Chlamydia trachomatis plasmid-encoded protein pORF5 up-regulates DJ-1 expression to activate the Nrf2/NQO1 pathway in the regulation of oxidative stress and apoptosis

Abstract:

[Background] The plasmid-encoded protein pORF5 is a pivotal virulence factor of Chlamydia trachomatis (Ct), capable of inhibiting oxidative stress and apoptosis. [Objective] To investigate whether pORF5 up-regulates the expression of DJ-1 protein to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)/NAD(P) H: quinone oxidoreductase 1 (NQO1) pathway in the regulation of lipopolysaccharide (LPS)-induced cellular oxidative stress and apoptosis. [Methods] HeLa cells were treated with different concentrations of pORF5 for different time periods. Western blotting was employed to determine the expression level of DJ-1. The reactive oxygen species (ROS) assay kit and flow cytometry were employed to examine the effects of pORF5 on LPS-induced oxidative stress and apoptosis. The expression of DJ-1 protein in HeLa cells was down-regulated by siRNA-DJ-1, and the cellular ROS fluorescence intensity was observed under a fluorescence microscope. Corresponding assay kits were used to measure the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in each group. After pretreatment with the Nrf2 inhibitor ML385 for 1 h, HeLa cells were stimulated with pORF5 alone or combined with LPS, and the expression levels of the apoptosis-related proteins B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were determined. Western blotting was employed to evaluate the effect of down-regulating the expression of DJ-1 on the Nrf2/NQO1 pathway. [Results] pORF5 up-regulated the expression of DJ-1, and the up-regulatory effect was the strongest after treatment with 10 μg/mL pORF5 for 18 h. pORF5 reduced LPS-induced ROS fluorescence intensity and cell apoptosis rate (P<0.001). The si-DJ-1 group showed increased ROS fluorescence intensity and MDA content but decreased SOD activity (P<0.001). ML385 inhibited pORF5-induced expression of Nrf2 and NQO1 (P<0.001), up-regulated the expression of the pro-apoptotic protein Bax (P<0.05), down-regulated the expression of the anti-apoptotic protein Bcl-2 (P<0.01). The expression of Nrf2 and NQO1 was down-regulated in the si-DJ-1 group (P<0.001, P<0.01). [Conclusion] pORF5 can up-regulate the expression of DJ-1 protein to activate the Nrf2/NQO1 pathway, thereby inhibiting LPS-induced oxidative stress and apoptosis.

Key words:Chlamydia trachomatis;pORF5;oxidative stress;apoptosis;DJ-1