熊果酸通过miR-224/NLRP3轴抑制牙龈卟啉单胞菌脂多糖刺激的RAW264.7细胞M1极化

Ursolic acid inhibits M1 polarization of RAW264.7 cells stimulated by porphyromonas gingivalis lipopolysaccharide via miR-224/NLRP3 axis

目的:探究熊果酸通过miR-224/NLRP3轴抑制牙龈卟啉单胞菌(Pg)脂多糖(LPS)刺激RAW264.7细胞M1极化的机制。方法:0、5、10、20、40、80μmol/L熊果酸处理RAW264.7细胞,MTT法检测细胞活力。RAW264.7细胞分为对照组、LPS组、熊果酸组、inhibitor control组、miR-224 inhibitor组。qRT-PCR检测miR-224表达,ELISA检测TNF-α、IL-1β、IL-10、Arg-1水平,流式细胞术检测CD11和CD206蛋白表达,双荧光素酶报告基因实验检测miR-224和NLRP3的靶向关系,Western blot检测NLRP3表达。结果:RAW264.7细胞存活率随熊果酸浓度的增加显著降低;与对照组相比,LPS组miR-224表达水平显著降低,NLRP3、TNF-α、IL-1β、CD11蛋白水平显著增加(P<0.05);与LPS组相比,熊果酸组可逆转上述指标变化;miR-224与NLRP3存在靶向结合位点;与熊果酸组相比,miR-224 inhibitor组TNF-α、IL-1β、CD11表达显著增加(P<0.05)。结论:熊果酸可能通过促进miR-224/NLRP3轴来抑制Pg-LPS刺激的RAW264.7细胞M1极化。

Objective: To explore the mechanism by which ursolic acid inhibits porphyromonas gingivalis lipopolysaccharide(Pg-LPS) stimulated M1 polarization of RAW264.7 cells through miR-224/NLRP3 axis. Methods: RAW264.7 cells were treated with 0, 5, 10, 20, 40 and 80 μmol/L ursolic acid respectively, the cell viability was detected by MTT assay. RAW264.7 cells were divided into control group, LPS group, ursolic acid group, inhibitor control group and miR-224 inhibitor group. The expression of miR-224 was detected by qRT-PCR;TNF-α, IL-1β, IL-10, Arg-1 level was detected by ELISA;CD11and CD206 protein expression was detected by flow cytometry. The targeting relationship between miR-224 and NLRP3 was detected by dual luciferase reporter gene experiment;NLRP3 expression was detected by Western blot. Results: The survival rate of RAW264.7 cells decreased significantly with the increase of ursolic acid concentration;compared with the control group, the expression level of miR-224 in the LPS group was significantly reduced, and the protein levels of NLRP3, TNF-α, IL-1β and CD11increased significantly(P<0.05). Compared with the LPS group, the ursolic acid group could reverse the changes of the above indicators. miR-224 had a targeted binding site with NLRP3. Compared with the ursolic acid group, the expression of TNF-α, IL-1β and CD11in the miR-224 inhibitor group increased significantly(P<0.05). Conclusion: Ursolic acid may inhibit the M1 polarization of RAW264.7 cells stimulated with Pg-LPS by promoting the miR-224/NLRP3 axis.