Abstract: Objective  To investigate whether β-hydroxybutyrate (BHB) alleviates stroke-related sarcopenia (SRS) by regulating protein synthesis and degradation homeostasis via the Tribbles homolog 3 (TRIB3) / protein kinase B (Akt) / mammalian target of rapamycin (mTOR) pathway. 
Methods  Eight-week-old male C57BL/6J mice were used to establish the transient middle cerebral artery occlusion (tMCAO) model by the suture method. After modeling, the mice were randomly divided into the BHB group and the control group: the BHB group was subcutaneously injected with BHB at a dose of 5 mg/kg, once every 8 hours for 3 consecutive days; the control group was injected with an equal volume of physiological saline. The cerebral infarction volume was measured. The skeletal muscle strength of mice was evaluated by the rotarod test, grip strength test, and hanging test. Bilateral tibialis anterior muscle tissue samples of mice were collected for hematoxylin and eosin staining to compare the cross-sectional area of muscle fibers. Bioinformatics analysis was used to screen the differential gene enrichment pathways in the tibialis anterior muscle tissue of tMCAO mice compared with the shame-operation group. Western blot and Real time quantitative PCR were used to detect the expression of Akt/mTOR pathway-related molecules and protein synthesis and degradation-related genes in different groups. The STRING database was used to predict Akt-interacting proteins, and co-immunoprecipitation was performed to verify the protein-protein interaction between TRIB3 and Akt. 
Results  After modeling, the cerebral infarction volume in mice of the BHB group was reduced compared with the control group (P<0.001), the rotarod duration, grip strength, and hanging time were increased (P<0.001) and the cross-sectional area of muscle fibers (P<0.001) were increased. The phosphoinositide 3-kinase (PI3K) / Akt pathway showed the most significant enrichment of differentially expressed genes. The protein expressions of phosphorylated mTOR (p-mTOR) and phosphorylated Akt (p-Akt) were increased in the BHB group (P<0.001). The expression levels of the protein degradation regulatory gene forkhead box protein O3a (FOXO3a) and protein degradation-related genes muscle ring-finger protein-1 (MuRF1) and muscle atrophy F-box (MAFbx) were decreased (P<0.001), while the expression level of the protein synthesis-related gene p70 ribosomal protein S6 kinase (p70S6K) was increased (P<0.001). Co-immunoprecipitation showed that TRIB3 interacted with Akt, and the expression level of TRIB3 decreased after BHB treatment (P<0.001). 
Conclusions  BHB can promote Akt phosphorylation by inhibiting TRIB3 expression, thereby activating the Akt/mTOR pathway, facilitating protein synthesis and inhibiting degradation, and ultimately improving SRS. 

Key words: Stroke-related sarcopenia; β-hydroxybutyrate; Tribbles homolog 3; Middle cerebral artery occlusion

摘要： 目的 探讨β-羟基丁酸（β-hydroxybutyrate，BHB）是否经Tribbles同源蛋白3（Tribbles homolog 3，TRIB3）/蛋白激酶B（protein kinase B，Akt）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）通路调控蛋白质合成和降解平衡，进而改善卒中相关性肌少症（stroke-related sarcopenia，SRS）。
方法 采用8周龄雄性C57BL/6J小鼠，通过线栓法建立短暂性大脑中动脉栓塞（transient middle cerebral artery occlusion，tMCAO）模型。将造模后小鼠随机分为BHB组与对照组：BHB组皮下注射5 mg/kg BHB，每8小时1次，持续3 d；对照组注射等体积生理盐水。测量小鼠脑梗死体积；采用转棒实验、抓握力强度测试和悬挂实验评估小鼠骨骼肌力量；取小鼠双侧胫骨前肌组织样本进行苏木精-伊红染色，比较肌纤维横截面积。通过生物信息学分析筛选tMCAO小鼠与假手术组小鼠相比，胫骨前肌组织中的差异基因富集通路；采用免疫印迹法、实时定量PCR检测Akt/mTOR通路及蛋白质合成和降解相关基因在不同组中的表达；采用STRING数据库预测Akt相互作用蛋白，并通过免疫共沉淀验证TRIB3与Akt蛋白-蛋白相互作用。
结果 与对照组相比，造模后BHB组小鼠的脑梗死体积下降（P<0.001）；转棒时间、握力及悬挂时间增加（P<0.001）；肌纤维横截面积增大（P<0.001）。差异表达基因在磷脂酰肌醇3激酶（phosphoinositide 3-kinase，PI3K）/Akt通路上富集最为显著。BHB组磷酸化哺乳动物雷帕霉素靶蛋白（phosphorylated mTOR，p-mTOR）及磷酸化蛋白激酶B（phosphorylated Akt，p-Akt）蛋白表达增加（P<0.001）；蛋白质降解调控基因叉头盒蛋白O3a（forkhead box protein O3a，FOXO3a）、蛋白质降解相关基因肌肉环指蛋白1（muscle ring-finger protein-1，MuRF1）和肌肉萎缩F盒（muscle atrophy F-box，MAFbx）表达水平降低（P<0.001），而蛋白质合成相关基因p70核糖体蛋白S6激酶（p70 ribosomal protein S6 kinase，p70S6K）表达水平升高（P<0.001）。免疫共沉淀证明TRIB3与Akt存在相互作用，且BHB处理后TRIB3表达水平下降（P<0.001）。
结论 BHB可通过抑制TRIB3表达，促进Akt磷酸化，从而激活Akt/mTOR通路，促进蛋白质合成并抑制降解，改善SRS。

关键词: 卒中相关性肌少症; β-羟基丁酸; Tribbles同源蛋白3; 大脑中动脉栓塞