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黄博,阮磊,王兰兰,薛惠天,孙梦龙,段苗苗,彭亮.推拿㨰法介导机械敏感性离子通道Piezo1对骨骼肌损伤模型大鼠细胞凋亡的影响[J].湖南中医药大学学报,2023,43(12):2249-2255[点击复制] |
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| 推拿㨰法介导机械敏感性离子通道Piezo1对骨骼肌损伤模型大鼠细胞凋亡的影响 |
| 黄博,阮磊,王兰兰,薛惠天,孙梦龙,段苗苗,彭亮 |
| (湖南中医药大学针灸推拿与康复学院, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 观察推拿㨰法对骨骼肌损伤模型大鼠的机械敏感性离子通道Piezo1及细胞凋亡的影响，探讨推拿㨰法治疗骨骼肌损伤的作用机制。方法 32只SD大鼠随机分成正常组、模型组、㨰法组、㨰法+抑制剂组，每组8只。采用肌内注射200μL虎蛇毒素（notexin，NTX）（10μg/mL）制备大鼠腓肠肌损伤模型。造模24 h后，㨰法组使用实验动物㨰法器模拟㨰法治疗，滚动频率为140次/min，每天2次，每次3 min，连续治疗3 d；㨰法+抑制剂组在实施㨰法治疗前，给予腹腔注射Piezo1抑制剂GsMTx4（270μg/kg），每天1次，连续注射3 d；正常组和模型组腹腔注射等体积生理盐水。采用平衡木测试进行行为学评估，HE染色观察腓肠肌组织显微结构变化，TUNEL染色计算腓肠肌组织细胞凋亡率，Western blot法检测大鼠腓肠肌组织Piezo1、B淋巴细胞瘤-2（B-lymphoma-2，Bcl-2）、B淋巴细胞瘤2相关X蛋白质（B-lymphoma-2 related X protein，Bax）、胱天蛋白酶-3（cysteine aspartic acid specific protease-3，Caspase-3）表达。结果 与正常组比较，模型组和㨰法+抑制剂组平衡木行走时间延长（P<0.01），滑爪次数增加（P<0.01），腓肠肌细胞凋亡率升高（P<0.01），腓肠肌组织Piezo1、Bcl-2蛋白表达降低（P<0.01），Bax、Caspase-3蛋白表达升高（P<0.05），肌细胞破裂、坏死、大小不一，排列稀疏、无规律，周围有大量中性粒细胞和淋巴细胞浸润。与模型组比较，㨰法组平衡木行走时间缩短（P<0.01），滑爪次数减少（P<0.01），腓肠肌细胞凋亡率降低（P<0.01），腓肠肌组织Piezo1、Bcl-2蛋白表达升高（P<0.01），Bax、Caspase-3蛋白表达降低（P<0.05），肌细胞病理情况好转，有少量细胞破裂、萎缩、坏死，细胞间隔缩小，轻度局灶性炎性细胞浸润。与㨰法组比较，㨰法+抑制剂组平衡木行走时间延长（P<0.01），滑爪次数增多（P<0.01），腓肠肌细胞凋亡率增高（P<0.01），腓肠肌组织Piezo1蛋白表达降低（P<0.05），Caspase-3蛋白表达升高（P<0.01），见较多肌细胞萎缩、坏死、大小不一，排列间隔较大，周围仍有一定数量中性粒细胞和淋巴细胞浸润。结论 推拿㨰法可促进骨骼肌运动功能恢复，缓解骨骼肌损伤，其作用机制可能与激活Piezo1，抑制细胞凋亡有关。 |
| 关键词: 骨骼肌损伤 推拿 㨰法 细胞凋亡 Piezo1 胱天蛋白酶-3 B淋巴细胞瘤-2 |
| DOI：10.3969/j.issn.1674-070X.2023.12.015 |
| 投稿时间：2023-09-14 |
| 基金项目:国家自然科学基金项目（82174521）；湖南中医药大学研究生创新课题立项项目（2022CX109）。 |
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| Effects of tuina rolling manipulation on mechanosensitive ion channel Piezo1 on apoptosis of skeletal muscle injury in model rats |
| HUANG Bo,RUAN Lei,WANG Lanlan,XUE Huitian,SUN Menglong,DUAN Miaomiao,PENG Liang |
| (School of Acupuncture-moxibustion, Tuina, and Rehabilitation, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To observe the effects of tuina rolling manipulation on mechanosensitive ion channel Piezo1 and cell apoptosis in skeletal muscle injury model rats, and to explore the mechanism of tuina rolling manipulation in treating skeletal muscle injury.Methods A total of 32 SD rats were randomly assigned into normal group, model group, tuina rolling manipulation group, and tuina rolling manipulation together with inhibitor group, with eight rats in each group. Rat gastrocnemius muscle injury model was prepared by intramuscular injection of 200 μL notexin(NTX)(10 μg/mL). After 24 h of modeling, experimental animal tuina roller was used in tuina rolling manipulation group for simulating the rolling manipulation, and the rolling frequency was 140times/min, twice a day, 3 min each time for 3 d; rolling manipulation together with inhibitor group was given intraperitoneal injection of Piezo1 inhibitor GsMTx4(270 μg/kg) before tuina rolling manipulation, once a day, for 3 d; the normal group and the model group were intraperitoneally injected with the same volume of normal saline. Behavioral evaluation was assessed by the balance beam test, microstructural changes in gastrocnemius muscle tissue were observed by HE staining, the apoptosis rate of gastrocnemius muscle tissue was calculated by TUNEL staining, and the expressions of Piezo1, B-lymphoma-2(Bcl-2), B-lymphoma-2 related X protein(Bax), and cysteine aspartic acid specific protease-3(Caspase-3) in gastrocnemius muscle tissue was measured by Western blot.Results Compared with the normal group, in the model group and tuina rolling manipulation together with inhibitor group, the balance beam walking time was prolonged(P<0.01), the number of paw sliping increases(P<0.01), the apoptosis rate of gastrocnemius muscle cells was higher(P<0.01), the expressions of Piezo1 and Bcl-2 protein in gastrocnemius muscle tissue were lower(P<0.01), and the expressions of Bax and Caspase-3 protein were higher(P<0.05); the muscle cells are broken, necrotic, of different sizes, arranged sparsely and irregularly, surrounded by a large number of neutrophil and lymphocyte infiltrating. Compared with model group, the tuina rolling manipulation group showed shorter balance beam walking time(P<0.01), less paws sliping(P<0.01), lower apoptosis rate of gastrocnemius muscle cells(P<0.01), higher expressions of Piezo1 and Bcl-2protein in gastrocnemius muscle tissue(P<0.01), and lower expressions of Bax and Caspase-3 protein(P<0.05); the pathological condition of muscle cells of rats in the tuina rolling manipulation group was improved, with a small number of cells ruptured, atrophic, necrotic, cell spacing narrowed, and mild focal inflammatory cell infiltration; compared with the tuina rolling manipulation group, the rats in the rolling manipulation together with inhibitor group had longer walking time on the balance beam(P<0.01), more paws sliping(P<0.01), higher apoptosis rate of gastrocnemius muscle cells(P<0.01), lower expression of Piezo1 protein in gastrocnemius muscle tissue(P<0.05), and higher expression of Caspase-3 protein(P<0.01); more muscle cells were atrophic, necrotic, of different sizes, with large arrangement intervals, and there were some neutrophils and lymphocytes infiltrating around.Conclusion Tuina rolling manipulation can promote the recovery of skeletal muscle motor function and alleviate skeletal muscle injury, and its mechanism may be related to the activation of Piezo1 mediated apoptosis. |
| Key words: skeletal muscle injury tuina rolling manipulation apoptosis Piezo1 cysteine aspartic acid specific protease-3 B-lymphoma-2 |
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