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花生四烯酸细胞色素P450ω羟化酶Cyp4a14基因敲除对骨骼肌损伤后再生的影响

张聪聪, 韩迎春, 程乃萱, 杜杰^*

首都医科大学附属北京安贞医院血管生物研究室，北京市心肺血管疾病研究所，北京 100029

摘要

本研究旨在探讨花生四烯酸细胞色素P450ω羟化酶CYP4A14在骨骼肌损伤后再生中的作用及其机制。在野生型(wild-type, WT)对照小鼠和Cyp4a14基因敲除(Cyp4a14 knockout, A14−/−)小鼠胫骨前肌注射心脏毒素(cardiotoxin, CTX)制备骨骼肌损伤模型。损伤后0、3、5和15天取双侧胫骨前肌，麦胚凝集素(wheat germ agglutinin, WGA)染色和天狼猩红染色观察损伤骨骼肌再生和纤维化程度，免疫组织化学染色观察细胞增殖相关蛋白Ki-67和巨噬细胞标志蛋白Mac-2的表达，real-time PCR检测骨骼肌中再生相关基因和炎症相关基因的表达。结果显示，损伤后15天时A14−/−小鼠的新生肌纤维横截面面积显著小于WT小鼠(P < 0.05)，间质纤维化多于WT小鼠(P < 0.05)。损伤后5天时A14−/−小鼠骨骼肌中Ki-67+增殖细胞的比例少于WT小鼠，与肌母细胞分化相关基因Myod1和Myog的表达也显著低于WT小鼠(P < 0.05)。损伤后3天时A14−/−小鼠骨骼肌中CD45和CD11b基因mRNA表达水平以及Mac-2+巨噬细胞比例显著低于WT小鼠(P < 0.05)，同时巨噬细胞分泌的促进肌母细胞增殖和分化的细胞因子IL-1β、IGF-1和SDF-1的表达也显著低于WT小鼠(P < 0.05)。这些结果表明花生四烯酸细胞色素P450ω羟化酶CYP4A14在骨骼肌损伤后再生的过程中发挥重要作用。

关键词： 骨骼肌再生; 花生四烯酸; 细胞色素P450ω羟化酶; 肌母细胞; 炎症反应

分类号：R339.4

Effect of arachidonic acid cytochrome P450ω hydroxylase Cyp4a14 gene knockout on skeletal muscle regeneration after injury

ZHANG Cong-Cong, HAN Ying-Chun, CHENG Nai-Xuan, DU Jie^*

Vascular Biology Department of Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart, Lung and Vascular Diseases, Beijing 100029, China

Abstract

The objective of this study was to explore the roles of arachidonic acid cytochrome P450ω hydroxylase CYP4A14 in skeletal muscle regeneration after injury. Wild-type (WT) control mice and Cyp4a14 knockout (A14−/−) mice were used to establish the muscle injury and regeneration model by intramuscular injection with cardiotoxin (CTX) on the tibial anterior (TA) muscle. The TA muscles were harvested at the time points of 0, 3, 5 and 15 days after injury. The changes in skeletal muscle regeneration and fibrosis were assessed by wheat germ agglutinin (WGA) staining and Sirius Red staining. Immunohistochemical staining was used to observe the expression of proliferation-related protein Ki-67 and macrophage marker protein Mac-2. The mRNA levels of regeneration and inflammation associated genes were analyzed by real-time PCR. The results showed that the cross-section area (CSA) of regenerated myofibers in A14−/− mice was significantly smaller (P < 0.05), while the percentage of fibrosis area was significantly higher than those in WT mice at 15 days after injury (P < 0.05). In A14−/− muscles, both the ratio of Ki-67 positive proliferating cells and the mRNA levels of differentiation associated genes Myod1 and Myog were significantly lower than those in WT muscles (P < 0.05). At 3 days after injury, the mRNA expression of inflammatory cells marker genes CD45 and CD11b and Mac-2 positive macrophages in A14−/− muscles were significantly lower than those in WT skeletal muscle (P < 0.05). Macrophages derived pro-regeneration cytokines IL-1β, IGF-1 and SDF-1 were also significantly decreased in A14−/− muscles (P < 0.05). These results suggest that arachidonic acid cytochrome P450ω hydroxylase CYP4A14 plays a critical role in skeletal muscle regeneration after injury.

Key words: skeletal muscle regeneration; arachidonic acid; cytochrome P450ω hydroxylase; myoblast; inflammation

收稿日期：2020-12-08　　录用日期：2021-03-11

通讯作者：杜杰　　E-mail: jiedu@ccmu.edu.cn

DOI: 10.13294/j.aps.2021.0049

引用本文：

张聪聪, 韩迎春, 程乃萱, 杜杰. 花生四烯酸细胞色素P450ω羟化酶Cyp4a14基因敲除对骨骼肌损伤后再生的影响[J]. 生理学报 2021; 73 (4): 577-583.

ZHANG Cong-Cong, HAN Ying-Chun, CHENG Nai-Xuan, DU Jie. Effect of arachidonic acid cytochrome P450ω hydroxylase Cyp4a14 gene knockout on skeletal muscle regeneration after injury. Acta Physiol Sin 2021; 73 (4): 577-583 (in Chinese with English abstract).