广西医学

目的探讨甲基转移酶样蛋白16（METTL16）靶点调控小分子化合物根皮素和表没食子儿茶素没食子酸酯（EGCG）对HIV⁃1复制的影响。方法（1）采用高通量计算机生物信息筛选技术筛选出与METTL16靶点相关的两种小分子化合物，即根皮素和EGCG。（2）利用非小细胞肺癌细胞A549对化合物进行初筛实验。将A549细胞分为空白组（仅含培养基）、对照组（含培养基和A549细胞）、实验组（含培养基、A549细胞、20 μmol/L根皮素/EGCG），干预24 h后采用CCK⁃8法测定各组A549细胞的相对活性。（3）将MT⁃2细胞和TZM⁃bl细胞分为空白组（含培养基）、对照组（含培养基、细胞）、实验组（含培养基、细胞、5种浓度根皮素/EGCG）。干预24 h后，通过ATP荧光定量分析法来检测活细胞数量。（4）将MT⁃2细胞和TZM⁃bl细胞分为阴性对照组（未感染病毒且未干预）、阳性对照组、实验组。在阳性对照组、实验组中，通过感染HIV⁃1 89.6构建HIV⁃1 89.6/TZM⁃bl模型、HIV⁃1 89.6/MT⁃2模型。染毒后，在实验组TZM⁃bl细胞中立即加入5种浓度根皮素或EGCG，在实验组MT⁃2细胞中立即加入5种浓度根皮素。通过检测荧光素酶报告基因的表达来评估各组细胞中相对病毒水平。结果（1）实验组A549细胞的相对活性低于对照组（P<0.05），即根皮素和EGCG对A549细胞有不同程度的杀伤抑制效果。（2）在5种浓度的根皮素干预下，实验组MT⁃2细胞的相对活性均>80%；在1.25 μmol/L、2.5 μmol/L的根皮素干预下，实验组TZM⁃bl细胞的相对活性均>80%；在5种浓度的EGCG干预下，实验组TZM⁃bl细胞的相对活性>80%。（3）与阳性对照组比较，在10 μmol/L、20 μmol/L根皮素干预下的实验组MT⁃2细胞上清液中相对病毒水平降低，在5 μmol/L、10 μmol/L、20 μmol/L根皮素干预下的实验组TZM⁃bl细胞中的相对病毒水平降低，在5种浓度EGCG干预下的实验组TZM⁃bl细胞中的相对病毒水平均降低（P<0.05）。（4）根皮素在HIV⁃1 89.6/MT⁃2模型、HIV⁃1 89.6/TZM⁃bl模型中的治疗指数分别为>2.064、2.078；EGCG在HIV⁃1 89.6/TZM⁃bl模型中的治疗指数>3.587。结论根皮素和EGCG可能通过调节甲基化转移酶中的METTL16靶点来调控N6⁃甲基腺苷修饰，从而抑制宿主细胞中HIV⁃1的感染和复制。

Objective To explore the effect of methyltransferase like protein 16 (METTL16) target⁃regulating small molecule compound phloretin and epigallocatechin gallate (EGCG) on HIV⁃1 replication. Methods (1) Two categories of small molecule compounds related to METTL16 target were screened by employing the high throughput computer bioinformation screening technique, namely, phloretin and EGCG. (2) Non⁃small cell lung cancerous cells A549 were used to perform screening experiment on compounds. A549 cells were assigned to blank group (only containing medium), control group (containing medium and A549 cells), or experiment group (containing medium, A549 cells, and 20 μmol/L phloretin/EGCG). After 24⁃hour intervention, the CCK⁃8 method was used to measure relative activity of A549 cells in various groups. (3) MT⁃2 and TZM⁃bl cells were assigned to blank group (containing medium), control group (containing medium, cells), or experiment group (containing medium, cells, and 5 concentrations of phloretin/EGCG). After 24⁃hour intervention, the number of living cells was detected through the ATP fluorescent quantitative analysis method. (4) MT⁃2 and TZM⁃bl cells were assigned to negative control group (without virus infection and without intervention), positive control group, or experiment group. In the positive control group and the experiment group, the HIV⁃1 89.6/TZM⁃bl model and HIV⁃1 89.6/MT⁃2 model were established by infecting HIV⁃1 89.6. After virus infection, 5 concentrations of phloretin or EGCG were immediately added to TZM⁃bl cells of the experiment group, and 5 concentrations of phloretin were immediately added to MT⁃2 cells of the experiment group. The relative virus levels in various groups were evaluated through detecting the expressions of luciferase reporter genes. Results (1) The relative activity of A549 cells of the experiment group was lower than that of the control group (P<0.05), namely, phloretin and EGCG had different kill and inhibitory effects on A549 cells. (2) The relative activities were all >80% in MT⁃2 cells of the experiment group via the intervention of 5 concentrations of phloretin. The relative activities were all >80% in TZM⁃bl cells of the experiment group via 1.25 μmol/L, 2.5 μmol/L of phloretin intervention. The relative activity was >80% in TZM⁃bl cells of the experiment group via 5 concentrations of EGCG intervention. (3) Compared with the positive control group, the relative virus level was declined in MT⁃2 cells supernate of the experiment group via 10 μmol/L and 20 μmol/L of phloretin intervention, the relative virus level was declined in TZM⁃bl cells of the experiment group via 5 μmol/L, 10 μmol/L, and 20 μmol/L of phloretin intervention, and the relative virus level was declined in TZM⁃bl cells of the experiment group via 5 concentrations of EGCG intervention (P<0.05). (4) The therapeutic index of phloretin in the HIV⁃1 89.6/MT⁃2 model and HIV⁃1 89.6/TZM⁃bl model was >2.064 and 2.078, respectively. The therapeutic index of EGCG in the HIV⁃1 89.6/TZM⁃bl model was >3.587. Conclusion Phloretin and EGCG may regulate N6⁃methyladenosine modification, so as to inhibit HIV⁃1 infection and replication in host cells through regulating METTL16 target in methyltransferase.