Center for Reproductive Medicine, Department of Pediatrics, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.
Center for Reproductive Medicine, Department of Pediatrics, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China
Ann Clin Lab Sci. 2024 Jul;54(4):489-497.
To investigate the molecular mechanism by which DNA damage induced apoptosis suppressor (DDIAS) regulates STAT3/CCL2 to enhance macrophage polarization to M1 type in Kawasaki disease (KD).
A KD vascular model was established by culturing human coronary artery endothelial cells (HCAECs) . Small interfering RNA of DDIAS (si-DDIAS) was transfected into the KD cell model. The human macrophage cell line THP-1 was induced into M1 macrophages using phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) and co-cultured with the endothelial cells using the HCAECs medium. Western blot analysis was utilized to assess cellular DDIAS, p-STAT3, STAT3, and CCL2 protein expression. MTT was utilized to detect cell proliferation. ELISA was utilized to assess the expression levels of TNF-, IL-4, IL-6, IL-8 and CCL2 in cell supernatants. Flow cytometry was utilized to examine cell apoptosis and the expression of M1 macrophage surface marker CD86.
The expression level of DDIAS was elevated in the KD group compared to the Control group. Serum inhibition of HCAEC proliferation in the KD group was concentration-dependent and pro-inflammatory cytokines were substantially elevated, while the anti-inflammatory cytokines were substantially reduced (<0.05). Compared to the si-NC group, cell proliferation was considerably enhanced; pro-inflammatory cytokines were substantially reduced; anti-inflammatory cytokines were substantially elevated, and the expression of p-STAT3 and CCL2 was lowered in the si-DDIAS group (<0.05). The percentage of M1 macrophages was substantially elevated in the THP-1+LPS group compared to the THP-1 group (<0.05). Compared to the THP-1+LPS+si-NC group, macrophage CCL2 expression was decreased in the THP-1+LPS+si-DDIAS group; the percentage of M1 macrophages was substantially lowered (<0.05); and the levels of pro-inflammatory cytokines and CCL2 in the cell supernatant were substantially reduced. Incubation of macrophages with STAT3 agonist reversed these changes, which were exacerbated by the addition of neutralizing antibody CCL2.
Downregulation of DDIAS inhibits macrophage polarization toward the M1 type through inhibition of the STAT3/CCL2 signaling pathway and can ameliorate vascular injury and inflammation in KD coronary arteries.
研究 DNA 损伤诱导凋亡抑制因子（DDIAS）通过调节 STAT3/CCL2 增强川崎病（KD）中巨噬细胞向 M1 型极化的分子机制。
培养人冠状动脉内皮细胞（HCAEC）建立 KD 血管模型。将 DDIAS 的小干扰 RNA（si-DDIAS）转染至 KD 细胞模型。用佛波醇肉豆蔻酸酯（PMA）和脂多糖（LPS）诱导人巨噬细胞系 THP-1 成为 M1 巨噬细胞，并在 HCAEC 培养基中与内皮细胞共培养。Western blot 分析用于评估细胞内 DDIAS、p-STAT3、STAT3 和 CCL2 蛋白表达。MTT 用于检测细胞增殖。ELISA 用于评估细胞上清液中 TNF-α、IL-4、IL-6、IL-8 和 CCL2 的表达水平。流式细胞术用于检测细胞凋亡和 M1 巨噬细胞表面标志物 CD86 的表达。
与对照组相比，KD 组中 DDIAS 的表达水平升高。KD 组血清抑制 HCAEC 增殖呈浓度依赖性，促炎细胞因子显著升高，而抗炎细胞因子显著降低（<0.05）。与 si-NC 组相比，细胞增殖明显增强；促炎细胞因子显著减少；抗炎细胞因子显著升高，p-STAT3 和 CCL2 的表达降低（<0.05）。与 THP-1 组相比，THP-1+LPS 组的 M1 型巨噬细胞百分比明显升高（<0.05）。与 THP-1+LPS+si-NC 组相比，THP-1+LPS+si-DDIAS 组巨噬细胞 CCL2 表达降低；M1 型巨噬细胞百分比明显降低（<0.05）；细胞上清液中促炎细胞因子和 CCL2 的水平明显降低。用 STAT3 激动剂孵育巨噬细胞可逆转这些变化，并用中和抗体 CCL2 可加重这些变化。
下调 DDIAS 通过抑制 STAT3/CCL2 信号通路抑制巨噬细胞向 M1 型极化，可改善 KD 冠状动脉中的血管损伤和炎症。
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