青蒿琥酯与重组蛋白IL-33联合调控小鼠脑型疟免疫相关指标研究

doi:10.12140/j.issn.1000-7423.2020.02.002

摘要/Abstract

摘要：

目的探讨青蒿琥酯（ART）和重组白细胞介素-33（rIL-33）联合治疗伯氏疟原虫ANKA（PbA）感染小鼠脑型疟的效果及其对免疫相关指标的影响。方法

40只雌性C57BL/6小鼠随机分为5组：PbA感染未治疗组（PbA组）,PbA感染治疗组（PbA+rIL-33组、PbA+ART组、PbA+rIL-33+ART组）和健康对照组,每组8只。PbA组及各治疗组小鼠经腹膜内注射1 × 10 ⁶ 个PbA感染的红细胞;感染后第2~4天,PbA+rIL-33组小鼠经腹膜内注射rIL-33（0.2 μg/鼠,连续3 d）,PbA+ART组灌胃ART[40 mg/（kg·d）,连续3 d],PbA+rIL-33+ART组灌胃ART[40 mg/(kg·d)]并经腹膜内注射rIL-33（0.2 μg/鼠,连续3 d）;PbA组不作处理;健康对照组在相同时间点注射等体积的PBS。感染后第3天起每隔1天采尾静脉血制作血涂片,吉氏染色后计数感染红细胞并计算感染率,记录小鼠死亡情况和生存期。感染后第5天各组取1只小鼠,尾静脉注射伊文思蓝溶液,根据脑组织上清液的吸光度（A₆₃₀值）检测通过血脑屏障的伊文思蓝含量,评估血脑屏障完整性。感染后第5天每组各处死4只小鼠,取脾组织,流式细胞术检测脾细胞中的Th1/Th2、调节性T细胞（Treg）、巨噬细胞和Toll样受体4（TLR4）细胞的百分率和绝对数。结果 PbA组小鼠在感染后第6天出现神经症状,死亡发生在感染后第6天至第13天;PbA+rIL-33组和PbA+ART组死亡均发生于感染后第8天;PbA+rIL-33+ART组小鼠神经系统的症状发生明显被抑制,在感染后第12天开始死亡,第23天2/7的小鼠死于贫血。感染后第3~13天,PbA+rIL-33+ART组的红细胞感染率从0.30%升至19.67%,均低于PbA组（0.93%~20.00%）和PbA+rIL-33组（0.46%~19.67%）（P < 0.05）。血脑屏障完整性检测结果显示,PbA+rIL-33+ART组小鼠脑组织上清液的A₆₃₀值为（0.11 ± 0.01）,低于PbA组（0.44 ± 0.01）（P < 0.01）、PbA+rIL-33组（0.19 ± 0.01）（P < 0.05）和PbA+ART组（0.27 ± 0.02）（P < 0.01）。流式细胞术分析结果显示,PbA+rIL-33+ART组脾细胞中Th1细胞百分率为（7.51 ± 0.26）%,低于PbA组[（14.27 ± 0.91）%]（P < 0.01）,但与PbA+ART组[（9.56 ± 1.75）%]、PbA+rIL-33组[（8.67 ± 0.26）%]的差异无统计学意义（P > 0.05）;PbA+rIL-33+ART组Th2细胞百分率为（2.63 ± 0.27）%,高于PbA组[（0.80 ± 0.13）%]（P < 0.01）和PbA+ART组[（0.88 ± 0.12）%]（P < 0.01）,但与PbA+rIL-33组[（1.70 ± 0.54）%]的差异无统计学意义（P > 0.05）;PbA+rIL-33+ART组巨噬细胞百分率为（3.85 ± 0.32）%,高于PbA组[（2.89 ± 0.89）%]（P < 0.05）,但与PbA+ART组[（3.15 ± 0.46）%]和PbA + rIL-33组[（4.11 ± 0.68）%]的差异无统计学意义（P > 0.05）;PbA+rIL-33+ART组Treg细胞百分率为（11.05 ± 1.50）%,高于PbA组[（6.44 ± 0.09）%]（P < 0.05）和PbA+ART组[（6.27 ± 0.39）%]（P < 0.01）,但与PbA+rIL-33组[（9.34 ± 0.61）%]的差异无统计学意义（P > 0.05）;PbA+rIL-33+ART组TLR4细胞百分率为（1.43 ± 0.21）%,高于PbA组[（3.76 ± 0.41）%]（P < 0.01）,但与PbA+ART组[（1.69 ± 0.26）%]和PbA+rIL-33组[（1.61 ± 0.15）%]的差异无统计学意义（P > 0.05）。

结论 rIL-33联合ART治疗可保护鼠疟模型的脑组织,通过平衡Th1/Th2免疫应答进而改善ART治疗脑型疟的结局及免疫效应。

关键词: 伯氏疟原虫ANKA, 白细胞介素-33, 青蒿琥酯, 脑型疟

Abstract:

Objective To investigate the effects of artesunate (ART) in combination with recombinant interleukin-33 (rIL-33) in the treatment of cerebral malaria in mice infected with Plasmodium berghei ANKA (PbA) and its effects on immune responses of mice.Methods Forty female C57BL/6 mice were randomly divided into 5 groups (n = 8): PbA infection without treatment (PbA group), PbA infection with treatment (PbA+rIL-33 group, PbA+ART group, and PbA+rIL-33+ART group), and normal control group. Mice in the PbA infection groups were injected intraperitoneally (i.p.) with 1 × 10 ⁶ PbA-infected red cells. Two to four days post-infection, mice in the PbA+rIL-33 group received daily i.p. injections of rIL-33 (0.2 μg/mouse) for 3 consecutive days, those in the PbA+ART group received 40 mg/kg ART (once daily for 3 days) by gavage, and those in the PbA+rIL-33+ART group received ART and rIL-33 injection for 3 consecutive days. The PbA group received no treatment. The control group received the same volume of PBS at the same time points as above. From day 3 after infection, tail vein blood was collected every other day, blood smears were made for Giemsa staining to examine the infection rate of red cells, and the death and survival time span were recorded as well. To assess the integrity of the blood-brain barrier (BBB), on day 5 after infection, evans blue solution was given to the mice intravenously, then the brain tissue eluent was examined to detect penetrated blue dye passing through the barrier by measuring absorbance at 630 nm (A₆₃₀). On day 5 after infection, four mice of each group were sacrificed to determine the percentage and absolute number of Th1/Th2, Tregs, macrophages and Toll-like receptor 4 (TLR4) cells in spleen by flow cytometry. Results In the PbA group, neurological symptoms first appeared on day 6, and deaths occurred from day 6 to day 13, while in the PbA+rIL-33 and the PbA+ART groups deaths occurred on day 8. The neurological symptoms of the PbA+rIL-33+ART group were significantly suppressed, and deaths occurred on day 12 post infection. Two of 7 mice died of anemia on day 23 post infection. The infection rate of red cells in the PbA+rIL-33+ART group increased from 0.30% to 19.67% during day 3 to 13 after infection, remaining consistently lower than those of the PbA group (0.93%-20.00%) and PbA+rIL-33 group (0.46%-19.67%) (P < 0.05). In addition, BBB integrity assessment showed that the A₆₃₀ in the PbA+rIL-33+ART group was (0.11 ± 0.01), significantly lower than those in the PbA group (0.44 ± 0.01) (P < 0.01), the PbA+rIL-33 group (0.19 ± 0.01) (P < 0.01) and the PbA+ART group (0.27 ± 0.02) (P < 0.01). Flow cytometry assay showed that the percentage of Th1 cells in the PbA+rIL-33+ART group was (7.51 ± 0.26)%, which was significantly lower than that in the PbA group [(14.27 ± 0.91)%, P < 0.01], but did not differ significantly from those in the PbA+ART group [(9.56 ± 1.75)%] and PbA+rIL-33 group [(8.67 ± 0.26)%] (P > 0.05). The percentage of Th2 cells in the PbA+rIL-33+ART group was (2.63 ± 0.27)%, which was significantly higher than those in the PbA group [(0.80 ± 0.13)%, P < 0.01] and the PbA+ART group [(0.88 ± 0.12)%, P < 0.01], but did not differ significantly from that in the PbA+rIL-33 group [(1.70 ± 0.54)%] (P > 0.05). The percentage of macrophages in the PbA+rIL-33+ART group was (3.85 ± 0.32)%, which was significantly higher than that in the PbA group [(2.89 ± 0.89)%] (P < 0.05), but did not differ significantly from those in the PbA+ART group [(3.15 ± 0.46)%] and the PbA+rIL-33 group [(4.11 ± 0.68)%] (P > 0.05). The percentage of Treg cells in the PbA+rIL-33+ART group was (11.05 ± 1.50)%, which was significantly higher than those in the PbA group [(6.44 ± 0.09)%, P < 0.05] and the PbA+ART group [(6.27 ± 0.39)%, P < 0.01], but did not differ significantly from that in the PbA+rIL-33 group [(9.34 ± 0.61)%] (P > 0.05). The percentage of TLR4 cells in the PbA+rIL-33+ART group [(1.43 ± 0.21)%] was significantly higher than that in the PbA group [(3.76 ± 0.41)%] (P < 0.01), but did not differ significantly from those in the PbA+ART group [(1.69 ± 0.26)%] and the PbA+rIL-33 group [(1.61 ± 0.15)%](P > 0.05).Conclusion rIL-33 combined with ART can protect the mouse brain from experimental cerebral malaria damage, and improve the therapeutic outcome through balancing Th1/Th2 immune responses.

Key words: Plasmodium berghei ANKA, Interleukin-33, Artesunate, Cerebral malaria

中图分类号:

R531.33

杜云婷, 赵薇, 曹雅明, 徐兰. 青蒿琥酯与重组蛋白IL-33联合调控小鼠脑型疟免疫相关指标研究[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 139-145.

Yun-ting DU, Wei ZHAO, Ya-ming CAO, Lan XU. Regulations of immune responses by artesunate in combination with rIL-33 in the treatment of cerebral malaria in mice[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(2): 139-145.

图/表 3

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