Study On The Mechanism Of AMPK-PGC1α-SIRT3 Signaling Pathway Involved In Sepsis-related Acquired Myasthenia

Objective:A rat sepsis-related acquired myasthenia model was constructed by intraperitoneal injection of lipopolysaccharide and immobilization,and the possible mechanism of AMPK-PGC1α-SIRT3 signaling pathway in the occurrence and development of sepsisrelated acquired myasthenia was explored from the in vivo level.Method :1.Serum samples of 8 patients with sepsis-related myasthenia in the intensive care unit of each affiliated hospital of Zunyi Medical University and 8 normal volunteers from the physical examination center of Zunyi Medical University were collected.The levels of SIRT3 and CAF in the samples were detected by ELISA.2.108 SPF adult male SD rats weighing 200-250 g were randomly divided into 6 groups(n=18):(1)N group(normal group);(2)NS group(normal saline group);(3)S group(sepsis group);(4)I(immobilization group);(5)S+I group(sepsis + immobilization group);(6)H group(SIRT3 agonist group),and then 18 rats in each group were randomly divided into 3,6,9 days group(n=6),a total of 18 groups of rats;ELISA detection of rat serum IL-6,TNF-α to assess inflammation indicators,and use HE staining to observe the morphological structure of rat skeletal muscle to assess sepsisrelated myasthenia model;After the model was successfully established,SIRT3 and CAF in rat serum were detected on 3,6,and 9 days,and ATP,ROS and mitochondrial membrane potential of skeletal muscle tissue were detected to evaluate the mitochondrial function of rats in the model group.Immunohistochemistry was used to observe the transition of muscle fiber types.Western The blot method was used to detect the expression changes of AMPK,SIRT3,PGC1α,SOD2,Beclin-1,P62,ATG7 and Calpain-1 in skeletal muscle tissue.Electron microscope observation of skeletal muscle mitochondrial ultrastructure and autophagosomes.Result:1.Compared with normal volunteers,serum SIRT3 levels in patients with sepsis-related myasthenia decreased(P < 0.05),and CAF levels increased(P < 0.05).2.Compared with the N group and the NS group,the levels of serum IL-6 and TNF-αin the S+I group were significantly increased(P < 0.05),and the body weight and skeletal muscle weight of the S+I group were significantly decreased(P < 0.05),HE staining showed that the S+I group rats muscle fiber atrophy,the fiber interval widened to varying degrees,and the muscle fiber transverse diameter value was significantly reduced(P < 0.05),and the rats showed obvious retardation,standing instability,lethargy,oronasal secretion The increase in blood,purulent urine and watery stools,it is determined that the sepsis-related myasthenia model has been successfully established.3.After establishing the sepsis-related myasthenia model,compared with the N group and the NS group,the Western blot of the rat skeletal muscle tissue in the S+I group showed: AMPK,PGC1α,SIRT3,SOD2,Beclin-1,P62,ATG7 expression was significantly down-regulated(P < 0.05),and Calpain-1 expression was significantly increased(P < 0.05);skeletal muscle mitochondrial membrane potential was significantly reduced,ROS production was significantly increased and ATP production was decreased(P < 0.05);immunohistochemical results showed : The type of muscle fiber of tibialis anterior muscle changes from fast muscle to slow muscle;under electron microscope,the fiber breaks,mitochondrial edema,vacuoles expand,and the number of mitochondria decreases.4.After the addition of SIRT3 agonist(Honokiol),compared with S+I group,Western blot of rat skeletal muscle in H group showed that SIRT3,SOD2,Beclin-1,P62,ATG7 were up-regulated(P < 0.05),Calpain-1 expression decreased(P < 0.05);membrane potential increased,ROS production decreased,and ATP production increased(P <0.05);there was no significant difference between the type of tibial anterior muscle muscle fiber and the N group and the NS group;only observed under electron microscope The mitochondria were slightly swollen,the muscle fiber structure was complete,the number of mitochondria was not significantly reduced compared with the N group and the NS group,and the autophagosomes increased.Conclusion: When sepsis-related acquired myasthenia occurs,skeletal muscle mitochondrial function is impaired,muscle fiber morphology is changed,autophagy levels are down-regulated,and calpain is increased.AMPK-PGC1α-SIRT3 signaling pathway may be involved in sepsis-related acquired muscle Weakness occurs and develops.