Excretory/secretory products of Angiostrongylus cantonensis fifth-stage larvae induce endoplasmic reticulum stress via the Sonic hedgehog pathway in mouse astrocytes

Abstract

Background: Angiostrongylus cantonensis is an important food-borne zoonotic parasite. Humans are non-permissive hosts, and this parasite develops into fifth-stage larvae (L5) in the brain and subarachnoid cavity and then induces eosinophilic meningitis and eosinophilic meningoencephalitis. Excretory/secretory products (ESPs) are valuable targets for the investigation of host-parasite interactions. These products contain a wide range of molecules for penetrating defensive barriers and avoiding the immune response of the host. Endoplasmic reticulum (ER) stress has been found to be associated with a wide range of parasitic infections and inflammation. ER stress can increase cell survival via the activation of downstream signalling. However, the mechanisms of ER stress in A. cantonensis infection have not yet been clarified. This study was designed to investigate the molecular mechanisms of ER stress in astrocytes after treatment with the ESPs of A. cantonensis L5.

Results: The results demonstrated that A. cantonensis infection activated astrocytes in the mouse hippocampus and induced the expression of ER stress-related molecules. Next, the data showed that the expression of ER stress-related molecules and the Ca²⁺ concentration were significantly increased in activated astrocytes after treatment with the ESPs of L5 of A. cantonensis. Ultimately, we found that ESPs induced GRP78 expression via the Sonic hedgehog (Shh) signalling pathway.

Conclusions: These findings suggest that in astrocytes, the ESPs of A. cantonensis L5 induce ER stress and that the Shh signalling pathway plays an important role in this process.

Keywords: Angiostrongylus cantonensis; Astrocytes; Endoplasmic reticulum stress; Excretory/secretory products; Fifth-stage larvae; Sonic hedgehog pathway.

Fig. 1

Astrocyte activation was induced in the brains of Angiostrongylus cantonensis-infected mice. The expression of GFAP was detected in the hippocampus in the absence of infection (a) and on days 7 (b), 14 (c) and 21 (d) post-infection after infection with 25 third-stage larvae. e The expression level of GFAP in the brain was quantified by ImageJ software (**P < 0.01). f The mRNA expression level of GFAP in the brain was detected by microarray analysis

Fig. 2

Angiostrongylus cantonensis infection stimulates the expression of ER stress-related molecules in the mouse brain. Mouse brains were collected from A. cantonensis-infected mice on days 0, 7, 14 and 21 post-infection. Then, the mRNA expression levels of ER stress-related molecules were detected by cDNA microarray analysis

Fig. 3

Excretory/secretory products of Angiostrongylus cantonensis L5 stimulate astrocyte activation and GRP78 expression. Cells were treated with 0, 31.3, 62.5, 125 or 250 μg/ml excretory/secretory products (ESPs) of A. cantonensis L5 for 12 h. The mRNA (a) and protein (b) expression levels of GFAP and GRP78 were detected by real-time qPCR and western blotting. The data are expressed as the mean ± SD from three independent experiments (n = 3). *P < 0.05 and **P < 0.01 compared with cells treated with 0 μg/ml ESPs of A. cantonensis L5 for 12 h

Fig. 4

Excretory/secretory products of Angiostrongylus cantonensis L5 stimulate the activation of ER stress-related pathways in astrocytes. Cells were treated with 0, 31.3, 62.5, 125 or 250 μg/ml excretory/secretory products (ESPs) of A. cantonensis L5 for 12 h. The mRNA (a) and protein (b) expression levels of ER stress-related molecules were detected by real-time qPCR and western blotting. The data are expressed as the mean ± SD from three independent experiments (n = 3). *P < 0.05 and **P < 0.01 compared cells treated with 0 μg/ml ESPs of A. cantonensis L5 for 12 h

Fig. 5

Excretory/secretory products of Angiostrongylus cantonensis L5 stimulate calcium secretion in astrocytes. Cells were treated with 0, 31.3, 62.5, 125 or 250 μg/ml excretory/secretory products (ESPs) of A. cantonensis L5 for 12 h. The concentration of calcium was measured by the Calcium Assay Kit. The data are expressed as the mean ± SD from three independent experiments (n = 3). **P < 0.01 compared with cells treated with 0 μg/ml ESPs of A. cantonensis L5 for 12 h

Fig. 6

Evaluation of the effectiveness of an activator or inhibitor on the Shh signalling pathway. Cells were pre-treated with recombinant Shh (r-Shh), Shh agonist (SAG) and cyclopamine (Cyclo) for 1 h, then incubated with 250 μg/ml excretory/secretory products (ESPs) of A. cantonensis L5 for 12 h. The protein expression levels were detected by western blotting. The data are expressed as the mean ± SD from three independent experiments (n = 3). ^#P < 0.01 compared with the control; **P < 0.01 compared with cells exposed to ESPs

Fig. 7

Excretory/secretory products induce GRP78 expression through the Shh signalling pathway. Cells were pre-treated with recombinant Shh (r-Shh), Shh agonist (SAG) and cyclopamine (Cyclo) for 1 h, then incubated with 250 μg/ml excretory/secretory products (ESPs) of A. cantonensis L5 for 12 h. The mRNA (a) and protein (b) expression levels were detected by real-time qPCR and 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 cells exposed to ESPs