Fig 1.
Histopathological changes and blood-brain barrier dysfunction are induced in Angiostrongylus cantonensis-infected mice.
Fifth-stage larvae of A. cantonensis were observed in the (A) anterior cerebral fissure, (B) hippocampus, (C) posterior cerebral fissure, and (D) cerebellar fissure of mice infected with 25 third-stage larvae on day 14 postinfection (stained with hematoxylin-eosin). Inflammatory cells were found surrounding these larvae. (E) Blood-brain barrier dysfunction and breakdown was shown by positive staining after the intravenous infusion of 2% Evans blue into the brains of mice infected with Angiostrongylus cantonensis on days 0, 7, 14, 21 and 28 postinfection.
Fig 2.
Excretory/secretory products of A. cantonensis L5 stimulate autophagy in astrocytes.
(A) Mouse brains were collected from A. cantonensis-infected mice on days 0, 7, 14, and 21 postinfection. Then, the mRNA expression levels of autophagy-related molecules were detected by cDNA microarray analysis. (B) Cells were treated with 0, 62.5, 125, 250, and 500 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. The protein expression levels of autophagy-related molecules were detected by Western blotting. The data are expressed as the means ± SD from three independent experiments (n = 3). *P<0.05, #P<0.01, compared with the respective values of cells treated with 0 μg/ml A. cantonensis L5 ESPs for 12 h.
Fig 3.
Excretory/secretory products of A. cantonensis L5 stimulate autophagic-like vacuole formation.
Cells were (A) left untreated or (B) treated with 500 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. The formation of autophagic-like vacuoles was detected by using transmission electron microscopy (TEM) (A: autophagosomes. M: mitochondria. Ly: lysosomes. RER: rough endoplasmic reticulum. N: nucleus). (C) TEM images of autophagic structures upon ESPs treatment ((I) (II) autolysosomes with dark undigested content; (III) autophagosomes; (IV) autolysosomes filled with undigested lipids; (V) phagophores; and (VI) empty autophagic-like vacuoles).
Fig 4.
Rapamycin induces autophagy in excretory/secretory product-treated astrocytes.
(A) Cells were pretreated with 100 or 500 nM rapamycin for 1 h and then incubated with 500 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. Western blotting was used to analyze the expression levels of the autophagy-related proteins LC3-I, LC3-II, Beclin, and p62. β-actin is shown as the control. (B) Cells were pretreated with 5 or 10 mM 3-methyladenine (3-MA) for 1 h and then incubated with 500 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. Western blotting was used to analyze the expression levels of the autophagy-related proteins LC3-I, LC3-II, Beclin, and p62. β-actin is shown as the control. The data are expressed as the means ± SD from three independent experiments (n = 3). ※P<0.05, *P<0.01, compared with the respective values of the control. #P<0.01, compared with the cells exposed to ESPs.
Fig 5.
Immunofluorescence staining of autophagy-related proteins in excretory/secretory product-treated astrocytes.
Cells were pretreated with 100 nM rapamycin for 1 h and then incubated with 500 μg/ml of A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. Immunofluorescence staining was used to detect the expression of autophagy-related proteins (blue: nucleus; green: autophagy).
Fig 6.
Rapamycin protects astrocytes upon excretory/secretory product treatment via autophagy induction.
Cells were pretreated with rapamycin, 3-methyladenine (3-MA), chloroquine (CQ), or bafilomycin A1 (BF) for 1 h and then incubated with 500 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. The viability of astrocytes was analyzed by the CCK-8 assay. The data are expressed as the means ± SD from three independent experiments (n = 3). *P<0.01, compared with the control. #P<0.01, compared with the cells exposed to ESPs. ※P<0.01, compared with the cells exposed to ESPs+100 nM rapamycin. ◎P<0.01, compared with the cells exposed to ESPs+500 nM rapamycin.
Fig 7.
Excretory/secretory products induce gene expressions of Shh signaling pathway.
Cells were treated with 0, 31.3, 62.5, 125, and 250 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. The mRNA expression levels of Shh signaling pathway-related molecules (Shh, Ptch, Smo, and Gli-1) were detected by Real-Time qPCR. The data are expressed as the means ± SD from three independent experiments (n = 3). #P<0.01, compared with the respective values of cells treated with 0 μg/ml A. cantonensis L5 ESPs for 12 h.
Fig 8.
Excretory/secretory products induce protein expressions of Shh signaling pathway.
Cells were treated with 0, 31.3, 62.5, 125, and 250 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. The protein expression levels of Shh signaling pathway-related molecules (Shh-N, Shh-C, Ptch, Smo, and Gli-1) were detected by Western blotting. The data are expressed as the means ± SD from three independent experiments (n = 3). #P<0.01, compared with the respective values of cells treated with 0 μg/ml A. cantonensis L5 ESPs for 12 h.
Fig 9.
Excretory/secretory products induce autophagy through the Shh signaling pathway.
Cells were pretreated with recombinant Shh (r-Shh), Shh agonist (SAG), and cyclopamine (Cyclo) for 1 h and then incubated with 500 μg/ml A. cantonensis L5 excretory/secretory products (ESPs) for 12 h. The protein expression levels of LC3-I and LC3-II were detected by Western blotting. The data are expressed as the means ± SD from three independent experiments (n = 3). *P<0.01, compared with the control. #p<0.01, compared with the cells exposed to ESPs.