南京医科大学第一附属医院
国家自然科学基金项目（面上项目）;国家自然科学基金项目（青年项目）；南京医科大学第一附属医院青年基金培育计划
目的：初步探讨liraglutide（利拉鲁肽，Li）对SiO2（二氧化硅，silica）诱导的培养巨噬细胞极化作用及其机制。方法：将巨噬细胞分为5组，分别为：Control组、SiO2组、SiO2+Li（10 nM）组、SiO2+Li（100 nM）组及SiO2+Li（1000 nM）组。CCK-8法检测细胞活性；ELISA法测定各组IL-1β、IL-10、TGF-β1；Western blot测定NLRP3、Caspase-1 p20、精氨酸酶（arginase，Arg）-1；JC-1荧光探针染色法测量线粒体膜电位；DCFH-DA探针检测细胞内ROS水平。结果：SiO2组NLRP3、Caspase-1 p20、Arg-1表达水平较Control组明显增加，差异具有统计学意义；与SiO2组相比，SiO2+Li（10 nM）组、SiO2+Li（100 nM）组及SiO2+Li（1000 nM）组NLRP3、Caspase-1 p20、Arg-1表达水平降低，差异具有统计学意义。SiO2组IL-1β水平为（127.00±17.05）pg/mL，较Control组（37.72±10.23）pg/mL明显增加，差异具有统计学意义；SiO2+Li（10 nM）组、SiO2+Li（100 nM）组及SiO2+Li（1000 nM）组IL-1β水平分别为（77.40±12.55）pg/mL、（64.74±6.86）pg/mL、（41.55±12.74）pg/mL，较SiO2组降低，差异具有统计学意义。SiO2组IL-10水平为（212.70±6.97）pg/mL，较Control组（70.88±3.21）pg/mL明显增加，差异具有统计学意义；SiO2+Li（10 nM）组、SiO2+Li（100 nM）组及SiO2+Li（1000 nM）组IL-10水平分别为（127.80±7.70）pg/mL、（110.80±6.53）pg/mL、（85.13±7.17）pg/mL，较SiO2组明显降低，差异具有统计学意义。SiO2组TGF-β1水平为（854.60±98.00）pg/mL，较Control组（268.40±19.29）pg/mL明显增加，差异具有统计学意义；SiO2+Li（10 nM）组、SiO2+Li（100 nM）组及SiO2+Li（1000 nM）组TGF-β1水平分别为（511.20±66.53）pg/mL、（386.70±32.30）pg/mL、（308.00±89.20）pg/mL，较SiO2组明显降低，差异具有统计学意义。SiO2组细胞内线粒体膜电位较Control组下降；与SiO2组相比，SiO2+Li（10 nM）组、SiO2+Li（100 nM）组及SiO2+Li（1000 nM）组细胞内线粒体膜电位上升。SiO2组ROS相对荧光强度较Control组增加，差异具有统计学意义；与SiO2组相比，SiO2+Li（10 nM）组、SiO2+Li（100 nM）组及SiO2+Li（1000 nM）组ROS相对荧光强度逐渐下降，差异具有统计学意义。结论：Liraglutide可通过改善SiO2诱导的线粒体功能障碍，减轻ROS氧化应激，阻止NLRP3炎症小体活化，抑制SiO2诱导的M2型巨噬细胞极化。
Objective: This study aims to investigate the effects and potential mechanisms of liraglutide (Li) on macrophage polarization induced by silica (SiO2). Methods: THP-1 cells were induced to adhere to macrophages, and five groups were established: Control group, SiO2 group, SiO2+Li group (with Li concentrations of 10 nM, 100 nM, 1000 nM). The cytotoxicity of Li on the macrophages was assessed by using a cell counting kit (CCK)-8 assay. ELISA was employed to analyze the levels of interleukin (IL) -1β, IL-10 and transforming growth factor-β1 (TGF-β1) in the macrophage culture. Western blot assay was used to determine the expression levels of NLRP3, Caspase-1 p20, Arg-1. Mitochondrial membrane potential in the macrophages was detected by using JC-1 fluorescent probe staining. Reactive oxygen species (ROS) levels in the macrophages from all groups were measured by using DCFH-DA probe. Results: Compared to Control group, the expression levels of NLRP3, Caspase-1 p20 and Arg-1 were significantly elevated in SiO2 group. In comparison to SiO2 group, there was a significant decrease in the expression levels of NLRP3, Caspase-1 p20 and Arg-1 in SiO2+Li (10 nM) group, SiO2+Li (100 nM) group and SiO2+Li (1000 nM) group. The level of IL-1β in SiO2 group was significantly higher than that in Control group [(127.00±17.05) pg/mL vs. (37.72±10.23) pg/mL]. In the SiO2+Li groups with different concentrations (10 nM, 100 nM, 1000 nM), IL-1β levels were (77.40±12.55) pg/mL, (64.74±6.86) pg/mL and (41.55±12.74) pg/mL respectively, showing statistically significant differences compared with SiO2 group . The level of IL-10 in SiO2 group was significantly higher than that in Control group [(212.70±6.97) pg/mL vs. (70.88±3.21) pg/mL]. In the SiO2+Li groups with different concentrations (10 nM, 100 nM, 1000 nM), IL-10 levels were (127.80±7.70) pg/mL, (110.80±6.53) pg/mL and (85.13±7.17) pg/mL respectively, showing statistically significant differences compared with SiO2 group. The level of TGF-β1 in SiO2 group was significantly higher than that in Control group [(854.60±98.00) pg/mL vs. (268.40±19.29) pg/mL]. In the SiO2+Li groups with different concentrations (10 nM, 100 nM, 1000 nM), TGF-β1 levels were (511.20±66.53) pg/mL, (386.70±32.30) pg/mL and (308.00±89.20) pg/mL respectively, showing statistically significant differences compared with SiO2 group. The intracellular mitochondrial membrane potential decreased in SiO2 group when compared to Control group. However, it increased in SiO2+Li (10nM) group, SiO2+Li (100 nM) group and SiO2+Li (1000 nM) group when compared to SiO2 group. The relative fluorescence intensity of ROS was higher in SiO2 group than that in Control group with statistical significance observed. Additionally, the relative fluorescence intensity of ROS was lower in SiO2+Li (10 nM) group, SiO2+Li (100 nM) group and SiO2+Li (1000 nM) group than that observed for SiO2 group. Conclusions: Liraglutide exhibits the potential to ameliorate mitochondrial dysfunction, alleviate ROS oxidative stress, inhibit NLRP3 inflammasome activation, and suppress M2-type macrophage polarization induced by SiO2.
