Abstract:

Objective To explore whether Paragonimus proliferus infection in rats can cause autophagy in lung tissue cells, by detecting the expression of factors related to the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Methods Forty SD rats were randomly divided into 4 groups (n = 10 in each group). The rats in all groups (except the control group) were intraperitoneally injected with 6 P. proliferus metacercaria, and were sacrificed on days 3, 7 and 14 after injection, respectively. Serum samples collected from the rats were used to detect the levels of IL-1 and IL-6 by ELISA. The lung tissues were examined for autophagosome formation by transmission electron microscopy (TEM), pathological changes by HE staining, and protein expression of Akt, mTOR, Beclin 1 and LC3Ⅱ by Western blotting and immunohistochemistry. Statistical analysis was performed with the SPSS 19.0 software. Results The ELISA results showed that the expression levels of IL-1 on days 3, 7 and 14 after infection was (1 558.0 ± 123.6), (1 511.0 ± 213.1) and (1 448.0 ± 176.8) pg/ml, respectively, all significantly higher than the normal group (1 222.0 ± 112.8) pg/ml (P < 0.05); the expression levels of IL-6 on days 3 and 7 after infection was (1 481.0 ± 197.9) and (1 423.0 ± 210.0) pg/ml, respectively, both higher than the control group (1 221.0 ± 138.9) pg/ml (P < 0.05). TEM revealed autophagy in mitochondria at different stages of infection. HE staining results showed that the cells in each infection group were arranged disorderly and the alveolar structure showed varied degrees of impairment. There was no significant difference in the protein level of Akt in the infection groups, compared to the control group (P > 0.05); the protein levels of p-Aktser ⁴⁷³ on days 3 and 7 after infection were (1.288 ± 0.109) and (1.619 ± 0.132), respectively, both higher than that of the normal group (0.733 ± 0.135) (P < 0.01). The protein levels of p-mTORser ²⁴⁴⁸ in the infection groups were (1.574 ± 0.278), (2.384 ± 0.125) and (1.808 ± 0.121), all higher than the normal group (1.260 ± 0.087) (P < 0.05). The protein levels of mTOR and Beclin 1 on day 3 after infection were (1.714 ± 0.217) and (2.736 ± 0.333), respectively, both higher than (1.345 ± 0.067) and (1.974 ± 0.225) in the normal group (P < 0.01). The LC3Ⅱ protein level on day 14 after infection was (1.938 ± 0.191), higher than that of the normal group (1.401 ± 0.200) (P < 0.01). The results of IHC showed that the lung tissue cells of the negative control group were blue, while the positive staining was in brown yellow; the proteins stained positive were all located in the cell membrane and cytoplasm. Compared with the control group, Akt and mTOR in each infection group showed no obvious brown color in the lung tissue cells, and there was no significant difference in the optical density of Akt and mTOR between the infection groups and the control group (P > 0.05). The staining for p-Aktser ⁴⁷³, p-mTORser ²⁴⁴⁸ and Beclin 1 in the groups 3 d, 7 d, and 14 d after infection showed denser brown yellow signals compared with the control group, and the optical densities were (0.104 ± 0.010), (0.143 ± 0.022), (0.088 ± 0.013); (0.100 ± 0.007), (0.151 ± 0.006), (0.120 ± 0.012); and (0.129 ± 0.005), (0.047 ± 0.004), (0.050 ± 0.005), which were higher than those in the normal group brown yellow signals[(0.032 ± 0.001), (0.065 ± 0.002) and (0.031 ± 0.001)] (P < 0.05). The LC3Ⅱ showed significantly darker brown yellow signals than the control group on days 3 and 14 after infection, and the optical densities were (0.056 ± 0.006) and (0.120 ± 0.007), which were higher than that of the normal group (0.042 ± 0.004) (P < 0.05). Conclusion P. proliferus infection in rats causes lung injury and inflammatory response, which may induce autophagy of liver cells. The autophagy event could be detected by assessing the expression of factors related to the Akt/mTOR signaling pathway.

Key words: Paragonimus proliferus, Infection, Rat, Akt/mTOR signaling pathway, Lung tissue cells, Autophagy