The results of this study indicate, for the first time, that GSDMD and MLKL inhibitor, NSA, prevents hyperalgesia through inhibiting increased protein expression of caspase-11 p20, p30-GSDMD, IL-1β, HMGB1, and SEMA3A in addition to RIPK1, RIPK3, and MLKL phosphorylation associated with decreased myelin PLP expression in the CNS of LPS-treated mice. Consistent with our previous findings [36–40], the results of the present study provide the first evidence that NSA prevents LPS-induced inflammatory hyperalgesia by not only inhibiting caspase-11/GSDMD-mediated pyroptosis, RIPK1/RIPK3/MLKL necrosome-mediated necroptosis, and demyelination but also promoting remyelination in the CNS of mice (Fig. 6).
Systemic administration of NSA has been shown to be efficacious in several inflammatory rodent models in vivo including LPS-induced sepsis in mice [15], AlCl3-induced Alzheimer's disease in rats [26], ischemia-reperfusion injury in rats [27], spinal cord injury in mice [24] and rats [28], and post-resuscitation myocardial dysfunction in mice [25] through protective mechanisms potentially related to inhibition of GSDMD-dependent pyroptotic and MLKL-dependent necroptotic pathways. In only one study conducted to date, Rathkey et al. [15] demonstrated that NSA (20 mg/kg; i.p.) binds directly to GSDMD and can ameliorate GSDMD-driven mortality associated with increased serum IL-1β and IL-6 levels in murine sepsis induced by LPS (25 mg/kg; i.p.). Nevertheless, to the best of our knowledge, the effect of NSA on LPS-induced inflammatory hyperalgesia has not been investigated. Increasing evidence also suggests that caspase-11/GSDMD-mediated pyroptosis signaling pathway triggered by LPS plays an important role in the pathogenesis of neuroinflammatory disorders [44, 45]. Although NSA has been shown to reverse the effects of LPS on pyroptosis both in vitro and in vivo [15, 29, 30]; its effects on caspase-11/GSDMD-mediated pyroptosis in the CNS during LPS-induced inflammatory hyperalgesia remain unknown. In the present study, we showed that systemic administration of LPS into mice resulted in hyperalgesia as well as increased protein expression of caspase-11 p20, p30-GSDMD, IL-1β, and HMGB1 in the mouse brain and spinal cord. Therefore, in agreement with the results of previous studies [15, 46] and our previous findings [36–40], this study presents novel data suggesting that NSA prevents hyperalgesia through decreased formation of pro-inflammatory mediators and HMGB1 release by pyroptotic cells to promote inflammation as a result of cell rupture and pyroptosis as a result of suppressed expression and/or activity of the caspase-11/GSDMD-mediated pyroptotic pathway. These findings also suggest that not only binding directly to GSDMD to inhibit p30-GSDMD oligomerization and pyroptosis, but also directly or indirectly decreasing expression of caspase-11 p20, p30-GSDMD, IL-1β, and HMGB1 proteins in the CNS of LPS-treated mice contribute to the protective effect of NSA in the hyperalgesia model. However, additional studies should be done to prove the validity of these hypotheses.
Recent evidence also indicates that RIPK1/RIPK3/MLKL necrosome-mediated necroptosis plays a crucial role in the pathogenesis of LPS-induced models of neuroinflammation-related CNS disorders [47–51]. There are only a few studies in the literature showing that NSA reverses the effects of LPS on the changes in the expression and/or activity of key components of necroptosis, RIPK1, RIPK3, and MLKL, in vitro [31–33]. On the other hand, it is unknown whether NSA prevents LPS-induced RIPK1/RIPK3/MLKL necrosome-mediated necroptosis in the CNS during inflammatory hyperalgesia. In the current study, we demonstrated that systemic administration of LPS into mice also resulted in not only increased phosphorylation of RIPK1, RIPK3, and MLKL proteins but also HMGB1 protein expression in the brain and spinal cord tissues. These data suggest that HMGB1 released by necrotic cells to promote inflammation as a result of cell rupture and necrosis due to increased activity of the RIPK1/RIPK3/MLKL necrosome-mediated necroptotic pathway in the CNS of mice seems to be involved in inflammatory hyperalgesia induced by LPS. Our findings also suggest that not only binding directly to MLKL to inhibit MLKL oligomerization and necroptosis, but also directly or indirectly, decreasing phosphorylation of RIPK1, RIPK3, and MLKL proteins in the CNS of LPS-treated mice contributes to the protective effect of NSA in the hyperalgesia model. However, additional studies should be done to prove the validity of these hypotheses.
SEMA3A, one of the remyelination inhibitors, is reported to be increased in the mouse brain in response to intracerebroventricular injection of LPS [52]. In recent years, loss of myelin proteins such as PLP have also received major attention in the neuroinflammatory changes in the brain induced by systemic or intrauterin injection of LPS to rodents [53–55]. On the other hand, whether NSA prevents LPS-induced changes in the protein expression of SEMA3A and myelin PLP have not been investigated until recently. Consistent with the findings of previous studies [54, 55], we obtained additional evidence confirming that systemic administration of LPS into mice results in increased protein expression of SEMA3A associated with decreased myelin PLP expression in the mouse brain and spinal cords. Moreover, LPS-induced changes in the expression of SEMA3A and myelin PLP were reversed by NSA. These results suggest that NSA not only prevents demyelination but also promotes remyelination in the LPS-induced inflammatory hyperalgesia model in mice.
Collectively, in line with the above-mentioned studies and our previous findings, directly or indirectly decreased formation of pro-inflammatory mediators as a result of inhibition of caspase-11/GSDMD-mediated pyroptotic and RIPK1/RIPK3/MLKL necrosome-mediated necroptotic pathways at transcriptional and/or post-transcriptional level in the CNS seems to contribute to the preventive effect of NSA against LPS-induced enhanced pain sensitivity to thermal stimuli associated with demyelination. In addition, NSA may also exert its beneficial effects due to its synergistic effect on the signaling pathways that play a role in the pathogenesis of inflammatory hyperalgesia caused by LPS. Nevertheless, much more detailed experiments are needed to verify the validity of the proposed hypothesis. Hence, exploring molecular mechanisms of the effects of NSA on the LPS-induced inflammatory hyperalgesia associated with demyelination in the CNS will ensure the framework for the extension of this research into comprehension the involvement of caspase-11/GSDMD-mediated pyroptosis and RIPK1/RIPK3/MLKL necrosome-mediated necroptosis. Therefore, further investigation will contribute to the preclinical and clinical studies to develop GSDMD and MLKL inhibitors, such as NSA, as drugs for the treatment of hyperalgesia-related demyelinating inflammatory diseases.
In conclusion, we demonstrated for the first time that the GSDMD and MLKL inhibitor, NSA, can prevent LPS-induced inflammatory hyperalgesia by preventing caspase-11/GSDMD-mediated pyroptosis, RIPK1/RIPK3/MLKL necrosome-mediated necroptosis, and demyelination in the CNS of mice. Thus, we suggest that GSDMD and MLKL inhibitors such as NSA may be useful as analgesic/anti-inflammatory drugs in the prevention and treatment of acute and chronic painful conditions in which inflammation plays a role in the pathophysiology that may result from bacterial infections.