信号传导与转录激活子4 信号通路在脂肪酶/Toll 样受体4调控M1/M2 型巨噬细胞分化的作用及机制

王万虹; 史波; 张荣林; 丁浩; 李锦爽; 李志龙; 叶力硕

文章摘要

王万虹,史波,张荣林,等.信号传导与转录激活子4 信号通路在脂肪酶/Toll 样受体4调控M1/M2 型巨噬细胞分化的作用及机制[J].安徽医药,2022,26(8):1597-1601.

信号传导与转录激活子4 信号通路在脂肪酶/Toll 样受体4调控M1/M2 型巨噬细胞分化的作用及机制

Role and mechanism of STAT4 signaling pathway in LPS / TLR4 regulating M1 / M2 macro?phage differentiation

DOI：10.3969/j.issn.1009-6469.2022.08.026

中文关键词: 动脉硬化颈动脉信号传导与转录激活子4信号通路 M1/M2巨噬细胞机制

英文关键词: Arteriosclerosis Carotid arteries STAT4 signaling pathway M1/M2 macrophages Mechanism

基金项目:宿迁市科技计划项目(S201716)

作者	单位	E-mail
王万虹	南京鼓楼医院集团宿迁医院、徐州医科大学附属宿迁医院，江苏宿迁223800
史波	宿迁市中医院，江苏宿迁223800
张荣林	南京鼓楼医院集团宿迁医院、徐州医科大学附属宿迁医院，江苏宿迁223800	zhangronglin2004@hotmail.com
丁浩	南京鼓楼医院集团宿迁医院、徐州医科大学附属宿迁医院，江苏宿迁223800
李锦爽	南京鼓楼医院集团宿迁医院、徐州医科大学附属宿迁医院，江苏宿迁223800
李志龙	南京鼓楼医院集团宿迁医院、徐州医科大学附属宿迁医院，江苏宿迁223800
叶力硕	江西财经大学统计学院，江西南昌333000

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中文摘要:

目的探讨信号传导与转录激活子4(STAT4)信号通路在脂肪酶(LPS)/Toll样受体4(TLR4)调控M1/M2型巨噬细胞分化的作用及机制。方法用野生型和STAT4-KO小鼠建立动脉内膜拉伤-增生模型，在术后1、3、7和14 d用流式细胞术分析免疫细胞M1/M2型巨噬细胞在外周血内的百分比变化。用免疫荧光双染检测M1，M2型巨噬细胞的表达。用实时荧光定量PCR(q-RT PCR)检测血管组织中STAT4和TLR4表达；酶联免疫吸附试验(ELISA)检测血管生长因子VEGF、PDGF-BB变化。从STAT4KO和蛋白质印迹法小鼠骨髓分离CD11b+细胞，用集落刺激因子GM-CSF和LPS处理，细胞实验分为WT con组、WT+LPS刺激组、STAT4KO con组、STAT4KO+LPS刺激组。观察巨噬细胞的分化情况。结果与野生组相比，STAT4-KO组在术后1、3、7和14天时M1 型巨噬细胞F4/80+iNOS+，F4/80+IL-12的细胞百分比降低(P<0.05)，而M2型巨噬细胞F4/80+Arg-1+和F4/80+CD206+ 的细胞百分比明显升高(P<0.05)。STAT4-KO组的M1，M2型巨噬细胞阳性表达率(9.42±0.41)%、(89.48±10.43)%与野生组(11.14±1.49)%、(48.73±5.89)%比较，差异有统计学意义(P<0.05)。STAT4 和TLR4 在STAT4-KO 组［(0.23±0.04)、(0.47±0.06)］中的表达量明显较野生组［(1.31±0.07)、(0.89±0.08)］低(P<0.05)。VEGF、PDGF-BB 在STAT4-KO 组［(9.28±1.02)、(10.56±1.62)］中的表达量明显较野生组［(3.14±0.91)、(4.23±0.84)］高(P<0.05)。M2型巨噬细胞的诱导因子(IL-4)及分泌因子(IL-10)在STAT4KO+LPS 刺激组、WT+LPS 刺激组、STAT4KO con 组中的表达量明显较WT con 组高(F=13.412，F=15.012，P<0.05)，其中STAT4KO+LPS刺激组表达量最为显著。结论STAT4信号通路可以促进抑制M1型巨噬细胞和增强M2型巨噬细胞分化。其作用机制可能是通过LPS/TLR4的结合。

英文摘要:

Objective To probe into the role and mechanism of signal transduction and transcription activator 4 (STAT4) signaling pathway in the regulation of M1 / M2 macrophage differentiation by lipase/Toll-like receptor 4 (LPS/TLR4).Methods Wild-type and STAT4-KO mice were used to establish an intimal strain-proliferative model. Flow cytometry was used to analyze the percentage change of the immune cell M1/M2 type macrophages in peripheral blood at 1, 3, 7 and 14 days after surgery. The expressions of M1 and M2 macrophages were checked by immunofluorescence double staining. Real-time fluorescent quantitative PCR (q-RT PCR) was used to detect the expressions of STAT4 and TLR4 in vascular tissues. Enzyme linked immunosorbent assay (ELISA) was used to check the changes of vascular growth factors VEGF and PDGF-BB. CD11b+ cells were isolated from the bone marrow of STAT4KO and WT mice and treated with colony-stimulating factors GM-CSF and LPS. Cell experiments were assigned into WT con group, WT + LPS stimula?tion group, STAT4KO con group, and STAT4KO + LPS stimulation group. The differentiation of macrophages was observed.Results Compared with the wild group, the percentage of M1 macrophages F4/80+ iNOS+, F4/80+IL-12 decreased at 1, 3, 7 and 14 days after sur?gery(P<0.05), while the percentage of M2 macrophages F4/80 + Arg-1+ and F4/80+CD206+ increased significantly (P<0.05). The positive expression rates of M1 and M2 macrophages in the STAT4-KO group, [(9.42±0.41) %, (89.48±10.43) %, respectively] were statistically significant compared with the wild group [(11.14±1.49) %, (48.73±5.89) %, respectively] (P<0.05). The expression levels of STAT4 and TLR4 in the STAT4-KO group [(0.23±0.04), (0.47±0.06), respectively] were significantly lower than those in the wild group [(1.31±0.07), (0.89±0.08)] (P <0.05). The expression levels of VEGF and PDGF-BB in the STAT4-KO group [(9.28±1.02), (10.56±1.62)] were significantly higher than those in the wild group [(3.14±0.91), (4.23±0.84)] (P<0.05). M2 macrophages'induction factor (IL-4) and secre?tion factor (IL-10) were obviously higher in STAT4KO + LPS stimulation group, WT + LPS stimulation group, and STAT4KO con group than in WT con group (F=13.412, F=15.012, P<0.05), of which the expression level in the STAT4KO + LPS stimulation group was the most significant.Conclusion STAT4 signaling pathway can promote the inhibition of M1 macrophages and enhance the differentiation of M2 macrophages, whose mechanism of action may be through the combination of LPS/TLR4.

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