Establishment and evaluation of in vitro screen model for LTB4 receptor 1

The GPCR family component leukotriene B4 receptor 1 (LTB4R1) is the receptor of leukotriene B4 (LTB4), the metabolic product of ω6 fatty acid. LTB4R1 is a potential therapeutic target for the treatment of insulin resistance, chronic inflammation and type 2 diabetes. Here we established a LTB4R1 inhibitor screen model based on the GPCR family protein property that its activation causes the cytosolic escalation of calcium. The cytosolic calcium probe Fluo-8 represents the change of calcium ion. After adding LTB4, the fluorescent signal of Fluo-8 in the CHO cells which are co-transfected with LTB4R1 and Gα16 will change with the increase of cytosolic calcium, and LTB4R1 inhibitor blocked the effect of LTB4 on fluorescent signal of Fluo-8 in the CHO cells. Here, we used 0.2% DMSO as a negative control, and cp-105696 as a positive control in the screen model. After stimulation with LTB4, the Fluo-8 signal in 0.2% DMSO treated CHO cells increased 2 fold and fell back slowly, while the signal in inhibitor (cp-105696) treated cells was not induced by LTB4. The results showed that LTB4 increased the cytosolic calcium detected by Fluo-8 in a dose dependent manner. Similarly, cp-105696 inhibited the Fluo-8 signal dose dependently, indicating that this method can quantify the inhibitory activity of the compounds. The Z'-factor, reflecting the robustness of the screen model, was 0.777 with a series of experiments. In sum, we over-expressed LTB4R1α and Gα16 in CHO cell, used Fluo-8 to detect the calcium signal activated by LTB4, and established the in vitro screen model for LTB4 receptor 1.