In this study, we aimed to explore the therapeutic potential of DMF against LPS-induced ALI. Despite its well-documented anti-inflammatory and antioxidant properties, DMF has not yet been studied in experimental models of LPS-induced ALI. Our results indicated that DMF may attenuate LPS-induced ALI in terms of improved lung inflammatory and reduced pyroptosis due in part to preserve NLRP3 inflammasome activation via Nrf2-mediated signaling (Fig. 6).
Accumulating evidences show that multiple pathological processes, such as capillary permeability increase, extensive neutrophil infiltration, inflammatory mediators release and edema, are displayed in the pathogenesis of ALI [21, 22]. In accordance with previous studies, our data showed that LPS could induce pulmonary edema, coagulation and inflammation cells, reduced neutrophil numbers. In our study, we found that the elevation of lung W/D ratios and protein level in BALF accounting for an increased pulmonary permeability and lung edema in LPS-instillation lungs, were markedly also attenuated by DMF treatment. Our study strongly suggested that DMF could improve LPS-induced ALI in mice. Clinical evidences demonstrated the superiority of DMF in treating multiple sclerosis patients and preclinical studies have shown that DMF could treat several human diseases including lung disease. Cattani-Cavalieri et al. found that DMF could attenuate lung injury induced by chronic exposure to diesel exhaust particles in mice [23]. Grzegorzewska et al. reported that DMF ameliorates pulmonary arterial hypertension and bleomycin-induced lung fibrosis [11]. In combination of these results, our studies indicated that DMF may be a promised drug protecting from a variety of factors-induced lung injury not limiting to LPS.
NLRP3 inflammasome play an important role in the pathogenesis of ALI. Wu et al. reported that NLRP3 inflammasome of alveolar macrophage is associated with ALI/ARDS in mice [24]. Furthermore, Zhang et al. found that NLRP3 inflammasome contributed to LPS-induced ALI while NLRP3 deficiency or inhibition attenuated lung histopathological injury, inflammatory cell infiltration [17]. We found that NLRP3 inflammasome was markedly activated in lung tissue of ALI mice and the plasma level of IL-1β was significantly increased in ALI mice, which were consistent with previous studies [24]. Emerging evidences showed DMF inhibited NLRP3 inflammasome activation in colitis, thioacetamide-induced liver and diabetic aortas [14–16]. In line with these findings, we found that the administration of DMF significantly inhibited NLRP3 inflammasome activation in lung tissue of ALI, as evidenced by decreased expression of NLRP3, cleavage of caspase-1 and IL-1β. These results indicated that the protective effect of DMF on LPS-induced ALI was associated with the inhibition of NLRP3 inflammasome. Interestingly, DMF has also inhibited NF-κB expression both in LPS-induced ALI mice and pulmonary epithelial cells, suggesting that inhibiting both “priming” and “assembly” signals was related to the protective effect of DMF on LPS-induced ALI.
The assembly of NLRP3 inflammasome triggers the autocleavage of pro-caspase-1 into active caspase-1[25]. Followed NLRP3 inflammasome activation, caspase-1 cleaves GSDMD to yield GSDMD-NT, which critically determines the fate of pyroptotic cell death [26]. Driven by inflammasome activation, pyroptotic cell death in pulmonary epithelial cells exacerbated lung damage in ALI [27]. Consistent with previous reports, we found that LPS promoted the cleavage of GSDMD in lung tissue of ALI, indicating the presence of pyroptosis in the ALI.
Pyroptosis leads to the formation of pores in the plasma membrane that allow a membrane-impermeant dye like 7-AAD to enter pyroptotic cells but not apoptotic or viable cells [20]. Considering that pulmonary epithelial cells have a profound impact on the development and prognosis of ALI by releasing inflammatory cytokines, BEAS-2B cells was subjected to LPS stimulation were used to seek whether DMF treatment inhibited NLRP3 inflammasome and related pyroptosis in vitro. Accumulating evidences supported that NLRP3 inflammasome pathway was activated under LPS stimulation. Correspondingly, we confirmed that LPS activated NLRP3 inflammasome and induced pyroptosis in BEAS-2B cells, as evidenced by increased cleavage of GSDMD and active caspase-1+/7-AAD+ cells. Our further expriments indicated that DMF treatment not only inhibited LPS-induced the expression of NLRP3 inflammasome but also significantly reduced pyroptosis in BEAS-2B cells.
Oxidative stress created by LPS plays an important role in the pathology of LPS-induced ALI [28]. Txnip is involved in inflammation in response to oxidative stress [29]. In the resting state, Txnip is kept in an inactive form by binding with its endogenous inhibitor thioredoxin (TRX). Both Txnip and TRX regulate and maintain the balance of intracellular oxidation and anti-oxidation systems. When Txnip separates from TRX, it directly activates the NLRP3 inflammasome under oxidative stress [30]. Here we clearly presented that Txnip acted as an upstream factor regulated by LPS, and thus led to NLRP3 inflammasome activation and pyroptosis, which were all reversed by DMF.
Nrf2 is a pleiotropic transcription factor through antioxidant response elements within the regulatory region of many target genes such as HO-1 and NQO1, coordinates antioxidative allowing all types of cells adapt to detrimental conditions caused by intracellular or extracellular stress [28, 31]. In this study, we found that DMF obviously promoted the expression of Nrf2 and inhibited the production of ROS in LPS-induced BEAS-2B cells. We further inhibited Nrf2 activity to confirm seek whether DMF inhibited NLRP3 inflammasome activation by upregulaying Nrf2 signaling. Our results demonstrated that inhibition of Nrf2 abrogated the protective effects of DMF on inhibiting NLRP3 inflammasome activation and related pyroptosis in LPS-induced ALI in vitro and in vivo. Taken together, DMF directly activated LPS-induced Nrf2 expression, thus leading to inactivation of NLRP3 inflammasome and inhibition of pyroptosis.
Collectively, the results we presented here revealed that DMF exerts therapeutic effects in LPS-induced ALI by inhibiting NLRP3 inflammasome activation and related pyroptosis. Moreover, we uncovered its therapeutic effect at least partially through promoting Nrf2 expression. Our study identified the benefit of DMF for ALI and provided more evidences for therapeutical value of DMF on diseases which were associated with NLRP3 inflammasome and related pyroptosis.