Acute lung injury (ALI) is one kind of primary or secondary disease caused by different kinds of intrapulmonary insults and extrapulmonary factors (including but not limited to sepsis, shock, pneumonia, pancreatitis etc) . Despite many methods have been widely used for the treatment of AP-associated ALI, the mortality and morbidity remain high, with no approved effective therapies and specific drugs. Previous researches have revealed that emodin could alleviate the AP-induced lung inflammatory response syndrome and acute lung injury via Nrf2/HO-1 signaling [11, 28]. However, systematic elucidation of the underlying therapeutic mechanism is still further detected. In this study, we established two clinically relevant SAP-ALI models, including primary lung injury caused by caerulein combined with LPS (CER + LPS) and secondary lung injury induced by 3.5% sodium taurocholate (NaT). Based on the results, we demonstrated that emodin treatment ameliorated AP-associated lung injury and inflammatory response by inhibition of NLRP3/Caspase1/GSDMD-mediated AMs pyroptosis.
Consistent with previous findings , we found that emodin attenuated pancreatitis-associated lung injury and mitigated excessive inflammatory response, as assessed by H&E staining, inflammatory factors and MPO activity. IL-1β and IL-18, as important promoters of inflammation, have the ability to recruit immune cells like neutrophils and accelerate the release of other inflammatory mediators like TNF-α and IL-6, which trigger cascades of inflammation and amplify the inflammatory response during the course of AP [29, 30]. MPO is seen as a reliable marker for neutrophil activation. In this study, we found that emodin significantly downregulated the levels of IL-1β, IL-18, TNF-α, and MPO activity within the lung in two types of SAP-ALI models, an effect consistent with the protective role of emodin in oxidative stress and inflammasome signals . Therefore, the interaction between pulmonary and systemic inflammation enhances the cascade of inflammation within the lungs and therefore exaggerates the progression of SAP-ALI.
Pyroptosis, a caspase1-dependent highly inflammatory programmed cell death, a molecular pattern leading to activation of procaspase-1 and secretion of IL-1β and IL-18, which is inherently associated with the inflammasome activation . Cell death ways and activation of alveolar macrophages are considered major factors responsible for the progression of uncontrolled pulmonary inflammation during ALI [32, 33]. Accumulating evidence has suggested that inflammasome-dependent AMs pyroptosis is closely related to ALI induced by a variety of challenges (e.g., lipopolysaccharide, cardiopulmonary bypass, and ischemia-reperfusion et al) [19, 34, 35]. It has been reported that AMs are involved in the progression of AP from local pancreatic injury to pulmonary dysfunction, through the release of various substances like inflammatory cytokines, nitric oxide (NO), and arachidonic acid metabolites . Therefore, in this trial, AMs were accordingly selected as the target cells in the treatment strategy for AP-related lung injury. Moreover, Cheng et al reported that a large amount of NO produced by AMs in the lung tissues could be a cause of pulmonary inflammatory damage secondary to sodium taurocholate-induced AP rats . However, the specific regulatory role of AMs during SAP-ALI remains not fully clarified. To identify this type of pyroptotic cells, active caspase-1 and PI positivity, downstream inflammatory factors and the LDH level were all performed. In this study, the percentages of pyroptotic AMs were increased, which indicated AMs pyroptosis was related to the ALI that was induced in NaT-AP rats and CER + LPS-AP mice. Emodin was previously reported to effectively inhibit GSDMD-mediated pyroptosis induced by myocardial ischemia/reperfusion in cardiomyocytes . Our findings disclosed that the emodin could effectively inhibit AMs pyroptosis in two kinds of AP models, and its inhibitory effect was similar to that of AYC.
Activation of NLRP3 inflammasome in macrophages played critical roles in the pathogenesis during SAP-ALI . NLRP3 deficiency or inhibitor attenuated excessive local and systemic inflammation in experimental SAP-ALI model . In line with other findings, our results showed the NLRP3 inflammasome in AMs was significantly activated both in NaT-AP rats and CER + LPS-AP mice. Emerging evidence showed that emodin had an inhibitory effect on the activation of NLRP3 inflammasome in myocardial injury combined with cardiovascular dysfunction [38, 40, 41]. Importantly, the administration of emodin and AYC markedly inhibited AP-associated activation of NLRP3 inflammasome in AMs, which was supported by downregulated expression of NLRP3, ASC, and Caspase1 p10. The assembly of this multimeric protein complex triggers the automatic cleavage of pro-caspase-1, which converts into caspase-1. Additionally, N-terminal GSDMD fragment (GSDMD-NT) was generated in the process after gasdermin D (GSDMD) was cleaved by active caspase-1, thus furthermore promoting membrane pore formation and consequent inflammatory cascades. Initiated by activation of the inflammasome, pyroptosis did occur in AMs and exaggerated ALI during the progression of acute pancreatitis. Consistent with other findings, we similarly observed that the cleavage of GSDMD was promoted under acute pancreatitis. Moreover, emodin and AYC downregulated the expressions of GSDMD and GSDMD-NT in AMs. Taken together, all evidence suggested the therapeutic effect of emodin on NaT-AP rats and CER + LPS mice was tightly related to the inhibition of AMs pyroptosis by targeting the NLRP3-Caspase1-GSDMD pathway.