Ying Kaiyue, Hua Ning, Luo Yanping, Liu Xingyu, Liu Min, Yang Wei
Department of Biophysics, School of Basic Medical Sciences, Zhejiang University, Hangzhou 310058, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2024 Oct 25;53(5):604-614. doi: 10.3724/zdxbyxb-2024-0257.
To establish a cell line stably expressing the transient receptor potential melastatin 2 (TRPM2) channel for screening TRPM2 inhibitors based on PiggyBac transposition system.
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 ´ 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.
The HEK293T cells transfected with pPB-hTRPM2-EGFP showed high 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 (>0.05). With a ´ 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 (<0.05) and reduced the level of ROS (<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 <0.05).
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.
基于猪尾巴（PiggyBac）转座系统建立稳定表达瞬时受体电位香草酸亚家族成员2（TRPM2）通道的细胞系，用于筛选TRPM2抑制剂。
利用猪尾巴转座系统构建质粒猪尾巴-人TRPM2（pPB-hTRPM2）真核表达载体。将该质粒与辅助质粒共转染至人胚肾293T（HEK293T）细胞中以表达TRPM2，通过荧光和膜片钳检测进行鉴定。用Z'因子评估高通量筛选性能。采用钙成像和膜片钳技术评估11种化合物分子的初始活性，确认主要分子对TRPM2的抑制作用。使用氧糖剥夺/再灌注（OGD/R）损伤模型和CCK-8试剂盒验证筛选出的化合物对受损细胞的保护作用。通过流式细胞术检测细胞活性氧（ROS）水平。使用短暂性大脑中动脉闭塞（tMCAO）小鼠模型评估化合物的神经保护作用。
转染pPB-hTRPM2-EGFP的HEK293T细胞显示出高表达。经筛选获得的嘌呤霉素抗性细胞表现出强烈荧光。全细胞膜片钳结果显示，诱导细胞呈现出与对照组相当的典型TRPM2电流特征，差异无统计学意义（>0.05）。在钙成像中Z'因子为0.5416，该模型证明适用于TRPM2抑制剂的高通量筛选。钙成像和电生理实验表明化合物6显著抑制TRPM2通道。进一步实验表明，在OGD/R损伤下，1.0 μmol/L的化合物6可提高SH-SY5Y细胞活力（<0.05）并降低ROS水平（<0.05）。0.3和1.0 mg/kg的化合物6可减小tMCAO小鼠的脑梗死体积（均<0.05）。
本研究利用猪尾巴基因编辑成功建立了稳定表达TRPM2基因的细胞系。通过钙成像和膜片钳技术筛选出TRPM2通道抑制剂，并鉴定出抑制剂化合物6。该化合物可通过降低细胞ROS水平减轻OGD/R后的细胞损伤，并对小鼠脑缺血再灌注损伤具有保护作用。
