摘要
6脂肪酸代谢产物白三烯B4(LTB4)的受体白三烯B4受体1(LTB4R1)属于G蛋白耦联受体(GPCR)家族,是胰岛素抵抗、慢性炎症和2型糖尿病的潜在药物作用靶点。本文根据GPCR家族蛋白激活后可以引起细胞质内钙离子升高的原理,建立了96孔板体系LTB4R1抑制剂筛选模型。胞质内钙离子探针Fluo-8可以表征细胞质内的钙离子变化,当共转染LTB4R1和G蛋白α亚基16(Gα16)的仓鼠卵巢癌细胞(CHO)受到LTB4刺激后,胞浆钙离子浓度升高,Fluo-8的荧光信号随之增强。LTB4R1抑制剂处理细胞后,Fluo-8的荧光信号减弱。本文以0.2%DMSO为阴性对照,LTB4R1抑制剂cp-105696为阳性对照建立筛选模型。经LTB4刺激后,0.2%DMSO处理的细胞Fluo-8信号上升约2倍后缓慢回落,而cp-105696处理组则抑制LTB4的作用。LTB4和抑制剂cp-105696浓度梯度实验表明,Fluo-8的荧光信号升高幅度和抑制剂的浓度成反比,可以作为评价待测化合物的抑制效果的指标。多次重复实验计算所得阳性与阴性对照Z’因子为0.777,证明该筛选模型稳定可靠。本文采用蛋白过表达方式在CHO细胞中构建了LTB4R1-内质网钙的信号通路,并在此基础上建立了通过检测钙信号强度来筛选化合物对LTB4R1抑制活性的体系。本方法可用于LTB4R1抑制剂先导化合物的体外初步筛选,为后续体内研究奠定基础。
The GPCR family component leukotriene B4 receptor 1(LTB4R1)is the receptor of leukotriene B4(LTB4),the metabolic product ofω6 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 used0.2%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 LTB4R1αand 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.
作者
马春晓
万彦军
侯少聪
何淑旺
颜世强
李平平
MA Chun-xiao;WAN Yan-jun;HOU Shao-cong;HE Shu-wang;YAN Shi-qiang;LI Ping-ping(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;Shandong DYNE Marine Biopharmaceutical Co.,Ltd.,Weihai 264333,China)
出处
《药学学报》
CAS
CSCD
北大核心
2022年第11期3316-3321,共6页
Acta Pharmaceutica Sinica
基金
国家自然科学基金资助项目(81770800)
中国医学科学院医学与健康科技创新工程(2021-I2M-1-016)
中国医学科学院中央级公益性科研院所基本科研业务费(2018RC350004)
北京高校卓越青年科学家计划项目(BJJWZYJH01201910023028)。
