钴原卟啉诱导HO-1表达增加对老龄小鼠骨骼肌组织氧化应激和炎症反应的影响

Effect of cobalt protoporphyrin-induced heme oxygenase 1 un-regulation on oxidative stress and inflammatory responses in skeletal muscle of aged mice

目的探讨注射钴原卟啉(cobalt protoporphyrin,Co PP)诱导血红素加氧酶-1(heme oxygenase 1,HO-1)表达增加在老龄小鼠骨骼肌微环境包括氧化应激、炎症反应的调节作用。方法分别将正常成年(2月龄)和老年(18月龄)C57BL/6雄性小鼠按照简单随机抽样法分为对照组及注射Co PP组;Co PP组小鼠连续3 d腹腔注射Co PP溶液[5 mg/(kg·d)],对照组注射同体积生理盐水。在3、7、14 d后取新鲜腓肠肌组织,Western blot检测HO-1(3 d组)的表达,ELISA检测过氧化标志分子丙二醛(MDA)、过氧化氢(H2O2)含量及抗氧化酶类Cu Zn-超氧歧化酶(SOD)等活性变化。用老年C57BL/6小鼠建立腓肠肌损伤模型,采用简单随机抽样法分为Co PP处理组和对照组,分别在3、7、14 d后检测中性粒细胞和M2型巨噬细胞浸润。结果正常老年组HO-1表达量高于成年组,老年组注射Co PP后HO-1表达升幅显著低于成年组(P<0.05);注射Co PP后,成年和老年组MDA、H2O2含量均下降,SOD、Cu Zn-SOD、GSH-Px和CAT酶活性升高,且老年Co PP组过氧化标志分子含量高于成年Co PP组,抗氧化酶活性较低(P<0.05)。骨骼肌损伤后3、7 d,Co PP处理组中性粒细胞浸润均低于对照组;CD163阳性M2型巨噬细胞浸润出现较晚,且注射Co PP后,M2型巨噬细胞的浸润显著高于对照组。结论老年骨骼肌对Co PP的反应性下降,抗氧化应激能力下降。损伤后诱导HO-1表达增加能够降低早期中性粒细胞、促进M2型巨噬细胞的浸润,可能有助于骨骼肌损伤修复。

Objective To determine the role of systematic heine oxygenase 1 （HO-1） in the regulation of oxidative stress and inflammation in aged skeletal muscle after intraperitoneal injection of cobalt protoporphyrin （CoPP）. Methods Normal adult （2 months old） and aged （18 months old） male C57BL/6 mice were randomly divided into control group and injection group. The mice of the injection group were intraperitoneally injected with CoPP solution [ 5 mg/（ kg · d） ] for 3 consecutive days, and those of the control group were given normal saline at the same volume. Fresh gastrocnemius samples were harvested in 3, 7 and 14 d after injection. The expression of HO-1 was examined with Western blotting. The levels of oxidative stress markers, malondialdehyde （MDA） and hydrogen peroxide （H2O2 ）, and the activity of antioxidant enzyme CuZn-super oxide dismutase （SOD） was detected by ELISA. Then gastrocnemius injury model was established in aged male C57BL/6 mice, and the mice were randomly divided into CoPP treatment and control group. The gastrocnemius was harvested in 3,7 and 14 d after final injection for neutrophil counting and M2 macrophage infiltration. Results The expression of HO-1 was higher in the aged group than the adult group, but the increase of HO-1 induced by CoPP injection was much lesser in the aged mice than adult group （P 〈 0.05 ）. After induction of HO-1, the contents of MDA and H2 O2 were declined while the activities of SOD, CuZn- SOD, GSH-Px and CAT enzyme were enhanced in both adult and aged groups, whereas the level of oxidation markers were higher and the antioxidant enzyme activity was lower in aged than in adult CoPP group （P 〈 0. 05）. CoPP injection resulted in lower neutrophil infiltration than in the control group in 3 and 7 d after muscle injury. The CD163＋ M2 macrophage was infiltrated later, and more macrophage infiltrated in CoPP group than in the control. Conclusion Aged skeletal muscle has markedly lower reactivity to CoPP, and reduced ability to resist oxidative stress. Induction of HO-1 by Copp reduces early neutrophils and promotes M2 macrophage infiltration after injury, which may be helpful in skeletal muscle regeneration.