慢性间歇性低氧条件下靶向封闭大麻素受体1对小鼠脾脏免疫功能及炎症反应的调节作用

Regulation of targeted blocking cannabinoid receptor 1 on spleen immune function and inflammatory response in mice under chronic intermittent hypoxia

目的观察靶向封闭大麻素受体1(CB1R)对慢性间歇性低氧(CIH)小鼠脾脏免疫功能及炎症反应的影响,探讨其调节作用。方法2021年5—8月,于山西医科大学第二医院实验动物中心选取40只4~5周龄SPF级雄性C57BL/6小鼠,随机数字表法随机分为常氧对照组(NC组)、CIH 6周组(6w CIH组)、CIH 10周组(10w CIH组)、CIH+CB1R靶向封闭6周组(6w CIH+AM251组)、CIH+CB1R靶向封闭10周组(10w CIH+AM251组),使用高级可编程间歇低氧仓建立CIH小鼠模型。苏木精-伊红(HE)染色观察CIH小鼠脾脏组织的形态结构;免疫荧光检测小鼠脾脏M1、M2型巨噬细胞表面标志物CD86、CD206的表达,qRT-PCR技术检测脾脏组织中CB1R、CD86、CD206等基因的mRNA表达水平,以及辅助性T细胞(Th)17和调节性T细胞(Treg)特征性转录调节因子维甲酸相关核孤儿受体γt(RORγt)和叉头框蛋白p3(Foxp3)的相对表达量。ELISA法测定炎症因子IL-6、IL-10的表达。采用SPSS 26.0和Graphpad prism 8.3软件分析数据。结果(1)与NC组相比,CIH组小鼠脾脏组织结构紊乱,纤维组织增生,小动脉周围淋巴鞘中淋巴细胞增生、排列紊乱;CIH组CB1R表达较NC组高(P<0.05),且10w CIH组较6w CIH组表达高(P<0.05),CIH+AM251组CB1R表达均低于对应的CIH组(均P<0.05);(2)与NC组比较,CIH组巨噬细胞CD86表达水平高于NC组;6w及10w CIH组RORγt相对表达量分别为0.76±0.03、0.91±0.04,均高于NC组的0.65±0.06(均P<0.05),炎症因子IL-6的相对表达量分别为10.80±1.73、14.86±0.01,均高于NC组的6.69±0.23(均P<0.05);6w及10w CIH+AM251组巨噬细胞CD206表达水平均高于对应的CIH组(P<0.05),6w及10w CIH+AM251组Foxp3相对表达量分别为0.62±0.05、0.32±0.21,均高于6w CIH组的0.28±0.02及10w CIH组的0.02±0.01(均P<0.05),抑炎因子IL-10相对表达量分别为668.45±15.71、379.15±56.84,表达水平均高于对应的CIH组(均P<0.05)。结论靶向封闭CB1R可通过调节小鼠脾脏巨噬细胞极化和炎性相关因子的表达,减轻CIH诱导的炎症反应,对机体有一定的保护作用。

ObjectiveTo observe the effects of targeting and blocking cannabinoid receptor 1(CB1R)on mouse spleen immune function and inflammatory response under chronic intermittent hypoxia(CIH)conditions,and to explore its regulatory effort.MethodsForty SPF male C57BL/6 mice aged 4 to 5 weeks,from May 2021 to August 2021 in Experimental Animal Center of the Second Hospital of Shanxi Medical University,were randomly divided into normal oxygen control group(NC),6-week CIH group(6w CIH),10-week CIH group(10w CIH),6-week CIH+CB1R group(6w CIH+AM251)and 10-week CIH+CB1R group(10w CIH+AM251)according to the method of random number table.The advanced programmable intermittent low oxygen chamber was used to prepare the CIH mouse model.The morphological structure of spleen tissue of CIH mice was stained by hematoxylin-eosin(HE)staining.The expression levels of M1 and M2 macrophage surface markers CD86,CD206 were determined by immunofluorescence.The mRNA expression levels of CB1R,CD86,CD206 and the relative expression levels of RORγt and Foxp3,which are characteristic transcriptional regulators of T helper 17(Th17)and Treg cells were detected by quantitative reverse transcriptase PCR(qRT-PCR).The expression of inflammatory factors IL-6 and IL-10 was determined by ELISA.SPSS 26.0 and Graphpad prism 8.3 were used to analyze the data.Results(1)Compared with NC group,spleen tissue structure was disordered,fibrous tissue hyperplasia,lymphocyte proliferation and disordered arrangement in periarteriole lymphatic sheath in CIH group.The expression of CB1R in CIH group was higher than that in NC group(P<0.05),and with the prolongation of CIH time,the expression of 10w CIH group was higher than that in 6w CIH group(P<0.05).The expression of CB1R in CIH+AM251 group was lower than that in the corresponding CIH group(all P<0.05).(2)Compared with NC group,the expression level of CD86 in macrophages in CIH group was higher than that in NC group(all P<0.05).The relative expression of RORγt in 6w and 10w CIH groups was 0.76±0.03 and 0.91±0.04,respectively,which was higher than that in NC group(0.65±0.06)(all P<0.05).The relative expression levels of inflammatory factor IL-6 were 10.80±1.73 and 14.86±0.01,respectively,which were higher than 6.69±0.23 in the NC group(all P<0.05).The expression level of CD206 in macrophages in the CIH+AM251 group was higher than that in the CIH group(all P<0.05).The relative expression levels of Foxp3 in 6w and 10w CIH+AM251 groups were 0.62±0.05 and 0.32±0.21,respectively,which were higher than those in 6w CIH group(0.28±0.02)and 10w CIH group(0.02±0.01)(P<0.05).The relative expression levels of anti-inflammatory factor IL-10 were 668.45±15.71 and 379.15±56.84,respectively,which were higher than those in CIH group(all P<0.05).ConclusionTargeted sealing of CB1R may alleviate inflammatory response of mouse spleen under CIH conditions by regulating macrophage polarization and the expression of inflammatory factors,and may have some protective effect.