CB2R调控小胶质细胞功能对小鼠孤独症样行为的影响

doi:10.11852/zgetbjzz2024-1205

摘要/Abstract

摘要： 目的探讨大麻素受体2(CB2R)对小胶质细胞活化和焦亡的调控作用,以及对小鼠孤独症样行为的影响,为ASD的病因学研究提供科学依据。方法体外实验:将BV2小胶质细胞分为对照组、LPS组、AM1241组,采用RT-PCR法检测炎性因子mRNA表达水平,Western Blot法检测焦亡标记物蛋白表达。体内实验:自2023年10月—2024年7月,构建CB2R敲除鼠(CB2R^-/-),将8周龄的小鼠分为WT组和CB2R^-/-组,采用免疫荧光评估小胶质细胞活化情况,采用RT-PCR和Western Blot检测焦亡标记物mRNA和蛋白表达水平,通过旷场、自梳、埋珠、三箱实验来评估小鼠的焦虑样行为、重复刻板行为和社交功能。结果体外实验:与对照组相比,LPS组促炎因子TNF-α、IL-6和CCL2的mRNA水平升高,抗炎因子Arg-1和IL-13的mRNA水平降低(P＜0.05);与LPS组相比,AM1241组TNF-α、IL-6和CCL2的mRNA水平降低,Arg-1、IL-10和IL-13的mRNA水平增加(P＜0.000 1)。与对照组相比,LPS组中NLRP3、GSDMD、GSDMD-N、caspase-1和cleaved-caspase-1的蛋白表达明显增加,AM1241干预后,均显著降低(P＜0.05)。体内实验:与WT小鼠相比,CB2R^-/-小鼠海马CA1、CA3区的CD86⁺/Iba1⁺比值升高,而CD206⁺/Iba1⁺比值降低(P＜0.01);NLRP3、GSDMD、caspase-1、IL-1β、IL-18的mRNA水平明显升高(t=2.20、3.12、2.55、2.54、2.30,P＜0.05);NLRP3、GSDMD、GSDMD-N、caspase-1和cleaved-caspase-1的蛋白表达明显增加(t=7.43、2.87、3.86、3.99、6.55,P＜0.05);移动距离、自梳时间和埋珠个数均显著升高,休息时间和社交偏好指数显著降低(P＜0.05)。结论 CB2R参与调控小胶质细胞活化和焦亡,影响神经炎症反应,进而导致小鼠出现孤独症样行为,提示CB2R可能是ASD潜在治疗靶点。

关键词: 大麻素受体2, 孤独症谱系障碍, 小胶质细胞活化, 小胶质细胞焦亡, 神经炎性反应

Abstract: Objective To explore the regulatory effect of cannabinoid receptor 2 (CB2R) on microglial function and its impact on autism-like behaviors in mice, in order to provide scientific basis for the etiology of ASD. Methods In vitro experiment, BV2 microglia were divided into control group, LPS group, and AM1241 group. RT-PCR was used to detect mRNA expression levels of inflammatory factors. Western Blot was used to measure the proteins expression of pyroptosis marker. In vivo experiments, the 8-week-old mice were divided into WT group and CB2R knockout mice (CB2R^-/- group). Immunofluorescence staining was used to evaluate the activation of microglia. RT-PCR and western blot were used to detect mRNA and proteins expression levels of pyroptosis marker. Anxiety, restricted and repetitive behaviors, and social function were evaluated by open field, self-grooming, burying marble, and three-chamber test. Results In vitro experiments, compared with the control group, the mRNA levels of pro-inflammatory factors TNF-α, IL-6, and CCL2 in the LPS group were increased, while anti-inflammatory factors Arg-1 and IL-13 were decreased (P<0.05). Compared with the LPS group, the mRNA levels of TNF-α, IL-6, and CCL2 in the AM1241 group were decreased, while Arg-1, IL-10, and IL-13 were increased (P<0.000 1). Compared with the control group, the protein expression of NLRP3, GSDMD, GSDMD-N, caspase-1, and cleaved caspase-1 were significantly increased in the LPS group. After intervention with AM1241, the above protein levels were significantly reduced (P＜0.05). In vivo experiments, compared with the WT group, the CD86⁺/Iba1⁺ ratios in the hippocampal CA1 and CA3 regions were increased, while the CD206⁺/Iba1⁺ ratios were decreased in the CB2R^-/- group (P<0.01). The mRNA expression of NLRP3, GSDMD, caspase-1, IL-1β, and IL-18 in CB2R^-/- group was significantly increased (t=2.20, 3.12, 2.55, 2.54, 2.30, P＜0.05). The protein expression levels of NLRP3, GSDMD, GSDMD-N, caspase-1, and cleaved caspase-1 in CB2R^-/- group were significantly increased (t=7.43, 2.87, 3.86, 3.99, 6.55, P＜0.05). The CB2R^-/- group showed a significant increase in the numbers of moved distance, self-grooming time and marbles buried in mice, a significant decrease in the resting time and social preference index (P<0.05). Conclusion CB2R is involved in regulating the activation and pyroptosis of microglia, affecting neuroinflammatory responses, thereby leading to autism-like behaviors in mice, which suggests that CB2R might be a potential therapeutic target for ASD.

Key words: cannabinoid receptor 2, autism spectrum disorder, microglial activation, microglia pyroptosis, neuroinflammatory response

中图分类号:

R749.94

田文茹, 杨璧璘, 胡婧仪, 刘世丹, 邹明扬, 武丽杰, 孙彩虹. CB2R调控小胶质细胞功能对小鼠孤独症样行为的影响[J]. 中国儿童保健杂志, 2025, 33(1): 62-70.

TIAN Wenru, YANG Bilin, HU Jingyi, LIU Shidan, ZOU Mingying, WU Lijie, SUN Caihong. Effect of cannabinoid receptor 2 on autism-like behaviors inmice by regulating microglial function[J]. Chinese Journal of Child Health Care, 2025, 33(1): 62-70.

参考文献

[1] Sahu P, Mudgal J, Arora D, et al. Cannabinoid receptor 2 activation mitigates lipopolysaccharide-induced neuroinflammation and sickness behavior in mice[J]. Psychopharmacology, 2019, 236(6): 1829-1838.
[2] Carrera J, Tomberlin J, Kurtz J, et al. Endocannabinoid signaling for GABAergic-Microglia (Mis) Communication in the brain aging[J]. Front Neurosci, 2021, 14: 606808.
[3] Hsieh GC, Pai M, Chandran P, et al. Central and peripheral sites of action for CB2 receptor mediated analgesic activity in chronic inflammatory and neuropathic pain models in rats[J]. Br J Pharmacol, 2011, 162(2): 428-440.
[4] Komorowska-Müller JA, Schmöle AC. CB2 Receptor in Microglia: The guardian of Self-Control[J]. Int J Mol Sci, 2020, 22(1): 19.
[5] Xin Q, Xu F, Taylor DH, et al. The impact of cannabinoid type 2 receptors (CB2Rs) in neuroprotection against neurological disorders[J]. Acta Pharmacol Sin, 2020, 41(12): 1507-1518.
[6] Hsu SK, Li CY, Lin IL, et al. Inflammation-related pyroptosis, a novel programmed cell death pathway, and its crosstalk with immune therapy in cancer treatment[J]. Theranostics, 2021, 11(18): 8813-8835.
[7] Shao BZ, Wei W, Ke P, et al. Activating cannabinoid receptor 2 alleviates pathogenesis of experimental autoimmune encephalomyelitis via activation of autophagy and inhibiting NLRP3 inflammasome[J]. CNS Neurosci Ther, 2014, 20(12): 1021-1028.
[8] Kibret BG, Ishiguro H, Horiuchi Y, et al. New insights and potential therapeutic targeting of CB2 cannabinoid receptors in CNS disorders[J]. Int J Mol Sci, 2022, 23(2), 975.
[9] Saghazadeh A, Ataeinia B, Keynejad K, et al. A meta-analysis of pro-inflammatory cytokines in autism spectrum disorders: Effects of age, gender, and latitude[J]. J Psychiatr Res, 2019, 115: 90-102.
[10] Zou M, Liu Y, Xie S, et al. Alterations of the endocannabinoid system and its therapeutic potential in autism spectrum disorder[J]. Open Biol, 2021, 11(2): 200306.
[11] Sánchez AJ, García-Merino A. Neuroprotective agents: Cannabinoids[J]. Clin Immunol, 2012, 142(1): 57-67.
[12] Palazuelos J, Aguado T, Pazos MR, et al. Microglial CB2 cannabinoid receptors are neuroprotective in Huntington's disease excitotoxicity[J]. Brain, 2009, 132(11): 3152-3164.
[13] Li J, Wang H, Liu D, et al. CB2R activation ameliorates late adolescent chronic alcohol exposure-induced anxiety-like behaviors during withdrawal by preventing morphological changes and suppressing NLRP3 inflammasome activation in prefrontal cortex microglia in mice[J]. Brain Behav Immun, 2023, 110: 60-79.
[14] Çakir M, Tekin S, Doğanyiğit Z, et al. Cannabinoid type 2 receptor agonist JWH-133, attenuates Okadaic acid induced spatial memory impairment and neurodegeneration in rats[J]. Life Sci, 2019, 217: 25-33.
[15] Zhang B, Zheng F, Liu A, et al. Activation of CB2 receptor inhibits pyroptosis and subsequently ameliorates cecal ligation and puncture-induced sepsis[J]. Int Immunopharmacol, 2021, 99: 108038.
[16] Yamaori S, Ishii H, Chiba K, et al. Δ8-Tetrahydrocannabinol induces cytotoxicity in macrophage J774-1 cells: Involvement of cannabinoid receptor 2 and p38 MAPK[J]. Toxicology, 2013, 314(2-3): 254-261.
[17] Hughes HK, Moreno RJ, Ashwood P. Innate immune dysfunction and neuroinflammation in autism spectrum disorder (ASD)[J]. Brain Behav Immun, 2023, 108: 245-254.
[18] Mecha M, Feliú A, Carrillo-Salinas FJ, et al. Endocannabinoids drive the acquisition of an alternative phenotype in microglia[J]. Brain Behav Immun, 2015, 49: 233-245.
[19] 张雪原,孔亚敏,马丙祥,等.孤独症谱系障碍动物模型相关神经生物学研究进展[J].中国实验动物学报,2022,30(8):1141-1149.
Zhang XY, Kong YM, Ma BX, et al. Research progress in the neurobiology of animal models of autism spectrum disorder[J]. Acta Lab Anim Sci Sin, 2022, 30(8): 1141-1149.(in Chinese)
[20] Arteaga-Henríquez G, Gisbert L, Ramos-Quiroga JA. Immunoregulatory and/or anti-inflammatory agents for the management of core and associated symptoms in individuals with autism spectrum disorder: A narrative review of randomized, placebo-controlled trials[J]. CNS Drugs, 2023, 37(3): 215-229.
[21] Szabo A, O'connell KS, Akkouh IA, et al. Elevated levels of peripheral and central nervous system immune markers reflect innate immune dysregulation in autism spectrum disorder[J]. Psychiatry Res, 2024, 342: 116245.
[22] Zhao P, Fu H, Cheng H, et al. Acupuncture at ST36 Alleviates the behavioral disorder of autistic rats by inhibiting TXNIP-mediated activation of NLRP3[J]. J Neuropathol Exp Neurol, 2022, 81(2): 127-134.
[23] Komorowska-Müller JA, Ravichandran KA, Zimmer A, et al. Cannabinoid receptor 2 deletion influences social memory and synaptic architecture in the hippocampus[J]. Sci Rep, 2021, 11(1): 16828.
[24] Komorowska-Müller JA, Rana T, Olabiyi BF, et al. Cannabinoid receptor 2 alters social memory and microglial activity in an age-dependent manner[J]. Molecules, 2021, 26(19): 5984.
[25] Canseco-Alba A, Schanz N, Sanabria B, et al. Behavioral effects of psychostimulants in mutant mice with cell-type specific deletion of CB2 cannabinoid receptors in dopamine neurons[J]. Behav Brain Res, 2019, 360: 286-297.
[26] Cortez IL, Silva NR, Rodrigues NS, et al. HU-910, a CB2 receptor agonist, reverses behavioral changes in pharmacological rodent models for schizophrenia[J]. Prog Neuro-Psychopharmacol Biol Psychiatry, 2022, 117: 110553.
[27] Fyke W, Alarcon JM, Velinov M, et al. Pharmacological inhibition of the primary endocannabinoid producing enzyme, DGL-α, induces autism spectrum disorder-like and co-morbid phenotypes in adult C57BL/J mice[J]. Autism Res, 2021, 14(7): 1375-1389.