柚皮甙通过Akt信号通路降低肌细胞ROS治疗废用性肌少症

Naringin reduces muscle cell ROS through Akt signaling pathway to treat disuse sarcopenia

目的通过细胞、分子生物学实验明确废用性肌少症的发病机制,进一步探讨柚皮甙治疗废用性肌少症的分子机制。方法通过鼠尾悬吊的方法制作废用性肌少症小鼠模型(模型组),原代培养肌细胞,使用CCK-8检测柚皮甙对肌细胞增殖能力的影响;2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)和荧光显微镜检测细胞内活性氧(ROS)水平;实时荧光定量PCR(RT-PCR)和Western blot检测蛋白激酶B(Akt)信号通路相关蛋白及mRNA表达情况。结果与对照组(柚皮甙0 ng/mL)比较,柚皮甙能促进肌细胞的增殖(P<0.05),且浓度在500 ng/mL时其促进肌细胞增殖的能力最强。模型组的原代肌细胞内ROS水平较对照组显著增高(P<0.05);而免疫荧光结果也发现柚皮甙干预能降低模型组肌细胞的ROS水平。同时与对照组比较,模型组肌细胞的Sod2 mRNA水平显著下降(P<0.05);Western blot结果显示:与对照组比较,双氧水组(模拟模型组ROS增高)的p-AKT水平显著降低(P<0.05);而与双氧水组比较,双氧水+柚皮甙组p-AKT水平显著增高(P<0.05)。另外与模型+柚皮甙组比较,模型+甙柚皮+曼青霉素组的Sod2 mRNA表达水平显著降低(P<0.05)。结论柚皮甙是一种良好的治疗废用性肌少症的药物,其治疗废用性肌少症的机制可能是通过激活Akt信号通路降低废用性肌少症肌细胞内ROS的水平并促进肌细胞增殖。

Objective To clarify the pathogenesis of disuse sarcopenia by cell and molecular biology experiments,and to further explore the molecular mechanism of naringin in the treatment of disuse sarcopenia.Methods The mouse model of disuse sarcopenia was made,and the primary muscle cells were cultured.CCK-8 was used to detect the effect of naringin on the proliferation of muscle cells.DCFH-DA and fluorescence microscopy were used to detect the intracellular reactive oxygen species(ROS)levels.Western blot and RT-PCR was used to detect the expression leves of protein and mRNAs of Akt signaling pathway related proteins.Results Compared with the control group,the naringin intervention group could promote the proliferation of muscle cells(P<0.05),and its concentration at 500 ng/mL showed strongest ability to promote the proliferation of muscle cells.The ROS level in the muscle cells of the experimental group(rat tail suspension group)was significantly higher than that of the control group(P<0.05);when compared with the control group,the Sod2 mRNA level in the model group was significantly reduced(P<0.05).Western blot results showed that the level of p-AKT in the hydrogen peroxide group was significantly reduced(P<0.05),when compared with the control group,while when compared with the model group,the level of p-AKT in the hydrogen peroxide+naring in group was significantly increased(P<0.05).In addition,compared with the model+naringingroup,the expression levels of Sod2 mRNA in the model+naringin+wortmannin group was significantly reduced(P<0.05).Conclusion Naringin is a good drug for the treatment of disuse sarcopenia.The mechanism may be through reducing the level of ROS in muscle cells of suspended mice and promoting the proliferation of muscle cells by activating the Akt signaling pathway.