of synovial damage, it was possible to demonstrate that the PGAL molecule has cytoprotective properties because it significantly reduces cell death caused by MSU and maintains the viability of cells exposed to these crystals. Sánchez-Sánchez et al. [16] reported protective effects of PGAL against the damage caused by UV radiation in fibroblasts, maintaining a good antioxidant capacity, which is unlikely using other polyphenols such as GA under these conditions. This effect was also shown in our model by inhibiting the generation of ROS in cells exposed to MSU. In another in vitro model of gout studied by Oliviero et al. [21], THP-1 cells stimulated with MSU for 24 h increased ROS production up to 5.5 times more than baseline levels. However, the use of polyphenols such as resveratrol and polydatin were effective in inhibiting ROS when they were added together with the crystals; however, the only polyphenol that facilitated a decrease in the PI was resveratrol. Resveratrol suppresses the activation of NLRP3 both in vitro and in vivo, and a possible mechanism for this action is through the suppression of mitochondrial ROS [22].
The antioxidant and anti-inflammatory properties of PGAL are attributed to its chemical structure, described as a multiradical polyanion (approximately 40-60 GA units with CC bonds) in a helical structure due to repulsion between its benzene rings. The rotation of this helix on its axis establishes intramolecular and intermolecular hydrogen bonds [23]. PGAL, by having stable free radicals in its structure, exerts an antioxidant mechanism of action mainly via single electron transfer (SET) instead of the most common mode of electron transfer in polyphenols and other antioxidants, i.e., hydrogen atom transfer (HAT) [24]. Both mechanisms lead to the elimination of free radicals [25]; however, SET may have interesting implications for certain treatments. HAT is dependent on the environment, and its effectiveness is greater for protonated forms of polyphenolic acids, which can be affected by the acid-base balance under physiological conditions or at an alkaline pH. Romero-Montero et al. [17] demonstrated that the hydroxyl group of the molecule makes it particularly available for the prevention and control of cell membrane lipoperoxidation.
In addition to the ability to inhibit ROS, the polyanionic structure in PGAL suggests a barrier mechanism that prevents the recognition of crystals by cell membrane receptors, inhibiting their phagocytosis and thus inhibiting the internalization of MSU by synovial cells. Within cells, PGAL interferes in the formation of vesicles or vacuoles, which are associated with crystal phagocytosis (Figure 7).
These vesicles are characteristic of cells that carry out phagocytosis [26]; therefore, our results corroborate the hypothesis that PGAL interferes with this primary mechanism for the activation of inflammation in gout by inhibiting IL-1β. In addition, an in vitro study indicated that PGAL has greater hydroxyl radical capture activity than does GA and a more significant protective effect on cellular damage induced by H2O2 [17].
The mechanisms of inflammation and OS that MSU activate in cells when recognized by TLR receptors through the plasma membrane involve NF-kB. A preliminary study indicated that PGAL reduces the expression of NF-kB (data not shown) and other components in THP-1 cells for inflammasome activation, thus inducing caspase-1 and IL-1β release, or activates gasdermin D, which promotes the formation of pores in the membrane, inducing pyroptosis [27].
Pyroptosis occurs after the intracellular detection of damage signals, which can be induced by MSU. Pyroptotic cells present membrane pore formation and plasma membrane rupture and release inflammatory mediators and cytoplasmic content into the extracellular space [28, 29]. Therefore, the formation of vesicles identified in synoviocytes could be associated with the activation of pyroptosis, and in this sense, PGAL, by inhibiting the formation of these vesicles, would exert a possible mechanism of action through the inhibition of inflammasomes or of pyroptosis. Similar to its precursor, GA, which was studied in in vitro and in vivo models of gout [30], was shown to inhibit pyroptosis in macrophages stimulated by MSU, block the activation of NLRP3, inhibit caspase-1 activation and IL-1β secretion and promote the expression of factor 2 related to nuclear factor E2 (Nrf2), reducing mitochondrial ROS, which supports our hypothesis that a similar mechanism occurs for PGAL.
Our results demonstrate the antioxidant and anti-inflammatory properties of PGAL in synoviocytes exposed to MSU in a model that mimics an acute attack of gout. Therefore, this research is relevant, and more studies are being carried out to elucidate the molecular pathways by which PGAL regulates phagocytic activity in cells as well as the inflammatory and oxidative states in gout. In vivo molecular studies are of interest to elucidate the mechanisms of action of PGAL in gout, in particular, those aimed at the inhibition of MSU recognition in synoviocytes, and its potential therapeutic role for the treatment of this disease.