阿仑膦酸钠治疗失神经骨骼肌萎缩的实验研究  

Experimental study of alendronate sodium in the treatment of denervated skeletal muscle atrophy

作　　者：田爱现 马剑雄[1,2] 马信龙 李岩[1,2] Tian Aixian;Ma Jianxiong;Ma Xinlong;Li Yan(Department of Orthopaedic Institute,Tianjin Hospital,Tianjin University,Tianjin 300050,China;Tianjin Key Laboratory of Orthopaedic Biomechanics and Medical Engineering,Tianjin 300050,China)

机构地区：[1]天津市天津医院(天津大学天津医院)骨科研究所,天津300050 [2]天津市骨科生物力学与医学工程重点实验室,天津300050

出　　处：《中华骨科杂志》2022年第13期856-863,共8页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金面上项目(81871777, 11772226);天津市卫生健康委科技项目(RC20204);天津市骨科研究所科研基金(2019TJGYSKY03)。

摘　　要：目的通过观察阿仑膦酸钠(alendronate, ALN)对小鼠失神经骨骼肌萎缩肌肉组织中自噬信号通路相关蛋白LC3、Beclin-1和P62表达的影响, 探讨其治疗骨骼肌萎缩的分子生物学机制。方法使用随机数方法将30只雄性C57BL/6小鼠分为3组(每组10只):空白对照组, 暴露坐骨神经不切除;模型组, 暴露坐骨神经并切除;ALN组, 切除坐骨神经+ALN干预。造模阶段采用坐骨神经切断术建立失神经骨骼肌萎缩小鼠模型;干预阶段给予小鼠1 mg/kg ALN灌胃治疗。采用湿重称量法称量小鼠腓肠肌质量;采用肌球蛋白ATP酶染色区分肌纤维类型;采用HE染色观察各组腓肠肌肌纤维排列和横截面积, 并通过Image J进一步定量计算分析;行RT-qPCR、Western blotting和免疫组织化学染色检测各组腓肠肌组织中MHC、MuRF1表达以及LC3Ⅰ、LC3Ⅱ、Beclin-1和P62表达情况。结果与空白对照组比较, 模型组小鼠腓肠肌质量显著降低, 证明小鼠失神经骨骼肌萎缩模型造模成功。与模型组比较, ALN组小鼠腓肠肌质量明显升高。HE染色示模型组肌纤维排列松散, 肌纤维间出现较多蓝染, 横截面积显著小于空白对照组;ATP酶染色示模型组Ⅱ型肌纤维分布较空白对照组增多, ALN组Ⅱ型肌纤维分布较模型组减少, 但高于空白对照组。Image J定量计算肌纤维横截面积结果示模型组肌纤维横截面积较空白对照组显著减小, ALN处理后肌纤维横截面积有所恢复。RT-qPCR和Western blotting示与空白对照组比较, 模型组小鼠腓肠肌组织中MHC、LC3、Beclin-1表达明显降低, 差异有统计学意义(均P<0.05), 而MuRF1、P62蛋白明显升高, 差异有统计学意义(均P<0.05)。ALN组MHC、LC3、Beclin-1明显高于模型组, 而ALN组MuRF1、P62明显低于模型组。免疫组织化学染色表明, 与空白对照组比较, 模型组小鼠腓肠肌中MHC表达显著降低, ALN组较模型组腓肠肌中MHC表达升高(P<0.05)。结论 ALN对骨Objective To observe the effects of alendronate(ALN)on the expression of autophagy signaling pathway related proteins LC3,Beclin-1 and P62 in the muscle tissue of mice with denervated skeletal muscle atrophy,and to explore the potential molecular biological mechanism of ALN in the treatment of skeletal muscle atrophy.Methods Thirty males C57BL/6 mice were divided into three groups with 10 mices in each group by random number method,including blank control group:sciatic nerve exposed without resection,model group:sciatic nerve exposed and resection,ALN group:sciatic nerve resection+ALN intervention.At the intervention stage,mices were given 1 mg/kg ALN by intragastric administration.The weight of gastrocnemius muscle was weighed by wet weight method.Atpase staining was used to distinguish muscle fiber types.HE staining was used to observe the arrangement and cross-sectional area of gastrocnemius muscle fibers in each group,and further quantitative analysis was performed by Image J 1.48 software.Western blotting and immunohistochemical staining were performed to detect the expressions of MHC and MuRF1 as well as LC3,Beclin-1 and P62 in gastrocnemius tissues of each group.Results The weight of gastrocnemius muscle in the model group 137±7.80 mg was significantly lower than that in the blank control group 203±10.34 mg,which proved that the denervation muscle atrophy mouse model was successfully established.After intervention,the gastrocnemius muscle weight of ALN group 177±11.65 mg was significantly higher than that of model group,and the muscle mass was significantly improved.HE staining showed that muscle fibers in the model group were loosely arranged and the cross-sectional area was significantly smaller than that in the blank control group,and there were more blue stains among muscle fibers.Atpase staining showed that the distribution of type II muscle fibers in the model group was increased compared with that in the blank control group,and the distribution of type II muscle fibers in the ALN group was decrea

关 键 词：阿屈膦酸盐 肌萎缩 自噬 对比研究 

分 类 号：R681[医药卫生—骨科学]