Abstract:

Objective To investigate the influence of Echinococcus multilocularis on the p38MAPK signaling pathway in liver tissue and the affect of p38MAPK expression level on E. multilocularis metacestodes. Methods E. multilocularis protoscolices were collected from the abdominal cavity of preserved gerbils. C57BL/6J mice aged 4-6 weeks were randomly assigned into three groups as infected group, uninfected group, and inhibitory group, with 10 mice each group. The inhibitory group mice were injected via liver portal vein with SB202190 solution (5 mg/kg, in mixed volvent of 20% DMSO and 80% sterile saline), while the uninfected and infected groups received the solvent only, once a week, for total 4 weeks. Subsequently, both the infected and inhibitory groups were given by liver subcapsular injection with 500 protoscolices and raised for 8 weeks. After euthanasia, the mice livers were collected and weighed to calculate weight ratio (liver mass/ body mass). Liver tissues adjacent to (within 0.3 cm) and distant from (1 cm away) the metacestode vesicles were collected. These liver samples were embedded with paraffin and sectioned, and then stained with hematoxylin-eosin (HE), periodic acid-schiff (PAS), and Masson’s trichrome for analyzing the range of inflammatory bands and hepatic fibrosis. Utilizing the TRIzol method, RNA was extracted from the liver tissues to detect the expression levels of MAPK14 gene by quantitative PCR (qPCR). Subsequently, liver tissues adjacent to the vesicles from each group were examined for the expression levels of p38MAPK and p-p38MAPK using Western blotting and immunohistochemical methods, respectively. Results The livermass/body mass weight ratio in the uninfected group was (6.49 ± 0.19)%, while it was increased to (6.80 ± 0.33)% in the infected group (t = 2.74, P < 0.05). The liver size in the uninfected group was (5.27 ± 0.34) cm³, whereas it increased to (5.80 ± 0.49) cm³ in the infected group (t = 2.83, P < 0.05). HE staining indicated the absence of inflammatory bands in the uninfected group, with the infected and inhibitory groups showing inflammatory band areas of (51.2 ± 14.0)% and (23.8 ± 9.8)%, respectively. The inhibitory group displayed a significant decrease in hepatic necrotic foci and inflammatory bands compared to the infected group (t = 3.92, P < 0.01). PAS staining revealed that the area of purple-red stained regions accounted for (1.3 ± 0.3)% in the uninfected group, (7.4 ± 1.8)% in the infected group, and (4.5 ± 0.4)% in the inhibitory group. The inhibitory group showed a significant reduction in the area of purple-red staining compared to the infected group (t = 3.82, P < 0.05). Masson’s trichrome staining demonstrated an absence of blue-stained areas in the uninfected group, indicative of the absence of collagen deposition. In contrast, the infected and inhibitory groups exhibited blue-stained areas representing (34.9 ± 4.1)% and (16.3 ± 2.8)% of the total tissue area, respectively. The extent of the blue-stained areas was significantly reduced in the inhibitory group compared to the infected group (t = 9.16, P < 0.01). qPCR analysis indicated that the relative expression of MAPK14 in liver tissues adjacent to E. multilocularis lesions in the infected group was significantly higher, with a level of 7.14 ± 2.23 (t = 6.13, P < 0.01), compared to tissues distant from the lesions (1). The infected group also demonstrated an elevated MAPK14 expression level of 3.17 ± 0.68 (t = 7.14, P < 0.01) relative to the uninfected group (1). The inhibitory group displayed a substantial decrease in MAPK14 expression levels adjacent to the lesions, with a relative expression of 0.07 ± 0.01, significantly lower than that of the infected group (1) (t = 126.83, P < 0.01). Western blotting data indicated that the ratio of p38MAPK to its phosphorylated form, p-p38MAPK, was significantly different among the groups. In the uninfected group, the relative expression levels of p38MAPK and p-p38MAPK, as well as p38MAPK/p-p38MAPK values were 0.80 ± 0.08, 0.67 ± 0.11, and 0.74 ± 0.09, respectively. For the infected group, the ratio increased to 0.97 ± 0.14, 0.87 ± 0.09, and 0.91 ± 0.14, respectively. The inhibitory group exhibited a markedly reduced ratio to 0.41 ± 0.07, 0.20 ± 0.07, and 0.49 ± 0.21, respectively, when compared to the infected group (t = 7.97, 13.32, 3.74, all P < 0.01). Immunohistochemical results showed p38MAPK scores of 4 (4, 4) for the uninfected group, 6 (6, 9) for the infected group, and 4 (2, 4) for the inhibitory group. The p-p38MAPK scores were 4 (4, 6), 9 (8, 9.75), and 6 (6, 6), respectively. Compared to the infected group, the inhibitory group exhibited a significant decrease in the molecular expression levels of both p38MAPK and p-p38MAPK in liver tissues adjacent to the lesions (Z = -3.00, -3.11, both P < 0.01). Conclusion E. multilocularis metacestode could promote the expression of p38MAPK and its phosphorylation in the liver tissue adjacent to the vesicles, while the p38MAPK signaling pathway in adjacent liver tissue may involve in maintaining the growth and invasive ability of metacestode.

Key words: Echinococcus multilocularis, Alveolar echinococcosis, p38MAPK, p-p38MAPK