基于FOXO3a/Nrf2信号通路探讨尿毒症骨骼肌自噬的分子机制

The molecular mechanism of skeletal muscle autophagy in uremia:a study based on the FOXO3a/Nrf2 signaling pathway

目的:探讨叉头盒(forkhead box,FOX)O蛋白3a(forkhead box O3a,FOXO3a)/转录因子NF-E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)信号通路的调节尿毒症骨骼肌自噬的机制。方法:24只小鼠随机分为假手术组(Sham组)、慢性肾病(chronic kidney disease,CKD)组和CKD+AST-120(AST)组,每组8只。通过5/6肾切除术建立CKD模型。在肾切除术后4周,CKD+AST组小鼠给予含AST-120粉末(尿毒毒素的木炭吸附剂)饮食,持续8周。透射电子显微镜观察腓肠肌组织中的自噬溶酶体。C2C12小鼠成肌细胞分为对照(Control,Con)组、硫酸吲哚酚(indophenol sulfate,IS)组、IS+si-FOXO3a组。实时RT-PCR和蛋白质印迹法分析FOXO3a表达情况。Giemsa染色对C2C12肌管直径进行量化。结果:与CKD组相比,CKD+AST组血清IS水平和腓肠肌组织中FOXO3a表达、自噬溶酶体的数量明显降低(P<0.05),以及腓肠肌、胫骨前肌、比目鱼肌质量明显增加(P<0.05)。与IS组相比,IS+si-FOXO3a组C2C12细胞中FOXO3a、肌肉萎缩盒基因(Muscle Atrophy F-box,atrogin-1)肌肉环脂蛋白1(muscle RING-finger protein-1,MuRF-1)的表达和细胞自噬通量明显降低(P<0.05),以及细胞直径明显增加(P<0.05)。与Sham组相比,CKD组小鼠腓肠肌组织中Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1,KEAP1)表达明显升高(P<0.05),和NRF2表达明显降低(P<0.05)。AST治疗逆转了CKD小鼠腓肠肌组织中这些蛋白的变化。si-NRF2逆转了FOXO3a敲低对自噬流量的抑制作用。结论:IS可能通过持续刺激FOXO3a-KEAP1-NRF2轴促进骨骼肌过度自噬,参与尿毒症少肌症的发病机制。

Objective:To explore the role of the forkhead box O3a(FOXO3a)/nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway in regulating skeletal muscle autophagy in uremia.Methods:Twenty-four mice were randomly divided into sham group,chronic kidney disease(CKD)group,and CKD+AST-120(AST)group,with eight mice in each group.A CKD model was established by using the 5/6 nephrectomy method.Four weeks after nephrectomy,the mice in the CKD+AST group were fed with powder chow containing AST-120(a charcoal adsorbent for uremic toxin)for eight weeks.Autolysosomes in the gastrocnemius muscle were observed using a transmission electron microscope.C2C12 myoblasts were divided into control group,indophenol sulfate group(IS),and IS+si-FOXO3a group.The expression of FOXO3a was determined using real-time RT-PCR and Western blot.The diameter of C2C12 myotubes was measured with Giemsa staining.Results:Compared with the CKD group,the CKD+AST group showed significant decreases in serum IS level and FOXO3a expression and the number of autolysosomes in the gastrocnemius tissue and significant increases in the masses of the gastrocnemius muscle,anterior tibial muscle,and soleus muscle(all P<0.05).Compared with the IS group,the IS+si-FOXO3a group showed significant reductions in the expression of FOXO3a,atrogin-1,and muscle RING-finger protein-1 in C2C12 cells as well as the autophagic flux of the cells and a significantly increased cell diameter(all P<0.05).Compared with the sham group,the CKD group showed significantly increased expression of Kelch-like ECH-associated protein 1(KEAP1)and significantly decreased Nrf2 expression in the gastrocnemius tissue(both P<0.05).AST treatment reversed the changes in these proteins in the gastrocnemius tissue of CKD mice.Si-Nrf2 reversed the inhibitory effect of FOXO3a knockdown on autophagic flux.Conclusion:IS may promote skeletal muscle autophagy by continuously stimulating the FOXO3A-KEAP1-Nrf2 axis to participate in the pathogenesis of uremic sarcopenia.