OP-4 - Investigation of the effects of autophagy inhibitors, Bafilomycin and Chloroquine, for oxidative stress

Abstract

We have previously reported that autophagy inhibition suppresses trophoblast differentiation and function. On the other hand, hydroxychloroquine (CHQ), an autophagy inhibitor, has recently been used in pregnant women with systemic lupus erythematosus and has been reported to be safe and inhibit the onset of preeclampsia. In this study, we focused on the effect of CHQ to trophoblasts, and investigated how it affects the oxidative stress defense mechanism in trophoblast cells. We used BeWo cells, HchEpC1b cells, HTR8 cells, TCL1 cells, and primary human trophoblast (PHT) cells. H2O2 was used to induce oxidative stress, and Bafilomycin A1 (Baf) and CHQ were used for the autophagy inhibition. BeWo cells, and PHT cells, which resemble syncytiotrophoblast cells, showed higher expression of HO-1, compared with that in extravillous trophoblast-derived cell lines (HchEpC1b, HTR8, TCL1). In BeWo cells, treatment with Baf reduced HO-1, but CHQ didn’t influence HO-1 expression. As a mechanism to induce HO-1 expression, cells with H2O2 and Baf treatment were compared. As a result, both treatments increased the expressions of p62 and phosphorylated p62. However, NBR1 was reduced by Baf but not H2O2 in BeWo cells. In the comparative study between Baf and CHQ, CHQ sustained NRB1 and HO-1 expressions in BeWo cells and villous explant culture. Recently, it has been reported that the p62 protein is involved in Nrf2 activation. In our study, although Baf showed marked accumulation of phosphorylated p62 protein, the antioxidant enzymes were decreased. Mechanistic studies suggested that a decrease in NBR1 may lead to a decrease in antioxidant enzymes in respond to Baf. On the other hand, CHQ did not decrease NBR1 and antioxidant enzymes, suggesting that CHQ could be administered more safely to pregnant women with systemic lupus erythematosus.