Oxidative stress is defined as an imbalance between the production of oxygen radicals and the antioxidant system, which may be caused by increased production of reactive oxygen species and/or defective antioxidant mechanisms[16]. Oxidative stress is present in normal pregnancy, caused by the normal systemic inflammatory response, and the production of oxides is accompanied by an increase in antioxidants, which counteract each other[17]. However, in patients with PE, uteroplacental hypoperfusion further aggravates oxidative stress[18]. On the other hand, the activity of free radical scavengers and antioxidant enzymes is reduced in the maternal body, which in turn leads to a decrease in antioxidant defenses[19], which studies have shown to be an important enabler in the development of pregnancy-related disorders, which are likewise involved in the development of PE[4, 5, 20]. Therefore, amelioration of placental oxidative stress injury has the potential to be one of the potential therapeutic approaches for PE. Therefore, the present study focused on amelioration of placental oxidative stress injury to investigate whether HCQ can be used as a new way of treating PE and its related mechanisms.
To verify our hypothesis, an oxidative stress model was established in HTR8/SVneo cell line by H2O2 treatment to simulate the cellular microenvironment associated with PE, and we injected pregnant rats with L-NAME[21], an endothelial nitric oxide synthase inhibitor, to establish a PE-like rat model. As expected, our study found elevated MDA levels and decreased SOD levels in the serum of PE rats, which is consistent with the pathophysiology of PE. Similarly, in vitro experiments, the levels of intracellular ROS and MDA were significantly increased after H2O2 treatment, indicating that the oxidative stress model was successfully established. In this study, we chose 1.0ug/ml HCQ to intervene in the oxidative stress model with reference of our previous studies, and the results showed that HCQ was able to significantly attenuate the oxidative stress damage in both cellular and animal experiments, as shown by a decrease in ROS and MDA and an increase in SOD.
In this study, we found that HCQ exerted an antihypertensive effect on pregnant rat with L-NAME-induced PE and reduced proteinuria and ameliorated FGR. Moreover, HCQ similarly improves pathological changes in the placenta and kidneys of preeclamptic rats. Our results demonstrated that HCQ may be effective in treating PE and improving pregnancy outcomes by ameliorating oxidative stress damage.
Most patients with PE produce excessive amounts of placental antiangiogenic factor, soluble fms-like tyrosine kinase 1(sFlt-1), which antagonizes vascular endothelial growth factor and placental growth factor(PIGF), leading to endothelial dysfunction[22, 23]. Thus, the ratio of sFlt-1 to PIGF has already become a predictor of PE[24]. We found that HCQ was able to significantly increase serum levels of PIGF, but unfortunately, although HCQ was able to decrease the L-NAME-induced increase in sFlt-1, there was no statistically significant difference in this alteration. Some studies have demonstrated that HCQ improves endothelial dysfunction in PE[25, 26], and these results suggest that HCQ may improve endothelial dysfunction by promoting the production of PIGF, but not by decreasing sFlt-1.
The underlying molecular mechanism by which HCQ exerts a therapeutic effect on PE have not been investigated. However, oxidative stress is a promoter of autophagy, and our study is based on the relationship between oxidative stress, autophagy and PE. It has been found that ROS generated by oxidative stress could inhibit the PI3K/AKT/mTOR pathway[15]. Previous studies have found a strong correlation between PI3K/AKT/mTOR pathway and PE[27, 28]. Our results confirmed that the PI3K-AKT-mTOR pathway is inhibited in a cellular oxidative stress model and in preeclamptic rats. Surprisingly, we also observed that HCQ reactivates the over-inhibited PI3K/AKT/mTOR pathway both in vivo and in vitro. Therefore, we used a PI3K inhibitor (LY294002) in our cell model to verify whether HCQ exerted therapeutic effects by regulating the PI3K/AKT/mTOR pathway. We found that LY294002 removed the alleviating effect of HCQ on oxidative stress damage induced by H2O2, and LY294002 able to suppress the HCQ activated PI3K/AKT/mTOR pathway. The above results demonstrated that HCQ alleviated oxidative stress injury in PE by inhibiting the PI3K/AKT/mTOR pathway.
Autophagy is considered to be a conserved survival process prompted by several cellular stress conditions, which prevents cell damage, promotes cell survival in the event of nutrient scarcity, and responds to cytotoxic stimuli[29]. During normal pregnancies, placental autophagy is vital for maintenance of cellular homeostasis that is needed for embryo development[30]. However, excessive activation of autophagy may be involved in the occurrence and development of PE and FGR[31]. Many studies have found that the presence of excessive autophagy in placenta of patients with PE[32, 33]. Similarly, in certain animal and cell models of PE, it has been observed that autophagy-related proteins (LC3 and Beclin1) exhibit an increase in expression, while the autophagy substrate (p62) shows a decrease, suggesting autophagy activity is enhanced in these models07:40 PM.
In this research, we found that autophagy activity was significantly enhanced in HTR8/SVneo cells induced by H2O2, mainly manifested as LC3II/I protein expression was increased, p62 protein expression was decreased, red and yellow spots were significantly increased after transfection with mCherry-GFP-LC3, and autophagic lysosomes were significantly increased under transmission electron microscopy. In addition, increased LC3II/I protein expression and decreased p62 expression were detected in the placenta of the PE rat model. Furthermore, our results show that HCQ inhibited excessive autophagy in the trophoblasts exposed to H2O2 and PE-like rat model.
To further determine the causal relationship between oxidative stress, PI3K/AKT/mTOR pathway and autophagy, we co-administered LY294002 with HCQ in our oxidative stress cell model. Notably, LY294002 inhibited the phosphorylation level of PI3K/AKT/mTOR pathway, increased the expression of LC3II/I, decreased the expression of p62, and activated autophagy. At the same time, treatment of H2O2-induced cells with HCQ and LY294002 further increased LC3II/I expression by western blot and increased autophagosomes by transmission electron microscopy. Curiously, the intracellular expression of p62 was elevated even though there was no statistical difference. The increase in the intracellular expression of p62 may be due to HCQ inhibiting the binding of autophagosomes and lysosomes, and the accumulation of autophagic substrates in cells is difficult to metabolize[36].
Based on the above results, we conclude that HCQ may inhibit autophagy by activating the PI3K-AKT-mTOR pathway, which in turn ameliorates oxidative stress injury and ultimately treat PE and improve pregnancy outcomes. (Fig. 6). In our study, in vitro and in vivo experiments were combined to verify the validity of our hypothesis. In future study, we need to further design a clinical trial to explore HCQ therapeutic effect in PE patients. In general, our study provides important basic research basis for the application of HCQ in the treatment of PE.