收稿日期: 2025-04-07
修回日期: 2025-05-07
网络出版日期: 2025-06-18
基金资助
国家自然科学基金面上项目(82272369);国家自然科学基金面上项目(82372283)
Mechanism of cGAS-STING regulation of immune responses against Cryptosporidium parvum infection in intestinal epithelial cells
Received date: 2025-04-07
Revised date: 2025-05-07
Online published: 2025-06-18
Supported by
National Natural Science Foundation of China(82272369);National Natural Science Foundation of China(82372283)
目的 探索肠上皮细胞环鸟苷酸-腺苷酸合成酶（cGAS）-干扰素基因刺激蛋白（STING）在调控微小隐孢子虫感染免疫中的作用机制。 方法 用微小隐孢子虫卵囊感染人结直肠癌细胞系HCT-8细胞（卵囊:细胞2:1），于感染后0、4、8、12、24、36和48 h提取各组细胞总蛋白，利用蛋白质免疫印迹（Western blotting）检测STING的相对表达水平。利用小干扰RNA（siRNA）分别建立cGAS和STING敲低的细胞模型并感染微小隐孢子虫虫卵，于感染后24 h用Western blotting检测HCT-8细胞cGAS、STING、TANK结合激酶1（TBK1）、p-TBK-1、干扰素调节因子3（IRF3）、p-IRF3、核转录因子κB（NF-κB）、p-NF-κB、干扰素β（IFN-β）、肿瘤坏死因子α（TNF-α）的相对表达水平。流式细胞术检测STING敲低前后的细胞凋亡水平。实时荧光定量PCR（qPCR）检测STING敲低前后微小隐孢子虫18S mRNA的相对转录水平。数据采用独立样本t检验进行统计学分析。 结果 Western blotting检测结果显示，微小隐孢子虫感染后4、8、12、24、36和48 h，STING的相对表达水平分别为3.000 ± 0.743、2.756 ± 0.847、2.397 ± 0.701、3.645 ± 0.306、3.773 ± 0.471、3.982 ± 0.468，均高于未感染组（0 h）的1.000 ± 0.039（t = 4.655、3.587、3.448、14.870、10.160、11.000，均P < 0.05）。STING敲低感染HCT-8细胞中，STING、p-NF-κB/NF-κB、TNF-α、p-TBK-1/TBK-1、p-IRF3/IRF3、IFN-β相对表达水平分别为1.024 ± 0.093、1.042 ± 0.160、1.060 ± 0.108、0.665 ± 0.297、0.929 ± 0.207、0.740 ± 0.104，均较感染组（1.757 ± 0.332、2.329 ± 0.336、1.522 ± 0.230、1.339 ± 0.088、1.332 ± 0.036）降低（t = 10.250、3.360、6.231、3.949、3.159、9.362，均P < 0.05）。cGAS敲低感染HCT-8细胞中，cGAS和STING的相对表达水平分别为0.771 ± 0.038和0.696 ± 0.094，均较感染组（1.231 ± 0.074和1.238 ± 0.023）降低（t = 9.608、9.674，均P < 0.05）。流式细胞术检测结果显示，STING敲低感染组细胞的凋亡水平为（18.90 ± 0.75）%，较感染组的（23.72 ± 2.55）%降低（t = 3.141，P < 0.05）。qRT-PCR结果显示，STING敲低感染后细胞中微小隐孢子虫18S mRNA相对转录水平为1.335 ± 0.037，较感染组（1.002 ± 0.071）增加（t = 7.195，P < 0.05）。 结论 cGAS-STING在微小隐孢子虫感染中通过激活TBK1和NF-κB信号通路，参与宿主抗微小隐孢子虫免疫应答。
关键词： 微小隐孢子虫; 环鸟苷酸-腺苷酸合成酶; 干扰素基因刺激蛋白; I型干扰素; 宿主免疫应答
崔丽君 , 李腾 , 沈玉娟 , 张璟 , 周浩 , 曹建平 . 肠上皮细胞cGAS-STING在抗微小隐孢子虫感染免疫中的作用机制[J]. 中国寄生虫学与寄生虫病杂志, 2025 , 43(3) : 311 -316 . DOI: 10.12140/j.issn.1000-7423.2025.03.001
Objective To investigate the mechanism of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) in regulation of immune responses against Cryptosporidium parvum infections in intestinal epithelial cells. Methods Human colorectal cell line HCT-8 was infected with C. parvum oocysts (oocysts to cell ratio of 2:1) for 0, 4, 8, 12, 24, 36 and 48 h. Then, total protein was extracted from cells, and the relative STING protein expression was determined using Western blotting. Cell models of cGAS and STING knockdown were generated using small interfering RNA (siRNA). The relative expression of cGAS, STING, TANK-binding kinase 1 (TBK-1), p-TBK-1, interferon regulatory factor (IRF3), p-IRF3, nuclear factor-κB (NF-κB), p-NF-κB, interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α) was determined using Wester blotting in HCT-8 cells 24 h post-infection, and the apoptosis of HCT-8 cells was detected using flow cytometry before and after STING knockdown. In addition, the relative expression level of C. parvum 18S mRNA was quantified using quantitative fluorescent PCR (qPCR) before and after STING knockdown. Statistical analysis of the data was conducted using the independent samples t-test. Results Western blotting assay determined that the relative STING protein expression was 3.000 ± 0.743, 2.756 ± 0.847, 2.397 ± 0.701, 3.645 ± 0.306, 3.773 ± 0.471, 3.982 ± 0.468 in HCT-8 cells 4, 8, 12, 24, 36 and 48 h post-infection with C. parvum, which was all higher than in uninfected cells (0 h) (1.000 ± 0.039) (t = 4.655, 3.587, 3.448, 14.870, 10.160, 11.000; all P < 0.05). The relative expression of cGAS, STING, p-NF-κB/NF-κB and TNF-α, p-TBK-1/TBK-1, p-IRF3/IRF3, IFN-β proteins was 1.024 ± 0.093, 1.042 ± 0.160, 1.060 ± 0.108, 0.665 ± 0.297, 0.929 ± 0.207, 0.740 ± 0.104 in C. parvum infected HCT-8 cells with STING knockdown, which was all lower than in infected cells (1.757 ± 0.332, 2.329 ± 0.336, 1.522 ± 0.230, 1.339 ± 0.088, 1.332 ± 0.036) (t = 10.25, 3.360, 6.231, 3.949, 3.159, 9.362; all P < 0.05), and the relative expression of cGAS and STING proteins was 0.771 ± 0.038 and 0.696 ± 0.094 in C. parvum infected HCT-8 cells with cGAS knockdown, which was all lower than in infected cells (1.231 ± 0.074, 1.238 ± 0.023) (t = 9.608, 9.674; both P < 0.05). Flow cytometry detected a lower apoptotic rate in C. parvum infected HCT-8 cells with STING knockdown [(18.90 ± 0.75)%] than in infected cells [(23.72 ± 2.55)%] (t = 3.141, P < 0.05), and qPCR assay quantified higher relative expression of C. parvum 18S mRNA in C. parvum infected HCT-8 cells with STING knockdown (1.335 ± 0.037) than in infected cells (1.002 ± 0.071) (t = 7.195, P < 0.05). Conclusion cGAS-STING contributes to host immune responses against C. parvum infections via activation of TBK1 and NF-κB signaling pathways.
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