Objectives: To establish a cell line stably expressing the transient receptor potential melastatin 2 (TRPM2) channel for screening TRPM2 inhibitors based on PiggyBac transposition system.
Methods: A plasmid PiggyBac-human TRPM2 (pPB-hTRPM2) eukaryotic expression vector was constructed using PiggyBac transposition system. The plasmid and a helper plasmid were co-transfected into HEK293T cells to express TRPM2, which was identified by fluorescence and patch-clamp assays. The high throughput screening performance was assessed with the Z ´ factor. Calcium imaging and patch clamp techniques were employed to assess the initial activity of eleven compound molecules, confirming the inhibitory effects of the primary molecules on TRPM2. The protective effect of the screened compounds on damaged cells was validated using the oxygen-glucose deprivation/reperfusion (OGD/R) injury model and CCK-8 kit. The level of cellular reactive oxygen species (ROS) was detected by flow cytometry. The neuroprotective effects of the compounds were evaluated using a transient middle cerebral artery occlusion (tMCAO) mouse model.
Results: The HEK293T cells transfected with pPB-hTRPM2-EGFP showed high TRPM2 expression. Puromycin-resistant cells, selected through screening, exhibited robust fluorescence. Whole-cell patch results revealed that induced cells displayed classical TRPM2 current characteristics comparable to the control group, showing no significant differences (P>0.05). With a Z ´ factor of 0.5416 in calcium imaging, the model demonstrated suitability for high-throughput screening of TRPM2 inhibitors. Calcium imaging and electrophysiological experiments indicated that compound 6 significantly inhibited the TRPM2 channel. Further experiments showed that 1.0 μmol/L of compound 6 enhanced cell viability (P<0.05) and reduced the level of ROS (P<0.05) of SH-SY5Y under OGD/R injury. 0.3 and 1.0 mg/kg of compound 6 reduced the cerebral infarction volume in tMCAO mice (both P<0.05).
Conclusions: A stable TRPM2 gene expressing cell line has been successfully established using PiggyBac gene editing in this study. TRPM2 channel inhibitors were screened through calcium imaging and patch clamp techniques, and an inhibitor compound 6 was identified. This compound can alleviate cell damage after OGD/R by reducing cellular ROS levels and has a protective effect against cerebral ischemia-reperfusion injury in mice.
目的: 使用PiggyBac（PB）转座系统建立稳定表达瞬时受体电位M2（TRPM2）通道的人胚胎肾细胞HEK293T，并进行TRPM2通道抑制剂筛选，为治疗脑缺血等疾病寻找潜在的药物。方法: 根据PB转座原理，构建pPB-hTRPM2真核表达载体。将重组质粒和辅助质粒共同转染至HEK293T细胞中，利用系统携带的荧光与膜片钳鉴定TRPM2基因表达，通过Z ´ 因子评估细胞模型是否适用于钙成像法的高通量筛选。利用钙成像法和膜片钳对11个先导化合物分子进行初步活性评价，测定化合物分子对TRPM2通道的抑制活性。利用氧糖剥夺/再灌注（OGD/R）损伤模型和细胞计数试剂盒8（CCK-8）方法验证筛选得到的化合物分子对损伤细胞的保护作用。利用流式细胞术检测细胞活性氧水平。利用小鼠短暂性大脑中动脉栓塞（tMCAO）模型评价化合物的神经保护作用。结果: 成功构建pPB-hTRPM2真核表达载体，构建出可高效表达TRPM2基因的HEK293T细胞系，并同时表达增强型绿色荧光蛋白（EGFP）。在筛选获得的嘌呤霉素抗性细胞中，所有细胞荧光明亮可见，诱导后细胞表现出经典的TRPM2通道电流特征，TRPM2通道电流值与瞬时转染对照组差异无统计学意义（P>0.05）。该筛选系统进行钙成像实验的Z ´ 因子为0.5416，表明其适用于高通量筛选。通过钙成像法结合膜片钳筛选出化合物6，其具有显著的TRPM2通道抑制作用，且1.0 μmol/L的化合物6能够显著提高OGD/R后SH-SY5Y细胞的存活率（P<0.05），降低活性氧水平（P<0.05），0.3、1.0 mg/kg的化合物6能降低tMCAO小鼠脑梗死体积百分比（均P<0.05）。结论: 利用PiggyBac基因编辑技术成功构建了TRPM2基因稳定表达细胞系，利用钙成像法和膜片钳在候选化合物中成功筛选出一个高亲和力的新的TRPM2通道抑制剂。该抑制剂可以通过降低细胞活性氧水平缓解 OGD/R 后细胞损伤，并对小鼠脑缺血再灌注损伤具有保护作用。.
Keywords: High-throughput screening; Mice; Oxygen-glucose deprivation/reperfusion injury; Patch clamp; PiggyBac transposition system; Transient middle cerebral artery occlusion; Transient receptor potential melastatin 2.