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。
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.
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
Key words
cannabinoid receptor 2 /
autism spectrum disorder /
microglial activation /
microglia pyroptosis /
neuroinflammatory response
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基金
国家自然科学基金(82073577);黑龙江省普通本科高等学校青年创新人才计划(UNPYSCT-2020168)
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