Establishment and evaluation of in vitro screen model for LTB4 receptor 1
10.16438/j.0513-4870.2022-0129
- VernacularTitle:LTB4R1抑制剂筛选方法的建立及评价
- Author:
Chun-xiao MA
1
;
Yan-jun WAN
1
;
Shao-cong HOU
1
;
Shu-wang HE
2
;
Shi-qiang YAN
2
;
Ping-ping LI
1
Author Information
1. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
2. Shandong DYNE Marine Biopharmaceutical Co., Ltd., Weihai 264333, China
- Publication Type:Research Article
- Keywords:
leukotriene B4 receptor 1;
screen;
chronic inflammation;
calcium signal;
Fluo-8
- From:
Acta Pharmaceutica Sinica
2022;57(11):3316-3321
- CountryChina
- Language:Chinese
-
Abstract:
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.
