Ding Yang, Wan Shengyun, Ma Long, Wei Kaikai, Ye Kun
Department of Vascular Surgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, Anhui, China.
Department of Vascular Surgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, Anhui, China.
Biochem Biophys Res Commun. 2023 Feb 12;644:62-69. doi: 10.1016/j.bbrc.2023.01.001. Epub 2023 Jan 2.
Hindlimb ischemia (HLI) is an arterial occlusive disease that exposes the patients to the risk of limb gangrene and loss. Polarization of macrophages is related to HLI-induced inflammation. Period circadian regulator 1 (PER1) is a core component of the circadian clock. We first showed, based upon bioinformatics analysis of microarray data, that PER1 expression was reduced in monocytes from patients with critical limb ischemia. The proximal femoral artery in the left hindlimb of male mice was ligated and then the femoral artery and its collateral branches were removed to establish the HLI mouse model. After modeling, a single intramuscular injection of 1 × 10 pfu Ad-PER1 was performed at the adductor and gastrocnemius muscles. The gastrocnemius muscle tissues were collected at day 0, 3, 7, 14, 21 post-HLI. There was obvious pathological necrosis, accompanied with reduced expression of PER1 in the muscle tissues of HLI mice. Expression of CD68 and CD31 seemed to be corresponded to PER1 in gastrocnemius muscle, implying the potential of PER1 in regulating macrophage-related inflammation and angiogenesis. PER1 overexpression diminished myocyte damage, promoted blood flow restoration and improved behavioral scores of HLI mice. Immunostaining of CD31 and α-SMA revealed that PER1 upregulation reversed HLI-induced decreases in capillary and arteriole density. In vitro, RAW264.7 cells were cultured in hypoxia (1% O) for 24 h. The percentage of pro-inflammatory CD86 macrophages (M1 type) was decreased and that of anti-inflammatory CD206+ macrophages (M2 type) was increased when PER1 was overexpressed. Moreover, the expression levels of TNF-α, IL-6 and M1-type marker iNOS were decreased, and levels of IL-10 and M2-type marker Arg-1 were increased by PER1 in gastrocnemius muscle of HLI mice and hypoxia-treated RAW264.7 cells. PER1 might reduce M1 macrophage polarization and promote M2 macrophage polarization, and thus exert anti-inflammatory and pro-angiogenic actions. Our findings suggest that PER1 overexpression promotes functional recovery of mice with HLI through regulating macrophage polarization.
后肢缺血（HLI）是一种动脉闭塞性疾病，会使患者面临肢体坏疽和丧失的风险。巨噬细胞的极化与HLI诱导的炎症有关。周期昼夜节律调节因子1（PER1）是生物钟的核心组成部分。基于对微阵列数据的生物信息学分析，我们首次发现，严重肢体缺血患者单核细胞中的PER1表达降低。结扎雄性小鼠左后肢的股动脉近端，然后切除股动脉及其侧支，以建立HLI小鼠模型。建模后，在内收肌和腓肠肌单次肌内注射1×10 pfu的Ad-PER1。在HLI术后第0、3、7、14、21天收集腓肠肌组织。HLI小鼠的肌肉组织出现明显的病理坏死，同时PER1表达降低。腓肠肌中CD68和CD31的表达似乎与PER1相关，这意味着PER1在调节巨噬细胞相关炎症和血管生成方面具有潜力。PER1过表达减少了HLI小鼠的心肌细胞损伤，促进了血流恢复并改善了行为评分。CD31和α-SMA的免疫染色显示，PER1上调逆转了HLI诱导的毛细血管和小动脉密度降低。在体外，将RAW264.7细胞在低氧（1% O）条件下培养24小时。PER1过表达时，促炎CD86巨噬细胞（M1型）的百分比降低，抗炎CD206+巨噬细胞（M2型）的百分比增加。此外，HLI小鼠腓肠肌和低氧处理的RAW264.7细胞中，PER1使肿瘤坏死因子-α、白细胞介素-6和M1型标志物诱导型一氧化氮合酶的表达水平降低，白细胞介素-10和M2型标志物精氨酸酶-1的水平升高。PER1可能减少M1巨噬细胞极化并促进M2巨噬细胞极化，从而发挥抗炎和促血管生成作用。我们的研究结果表明，PER1过表达通过调节巨噬细胞极化促进HLI小鼠的功能恢复。
