The Role And Mechanism Of Pannexin1 In Regulating Mitochondrial Dynamics In Sepsis-induced Acute Lung Injury

Background Sepsis-induced acute lung injury（ALI）and/or acute respiratory distress syndrome（ARDS）are leading causes of mortality in septic patients.The underlying mechanisms of injury remain elusive.Dysregulation of mitochondrial dynamics is implicated in apoptosis regulation and the pathogenesis of numerous diseases,suggesting that it may represent a novel therapeutic avenue for sepsis-induced ALI.Pannexin（Panx）1,the largest known channel protein,mediates ATP release,promotes Ca²⁺influx,and modulates apoptosis and necroptosis.However,the role of Panx1 in mitochondrial dynamics regulation remains unexplored.Objective The objective of this study was to investigate the precise function and mechanism of Panx1 in sepsis-induced ALI with a focus on mitochondrial dynamic imbalances.The aim was to offer novel research avenues and potential therapeutic targets for the management of sepsis-induced ALI/ARDS.MethodsPart Ⅰ:A sepsis-induced ALI model was established by caecal ligation and puncture（CLP）in C57BL/6J wild-type mice.The model was evaluated based on lung histopathological damage,alteration of the alveolar-capillary barrier,an inflammatory response,and evidence of physiological dysfunction.In addition,we analyzed mitochondrial morphology,protein expression related to mitochondrial dynamics,apoptosis,mitochondrial membrane potential（MMP）,reactive oxygen species（ROS）levels,and adenosine triphosphate（ATP）levels in bronchoalveolar lavage fluid（BALF）to investigate the changes in mitochondrial dynamics during sepsis-induced ALI and the correlation with Panx1.Part Ⅱ:To evaluate the function of Panx1 in sepsis-induced acute lung injury（ALI）and the regulation of mitochondrial dynamics,a murine model of sepsis-induced ALI was established utilizing Panx1 knockout mice,and the aforementioned parameters were monitored.Furthermore,to investigate the potential involvement of Panx1 in modulating the calcineurin（CaN）-dynamin-related protein 1（DRP1）signaling pathway,the expression and activity of CaN,as well as the degree of serine phosphorylation at site 637 of DRP1(p-DRP1^S637),were also evaluated.Part Ⅲ:The CaN inhibitor,FK506,was used to treat wild-type C57BL/6J mice to inhibit CaN activity,and a model of sepsis-induced ALI was constructed to detect changes in the above indicators and evaluate the role of CaN in sepsis-induced ALI,as well as the regulation of mitochondrial dynamics.Part Ⅳ:In this study,an in vitro ALI model was established using lipopolysaccharide-stimulated murine lung epithelial cells（MLE-12）.Panx1expression was silenced through small interfering RNA（si RNA）technology,allowing for the investigation of mitochondrial morphology,expression of mitochondrial dynamics-related proteins,p-DRP1^S637 expression,apoptosis,MMP,ROS levels,CaN activity,intracellular Ca²⁺concentration,and ATP content in the cell culture medium of MLE-12 cells.The functional role of Panx1 in septic ALI was assessed at the cellular level,in addition to its regulatory impact on mitochondrial dynamics.Moreover,the study explored the mechanism by which Panx1 regulates the CaN-DRP1 signaling pathway.In a subsequent set of experiments,plasmid transfection techniques were employed to overexpress Panx1.FK506 and Ca²⁺（4m M）were used to inhibit and activate CaN activity,respectively.By monitoring changes in the aforementioned indicators,the study aimed to elucidate the mechanism underlying the involvement of the CaN-DRP1 signaling pathway in the regulation of mitochondrial dynamics by Panx1 in the sepsis-induced ALI modelResultsPart Ⅰ:CLP method can successfully establish ALI models.Compared with the control group,the model mouse showed increased lung injury scores,lung wet-to-dry（W/D）ratio,and BALF protein content with a down-regulation of tight junction protein expression,significantly increased pro-inflammatory cytokines（IL-6,IL-1β,and TNF-α）levels in BALF and serum,decreased lung compliance,increased airway resistance,increased alveolar epithelial cells（AEC）apoptosis,enlarged and vacuolated mitochondria,decreased MMP,and increased ROS production,Downregulation of mitochondrial fusion proteins and upregulation of fission proteins were also observed.These changes were accompanied by increased Panx1 expression and ATP in BALF,suggesting an imbalance of mitochondrial dynamics in the sepsis-induced ALI model,potentially associated with the overexpression of Panx1.Part Ⅱ:Knockdown of Panx1 resulted in increased survival rates at 72 hours,decreased lung histopathological damage,a reduction in lung W/D ratio,lowered ATP levels and pro-inflammatory cytokine concentrations（IL-6,IL-1β,and TNF-α）in BALF,increased expression of tight junction proteins,improved lung compliance,reduced airway resistance,attenuated AEC apoptosis,restored mitochondrial morphology,elevated MMP,decreased ROS production,and promoted mitochondrial fusion.These outcomes were concurrent with the inhibition of the CaN-DRP1signaling pathway.Collectively,these results indicate that Panx1 knockdown can restore mitochondrial dynamics,reduce apoptosis,and exert a protective effect against sepsis-induced ALI.Moreover,these protective effects may be mediated through the inhibition of the CaN-DRP1 signaling pathway.Part Ⅲ:Administration of FK506 resulted in the inhibition of calcineurin（CaN）activity,leading to decreased lung injury scores,a reduction in the lung W/D ratio,lowered concentrations of pro-inflammatory cytokines（IL-6,IL-1β,and TNF-α）in BALF,increased expression of tight junction proteins,improved lung compliance,elevated MMP,decreased airway resistance,attenuated alveolar epithelial cells apoptosis,and reduced ROS production.Additionally,FK506 promoted mitochondrial fusion and restored mitochondrial morphology.Taken together,these findings suggest that inhibition of the CaN-DRP1 signaling pathway may attenuate mitochondrial dynamic imbalance and protect against sepsis-induced ALI.Part Ⅳ:An ex vivo ALI model was successfully constructed using lipopolysaccharide-stimulated MLE-12 cells,and significant upregulation of Panx1expression and increased channel opening was found.Panx1 silencing led to the inhibition of the CaN-DRP1 signaling pathway,reducing the release of ATP and Ca²⁺influx,increasing transepithelial electric resistance,expression of tight junction proteins,MMP,mitochondrial fusion,and the restoration of mitochondrial morphology.This indicates that Panx1 suppression can restore mitochondrial homeostasis and mitigate apoptosis by blocking the CaN-DRP1 signaling cascade,likely through decreased ATP release and Ca²⁺influx.Further studies revealed that Panx1 overexpression exacerbates mitochondrial dynamics dysregulation,disrupts mitochondrial structure and function,and elevates apoptosis,whereas CaN inhibition enhances mitochondrial dynamics regulation,reinstates mitochondrial morphology and function,and attenuates apoptosis.This suggests that inhibiting the CaN-DRP1pathway can alleviate the damage caused by the overexpression of Panx1.Moreover,activation of CaN activity aggravated the imbalance in mitochondrial dynamics,disrupting mitochondrial morphology and function,increasing apoptosis,and blunting the protective effect of Panx1 silencing on cells.Conclusions Panx1 activates the CaN-DRP1 pathway,leading to the imbalance in mitochondrial dynamics,increased AEC apoptosis,tight junction disruption,and exacerbated lung injury,providing a novel theoretical basis for investigating the mechanism of sepsis-induced ALI.