目的 探讨N⁶-甲基腺嘌呤（m⁶A）修饰在刚地弓形虫感染后调控巨噬细胞瞬时受体电位M8型通道蛋白（TRPM8）中的作用及机制。方法 转录组测序（RNA-seq）和甲基化RNA免疫共沉淀测序（MeRIP-seq）数据来源于NCBI基因表达综合数据库中的数据集（GSE288205），采用OmicStudio对TRP通道蛋白家族基因进行生物信息学分析。使用RStudio软件中的ggplot2包，采用MeRIP-seq数据绘制TRP通道相关因子1（TCAF1）和TRPM8 mRNA m⁶A修饰火山图。将人单核细胞系THP-1源性巨噬细胞、人外周血单个核细胞（PBMC）、小鼠RAW264.7细胞接种于6孔板中（1 × 10⁶个/孔），1∶1（细胞∶虫）加入弓形虫速殖子作为感染组，对照组加入等量培养基。TRIzol法提取各组细胞的总RNA，逆转录为cDNA，实时荧光定量PCR（qPCR）检测各组的TRPM8（人）/Trpm8（小鼠），以及THP-1源性巨噬细胞感染组和对照组TCAF1、TCAF2的mRNA相对转录水平。流式细胞术检测各组细胞Ca²⁺内流情况。将THP-1源性巨噬细胞、脂肪量和肥胖相关蛋白（FTO）敲低的THP-1源性巨噬细胞（shFTO-THP-1）及携带非靶向对照shRNA的THP-1源性巨噬细胞（shNC-THP-1）接种于6孔板中（1 × 10⁶个/孔），1∶1（细胞∶虫）加入弓形虫速殖子作为感染组，对照组加入等量培养基。利用m⁶A甲基化RNA免疫共沉淀-qPCR（m⁶A-IP-qPCR）验证THP-1源性巨噬细胞TCAF1 mRNA上m⁶A修饰变化。RNA免疫沉淀实验（RIP）检测THP-1源性巨噬细胞感染组与m⁶A识别蛋白YTH结构域家族蛋白2（YTHDF2）结合的TCAF1 mRNA的相对转录水平。qPCR检测shFTO-THP-1和shNC-THP-1组在加入放线菌素D处理0、2、4、6 h后的TCAF1 mRNA降解速率。两组之间比较采用独立样本t检验。结果 RNA-seq生物信息学分析显示，TRP通道蛋白家族基因中除了瞬时受体电位阳离子通道亚家族C成员4相关蛋白表达上调外，其他TRP通道蛋白表达水平均显著下调。其中TRPM8蛋白的下调幅度最大，其转录水平下降至对照组的0.16。qPCR结果表明，THP-1源性巨噬细胞、PBMC和RAW264.7细胞感染组TRPM8（人）/Trpm8（小鼠）mRNA的相对转录水平分别为0.445 ± 0.118、0.302 ± 0.040、0.365 ± 0.234，低于其对照组的1.000 ± 0.036、1.042 ± 0.381、1.004 ± 0.105（t = 7.828、3.345、4.312，均P < 0.05）。流式细胞术结果显示，THP-1源性巨噬细胞、PBMC和RAW264.7细胞感染细胞内Ca²⁺平均荧光强度分别为19 500.0 ± 2 427.0、3 569.0 ± 313.9、6 513.0 ± 348.0，低于其对照组的23 500.0 ± 264.6、13 050.0 ± 1 072.0、7 683.0 ± 245.2（t = 2.838、16.970、4.762，均P < 0.05）。qPCR结果表明，THP-1源性巨噬细胞感染组TCAF1 mRNA转录水平为0.617 ± 0.132，低于其对照组的1.005 ± 0.115（t = 3.832，P < 0.05）；TCAF2 mRNA转录水平为0.973 ± 0.030，与对照组（1.015 ± 0.209）相比，差异无统计学意义（t = 0.343，P > 0.05）。MeRIP-seq数据的火山图结果显示，THP-1源性巨噬细胞感染组TCAF1 mRNA的3′非翻译区（3′UTR）区m⁶A修饰较对照组上调了552倍。m⁶A-IP-qPCR结果表明，THP-1源性巨噬细胞感染组TCAF1 mRNA 3′UTR区m⁶A修饰水平为对照组（1.000 ± 0）的（4.794 ± 0.854）倍，显著上调（t = 7.696，P < 0.05）。YTHDF2-RIP结果表明，THP-1源性巨噬细胞感染组与YTHDF2结合的TCAF1 mRNA相对转录水平为2.423 ± 0.782，高于对照组的1.010 ± 0.180（t = 3.048，P < 0.05）。与shNC-THP-1感染组相比，shFTO-THP-1感染组TCAF1 mRNA降解速率更快。结论 弓形虫感染后，m⁶A修饰参与了人和小鼠巨噬细胞中冷感受器TRPM8表达的下调。

Objective To investigate the role of N⁶-methyladenosine (m⁶A) modification in regulation of transient receptor potential cation channel subfamily M member 8 (TRPM8) protein in macrophages following Toxoplasma gondii infection and unravel its underlying mechanisms. Methdos Transcriptome sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) data were sourced from the NCBI Gene Expression Omnibus (GEO) dataset GSE288205. Transient receptor potential (TRP) channel protein family-coding genes were subjected to bioinformatics analyses with the OmicStudio platform, and volcano plots for m⁶A modifications of TRP channel-associated factor 1 (TCAF1) and TRPM8 mRNA were generated based on MeRIP-seq data using ggplot2 in the RStudio package. THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs) and mouse RAW264.7 cells were seeded onto 6-well plates at a density of 1 × 10⁶ cells per well. Cells infected with T. gondii RH strain tachyzoites at a cell-to-parasite ratio of 1∶1 served as the infection group, while cells in the control group received an equal volume of parasite-free medium. Total RNA was extracted from cells using the TRIzol reagent, reversely transcribed into cDNA. The relative mRNA expression of TRPM8 (human) and Trpm8 (mouse) was quantified in cells using quantitative fluorescent real-time PCR (qPCR) assay, and the relative TCAF1 and TCAF2 mRNA levels were detected in THP-1-derived macrophages with and without infected with T. gondii tachyzoites infections using qPCR assay. Intracellular Ca²⁺ influx was detected in cells using flow cytometry. THP-1-derived macrophages, fat mass and obesity-associated protein (FTO)-knockdown THP-1-derived macrophages (shFTO-THP-1), and THP-1-derived macrophages carrying non-targeted control shRNA (shNC-THP-1) were seeded onto 6-well plates at a density of 1 × 10⁶ cells per well. Cells in the infection group were infected with T. gondii RH strain tachyzoites at a cell-to-parasite ratio of 1∶1, while cells in the control group received an equal volume of parasite-free medium. Changes in m⁶A modification on TCAF1 mRNA in THP-1-derived macrophages were validated using m⁶A methylated RNA immunoprecipitation-real time quantitative PCR (m⁶A-IP-qPCR) assay. The expression of TCAF1 mRNA binding the m⁶A recognition protein YTH domain family protein 2 (YTHDF2) was detected in THP-1-derived macrophages infected with T. gondii tachyzoites using RNA immunoprecipitation (RIP) assay. The rate of TCAF1 mRNA degradation was compared between the shFTO-THP-1 and shNC-THP-1 groups using qPCR assay 0, 2, 4, and 6 hours post-treatment with actinomycin D. Differences of means between groups were tested for statistical significance with independent-sample Student’s t-test. Results RNA-seq revealed significant downregulation of TRP channel protein-coding genes except transient receptor potential cation channel subfamily C member 4-related protein, which was upregulated. Notably, TRPM8 exhibited the most pronounced downregulation, with its transcription level at 0.16-fold of the control group. qPCR assay quantified lower TRPM8 (human) and Trpm8 (mouse) mRNA expression in THP-1-derived macrophages [(0.445 ± 0.118) vs. (1.000 ± 0.036); t = 7.828, P < 0.05], PBMCs [(0.302 ± 0.040) vs. (1.042 ± 0.381); t = 3.345, P < 0.05], and RAW264.7 cells [(0.365 ± 0.234) vs. (1.004 ± 0.105); t = 4.312, P < 0.05] infected with T. gondii tachyzoites than in controls, and flow cytometry detected lower intracellular fluorescence intensities of Ca²⁺ in THP-1-derived macrophages [(19 500.0 ± 2 427.0) vs. (23 500.0 ± 2 64.6); t = 2.838, P < 0.05], PBMCs [(3 569.0 ± 313.9) vs. (13 050.0 ± 1 072.0); t = 16.970, P < 0.05], and RAW264.7 cells [(6 513.0 ± 348.0) vs. (7 683.0 ± 245.2); t = 4.762, P < 0.05] infected with T. gondii tachyzoites than in controls. qPCR assay quantified lower TCAF1 mRNA expression in THP-1-derived macrophages [(0.617 ± 0.132) vs. (1.005 ± 0.115); t = 3.832, P < 0.05] infected with T. gondii tachyzoites than in controls, and no significant differences in TCAF2 mRNA expression between the infection and control groups [THP-1-derived macrophages: (0.973 ± 0.030) vs. (1.015 ± 0.209), t = 0.343, P > 0.05]. Volcano plots of MeRIP-seq data displayed that m⁶A modification in the 3′ untranslated region (3′UTR) of TCAF1 mRNA was upregulated by 552 folds in THP-1-derived macrophages infected with T. gondii tachyzoites than in controls, and m⁶A-IP-qPCR assay detected a higher m⁶A modification level in the 3′ UTR region of TCAF1 mRNA in THP-1-derived macrophages infected with T. gondii tachyzoites than in controls [(4.794 ± 0.854) vs. (1.000 ± 0); t = 7.696, P < 0.05]. YTHDF2-RIP analysis showed a higher transcriptional level of TCAF1 mRNA binding to YTHDF2 in THP-1-derived macrophages infected with T. gondii tachyzoites than in controls [(2.423 ± 0.782) vs. (1.010 ± 0.180); t = 3.048, P < 0.05]. In addition, a higher rate of TCAF1 mRNA degradation was seen in shFTO-THP-1 cells infected with T. gondii tachyzoites than in shNC-THP-1 cells infected with T. gondii tachyzoites. Conclusion m⁶A modification contributes to downregulation of the cold receptor TRPM8 in both human and mouse macrophages following T. gondii infection.