トリプトライドとメトトレキサートの併用がcGAS-STINGシグナル伝達経路に基づいてLPS誘発RAW264.7細胞に及ぼす相乗効果

This study aims to explore the synergistic effect of triptolide(TP) combined with methotrexate(MTX) on lipopolysaccharide(LPS)-induced RAW264.7 macrophages and its potential mechanism based on the cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)-stimulator of interferon genes(STING) signaling pathway, in order to infer the therapeutic effect of the combination of the two drugs on rheumatoid arthritis. RAW264.7 cells were cultured in vitro, and cell counting kit-8(CCK-8) assay was used to detect cell survival rates under the intervention of different concentrations of drugs and STING activator 5,6-dimethylxanthenone-4-acetic acid(DMXAA) and to evaluate the synergistic effect of the combination of TP and MTX by the combination index(CI). RAW264.7 cells were divided into a control group(normal cells), a model group(1 μg·mL~(-1) LPS), a TP group(0.025 μmol·L~(-1)), a MTX group(0.1 μmol·L~(-1)), combination groups(TP+MTX), and an activator group(TP+MTX+DMXAA). Cell migration and invasion abilities were detected by cell scratch and Transwell invasion assays. Levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in cell supernatants were determined by enzyme-linked immunosorbent assay(ELISA). cGAS and STING protein expression levels were analyzed in cells by Western blot. The results showed that the expression levels of IL-6, TNF-α, cGAS, and STING increased, and the cell migration and invasion abilities were enhanced in the model group compared with the control group. CCK-8 results showed that TP and MTX had a certain inhibitory effect on the proliferation of RAW264.7 cells, and there was a trend of concentration-and time-dependence. The six combination groups had a synergistic inhibitory effect on the proliferation of RAW264.7 cells(CI<1). The results of cell scratch and Transwell invasion assays showed that the migration and invasion of cells were decreased in each drug-treated group compared with the model group, and the decrease was more obvious in the combination groups(P<0.05). The results of ELISA and Western blot showed that the expression of IL-6, TNF-α, cGAS, and STING was down-regulated in the cells of each drug-treated group compared with the model group, and the effect of the combination groups was more favorable(P<0.05). In addition, the STING activator DMXAA attenuated the inhibitory effect of TP combined with MTX on LPS-induced RAW264.7 cell viability and inflammatory response(P<0.05). In conclusion, TP combined with MTX synergistically inhibited LPS-induced proliferation of RAW264.7 cells, and its mechanism of action may be related to the down-regulation of the cGAS-STING pathway and the influence on macrophage migration and invasion, which provides further evidence for the treatment of rheumatoid arthritis with TP combined with MTX.