The Role And Mechanism Of TLR4/MyD88/ERK-mediated Schwann Cell Activation In Sepsis-induced Acquired Muscle Weakness

Objective:To investigate the effects of sepsis on Schwann cells（SCs）and neuromuscular function,and to explore the role and mechanism of TLR4/MyD88/ERK-mediated SCs activation in sepsis induced acquired muscle weakness.Methods:In the first part,30 healthy male wild-type C57BL/6J mice（age:9 to 12 weeks,weight:20 to 25g）were divided into two groups randomly:Sham group（n=10）and CLP group（n=20）.In Sham group,laparotomy was performed without caecal ligation and puncture（CLP）.CLP group was treated with CLP to establish sepsis model.Detection and samples collection were performed at Day 7 after modeling,and SCs were extracted for primary culture.In the second part,all mice were performed by CLP to establish sepsis,including 12 SPF healthy male mice with MyD88 gene knockout(MYD88^-/--CLP group,n=12)and 12 healthy male wild-type C57BL/6J mice（WT-CLP group,n=12）.Detection and samples collection were performed at Day 7 after modeling,sciatic nerve SCs were extracted for primary culture,and macrophages（cell line RAW264.7）were treated with supernatant of primary culture medium.Compound muscle action potential（CMAP）was measured by electromyography in all groups.The sciatic nerve was examined by electron microscopy.Immunofluorescence was used to detect the expression of SCs and macrophages.TLR4,MyD88,ERK and p-ERK protein expression levels of SCs were detected by Western-blot.Cell cycle of SCs was detected by flow cytometry.The macrophages aggregation was detected by migration assay.Results:（1）The neuromuscular function:the amplitude of CMAP in CLP group was significantly decreased compared with Sham group（P<0.05）,and the latency of CMAP was prolonged in CLP group（P<0.05）.Compared with WT-CLP group,the amplitude of CMAP was increased significantly（P<0.05）and latency of CMAP was shortened in MyD88^-/--CLP group（P<0.05）.（2）Electron microscopy:compared with Sham group,the sciatic nerve in CLP group was edema and the sheath thickened.The sciatic nerve edema in WT-CLP group was heavier than that in MyD88^-/--CLP group,and the sheath thickened significantly.（3）Immunofluorescence:compared with Sham group,SCs and macrophages were increased and the proliferation ratio of SCs was increased in CLP group.Activated SCs in MyD88^-/--CLP group were less than that in WT-CLP group,and macrophages in MYD88^-/--CLP group were less.（4）Western-blot:compared with Sham group,TLR4,MyD88 and p-ERK/ERK protein expressions of SCs in CLP group were increased significantly（P<0.05）.The expression level of p-ERK/ERK protein in MyD88^-/--CLP group was decreased significantly（P<0.05）.（5）Flow cytometry:G0/G1 in CLP group was lower than that in Sham group（P<0.05）,while the ratio of S stage was increased significantly（P<0.05）.There was no statistically significant difference in G2+M（P>0.05）.Compared with WT-CLP group,MyD88^-/--CLP group showed an obvious increase in G0/G1（P<0.05）,the ratio of S stage was decreased significantly（P<0.05）,and the ratio of G2+M was decreased（P<0.05）.（6）Migration test:the migration rate of macrophages in MyD88^-/--CLP group was lower than that in WT-CLP group（P<0.05）.Conclusion:1.Sepsis leads to neuromuscular dysfunction,activates SCs and causes aggregation of macrophages.2.The TLR4/MyD88/ERK-mediated activation of SCs plays an important role in sepsis-induced acquired muscle weakness.