脓毒症通过抑制AKT/GSK3β磷酸化导致骨骼肌细胞膜nAChRs聚集障碍

Sepsis impairs aggregation of nicotinic acetylcholine receptors on murine skeletal muscle cell membranes by inhibiting AKT/GSK3β phosphorylation

目的探究蛋白质丝氨酸苏氨酸蛋白激酶(AKT)/糖原合成酶激酶3β(GSK3β)介导的信号通路在脓毒症导致骨骼肌细胞膜上烟碱型乙酰胆碱受体(nAChRs)聚集障碍中的作用。方法小鼠源性C2C12肌原细胞经2%马血清诱导分化为肌管后随机分为4组:Sham组:假手术小鼠血清处理;Sepsis组:脓毒症小鼠血清处理;Sepsis+D组:脓毒症小鼠血清和二甲基亚砜(DMSO)处理;Sepsis+SB组:脓毒症小鼠血清和GSK3β抑制剂SB216763处理,各组在作相应处理前均加入Agrin蛋白诱导nAChRs聚集。血清处理5.5 h后采用Alexa Fluor 594-conjugatedα-bungarotoxin(α-BTX)标记肌管膜上nAChRs聚集簇,采用Western blotting检测AKT、磷酸化AKT(p-AKT)、GSK3β、磷酸化GSK3β(p-GSK3β)、细胞质连接蛋白相关蛋白2(CLASP2)的表达水平,采用免疫共沉淀(Co-IP)检测磷酸化CLASP2(p-CLASP2)以及CLASP2与微管蛋白α-tubulin的结合水平。结果与Sham组相比,Sepsis组C2C12肌管膜上nAChRs聚集簇面积和密度均降低,p-AKT、p-GSK3β水平下降,p-CLASP2水平升高,CLASP2与α-tubulin的结合减少;与Sepsis+D组相比,Sepsis+SB组C2C12肌管膜上nAChRs聚集簇面积和密度均增加,p-CLASP2水平降低,CLASP2与α-tubulin的结合水平增加。结论脓毒症小鼠血清通过抑制Akt/GSK3β磷酸化导致下游信号分子CLASP2与α-tubulin结合减少,引起C2C12肌管膜上nAChRs聚集障碍。

Objective To investigate the role of the protein-serine-threonine kinase(AKT)/glycogen synthase kinase 3β(GSK3β)signaling pathway in nicotinic acetylcholine receptors(nAChRs) aggregation disorder on skeletal muscle cell membranes induced by sepsis. Methods Mouse C2 C12 myoblasts were differentiated into myotubes by horse serum,and then C2 C12 myotubes were randomly divided into four groups: the Sham group treated with serum from sham-operated mice, the Sepsis group treated with serum from septic mice, the Sepsis+D group treated with serum from septic mice and dimethyl sulfoxide(DMSO), the Sepsis+SB group treated with serum from septic mice and GSK3β inhibitor SB216763. Agrin was added into the cell culture to induce nAChRs aggregation before the treatment. After serum treatment for 5.5 h, the myotubes were examined for nAChRs clusters using Alexa Fluor 594-conjugated α-bungarotoxin(α-BTX). The expression levels of AKT, GSK3β and CLIP-associated protein 2(CLASP2) and the phosphorylation of AKT, GSK3β were examined with Western blotting. The phosphorylation of CLASP2 and the interaction between CLASP2 and α-tubulin were detected with co-immunoprecipitation(Co-IP) assay. Results Compared with the serum from sham-operated mice, the serum from septic mice caused significant reduction in the area and density of nAChRs clusters on C2 C12 myotubes, lowered the levels of phosphorylated AKT(p-AKT)and phosphorylated GSK3β(p-GSK3β), increased the expression of phosphorylated CLASP2(p-CLASP2), and obviously reduced the binding between CLASP2 and α-tubulin. Compared with DMSO, SB216763 significantly increased the area and density of nAChRs clusters on C2 C12 myotubes treated with serum from septic mice, decreased the expression of p-CLASP2,and enhanced the interaction between CLASP2 and α-tubulin. Conclusion Septic mouse serum impairs nAChRs aggregation on C2 C12 myotubes possibly by suppressing AKT/GSK3β phosphorylation to cause reduced interaction between CLASP2 andα-tubulin.