MicroRNA-155在弓形虫感染介导小胶质细胞活化中的作用

doi:10.20199/j.issn.1672-2302.2025.02.008

摘要/Abstract

摘要：

目的探讨弓形虫感染对小胶质细胞microRNA-155（miR-155）表达的影响及miR-155对小胶质细胞活化的作用。方法将BV-2小鼠小胶质细胞分为PRU感染组（感染弓形虫PRU株速殖子）、miR-155 mimics组（转染miR-155 mimics）、PRU + miR-155 inhibitor组（感染后转染miR-155 inhibitor）和对照组（仅添加转染试剂）。收集各组细胞和细胞上清，采用qPCR检测各组细胞miR-155、IL-1β、IL-12和iNOS的mRNA表达，ELISA检测各组细胞上清中IL-1β、IL-12蛋白以及NO的表达；Western blotting检测各组细胞中miR-155的靶向基因细胞因子信号抑制物1（cytokine signal suppressor 1, SOCS1）蛋白表达。用各组细胞上清配制的条件培养基培养神经细胞N2a，流式细胞术检测凋亡率。结果与对照组和PRU + miR-155 inhibitor组相比，PRU感染组和miR-155 mimics组BV-2小鼠小胶质细胞中的miR-155、IL-1β、IL-12、iNOS以及NO表达均显著上调（P均<0.05），miR-155的靶向SOCS1蛋白表达显著下调（P均<0.05）。神经细胞凋亡实验显示，PRU感染组和miR-155 mimics组条件培养基处理的神经细胞N2a凋亡比例分别为（22.37±1.88）%和（29.87±0.67）%，高于对照组的（9.84±0.57）%；而PRU+miR-155 inhibitor组细胞凋亡比例为（10.91±2.01）%，与对照组相当。结论弓形虫感染小胶质细胞会导致细胞miR-155表达升高，抑制其靶向SOCS1蛋白表达，从而促进IL-1β、IL-12等促炎因子和iNOS表达，最终诱导神经细胞凋亡。

关键词: 弓形虫, 小胶质细胞, miR-155, 细胞凋亡

Abstract:

Objective To investigate the effects of Toxoplasma gondii infection on the expression of microRNA-155 (miR-155) in microglia and the role of miR-155 in the microglial activation. Methods BV-2 murine microglial cells were divided into four groups, namely, PRU infection group (infected with T. gondii PRU strain tachyzoites), miR-155 mimics group (transfected with miR-155 mimics), PRU + miR-155 inhibitor group (infection followed by miR-155 inhibitor transfection), and control group (transfection reagent only). Cellular miR-155, IL-1β, IL-12, and inducible nitric oxide synthase (iNOS) mRNA levels were detected by qPCR. Protein levels of IL-1β, IL-12, and nitric oxide (NO) in supernatants were measured by ELISA. Western blotting was performed to analyze the suppressor of cytokine signaling 1 (SOCS1) protein expression. Flow cytometry was used to assess the apoptosis in N2a neuronal cells cultured with conditioned medium prepared from each group′s supernatants. Results Compared with control group and PRU + miR-155 inhibitor group, both PRU-infected and miR-155 mimics groups showed significantly upregulated miR-155 expression, accompanied by increased IL-1β, IL-12, iNOS mRNA levels, and elevated NO production, whereas protein SOCS1 expression was markedly downregulated (all P < 0.05). Neuronal apoptosis rates in PRU-infected (22.37%±1.88%) and miR-155 mimics groups (29.87%± 0.67%) were significantly higher than those in the control group (9.84%±0.57%), yet the PRU + inhibitor group (10.91%±2.01%) showed similar apoptosis rates to the controls. Conclusion Toxoplasma gondii infection may result in upregulated miR-155 expression in the microglia, which further suppresses SOCS1 protein expression and subsequently promotes release of proinflammatory cytokines IL-1β, IL-12 and iNOS, ultimately inducing neuronal apoptosis.

Key words: Toxoplasma gondii, Microglia, miR-155, Apoptosis

中图分类号:

R382.5

金郁, 刘道华, 汪奇志, 杨荣笙, 杨少萌, 操治国. MicroRNA-155在弓形虫感染介导小胶质细胞活化中的作用[J]. 热带病与寄生虫学, 2025, 23(2): 105-109,121.

JIN Yu, LIU Daohua, WANG Qizhi, YANG Rongsheng, YANG Shaomeng, CAO Zhiguo. Role of microRNA-155 in microglial activation mediated by Toxoplasma gondii infection[J]. Journal of Tropical Diseases and Parasitology, 2025, 23(2): 105-109,121.

图/表 6

表1

图1

图2

图3

图4

图5

参考文献 21

[1]	Schmidt M, Sonneville R, Schnell D, et al. Clinical features and outcomes in patients with disseminated toxoplasmosis admitted to intensive care:a multicenter study[J]. Clin Infect Dis, 2013, 57(11):1535-1541.
[2]	Fuglewicz AJ, Piotrowski P, Stodolak A. Relationship between toxoplasmosis and schizophrenia:a review[J]. Adv Clin Exp Med, 2017, 26(6):1031-1036.
[3]	Cai YH, Chen H, Jin L, et al. STAT3-dependent transactivation of miRNA genes following Toxoplasma gondii infection in macrophage[J]. Parasit Vectors, 2013,6:356.
[4]	Cai YH, Chen H, Mo XW, et al. Toxoplasma gondii inhibits apoptosis via a novel STAT3-miR-17-92-Bim pathway in macrophages[J]. Cell Signal, 2014, 26(6):1204-1212.
[5]	Jiang D, Wu SZ, Xu LQ, et al. Anti-infection roles of miR-155-5p packaged in exosomes secreted by dendritic cells infected with Toxoplasma gondii[J]. Parasit Vectors, 2022, 15(1):3. doi: 10.1186/s13071-021-05003-x pmid: 34986898
[6]	Hu RS, He JJ, Elsheikha HM, et al. Differential brain microRNA expression profiles after acute and chronic infection of mice with Toxoplasma gondii oocysts[J]. Front Microbiol, 2018,9:2316.
[7]	Freilich RW, Woodbury ME, Ikezu T. Integrated expression profiles of mRNA and miRNA in polarized primary murine microglia[J]. PLoS One, 2013, 8(11):e79416.
[8]	Moore CS, Rao VTS, Durafourt BA, et al. miR-155 as a multiple sclerosis-relevant regulator of myeloid cell polarization[J]. Ann Neurol, 2013, 74(5):709-720. doi: 10.1002/ana.23967 pmid: 23818336
[9]	Tarassishin L, Loudig O, Bauman A, et al. Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155[J]. Glia, 2011, 59(12):1911-1922. doi: 10.1002/glia.21233 pmid: 22170100
[10]	蔡亦红, 刘静, 洪露. microRNA-155在弓形虫感染调节巨噬细胞极化中的作用[J]. 中国血吸虫病防治杂志, 2018, 30(6):652-655.
[11]	Dincel GC. First description of enhanced expression of Glia maturation factor-beta in experimental toxoplasmic encephalitis[J]. J Int Med Res, 2017, 45(6):1670-1679.
[12]	Atmaca HT, Kul O, Karakuş E, et al. Astrocytes,microglia/macrophages, and neurons expressing Toll-like receptor 11 contribute to innate immunity against encephalitic Toxoplasma gondii infection[J]. Neuroscience, 2014, 269:184-191. doi: 10.1016/j.neuroscience.2014.03.049 pmid: 24704432
[13]	Dincel GC, Atmaca HT. Nitric oxide production increases during Toxoplasma gondii encephalitis in mice[J]. Exp Parasitol, 2015, 156:104-112.
[14]	Xiao JC, Li Y, Gressitt KL, et al. Cerebral complement C1q activation in chronic Toxoplasma infection[J]. Brain Behav Immun, 2016, 58:52-56.
[15]	Tanaka N, Ashour D, Dratz E, et al. Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis[J]. Microbes Infect, 2016, 18(7/8):496-504.
[16]	Zhang YH, Chen H, Chen Y, et al. Activated microglia contribute to neuronal apoptosis in Toxoplasmic encephalitis[J]. Parasit Vectors, 2014, 7:372.
[17]	Kim JH, Jou I, Joe EH. Suppression of miR-155 expression in IFN-γ-treated astrocytes and microglia by DJ-1: a possible mechanism for maintaining SOCS1 expression[J]. Exp Neurobiol, 2014, 23(2):148-154.
[18]	O’Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155 is induced during the macrophage inflammatory response[J]. Proc Natl Acad Sci USA, 2007, 104(5):1604-1609. doi: 10.1073/pnas.0610731104 pmid: 17242365
[19]	Wang DY, Tang MC, Zong PF, et al. MiRNA-155 regulates the Th17/treg ratio by targeting SOCS1 in severe acute pancreatitis[J]. Front Physiol, 2018,9:686.
[20]	Huffaker TB, O′Connell RM. miR-155-SOCS1 as a functional axis:satisfying the burden of proof[J]. Immunity, 2015, 43(1):3-4. doi: 10.1016/j.immuni.2015.06.020 pmid: 26200005
[21]	Han SR, Kang YH, Jeon H, et al. Differential expression of miRNAs and behavioral change in the cuprizone-induced demyelination mouse model[J]. Int J Mol Sci, 2020, 21(2):646.

基因	正向引物 (5′-3′)	反向引物 (5′-3′)
miR-155	ACACTCCAGCTGGGTTAATGCTAATTGTGAT	TGCGTGTC-GTGGAGTC
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
IL-1β	CTGAACTCAACTGTGAAATGC	TGATGTGCTGCTGCGAGA
IL-12	GATGTCACCTGCCCAACTG	TGGTTTGATGATGTCCCTGA
iNOS	TTCTCAGCCCAACAATACAAGA	GTGGACGGGTCGATGTCAC
Actb	AGAGGGAAATCGTGCGTGAC	CAATAGTGATGACCTGGCCGT