细胞外三磷腺苷对失神经大鼠腓肠肌细胞中FoxO3a通路的影响

The effect of extracellular ATP on the FoxO3a pathway of denervated gastrocnemius muscle cell in rats

目的探讨细胞外三磷腺苷（ATP）对失神经大鼠腓肠肌细胞中FoxO3a／MAFbx通路的影响。方法雌性成年Wistar大鼠54只随机分为3组：失神经对照组，ATP治疗组，健康对照组。其中失神经对照组和ATP治疗组在右侧梨状肌下缘切断坐骨神经，制作失神经动物模型。术后ATP治疗组于右侧腓肠肌内注射ATP0．1mg,／d，左侧腓肠肌内注射等量生理盐水；失神经对照组双下肢注射等量的生理盐水；健康对照组不做任何处理。在术后0,2、7、14、28d分别处死一组大鼠，取其两侧腓肠肌，称肌湿重，计算肌失重比（右侧／左侧），采用实时荧光定量PCR和Western Blot检测大鼠腓肠肌FoxO3a和ikFbx的mRNA和蛋白表达水平以及253位磷酸化的FoxO3a（p-FoxO3a）蛋白表达。结果ATP治疗组肌湿重比高于失神经对照组，差异有统计学意义（P〈0．01）；ATP治疗组FoxO3a的mRNA和蛋白表达略低于失神经对照组，差异无统计学意义（P〉0．05）；MAFbx的mRNA和蛋白表达低于失神经对照组，差异有统计学意义（P〈0．05）；p-FoxO3a的蛋白表达量较对照组明显增加，不同时段差异均有统计学意义（P〈0．05）。结论细胞外ATP可能通过磷酸化FoxO3a进而抑制MAFbx蛋白的表达来延缓大鼠骨骼肌失神经萎缩。

Objective To study the effect of extracellular ATP on the FoxO3a pathway of myocytes of denervated gastrocnemius muscle. Methods Fifty-four female adult Wistar rats were randomly divided into three groups. In the two denervation groups, gastrecnemius muscle denervation was created by transecting the fight sciatic nerve at the inferior border of piriformis. In the ATP treatment group, 0.1 mg/day ATP was injected into the right gastrocnemins muscle while the same amount of normal saline was injected into the left gastmcnemins for 4 weeks continuously. In the denervation control group, gastrecnemins muscle on both sides received normal saline injection. The third group of rats without any surgery and injections served as normal control. At 0, 2, 7, 14 and 28 days postoperatively, rats from each denervation groups were sacrificed to harvest gastrecnemius muscle of both sides. Wet muscles were weighed and wet muscle weight ratio （fight/loft） was calculated. The expression of p-FoxO3aS253, FoxO3a, MAFbx protein and mRNA were detected using Western Blot and quantitative RT-PCR respectively. Results The wet muscle weight ratio of the ATP treatment group was significantly higher than that of the denervation control group （ P 〈 0. 01）. The expression FoxO3a mRNA and protein in the ATP treatment group was lower than these of the denervation control group. The difference, however, was not statistically significant （ P 〉 0.05）. The expression of MAFbx mRNA and protein showed the same trend as that of FoxO3a, but the difference was statistically significant （ P 〈 0.05）. p-FoxO3a protein expression remarkably increased in the ATP treatment group, the difference being statistically significant at different time points when compared with the control group （ P 〈 0. 05）. Conclusion Extracellular ATP can potentially delay denervation muscle atrophy by inhibiting the expression of MAFbx protein via FoxO3a phosphorylafion.