白藜芦醇通过抑制TGF-β1、COL-Ⅰ过表达治疗骨骼肌损伤的实验研究

Resveratrol Inhibited Over-expressions of TGF-β,and COL-Ⅰin Treatment of Skeletal Muscle Injury

目的观测骨骼肌急性钝挫伤修复过程中转移生长因子(TGF-β1)、Ⅰ型胶原蛋白(COL-Ⅰ)的表达规律,探讨白藜芦醇(Res)促进受损骨骼肌结构与功能恢复之作用机制。方法33只新西兰兔随机分为3组:正常组(3只)、自然恢复组(15只)、Res组(15只)。除正常组外均制造骨骼肌钝挫伤模型,损伤后自然恢复组不予处理,Res组每天给予Res 15 mg/kg灌胃治疗。分别于伤后1、3、7、14、21天处死动物,采用免疫组织化学(IHC)、免疫印记法(Western Blot)检测骨骼肌中TGF-β1、COL-Ⅰ蛋白表达。结果一般结果:损伤组损伤区肿胀,12 h至肿胀峰值,72 h后肿胀消退至实验前状态。HE显示:正常组兔肌纤维多边形、形态规则、排列紧密,肌核均匀分布于肌膜下,无增生与固缩,肌膜完整;各损伤组第1天见血细胞渗出,第3天炎症细胞开始浸润至7天达峰值,伤后第21天肌纤维形态基本恢复正常,Res组在炎症细胞浸润,修复时间上整体优于自然恢复组。Masson染色显示:正常肌细胞中胶原纤维含量极少,损伤后随着瘢痕组织形成,胶原纤维逐渐增加,并于14天达高峰,Res组胶原纤维含量低于自然恢复组。IHC显示:TGF-β1、COL-Ⅰ主要为胞浆表达,其蛋白含量在损伤后呈现先升后降趋势,自然恢复组和Res组均于7 d达高峰,自然恢复组高于Res组,(P<0.05)。Western Blot显示:TGF-β1、COL-Ⅰ蛋白表达结果与IHC结果相似。结论TGF-β1和COL-Ⅰ蛋白在骨骼肌修复过程中呈现先升高后降低的变化规律,与骨骼肌修复一般规律相类;TGF-β1和COL-Ⅰ蛋白表达量变化与瘢痕组织出现有联系,出现明显瘢痕组织时TGF-β1和COL-Ⅰ蛋白含量也达最大值;Res能抑制TGF-β1和COL-Ⅰ蛋白表达,促进骨骼肌损伤修复,但并不改变骨骼肌损伤修复过程中蛋白表达量的整体变化规律。

Objective To observe the expressions of transforming growth factor(TGF-β1) and collagen typeⅠ(COL-Ⅰ) during the repair of skeletal muscle acute blunt contusion, and to study the effect of resveratrol(Res) in promoting injured muscle structure and function recovery. Methods Thirty-three New Zealand rabbits were randomly divided into three groups: normal group(n=3), natural recovery group(n=15), and Res group(n=15). Except for the normal group, skeletal muscle blunt contusion models were established. After the injury, rabbits in the natural recovery group were not treated. Rabbits in the Res group were given Res at 15 mg/kg per day. They were sacrificed at day 1, 3, 7, 14, and 21 after injury, and the expressions of TGF-β1 and COL-Ⅰ protein in skeletal muscle were detected by immunohistochemistry(IHC) and Western Blot. Results General states: Swelling in injured areas peaked at 12 h. After 72 h, the swelling subsided to the pre-experimental state. HE staining showed muscle fibers in rabbits of the normal group showed polygon, regular shape, and tight arrangement. The muscle nucleus were evenly distributed under the sarcolemma, with no hyperplasia or pyknosis. The sarcolemma was intact;Exudation of blood cells could be seen on the 1 st day in each injury group, and the inflammatory cells began to infiltrate from the 3 rd day and reach the peak on the 7 th day. The morphology of muscle fibers returned to normal basically at day 21. In the Res group, the infiltration of inflammatory cells and the repair time were better than the natural recovery group. Masson staining showed that the collagen fiber content was extremely little in normal muscle cells, and the collagen fiber gradually increased with the formation of scar tissue, and peaked at day 14. The collagen fiber content of the Res group was lower than that of the natural recovery group.. IHC showed that TGF-β1 and COL-Ⅰ were mainly expressed in cytoplasm, and their protein content increased first and then decreased after injury. They reached the peak at day 7 in the natural recovery group and Res group, and they were higher in the natural recovery group than the Res group(P<0.05). Western Blot showed that expressions of TGF-β1 and COL-Ⅰ protein were similar to the IHC results. Conclusions The expressions of TGF-β1 and COL-Ⅰ protein increased first and then decreased during skeletal muscle repair, which was similar to the general rule of skeletal muscle repair. The expressions of TGF-β1 and COL-Ⅰ protein were correlated with the appearance of scar tissue, and the contents of TGF-β1 and COL-Ⅰ protein also reached the maximum when scar tissue appeared. Res inhibited the expressions of TGF-β1 and COL-Ⅰ protein and promoted the repair of skeletal muscle injury, but it did not change the overall changing rules of protein expression during skeletal muscle injury repair.