Abstract:

Objective To evaluate the effect of recombinant protein P29 (rP29), a 29 kDa antigen identified in hydatid cyst fluid which induces protective immunity, on the transforming growth factor-β (TGF-β)/Smad signaling pathway in the liver of mice infected with Echinococcus granulosus. Methods A group of 12 female BALB/c mice were subcutaneously immunized with 10 μg of rP29 emulsified with Freund’s adjuvant, then boosted twice with two weeks interval. Another two groups of mice were given with Freund’s adjuvant or PBS only as controls. The three groups of mice were intraperitoneally challenged with 1 500 protoscolex of E. granulosus per mouse two weeks after the last immunization. Twelve weeks after infection, the mice were euthanized and the liver tissues were collected. The TGF-β1 mRNA expression level was detected by RT-PCR in the liver of each infected mouse. The expression levels of TGF-β/Smad signaling pathway regulated downstream proteins such as TGF-RI, TGF-RII, p-Smad2,3, Smad4 and Smad7 were measured by Western blotting in the liver tissues. Results RT-PCR results showed that the relative expression level of TGF-β1 mRNA in the liver tissues of mice immunized with rP29 (1.24 ± 0.56) was reduced compared to PBS control mice (1.71 ± 0.29) with significant difference (P < 0.01), however, it was higher than that in adjuvant control mice (0.98 ± 0.42). The changes of the TGF-β1 mRNA expression level in the liver tissues were consistent with the TGF-β1 protein expression level detected by Western blotting. The expression density of TGF-β1 protein in the rP29 immunized group was 372.04 ± 0.01 (measured by densitometry) which was lower than that in the PBS group (673.02 ± 0.06), but higher than that in the adjuvant group (213.69 ± 0.31). The TGF-β/Smad signaling pathway-regulated downstream proteins also showed the similar changes except for Smad 7. The expression levels of TGF-β-RI, TGF-β-RII, p-Smad 2,3 and Smad 4 in the liver of rP29 immunized mice (357.59 ± 0.83, 289.07 ± 0.21, 204.06 ± 0.19, 396.11 ± 0.01, respectively) were reduced compared with the PBS group (496.89 ± 0.09, 378.41 ± 0.01, 428.11 ± 0.29, 566.95 ± 0.21, respectively), but higher than that of the adjuvant control group (171.99 ± 0.82, 185.77 ± 0.09, 128.09 ± 0.08, 205.87 ± 0.59, respectively). However, Smad 7 level expressed in rP29 immunized mice (278.89 ± 0.12) was up-regulated compared with PBS group (142.32 ± 0.07), but significantly lower than that in adjuvant group (384.17 ± 0.51). Conclusion Immunization of recombinant P29 protein significantly reduced the expression of TGF-β1 at both mRNA and protein levels in the liver of mice infected with E. granulosus. Most of the TGF-β/Smad signaling pathway-regulated downstream proteins (TGF-β-RI, TGF-β-RII, p-Smad 2,3 and Smad 4) were also reduced except for Smad 7 which was up-regulated.

Key words: Echinococcus granulosus, Transforming growth factor-β1;, TGF-β/Smad signaling pathway;