Effect Of GSDMD-mediated Pyroptosis On Sepsis-induced ICU-acquired Weakness

Background:ICU-acquired weakness(ICU-AW)refers to the syndrome with neuromuscular dysfunction as the pathological basis and systemic fatigue as the clinical manifestation in the course of the disease of critically ill patients,especially those with severe sepsis.ICU-AW is the most common complication in ICU patients,which not only affects the prognosis of patients and seriously reduces the quality of life of critically ill survivors,but also increases the incidence of critically ill related complications,prolongs the time of ICU treatment and recovery,and greatly increases the cost of intensive care treatment worldwide.However,at present,the pathogenesis of ICU-AW is not yet clear,and finding effective methods for the prevention and treatment of sepsis related ICU-AW has become an urgent scientific problem in the field of critical care medicine.Aims:Based on previous researches,in view of the unknown pathogenesis of ICU-AW,this study intends to establish sepsis related ICU-AW mouse model and corresponding myotube injury model,observe the role of GSDMD and its mediated apoptosis in sepsis related ICUAW from the in vivo and cell level,explore the possible mechanism of GSDMD and its mediated apoptosis in the occurrence and development of sepsis related ICU-AW,and provide a new theoretical basis for the prevention and treatment of ICU-AW related to sepsis.Method:In order to explore the mechanism of sepsis related ICU-AW,we first established a mouse model of sepsis by intraperitoneal injection of LPS at different concentrations,and studied the survivors 96 h and 2wk after modeling by ICU-like interventions.The survival rate,the changes in body weight and spleen weight of 96h-2wk mice were observed,the inflammatory indexes of peripheral blood of mice were detected by routine blood tests,and the inflammatory cell infiltration of skeletal muscle and organs of mice were observed by hematoxylin-eosin(HE)staining.The sepsis mouse model was established successfully based on the above indexes.Subsequently,the m RNA expression levels of Atrogin-1 and Mu RF-1 in skeletal muscle of the model mice were detected by q PCR method,and the contractile function of skeletal muscle of the model mice was detected by biological muscle strength transducer(BL-420s)to evaluate whether the model mice suffered from pathological signs of ICU-AW.At the same time,in order to further explore pyroptosis associated with the sepsis-induced ICUAW,we detected pyroptosis related inflammatory factor IL-1β and IL-18 m RNA expression level in skeletal muscle of mice by q PCR.The release of IL-1β and IL-18 in serum were determined by Enzyme-linked Immunosorbent Assay(ELISA).Immunofluorescence and immunohistochemical staining indicated the expression of GSDMD,a signature protein of pyroptosis.The changes of mitochondrial structure in skeletal muscle of mice were observed by transmission electron microscopy.In addition,in order to observe the effect of pyroptosis on skeletal muscle atrophy at the cellular level,we differentiated and cultured myotubes in vitro and stimulated the myotubes with LPS/LPS+ATP to successfully construct the myotubes injury model.The expression of Atrogin-1,Mu RF-1 and IL-18 m RNA in myotubes of skeletal muscle of mice were detected by q PCR.The concentration of IL-18 in supernatant of myotubes culture was determined by ELISA.Western Blot was used to detect the expression of GSDMD in the myotubes of mice skeletal muscle.Result:1.In vivo study results: In the same modeling time,the survival rate of sepsis model mice injected intraperitoneally with different concentrations of LPS was related to the injection concentration of LPS.With the prolongation of modeling time,the survival rate of the model mice tended to be stable and the body weight showed an upward trend.The inflammatory indexes of the sepsis model mice were significantly higher than those of the control group.Morphological observation showed that the infiltration of inflammatory cells in skeletal muscle and tissues of various organs was increased,and the mitochondrial structure of skeletal muscle of the model mice were significantly abnormal compared with that of the control group.Compared with the control group,the expressions of Atrogin-1 and Mu RF-1 m RNA were significantly increased in the model mice,and the contractile function of skeletal muscle was decreased in model mice,which were consistent with the evaluation of sepsis related ICU-AW.In the skeletal muscle of sepsis related ICU-AW model mice,the m RNA expression levels of inflammatory cytokine IL-1β and IL-18 were significantly increased and released in large amounts in serum.Meanwhile,immunofluorescence and immunohistochemical results showed that the pyroptosis marker protein GSDMD was significantly expressed in skeletal muscle of the model mice.2.In vitro study results: The morphology of myotubes after differentiation was observed under light microscope.Western Blot analysis showed that the myotubes formation marker protein could be stably expressed in the myotubes in each group.After LPS/LPS+ATP intervention,the m RNA expression levels of Atrogin-1 and Mu RF-1 in the myotubes of each group were increased,and the expression of IL-18 m RNA and the concentration of IL-18 in supernatant of myotubes were significantly increased.At the same time,Western Blot analysis showed that the expression of GSDMD was increased in the myotubes of each group after LPS/LPS+ATP intervention.Conclusion:The above results in this study confirmed that in critically ill sepsis mice,pyroptosis may directly or indirectly regulate the inflammatory response,causing pathological changes such as decreased skeletal muscle systolic function,muscle atrophy and mitochondrial structure abnormality,thus promoting the occurrence and development of ICU acquired weakness.At the same time,the results of in vitro studies confirmed that pyroptosis occurred in the myotubes under the stimulation of LPS/LPS+ATP,and the severity of pyroptosis was related to the expression of myotubes atrophy gene,which further provided a theoretical basis for the pathogenesis of inflammation-induced muscular atrophy.In conclusion,this study confirmed that GSDMD and its mediated pyroptosis are involved in the occurrence and development of sepsis related ICU-AW,it may be propose a new direction for the follow-up study on the pathogenesis of sepsis related ICU-AW.