In the present study, we reported that progesterone inhibited the production of ROS, and promoted the activity of antioxidant enzymes to protect BEEC from LPS-induced oxidative damage. Progesterone activated the Nrf2/Keap1 pathway, and promoted the entry of Nrf2 into the nucleus. This antioxidant effect was most prominent in P4 of 1 and 3 ng/mL, not 5 ng/mL. The addition of progesterone receptor antagonist RU486 inhibited the antioxidant effect of P4.
The excessive accumulation of ROS is one of the important signs of oxidative injury. The antioxidant enzyme system is the first line of defense against damage . It has been found that LPS stimulated ROS production and inhibited the activities of antioxidant enzymes in BEEC and bovine mammary epithelial cells [30, 31]. Our data were consistent with these studies, indicating the oxidative damage of BEEC induced by LPS. We found that the production of ROS and MDA decreased after P4 treatment in LPS-stimulated BEEC. This was consistent with the results of previous reports in vitro that progesterone can eliminate MDA and protect against oxidative damage in mouse nerve cells . In order to study the relationship between ROS clearance and P4 antioxidant capacity, we detected the activities of some enzymes in the antioxidant enzyme system. The results showed that the activities of SOD, CAT, and T-AOC increased after the addition of P4. Therefore, we speculated that progesterone may relieve injury by enhancing the activity of antioxidant enzyme system and reducing the level of ROS.
Nrf2/Keap1 signaling pathway is a key pathway regulating the expression of antioxidant enzymes and protect cells from oxidation induced cytotoxicity [32, 33]. Under normal physiological conditions, Nrf2 is anchored in the cytoplasm through Keap1 dependent ubiquitination proteasome degradation to maintain antioxidant and cytoprotective enzymes at basal level and to keep cellular homeostasis [34, 35]. The modification of key cysteine thiols and/or the phosphorylation of electrophiles and oxidants leads to the activation of Nrf2, and prompts Keap1 to release Nrf2, and finally Nrf2 enters the nucleus . Nrf2 regulates the antioxidant defense system through a variety of mechanisms, including the regeneration of oxidative cofactors and proteins, the synthesis of reducing factors, the increase in redox transports, and the induction of stress response proteins. As a stress response protein, HO-1 can enhance antioxidant activity and maintain redox homeostasis. The role of HO-1 in redox transport, such as cystine/glutamate transport, is mainly supported by cystine transporter [37, 38]. HO-1 has the ability to regulate cellular redox defense to maintain redox homeostasis [39, 40]. NQO1 is an important reductant that regulates transcription through anti-oxidative response element (ARE)[41–44]. The combination of the activated Nrf2 and ARE promotes the expression of antioxidant genes such as NQO1 and HO-1, and further enhances the activity of antioxidant enzymes . In this study, we detected the decreased levels of HO-1, NQO1, and total Nrf2, the increased Keap1 level and translocation of nuclear Nrf2 in BEEC after LPS stimulation. The downregulation of the downstream antioxidant genes of Nrf2 pathway, including NFE2L2, HMOX1, NQO1, GPX2, GCLC, and GSTP1, was consistent with the Western blot result. HMOX1 and NQO1 have significant effects on maintaining the redox balance of BEEC. The activities of HMOX1 and NQO1 are regulated by NFE2L2 . It has been reported that Nrf2 is an important factor to promote the gene transcription of biosynthesis key enzymes (GSH -synthase and SLC7A11), GSH reductase and GPx4, and protect mouse nerve cells from ferroptosis caused by oxidative stress [46, 47]. Our results suggested the LPS-induced oxidative stress and the activation of Nrf2 pathway in BEEC. Similarly, it has been reported that LPS induced inflammation and oxidative stress in bovine endothelial cell line .
We observed that the addition of 1 and 3 ng/mL P4 promoted the activation Nrf2 pathway and the expressions of the downstream antioxidant genes and proteins in BEEC with oxidative stress response, which was in agreement with the report that progesterone reduced oxidative injury in experimental colitis in rats . These results revealed that 1 and 3 ng/mL P4 inhibited LPS-induced oxidative stress in BEEC. Progesterone plays its role through progesterone receptor (PR). Mifepristone (RU486) is a specific inhibitor of PR. RU486 was used to verify the effect of progesterone in BEEC. We observed that RU486 reversed the antioxidant effect of progesterone, indicating that the antioxidant effect of P4(1 and 3 ng/mL) was mediated by PR.
In our study, the addition of 5 ng/mL progesterone had no obvious antioxidative effect in BEEC. In the study of vascular endothelial progenitor cells, it has been found that low concentration of progesterone (3.2 ng/mL) enhanced the expression of progesterone receptors A and B, whereas high concentration of progesterone (5.68 ng/mL) inhibited the progesterone receptor expressions [49–51]. Kempisty et al. suggested that early rise in P4 concentration (> 5 ng/mL) has a negative effect on embryo development because serum progesterone concentration over 5 ng/mL increases the risk of bacterial infection. Hill et al. [53–55] sets the progesterone threshold at 1.53~4.36 ng/mL, and found that the progesterone concentration over 4.36 ng/mL has a significant negative effect on DNA hypermethylation on endometrium and induced apoptosis and oxidative stress in endometrial cell. Therefore, it is possible that 5 ng/mL P4 reduces the expression of progesterone receptor as compared with 1 and 3 ng/mL P4. In addition, 5 ng/ml P4 makes the uterus more susceptible to infection and induces apoptosis in BEEC and finally aggravates oxidative damage. Further research is needed to clarify the underlying mechanism.
In conclusion, 1 and 3 ng/mL P4 inhibited the LPS-induced oxidative damage in primary bovine endometrial epithelial cells through activating Nrf2/Keap1 signaling pathway. This antioxidative effect of P4 was probably mediated by PR.