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Abstract：
OBJECTIVE: To explore the effect of miR-370-3p on LPS triggering, in particular its involvement in disease progression by targeting the TLR4-NLRP3-caspase-1 cellular pyroptosis pathway in macrophages.
METHODS: Human macrophage RAW264.7 was divided into 6 groups: control, LPS, LPS + inhibitor-NC, LPS + miR-370-3p inhibitor, LPS + mimics-NC and LPS + miR-370-3p mimics. RT-qPCR was used to detect the expression level of miR-370-3p and analyzed comparatively. CCK-8 and flow cytometry assays were used to detect cell viability and apoptosis. ELISA assay was used to detect the levels of IL-1β and TNF-α in the supernatant of the cells. The WB assay was used to detect TLR4, NLRP3, Caspase-1 and GSDMD levels.
RESULTS: After LPS induction, macrophage miR-370-3p levels decreased, cell viability decreased, and apoptosis increased. At the same time, the levels of TLR4, NLRP3, Caspase-1 and GSDMD increased in the cells, and the levels of IL-1β and TNF-α increased in the cell supernatant. Compared with the LPS group, the significantly higher expression level of miR-370-3p in the cells of the LPS + miR-370-3p mimics group was accompanied by significantly higher cell viability, significantly lower apoptosis rate, significantly lower levels of TLR4, NLRP3, Caspase-1, and GSDMD in the cells, and significantly lower levels of IL-1β and TNF-α in the cell supernatant.
CONCLUSIONS: MiR-370-3p may be involved in anti-infective immune responses by targeting and inhibiting the macrophage TLR4-NLRP3-caspase-1 cellular pyroptosis pathway.
METHODS: Human macrophage RAW264.7 was divided into 6 groups: control, LPS, LPS + inhibitor-NC, LPS + miR-370-3p inhibitor, LPS + mimics-NC and LPS + miR-370-3p mimics. RT-qPCR was used to detect the expression level of miR-370-3p and analyzed comparatively. CCK-8 and flow cytometry assays were used to detect cell viability and apoptosis. ELISA assay was used to detect the levels of IL-1β and TNF-α in the supernatant of the cells. The WB assay was used to detect TLR4, NLRP3, Caspase-1 and GSDMD levels.
RESULTS: After LPS induction, macrophage miR-370-3p levels decreased, cell viability decreased, and apoptosis increased. At the same time, the levels of TLR4, NLRP3, Caspase-1 and GSDMD increased in the cells, and the levels of IL-1β and TNF-α increased in the cell supernatant. Compared with the LPS group, the significantly higher expression level of miR-370-3p in the cells of the LPS + miR-370-3p mimics group was accompanied by significantly higher cell viability, significantly lower apoptosis rate, significantly lower levels of TLR4, NLRP3, Caspase-1, and GSDMD in the cells, and significantly lower levels of IL-1β and TNF-α in the cell supernatant.
CONCLUSIONS: MiR-370-3p may be involved in anti-infective immune responses by targeting and inhibiting the macrophage TLR4-NLRP3-caspase-1 cellular pyroptosis pathway.
摘要：
目的：探讨miR-370-3p对LPS触发的影响,特别是其通过靶向巨噬细胞中的TLR4-NLRP3-caspase-1细胞焦亡途径参与疾病进展。
方法：人巨噬细胞RAW264.7分为6组：对照组，LPS,LPS+抑制剂-NC，LPS+miR-370-3p抑制剂，LPS+模拟-NC和LPS+miR-370-3p模拟物。采用RT-qPCR检测miR-370-3p的表达水平并进行比较分析。CCK-8和流式细胞术测定用于检测细胞活力和凋亡。ELISA法检测细胞上清液中IL-1β和TNF-α的含量。WB测定用于检测TLR4、NLRP3、Caspase-1和GSDMD水平。
结果：LPS诱导后，巨噬细胞miR-370-3p水平降低，细胞活力下降，细胞凋亡增加。同时,细胞中TLR4、NLRP3、Caspase-1和GSDMD水平升高,细胞上清液中IL-1β和TNF-α水平升高。与LPS组比拟，miR-370-3p在LPS+miR-370-3p模拟组细胞中显著较高的表达水平伴随着显著较高的细胞活力,显著降低细胞凋亡率，细胞中TLR4，NLRP3，Caspase-1和GSDMD的水平显着降低，并显著降低细胞上清液中IL-1β和TNF-α的水平。
结论：MiR-370-3p可能通过靶向抑制巨噬细胞TLR4-NLRP3-caspase-1途径参与抗感染免疫反应。
方法：人巨噬细胞RAW264.7分为6组：对照组，LPS,LPS+抑制剂-NC，LPS+miR-370-3p抑制剂，LPS+模拟-NC和LPS+miR-370-3p模拟物。采用RT-qPCR检测miR-370-3p的表达水平并进行比较分析。CCK-8和流式细胞术测定用于检测细胞活力和凋亡。ELISA法检测细胞上清液中IL-1β和TNF-α的含量。WB测定用于检测TLR4、NLRP3、Caspase-1和GSDMD水平。
结果：LPS诱导后，巨噬细胞miR-370-3p水平降低，细胞活力下降，细胞凋亡增加。同时,细胞中TLR4、NLRP3、Caspase-1和GSDMD水平升高,细胞上清液中IL-1β和TNF-α水平升高。与LPS组比拟，miR-370-3p在LPS+miR-370-3p模拟组细胞中显著较高的表达水平伴随着显著较高的细胞活力,显著降低细胞凋亡率，细胞中TLR4，NLRP3，Caspase-1和GSDMD的水平显着降低，并显著降低细胞上清液中IL-1β和TNF-α的水平。
结论：MiR-370-3p可能通过靶向抑制巨噬细胞TLR4-NLRP3-caspase-1途径参与抗感染免疫反应。
