Abstract: Objective　To investigate the temporal changes in pathological damage and macrophage polarization in liver and spleen tissues of mice infected with Angiostrongylus cantonensis, and to preliminarily unravel the peripheral immune responses during the early stage of A. cantonensis infection. Methods　Forty female BALB/c mice at ages of 6 to 8 weeks were randomly divided into four groups, including the control group and 7⁃, 14⁃, and 21⁃day infection groups, with 10 mice in each group. Each mouse in the infection groups was inoculated with 30 third⁃stage (L3) larvae of A. cantonensis by oral gavage, and five mice were randomly selected from each infection group on days 7, 14, and 21 post⁃infection, while mice in the control group were given the same volume of physiological saline and five mice were randomly selected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled. The histopathological changes of mouse liver and spleen tissues were observed using hematoxylin and eosin (HE) staining, and the percentage of positive staining area and the co⁃localization positive rates of the macrophage surface antigens F4/80, CD86, and CD206 were quantified in mouse liver and spleen tissues using immunohistochemical and immunofluorescence staining. In addition, five mice were collected from each infection group on days 7, 14, and 21 post⁃infection, and five mice were collected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled for detection of macrophage markers CD86 and CD206 and macrophage phenotyping using flow cytometry, and the expression of M1 macrophage markers, including inducible nitric oxide synthase (Nos2), tumor necrosis factor⁃α (TNF⁃α), interleukin⁃1β (IL⁃1β) and M2 markers, including arginase 1 (Arg1), mannose receptor C⁃type 1 (Mrc1) and chitinase⁃like protein 3 (Chil3) was quantified in mouse liver and spleen tissues using real⁃time quantitative PCR (RT⁃qPCR) assay. Results　Proliferative lesions of the hepatocyte were observed in mouse liver tissues and the follicular structures of the mouse spleen white pulp were disrupted 21 days post⁃infection with A. cantonensis. Immunohistochemical staining showed that there were significant differences in the percentages of F4/80, CD86 and CD206 positive staining areas in the liver and spleen tissues among the four groups of mice (F = 242.40, 197.14, 183.19, 157.65, 242.35 and 146.24; all P values < 0.001), and the percentages of positive staining in the liver and spleen tissues of mice in the 14⁃day infection group [(4.45 ± 0.51)%, (3.74 ± 0.67)%, (8.32 ± 0.72)%, (16.56 ± 1.14)%, (11.62 ± 0.52)%, and (8.29 ± 0.72)%, respectively] and the 21⁃day infection group [(3.70 ± 0.11)%, (3.22 ± 0.43)%, (11.53 ± 1.03)%, (12.59 ± 1.05)%, (9.02 ± 0.83)%, and (11.67 ± 1.10)%, respectively] were higher than in the control group [(0.35 ± 0.16)%, (0.40 ± 0.02)%, (0.93 ± 0.05)%, (2.78 ± 0.26)%, (2.33 ± 0.20)%, and (1.85 ± 0.20)%, respectively] (all P values < 0.05). Immunofluorescence staining showed significant differences in the positive rates of F4/80 co⁃localization with CD86 and CD206 in mouse liver and spleen tissues among the four groups (F = 24.42, 25.28, 54.51 and 130.55; all P values < 0.001). Flow cytometry detected significant differences in the proportions of CD86+ and CD206+ macrophages in mouse liver and spleen tissues among the four groups (F = 67.98, 18.41, 29.77, 172.80; all P values < 0.001), and the proportions of CD206+ macrophages in the liver and spleen of the 21⁃day infection group were significantly higher than those in the control group [(9.25 ± 2.55)% vs (3.83 ± 0.72)%, and (4.22 ± 0.56)% vs (0.47 ± 0.18)%, respectively] (both P values < 0.05). In addition, RT⁃qPCR assay quantified significant differences in the relative mRNA expression of M1 macrophage markers (IL⁃1β, TNF⁃α and Nos2) and M2 macrophage markers (Arg1, Chil3 and Mrc1) in mouse liver and spleen tissues among the four groups (F = 41.30, 31.82, 199.33, 19.96, 62.01, 119.76, 23.67, 95.90, 72.27, 82.59, 123.41 and 29.75; all P values < 0.05). Conclusions　A. cantonensis infection may cause progressive pathological damage in mouse liver and spleen tissues, accompanied by dynamic temporal changes in macrophage polarization. M1 macrophage polarization predominates at the early stage of A. cantonensis infection and shifts towards M2 polarization at the later stages, suggesting that M2 polarization may participate in immune regulation at late stages of A. cantonensis infection by suppressing excessive inflammatory responses and promoting tissue repair.

Key words: Angiostrongylus cantonensis, Liver, Spleen, Tissue, Macrophages, Polarization, Mouse

摘要： 目的　观察广州管圆线虫感染小鼠模型中小鼠肝脾组织病理损伤及巨噬细胞极化的时序性变化，初步揭示广州管圆线虫感染早期外周免疫反应。方法　40只6 ~ 8周龄雌性BALB/c小鼠随机分为对照组及感染7、14、21 d组，每组10只。各感染组每只小鼠灌胃感染30条广州管圆线虫Ⅲ期幼虫，于感染7、14、21 d各随机选取5只小鼠；对照组以等量生理盐水灌胃，于灌胃当天随机选取5只小鼠。收集各组所选取小鼠的肝、脾脏组织，采用苏木素⁃伊红（hematoxylin and eosin，HE）染色观察组织病理损伤变化，采用免疫组织化学染色法及免疫荧光染色法，定量检测巨噬细胞表面抗原F4/80、CD86、CD206在组织中的阳性染色面积百分比和共定位阳性率。此外，各感染组分别于感染7、14、21 d取5只小鼠，对照组于灌胃当天取5只小鼠；收集小鼠肝脏、脾脏组织，利用流式细胞术检测巨噬细胞抗原CD86、CD206，并进行分型分析。采用实时荧光定量PCR（real time fluorescent quantitative PCR，RT⁃qPCR）法检测小鼠肝脏与脾脏组织中M1型标志物诱导型一氧化氮合酶（inducible nitric oxide synthase，Nos2）、肿瘤坏死因子⁃α（tumor necrosis factor⁃α，TNF⁃α）、白细胞介素⁃1β（interleukin⁃1β，IL⁃1β）和M2型标志物精氨酸酶1（arginase1，Arg1）、巨噬细胞甘露糖受体（mannose receptor C⁃type1，Mrc1）、几丁质酶样蛋白（chitinase⁃like protein3，Chil3）等基因表达水平。结果　小鼠感染广州管圆线虫21 d后，肝脏组织出现肝细胞增生性病变，脾脏白髓滤泡结构破坏。免疫组织化学染色结果显示，4组小鼠肝脏、脾脏组织内F4/80、CD86、CD206阳性染色面积百分比差异均有统计学意义（F = 242.40、197.14、183.19、157.65、242.35、146.24，P均< 0.001），其中感染14 d组［（4.45 ± 0.51）%、（3.74 ± 0.67）%、（8.32 ± 0.72）%、（16.56 ± 1.14）%、（11.62 ± 0.52）%、（8.29 ± 0.72）%］、感染21 d组小鼠肝脏、脾脏组织内F4/80、CD86、CD206阳性染色面积百分比［（3.70 ± 0.11）%、（3.22 ± 0.43）%、（11.53 ± 1.03）%、（12.59 ± 1.05）%、（9.02 ± 0.83）%、（11.67 ± 1.10）%］均高于对照组［（0.35 ± 0.16）%、（0.40 ± 0.02）%、（0.93 ± 0.05）%、（2.78 ± 0.26）%、（2.33 ± 0.20）%、（1.85 ± 0.20）%］（P均< 0.05）。免疫荧光染色结果显示，4组小鼠肝脏、脾脏组织内F4/80与CD86、CD206共定位阳性率差异均有统计学意义（F = 24.42、25.28、54.51、130.55，P均< 0.001）。流式细胞术检测结果显示，4组小鼠肝脏、脾脏组织CD86+、CD206+巨噬细胞比例差异均有统计学意义（F = 67.98、18.41、29.77、172.80，P均< 0.001），其中感染21 d组肝脏、脾脏组织CD206+巨噬细胞比例［（9.25 ± 2.55）%、（4.22 ± 0.56）%］较对照组［（3.83 ± 0.72）%、（0.47 ± 0.18）%］均升高（P均< 0.05）。4组小鼠肝脏、脾脏组织巨噬细胞M1型（IL⁃1β、TNF⁃α、Nos2）及M2型标志物（Arg1、Chil3、Mrc1）mRNA相对表达量差异均有统计学意义（F = 41.30、31.82、199.33、19.96、62.01、119.76、23.67、95.90、72.27、82.59、123.41、29.75，P均< 0.05）。结论　广州管圆线虫感染可致小鼠肝脾组织进行性病理损伤，并伴随巨噬细胞极化的动态时序变化；感染早期以M1型为主，后期向M2型转变，提示M2型极化在感染后期可能通过抑制过度炎症反应、促进组织修复而参与免疫调节。

关键词: 广州管圆线虫, 肝脏, 脾脏, 组织, 巨噬细胞, 极化, 小鼠