目的 探讨左旋肉碱经Runx2/COL1A1通路抑制老年鼠肿瘤恶液质的骨骼肌纤维化。方法 用结肠癌MC38细胞在C57老 年鼠右侧腹股沟皮下植瘤构建肿瘤恶液质模型，实验分为非荷瘤组、荷瘤组和左旋肉碱组，并进行相应干预。实验结束后取一侧腓 肠肌称重，行苏木精⁃伊红染色（HE染色）和Masson染色后，分别测量腓肠肌横截面积和胶原纤维面积占比；对侧腓肠肌提取总蛋白， 蛋白质印迹法检测Runx2和COL1A1蛋白水平。体外实验用转化生长因子-β1（TGF-β1）诱导NIH/3T3细胞，建立体外纤维化模型， 经左旋肉碱干预后，分别提取总蛋白和mRNA，蛋白质印迹法检测Runx2和COL1A1蛋白水平，并用qRT⁃PCR检测COL1A1 mRNA水 平，转染cDNA⁃Runx2验证左旋肉碱通过Runx2调控COL1A1。结果 三组进行比较，荷瘤组腓肠肌重量明显低于非荷瘤组（[ 98.12± 17.04）mg 比 (122.18±6.91）mg（] P<0.05）；荷瘤组腓肠肌横截面积（207.46±54.55）µm2 显著低于非荷瘤组（488.61±46.72）µm2 和左旋肉 碱组（434.54±113.84）µm（2 P<0.05）；胶原纤维面积占比荷瘤组（9.69±1.55）%明显高于左旋肉碱组（5.48±1.19）%和非荷瘤组（3.88±0.86）% （P<0.05）。蛋白质印迹法结果显示，与其他两组相比，荷瘤组Runx2和COL1A1高表达（P<0.05）。体外研究表明，左旋肉碱可逆转 TGF⁃β1诱导的Runx2蛋白和COL1A1 mRNA高表达。过表达Runx2结果证实左旋肉碱通过抑制Runx2蛋白的表达下调COL1A1 mRNA。 结论 左旋肉碱通过降低Runx2/COL1A1改善老年鼠肿瘤恶液质的骨骼肌纤维化。

Objective To investigate the inhibitory effect of L⁃carnitine on skeletal muscle fibrosis in aged mice with cancer cachexia via Runx2/COL1A1 pathway. Methods A cancer cachexia model was constructed by subcutaneously implanting colon cancer MC38 cells in the right inguinal groin of C57 aged mice. The mice were divided into non⁃tumor⁃bearing group (NTB group), tumor⁃bearing group (TB group) and L⁃carnitine group (LC group), with respect to corresponding interventions. After the cessation of the intervention, one side of the gastrocnemius muscle (GM) was weighed. Hematoxylin and eosin (HE) staining and Masson staining were then performed, and the cross⁃sectional area of the GM and the percent of collagen fiber area were measured respectively. The total protein from the opposite side of the GM was extracted, and the levels of Runx2 and COL1A1 protein were detected by Western blotting. NIH/3T3 cells were induced using transforming growth factor⁃β1 (TGF⁃β1) to establish fibrosis model in vitro. Total protein and mRNA were extracted after L⁃carnitine intervention, respectively. Runx2 and COL1A1 protein levels were detected by Western blotting, and COL1A1 mRNA levels were detected by qRT⁃PCR. cDNA⁃Runx2 was transfected to verify that COL1A1 was regulated by the L⁃carnitine through Runx2. Results By comparing the three groups, the weight of the GM in the TB group was significantly lower than that of the NTB group [(98.12±17.04)mg vs (122.18±6.91)mg] (P<0.05); the cross⁃sectional area of the GM in the TB group (207.46±54.55)µm2 was significantly lower than the NTB group (488.61±46.72) µm2 and the LC group (434.54±113.84)µm2 (P<0.05); the area of collagen fibers in the TB group (9.69±1.55)% was significantly higher than that of the LC group (5.48±1.19)% and the NTB group (3.88±0.86)% (P<0.05). The results of Western blotting showed that compared with the LC group and the NTB group, Runx2 and COL1A1 were highly expressed in the TB group(P<0.05). In vitro studies showed that L⁃carnitine reversed the TGF⁃β1 induced high expression of Runx2 protein and COL1A1 mRNA. Overexpression of Runx2 confirmed that L⁃carnitine down⁃regulated COL1A1 mRNA by inhibiting Runx2 protein expression. Conclusion L⁃carnitine ameliorates skeletal muscle fibrosis in aged mice with cancer cachexia partly due to its down⁃regulating effects of Runx2 / COL1A1.