关键词: 广州管圆线虫, 血脑屏障, 紧密连接蛋白, 基质金属蛋白酶, 适宜宿主

Abstract:

Objective To investigate the characteristics and dynamic patterns of the blood-brain barrier (BBB) disruption in rats infected with Angiostrongylus cantonensis, so as to provide experimental evidence for elucidating the pathogenesis of angiostrongyliasis. Methods A. cantonensis infection was modeled in Sprague-Dawley (SD) rats, which were then divided into an uninfected group and six infection groups corresponding to 7, 14, 21, 28, 35 and 42 days post-infection (dpi), with 10 rats in each group. Brain tissues were collected from rats in each group. The alterations of BBB integrity were detected using Evans blue (EB) extravasation assay in rats at different time points post-infection, and the leakage of albumin was detected using immunofluorescence staining in brain tissues of infected rats. RNA and proteins were extracted from rat brain tissues. The mRNA expression of BBB tight junction proteins claudin-5, occludin, Zonula occludens-1 (ZO-1) and platelet endothelial cell adhesion molecule (CD31) was quantified using real-time fluorescent quantitative reverse transcription PCR (RT-qPCR) assay in rats infected with A. cantonensis, and the expression of claudin-5, occludin and CD31 proteins was determined using Western blotting assay. In addition, the transcriptional levels of BBB disruption-related genes were quantified using RT-qPCR assay in rats infected with A. cantonensis at different time points, including matrix metalloproteinase-2 (mmp2), mmp9, intercellular adhesion molecule-1 (icam-1), vimentin, tumor necrosis factor-α (tnf-α) and interleukin 1β (il-1β). Differences of means between groups were tested for statistical significance with Student’s t-test. Results EB extravasation assay revealed remarkable BBB disruption in rats at 28 dpi with higher amount of EB extravasation [(20.030 ± 5.580) μg/g vs. (1.000 ± 0.327) μg/g; t = -5.896, P < 0.01] and EB fluorescence intensity (2.452 ± 3.14 vs. 0.596 ± 0.485; t = -6.464, P < 0.01] than in uninfected rats. The amount of EB extravasation gradually decreased on 35 dpi (6.568 ± 2.072) and 42 dpi (4.237 ± 2.033)， and the EB fluorescence intensity gradually reduced on 35 dpi (2.843 ± 2.314) and 42 dpi (1.816 ± 0.697), indicating gradual recovery of BBB functions. Immunofluorescence staining demonstrated remarkable albumin extravasation from rat brain tissues into extravascular regions, notably during the period from 28 to 42 dpi. RT-qPCR assay quantified lower relative mRNA levels of occludin and CD31 in infected rats on 21 and 28 dpi (occludin: 0.729 ± 0.087, 0.664 ± 0.162; CD31: 0.700 ± 0.145, 0.584 ± 0.208) than in uninfected rate (occludin: 1.017 ± 0.189; CD31: 1.032 ± 0.296) (occludin: t = -2.834 and -2.999, both P < 0.05; CD31: t = -2.515 and -3.193, both P < 0.05), and the transcriptional expression of occludin and CD31 recovered on 35 dpi (occludin: 0.943 ± 0.139; CD31: 0.815 ± 0.205) and 42 dpi (occludin: 1.139 ± 0.531; CD31: 0.869 ± 0.107). The relative mRNA level of ZO-1 was significantly lower in infected rats on 14 dpi (0.756 ± 0.110) and 21 dpi (0.726 ± 0.109) than in uninfected rats (1.017 ± 0.219) (t = -2.602 and -2.919, both P < 0.05), and gradually increased on 28 dpi (0.824 ± 0.131), 35 dpi (0.928 ± 0.072) and 42 dpi (0.868 ± 0.111). No significant difference was observed in claudin-5 mRNA level between infected and uninfected rats across all time points dpi (t = 1.031, 1.429, 1.906, 0.798, 0.991 and 0.473, all P > 0.05). Western blotting assay determined lower relative occludin protein expression in brain tissues in rats infected with A. cantonensis (1.240 ± 0.176) on 21 dpi than in uninfected rats (0.734 ± 0.159) (t = 3.693, P < 0.05), and the protein expression gradually recovered to normal on 28 (t = 2.679, P > 0.05), 35 (t = 1.155, P > 0.05) and 42 dpi (t = 1.459, P > 0.05). The relative CD31 protein expression was lower in infected rats than in uninfected rats on 28 dpi [(0.693 ± 0.009) vs. (1.118 ± 0.104); t = ‒14.569, P < 0.01], and then recovered on 35 dpi (0.925 ± 0.294) and 42 dpi (1.068 ± 0.052). In addition, the relative mRNA levels of BBB disruption-related genes mmp2, mmp9, icam-1, vimentin, tnf-α, il-1β were all higher in infected rats (2.735 ± 0.560, 3.393 ± 2.066, 4.717 ± 2.037, 17.447 ± 5.129, 1.739 ± 0.572, 7.449 ± 1.905) than in uninfected rats (1.017 ± 0.225, 1.109 ± 0.647, 1.250 ± 0.266, 1.040 ± 0.354, 1.026 ± 0.252, 1.111 ± 0.593) on 28 dpi (t = 5.794, 2.504, 2.686, 6.381, 2.347, 6.648, P < 0.05 or P < 0.01), and exhibited a tendency towards a decline from 35 dpi to 42 dpi, with similar dynamic changes with the pattern of BBB disruption and recovery. Conclusion A. cantonensis infection may induce BBB disruption in rats, which peaks at 28 dpi and then gradually recovers. It is hypot underlying mechanism may be closely related to the reduced expression of tight junction proteins and the upregulation of BBB disruption-related genes such as mmp2 and mmp9.

Key words: Angiostrongylus cantonensis, Blood-brain barrier, Tight junction protein, Matrix metalloproteinase, Permissive host