地塞米松对实验性脑型疟小鼠的免疫保护作用

doi:10.12140/j.issn.1000-7423.2022.04.005

摘要/Abstract

摘要：

目的探讨地塞米松（DEX）对实验性脑型疟（ECM）小鼠的免疫保护作用。方法将C57BL/6小鼠随机分为感染组、DEX治疗组和对照组,每组20只。感染组和DEX治疗组小鼠经腹腔注射1 × 10⁶个伯氏疟原虫ANKA感染红细胞,对照组小鼠不作任何处理。DEX治疗组小鼠在感染后第1、2天腹腔注射地塞米松磷酸钠（80 mg/kg）进行治疗,对照组和感染组则注射等体积生理盐水。感染后第3 天起,取感染小鼠尾静脉血制备血涂片,吉氏染色后计数感染红细胞,观察原虫血症变化情况。每日记录小鼠的存活和与ECM相关的神经体征。感染后第3、5天,取小鼠脾脏制备脾细胞悬液,流式细胞术检测脾Th1细胞,调节性T细胞（Tregs）,表达程序性死亡受体-1（PD-1）、细胞毒性T淋巴细胞相关抗原-4（CTLA-4）的CD4⁺ T细胞以及M1、M2型巨噬细胞的变化情况。取脾细胞悬液体外培养48 h后取上清,ELISA检测肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和IL-10等促炎/抑炎相关因子的分泌水平。感染后第6天,伊文思蓝（EB）染色法评价DEX治疗后对小鼠血脑屏障的影响。流式细胞术和细胞因子检测结果的比较采用One-way ANOVA检验,小鼠原虫血症比较采用独立样本t检验,生存率的比较采用Log-Rank检验。结果感染后第3天,感染组和DEX治疗组小鼠外周血开始出现疟原虫感染红细胞,感染后第9天的原虫血症分别为（25.89 ± 1.97）%和（22.91 ± 3.21）%,二者差异无统计学意义（t = 1.12,P > 0.05）。感染组小鼠在感染后第6天开始出现死亡,伴有ECM神经症状,感染后第9天全部死亡;DEX治疗组小鼠在感染后第8天出现死亡,伴有ECM神经症状,第10天全部死亡。与感染组比较,DEX治疗延缓了脑型疟的发生（χ² = 4.31,P < 0.05）。感染后第3、5天,DEX治疗组小鼠表达PD-1的脾CD4⁺ T细胞比例分别为（18.10 ± 0.52）%和（39.13 ± 0.91）%,高于对照组的（11.53 ± 1.00）%和（13.72 ± 1.78）%以及感染组的（14.87 ± 1.02）%和（35.87 ± 0.74）%（F = 41.41、378.00,P < 0.05、0.01）;表达CTLA-4的脾CD4⁺ T细胞比例分别为（3.05 ± 0.12）%和（8.70 ± 1.01）%,高于对照组的（0.28 ± 0.02）%和（0.45 ± 0.10）%以及感染组的（0.44 ± 0.13）%和（0.68 ± 0.13）%（F = 730.20、191.80,P < 0.01）。感染后第3、5天,DEX治疗组小鼠脾细胞中Th1细胞的比例分别为（0.14 ± 0.04）%和（0.63 ± 0.06）%,低于感染组的（0.30 ± 0.06）%和（0.80 ± 0.04）%（F = 10.93、56.58,P < 0.05、0.01）。感染后第5天,DEX治疗组小鼠脾细胞中Tregs细胞的比例为（1.30 ± 0.08）%,高于对照组的（1.12 ± 0.08）%和感染组的（0.86 ± 0.03）%（F = 36.51,P < 0.01）。感染后第3天,DEX治疗组小鼠脾细胞中M1型巨噬细胞的比例为（0.43 ± 0.12）%,低于感染组的（1.03 ± 0.14）%（F = 29.81,P < 0.01）。感染后第5天,DEX治疗组小鼠脾细胞中M2型巨噬细胞比例为（1.56 ± 0.20）%,高于对照组的（0.49 ± 0.04）%和感染组的（0.87 ± 0.06）%（F = 60.21,P < 0.01）。感染后第3、5天,DEX治疗组小鼠脾细胞培养上清中TNF-α水平分别为（140.18 ± 27.38）和（328.23 ± 75.70）pg/ml,低于感染组的（247.34 ± 35.34）和（650.88 ± 43.56）pg/ml（F = 11.70、32.09,P < 0.01）;IL-6水平分别为（40.03 ± 9.09）和（1 270.52 ± 83.75）pg/ml,低于感染组的（511.97 ± 41.70）和（4 131.31 ± 645.16）pg/ml（F = 299.40、93.11,P < 0.01）;IL-10的水平分别为（283.06 ± 44.83）和（592.63 ± 51.69）pg/ml,高于感染组的（118.82 ± 8.47）和（424.49 ± 51.44）pg/ml（F = 36.04、73.95,P < 0.01）。感染后第6天,感染组小鼠的血脑屏障破坏严重,EB渗入脑组织,呈现深蓝色;对照组小鼠血脑屏障完好,未见明显的EB渗入;DEX治疗组小鼠仅有少量EB渗入脑组织。结论在ECM小鼠中,DEX通过提高CD4⁺ T细胞表面免疫抑制分子PD-1和 CTLA-4的水平调控Th1和Tregs细胞活化与应答强度,促进M1向M2型巨噬细胞极化,抑制促炎因子的分泌水平,减轻ECM小鼠血脑屏障的损伤,从而降低ECM的致病性。

关键词: 疟疾, 地塞米松, 脾脏, 促炎症反应

Abstract:

Objective To explore the immunoprotective effect of dexamethasone (DEX) on experimental cerebral malaria (ECM) in mice. Methods C57BL/6 mice were randomly assigned into infection group, DEX treatment group and control group with 20 mice in each group. The mice in the infection group and DEX treatment group were injected intraperitoneally with 1 × 10⁶ Plasmodium berghei ANKA-infected red blood cells, while mice in the control group did not receive any treatment. The mice in the DEX treatment group were treated with intraperitoneal dexamethasone sodium phosphate (80 mg/kg) on the 1st and 2nd days post-infection, while the control and infection groups were injected with an equal volume of normal saline. From the 3rd day on post-infection, the tail vein blood of infected mice was collected to prepare blood smears, and the infected erythrocytes were counted after Giemsa staining to observe the changes of parasitemia. The survival rate and ECM-related neurological symptoms of the mice were recorded daily. On the 3rd and 5th days post-infection, mice spleen cell suspensions were prepared, and flow cytometry was used to detect spleen Th1 cells, regulatory T cells (Tregs), the expression level of programmed death receptor-1 (PD-1), toxic T lymphocyte-associated antigen-4 (CTLA-4) on CD4⁺ T cells, M1 and M2 macrophages. The spleen cell suspensions were cultured for 48 hours, and the culture supernatant was tested by ELISA for secretion pro-inflammatory/anti-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10. On the 6th day post-infection, Evans blue (EB) staining was used to evaluate the effect of DEX treatment on the blood-brain barrier in mice. One-way ANOVA test was used to compare the results from flow cytometry and cytokine detection, independent sample t-test was used to compare the mice parasitemia, and Log-Rank test was used to compare survival rate. Results Plasmodium-infected red blood cells began to appear in the peripheral blood of mice in the infection group and DEX treatment group from the 3rd day post-infection, and the parasitemia was (25.89 ± 1.97)% and (22.91 ± 3.21)% on the 9th day post-infection, respectively. The difference was not statistically significant (t = 1.12, P > 0.05). Mice in the infection group began to die on the 6th day post-infection, accompanied by ECM neurological symptoms, and all died by the 9th day post-infection. Mice in the DEX treatment group began to die on the 8th day post-infection, accompanied by ECM neurological symptoms, and all died by the 10th day post-infection. Compared to the infection group, DEX treatment delayed the onset of cerebral malaria (χ² = 4.31, P < 0.05). On the 3rd and 5th days post-infection, the proportion of spleen CD4⁺ T cells expressing PD-1 in the DEX treatment group was (18.10 ± 0.52)% and (39.13 ± 0.91)%, respectively, higher than the control group [(11.53 ± 1.00)% and (13.72 ± 1.78)%] and the infection group [(14.87 ± 1.02)% and (35.87 ± 0.74)%]. The differences were statistically significant (F = 41.41, 378.00; P < 0.05, 0.01). The proportion of spleen CD4⁺ T cells expressing CTLA-4 were (3.05 ± 0.12)% and (8.70 ± 1.01)%, respectively, higher than the control group [(0.28 ± 0.02)% and (0.45 ± 0.10)%] and the infection group [(0.44 ± 0.13)% and (0.68 ± 0.13)%]. The differences were statistically significant (F = 730.20, 191.80; P < 0.01). On the 3rd and 5th days post-infection, the proportion of Th1 in the spleen cells of the mice in the DEX treatment group was (0.14 ± 0.04)% and (0.63 ± 0.06)%, respectively, which were lower than the infection group [(0.30 ± 0.06)% and (0.80 ± 0.04)%]. The differences were statistically significant (F = 10.93, 56.58; P < 0.05, 0.01). On the 5th day post-infection, the proportion of Tregs cells in the spleen cells of the DEX treatment mice was (1.30 ± 0.08)%, which was higher than the control group (1.12 ± 0.08)% and the infection group(0.86 ± 0.03)% (F = 36.51, P < 0.01). On the 3rd day post-infection, the proportion of M1 macrophages in the spleen cells of the mice in the DEX treatment group was (0.43 ± 0.12)%, which was lower than the infection group (1.03 ± 0.14)% (F = 29.81, P < 0.01). On the 5th day post-infection, the proportion of M2 macrophages in the spleen cells of the DEX treatment mice was (1.56 ± 0.20)%, which was higher than the control group (0.49 ± 0.04)% and the infection group (0.87 ± 0.06)% (F = 60.21, P < 0.01). On the 3rd and 5th days post-infection, the levels of TNF-α in the culture supernatant of spleen cells in the DEX treatment group were (140.18 ± 27.38) pg/ml and (328.23 ± 75.70) pg/ml, which were lower than those in the infection group [(247.34 ± 35.34) pg/ml and (650.88 ± 43.56) pg/ml] (F = 11.70, 32.09; P < 0.01). IL-6 levels were (40.03 ± 9.09) pg/ml and (1 270.52 ± 83.75) pg/ml, which were lower than the infection group (511.97 ± 41.70) pg/ml and the control group (4 131.31 ± 645.16) pg/ml. The difference was statistically significant (F = 299.40, 93.11; P < 0.01). The level of IL-10 was (283.06 ± 44.83) pg/ml and (592.63 ± 51.69) pg/ml, higher than (118.82 ± 8.47) pg/ml and (424.49 ± 51.44) pg/ml in the infection group (F = 36.04, 73.95; P < 0.01). On the 6th day post-infection, the blood-brain barrier of the mice in the infection group was dramatically damaged, and EB penetrated into the brain tissue, showing a dark blue stain. The blood-brain barrier of the mice in the control group was intact, and there was no obvious infiltration of EB. The mice in the DEX treatment group only had a small amount of EB infiltrated into the brain tissue. Conclusion In ECM mice, DEX regulates the activation and response intensity of Th1 and Tregs cells through enhancing the expression levels of immunosuppressive molecules PD-1 and CTLA-4 on the surface of CD4⁺ T cells, promotes the polarization of M1 to M2 macrophages, and inhibits the secretion levels of pro-inflammatory factors, alleviates blood-brain barrier damage in ECM mice, thereby reducing the pathogenicity of ECM.

Key words: Malaria, Dexamethasone, Spleen, Proinflammatory response

中图分类号:

R531.39

黄媛媛, 姚世杰, 卞致芳, 温易鑫, 郑丽, 曹雅明. 地塞米松对实验性脑型疟小鼠的免疫保护作用[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 446-453.

HUANG Yuan-yuan, YAO Shi-jie, BIAN Zhi-fang, WEN Yi-xin, ZHENG Li, CAO Ya-ming. Immunoprotective effect of dexamethasone on experimental cerebral malaria in mice[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(4): 446-453.

图/表 3

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