Beijing Advanced Innovation Center for Food Nutrition and Human Health, China-Canada Joint Lab of Food Nutrition and Health (Beijing), Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, China.
Burn Institute, the First Affiliated Hospital of PLA General Hospital, Beijing, China.
Shock. 2019 Nov;52(5):513-521. doi: 10.1097/SHK.0000000000001284.
Cell autophagy is an important material recycling process and is involved in regulating many vital activities under both physiological and pathological conditions. However, the mechanism of autophagy regulating burn-induced skeletal muscle wasting still needs to be elucidated.
The rat burn model with 30% total body surface area and L6 cell line were used in this study. An immunofluorescence assay was used to detect autophagic levels. MicroRNA array and real-time PCR were employed to measure miR-190b levels, and its influence on PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) protein translation was estimated using luciferase reporter assay. The expression levels of autophagy-related proteins were analyzed by Western blot. Skeletal muscle wasting was evaluated by the ratio of tibias anterior muscle weight to body weight.
Our study demonstrates that burn injury promotes expression of the autophagy-related proteins light chain 3 (LC3) and Beclin-1, suppresses expression of Akt and Forkhead box O (FoxO) 3a protein phosphorylation, and increases PHLPP1 protein level which is required for Akt dephosphorylation. miR-190b, the regulator of PHLPP1 protein translation, also significantly decreases after burn injury. Ectopic expression of miR-190b in L6 myoblast cell downregulates PHLPP1 protein expression, elevates Akt and FoxO3a phosphorylation, and subsequently reduces cell autophagy. Finally, suppressing autophagy with 3-methyladenine represses the protein expression of LC3 and Beclin-1 and mitigates burn-induced skeletal muscle wasting.
Burn injury induced skeletal muscle cell autophagy and subsequently resulted in skeletal muscle wasting via regulating miR-190b/PHLPP1/Akt/FoxO3a signaling pathway.
细胞自噬是一种重要的物质回收过程，参与调节生理和病理条件下的许多重要活动。然而，自噬调节烧伤诱导的骨骼肌萎缩的机制仍需要阐明。
本研究采用 30%总体表面积烧伤大鼠模型和 L6 细胞系。免疫荧光检测自噬水平。微 RNA 芯片和实时 PCR 用于测量 miR-190b 水平，并通过荧光素酶报告实验评估其对 PH 结构域和富含亮氨酸重复蛋白磷酸酶 1（PHLPP1）蛋白翻译的影响。Western blot 分析自噬相关蛋白的表达水平。通过胫骨前肌重量与体重的比值评估骨骼肌萎缩。
我们的研究表明，烧伤损伤促进自噬相关蛋白 LC3 和 Beclin-1 的表达，抑制 Akt 和 Forkhead box O（FoxO）3a 蛋白磷酸化，增加 Akt 去磷酸化所需的 PHLPP1 蛋白水平。烧伤后 miR-190b，PHLPP1 蛋白翻译的调节因子，也显著减少。L6 成肌细胞中 miR-190b 的异位表达下调 PHLPP1 蛋白表达，增加 Akt 和 FoxO3a 磷酸化，随后减少细胞自噬。最后，用 3-甲基腺嘌呤抑制自噬会抑制 LC3 和 Beclin-1 的蛋白表达，并减轻烧伤引起的骨骼肌萎缩。
烧伤诱导骨骼肌细胞自噬，进而通过调节 miR-190b/PHLPP1/Akt/FoxO3a 信号通路导致骨骼肌萎缩。
