维生素D对老龄大鼠超负荷诱导的骨骼肌肥大及维生素D受体表达的影响

Effect of vitamin D on overload-induced hypertrophy of skeletal muscle and expression of vitamin D receptor in aged rats

目的采用腓肠肌远端腱切术方案建立老龄大鼠骨骼肌肥大模型,探讨维生素D补充对老龄大鼠骨骼肌肥大的影响及可能机制。方法雄性大鼠(24个月龄)20只,手术切断左后肢单侧腓肠肌远端肌腱造成超负荷骨骼肌肥大模型(Overload,Ovld侧),另一侧行假手术(Sham侧)。造模完成后将大鼠随机分为对照组和维生素D组,维生素D组每天灌胃1 000IU/kg的维生素D,对照组灌胃等量大豆油(溶剂),干预1周后处死。无菌条件下取腹主动脉血待检;取左、右后肢跖肌称量,并液氮速冻,置低温冰箱保存待测。采用ELISA法检测血清25(OH)D含量和骨骼肌中维生素D受体(VDR)的含量;采用蛋白质印迹法检测mTOR、rpS6蛋白及其磷酸化水平的表达。结果在实验期间,2组大鼠摄食量和体质量差异无统计学意义。2组大鼠Ovld侧跖肌质量均较Sham侧增加(P<0.05),且维生素D Ovld侧跖肌的质量高于对照组(P<0.05),而2组Sham侧跖肌质量差异无统计学意义。ELISA结果显示,补充维生素D提高了大鼠血清25(OH)D水平(与对照组比较P<0.05),促进了Ovld侧肌组织VDR的表达(与Sham侧比较,P<0.05),而对照组Ovld侧肌组织VDR的表达与Sham侧比较差异无统计学意义。蛋白质印迹结果显示,2组Ovld侧肌组织中p-mTOR/mTOR、p-rpS6/rpS6比值均比Sham侧增加,但仅在维生素D组差异有统计学意义(P<0.05)。结论腓肠肌远端腱切术能够有效促进老龄大鼠骨骼肌肥大,而维生素D补充能进一步促进骨骼肌肥大,其机制可能与维生素D促进老龄大鼠骨骼肌中VDR的表达和蛋白质合成相关信号蛋白mTOR、rpS6的表达有关。

Objective To establish an overload-induced hypertrophy model in aged rats by severing the distal tendon of gastrocnemius muscle, and to investigate the effect of vitamin D on overload-induced hypertrophy and the related mechanism. Methods A total of 20 male rats （24 months old） underwent tenotomy of the achilles tendon of the gastrocnemius muscle in the left hind limb; and a control sham operation was performed on the right hind limb. The rats were randomly divided into control group and experimental group. Experimental group received 1 000 IU/kg of vitamin D by intragastric administration, and the control group was given soybean oil. The animals were sacrificed one week later, the blood samples were collected, and the left, right hind musculus plantaris tissues were weighed and kept in liquid nitrogen. ELISA assay was used to examine serum 25（OH）D level and vitamin D receptor （VDR） in the skeletal muscle. Western blotting analysis was used to examine mTOR, rpS6 protein and their phosphorylation. Results The food intake and body mass were not significantly different between the two groups. Compared with the control side, vitamin D supplement significantly increased the muscle mass of the overload side in both groups （P〈0.05）; and the mass of the overload side in the vitamin D supplement group was significantly higher than that in the control group （P〈0.05）, while the mases were not significantly different for the sham sides in the two groups. The results of ELISA assay showed that vitamin D supplement significantly increased serum 25（OH）D levels in rats compared with the control group （P〈0.05）, and significantly promoted the expression of VDR in the overload side compared with the Sham side （P〈0.05） , while there was no significant difference between the two sides in the control group. Western blotting analysis showed that p-rpS6/rpS6 and p-mTOR/mTOR ratios in the overload sides were higher than those in the Sham sides, but significant difference was only found for the vitamin D supplement group （P〈0.05）. Conclusion Tenotomy of the achilles tendon of the gastrocnemius muscle can effectively promote the skeletal muscle hypertrophy in aging rats, and vitamin D supplement can further enhance overload-induced skeletal muscle hypertrophy, which might be related to VDR expression in skeletal muscle and protein synthesis protein mTOR and rpS6.