背景:胶原蛋白是含量最为丰富的胞外基质成分,与骨骼肌细胞外基质的结构、功能密切相关,但补充由生物工程技术生产的重组人源胶原蛋白(recombinant human collagen,rhC)对骨骼肌细胞外基质的影响及机制尚不清楚. 目的:探讨补充rhC对离心运动后骨骼肌细胞外基质重塑的影响及作用机制. 方法:将104只8周龄健康雄性C57小鼠随机分为对照组(生理盐水)、rhC低剂量组(0.2 g/kg)、中剂量组(1.0 g/kg)和高剂量组(2.0 g/kg),连续7 d灌胃干预后每组选取2只小鼠,解剖各脏器进行苏木精-伊红染色判断炎性浸润情况,其余小鼠于第8天进行离心运动,分别在离心运动后即刻(0),24,48,96 h观察细胞外基质结构变化,检测小鼠抓握力、血清肌酸激酶活性、骨骼肌组织基质金属蛋白酶2,9,14和基质金属蛋白酶抑制剂2的蛋白水平. 结果与结论:①苏木精-伊红染色观察短期补充rhC对心脏、肝脏、脾、肾无显著影响;②一次性离心运动后,rhC中、高剂量组小鼠抓握力恢复率显著升高(P<0.01);③各组肌酸激酶变化趋势一致,组间无显著性差异;④rhC低剂量组细胞外基质恢复进程快于对照组,rhC中、高剂量组肌束膜较为完整;⑤rhC高剂量组基质金属蛋白酶9蛋白水平显著降低(P<0.05);⑥rhC中、高剂量组基质金属蛋白酶14蛋白水平显著降低(P<0.05);⑦rhC中、高剂量组基质金属蛋白酶2蛋白水平显著降低(P<0.05);⑧rhC中、高剂量组基质金属蛋白酶抑制剂2蛋白水平显著升高(P<0.05);⑨rhC各剂量组基质金属蛋白酶2/基质金属蛋白酶抑制剂2比值显著降低(P<0.05).该研究发现连续7 d预补充1.0,2.0 g/kg rhC可通过调节基质金属蛋白酶和基质金属蛋白酶抑制剂来抑制运动后骨骼肌细胞外基质降解,从而促进小鼠骨骼肌力量恢复.
BACKGROUND:Collagen is the most abundant extracellular matrix component,which is closely related to the structure and function of the extracellular matrix of skeletal muscle,but the effect and mechanism of recombinant human collagen(rhC)produced by bioengineering technology on the extracellular matrix of skeletal muscle are unclear. OBJECTIVE:To investigate the effect of rhC supplementation on the remodeling of skeletal muscle extracellular matrix after eccentric exercise,thereby revealing the possible mechanism by which rhC improves the injury of skeletal muscle extracellular matrix and promote the recovery after exercise. METHODS:A total of 104 healthy male C57 mice aged 8 weeks old were randomly divided into control group(normal saline),low-dose rhC group(0.2 g/kg),medium-dose rhC group(1.0 g/kg),and high-dose rhC group(2.0 g/kg).Two mice in each group were selected after continuous 7 days of intragastric intervention,and organs were dissected for hematoxylin-eosin staining to determine inflammatory infiltrates.On the 8th day,the remaining mice were subjected to eccentric exercise.The structural changes of the skeletal muscle extracellular matrix were observed under scanning electron microscopy immediately(0),24,48,and 96 hours after eccentric exercise.Meanwhile,grip strength,creatine kinase activity,and protein levels of matrix metalloproteinases 2,9,14 and tissue inhibitor of metalloproteinase-2 in skeletal muscle were detected by western blot assay. RESULTS AND CONCLUSION:Hematoxylin-eosin staining results indicated that short-term rhC supplementation showed no significant effects on the morphology of the heart,liver,spleen and kidney.After one-time eccentric exercise,the recovery rate of grip strength in the medium-and high-dose rhC groups was significantly increased(P<0.01).The trend of creatine kinase changes was consistent in all groups and there was no significant difference between groups.The recovery process of the extracellular matrix in the low-dose rhC group was faster than that in the control group,and the muscle tract membrane in the medium-and high-dose rhC groups was more complete.The protein level of matrix metalloproteinase 9 in the high-dose rhC group was significantly decreased(P<0.05).The protein levels of matrix metalloproteinase 14 in the medium-and high-dose rhC groups were significantly decreased(P<0.05).The protein levels of matrix metalloproteinase 2 in the medium-and high-dose rhC groups were significantly decreased(P<0.05).Tissue inhibitor of metalloproteinase-2 protein levels in the medium-and high-dose rhC groups were significantly increased(P<0.05).The ratio of matrix metalloproteinase 2 to tissue inhibitor of metalloproteinase-2 in each rhC group was significantly decreased(P<0.05).To conclude,pre-supplementation of 1.0 and 2.0 g/kg rhC for 7 days can inhibit extracellular matrix degradation in skeletal muscle after exercise by modulating matrix metalloproteinases and matrix metalloproteinase inhibitors,thereby promoting recovery of skeletal muscle strength in mice.