RN-1734 is selective TRPV4 channel antagonist(IC50 of 2.3 μM,5.9 μM,3.2 μM for hTRPV4, mTRPV4 and rTRPV4,respectively)

TRPV4 (human):2.3 μM , TRPV4 (rat):3.2 μM, TRPV4 (mouse):5.9 μM

体外实验中，RN-1734处理明显抑制了钙的流入，并通过抑制NF-κB P65的磷酸化，降低了脂多糖(LPS)激活的微胶质细胞中IL-1β和TNF-α的水平。RN-1734还减轻了LPS激活的微胶质细胞引起的少突胶质细胞凋亡。微胶质细胞中TRPV4的激活通过激活NF-κB信号通路，参与了少突胶质细胞的凋亡，从而揭示了中枢神经系统脱髓鞘新机制[1]。

LPS-treated microglial cells were further divided into a LPS group, a vehicle (DMSO treatment) group and a RN-1734 group.?The cells were treated with 1 μg/ml LPS (Sigma) or RN-1734 (10 μM) for 3 h .?Next, the cells were triple-washed with DMEM medium to remove LPS, and then incubated in DMEM with or without RN1734 for 24 h. Finally, the cells were collected for TRPV4 immunocytostaining, and the supernatant was collected separately for enzyme-linked immunosorbent assay (ELISA) or for the oligodendrocyte apoptosis experiment.?The supernatant of the microglial cells was mixed with DMEM/F12 at a ratio of 1:1.?The mixed medium without LPS was used for the control (Ctrl) group, the medium with LPS only was used for the conditional medium (CM) group, and the media with LPS and DMSO or RN-1734 were used for the CM-vehicle group and the CM-RN1734 group, respectively.?After oligodendrocytes grew for 2 days in 6-well plates in DMEM/F12 containing 10% FBS, the differentiation medium was replaced with the described Ctrl or different CM media for 24 h. Then, the cells were harvested for apoptosis analysis and western blot analysis separately[1].