Introduction

The climacteric period is commonly known as menopause, marking the end of the reproductive period for most women [1]. With the increased social pressure and the accelerated pace of life, the incidence of menopausal depression in women tends to be high [2]. Generally, depression is a broad and heterogenous diagnosis, with depressed mood and/or loss of pleasure in activities as the main diagnostic characteristics [3].

Many studies have revealed that neuroinflammation, triggered by the activation of microglia, is closely related to the pathogenesis of depression [4]. Microglia are important immune cells in the central nervous system (CNS), playing an important role in the occurrence and development of depression [5]. Under normal physiological conditions, microglia are in a quiescent state, monitoring CNS homeostasis. In response to stresses or abnormal neuronal activities, the morphology and functions of microglia are rapidly changed, presenting an activated state. According to the “gliocentric theory,” stress-induced inflammation resulting from microglia activation may trigger a cascade of glial dysfunctions that supports the development of depressive disorders [6].

Evidence suggests that the endogenous cannabinoid system is involved in the pathophysiology of depression [7]. Endocannabinoids are signaling lipids that activate cannabinoid receptors in the CNS and peripheral tissues. Cannabinoid receptors are divided into two classes, i.e., cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). The CB1 receptors in the brain are mainly distributed in presynaptic axons and nerve endings [8]. Certain genetic polymorphisms in CB1 and CB2 receptors are associated with major depression and bipolar disorder, while the CB1 knockout mice showed significant depression-like behaviors [9, 10]. Besides, the selective estrogen receptor modulators (SERMs), e.g., tamoxifen, are used to treat estrogen receptor (ER)-positive breast cancer and osteoporosis. Interestingly, as indicated as a potential ER-independent target, tamoxifen binds to cannabinoid receptors (CBRs) with affinity in the low concentration range and acts as an inverse agonist [11]. Suggesting that the estrogen receptor activation could promote CBRs to undergo the relevant responses.

Many of the health complications associated with menopause in women are directly related to decreased functions of the ovarian hormone, primarily 17β-Estradiol (E2) deficiency. Therefore, the importance of physiological hormone replacement therapies, mainly including E2 replacement therapy (ERT), has attracted increasing attention in the treatment of postmenopausal women [12].

Currently, hormone therapy in menopausal syndrome is still controversial. Firstly, in animal and cell experiments, the ERT has shown anti-inflammatory and antioxidant effects, stabilizing intracellular calcium levels, modulating the cholinergic system, and ultimately improving cognitive functions and depressive-like behaviors in ovariectomized (OVX) mice [37]. Several laboratory and clinical studies have reported that E2 showed no effect on these diseases in elderly postmenopausal women but increased the risk of the onset and even mortality of the patients of these diseases [38].

Xu et al. conducted animal studies to demonstrate that the administration of E2 effectively ameliorated the depressive behaviors through the inhibition of inflammation and the activation of indoleamino-2, 3-dioxygenase (IDO), ultimately regulating the levels of 5-hydroxytryptamine (5-HT) in the hippocampus [39]. However, the evidence for estrogenic antidepressant-like effects in humans is less conclusive compared to rodent studies, and in some cases, contradictory findings have been reported [40]. Therefore, the effective physiological doses of E2 supplementation for menopausal depression are still controversial. In fact, several studies have explored the medical effects of sE2. For example, Bronwyn et al. found that sE2 impaired the preestrus fear resolution in rats [41]. While Stephanie et al. found that high doses of E2 could prevent heart failure after myocardial infarction in rats [42].

In our study, the results revealed the potentially neurotoxic effect of sE2. To investigate the therapeutic effect of sE2 on OVX mice, the sE2 (0.5 mg/kg) was intraperitoneally injected into the OVX mice for 5 weeks. The results showed that sE2 treatment worsened depressive-like behaviors in OVX mice. It is necessary to note that more explorations of the cerebrotoxic effects of different E2 doses and durations of action are needed in the future. Although this study used different concentrations and time gradients to detect the effects of E2 on neurons and microglia in vitro, the in vivo experiment was a single-endpoint (single-dose) test and could not accurately reflect the effects of E2 on the mouse brain in a wider time range and concentration gradient. Furthermore, it is imperative to investigate the direct impact of ERT on the brains of healthy mice that have not undergone ovariectomy, considering the adverse cardiovascular and cerebrovascular effects associated with ERT. Consequently, it is noted that this study is deficient in experiments involving the administration of sE2 to sham-operated mice. Therefore, it would be worthwhile to include mouse models of primary ovarian failure or normal menopausal experience as research subjects in future studies, as the menopausal women differ from OVX mice in their physiological conditions.

Previous research showed that the exogenous E2 supplementation was frequently employed as a prevalent therapeutic approach for managing climacteric syndrome by sustaining the peripheral blood levels of estrogen at physiological ranges [1]. Typically, three methods are employed to maintain the physiological estrogen level in animals, i.e., subcutaneous or intraperitoneal injection of estradiol, the implantation of sustained-release capsules containing estradiol beneath the neck or oral gavage. However, the actual dose used was varied in different studies to maintain the normal physiological levels of estrogen. For instance, Zhu et al. administered a dosage of 0.36 mg/60-day E2 to address the abdominal obesity in OVX mice, while Adachi et al. employed a dosage of 0.05 mg/21-day E2 to treat psoriatic inflammation in OVX mice [44, 45]. Zhou et al. administered a dose of 0.3 mg/kg/d E2 orally to mice to improve the cognitive decline and depressive behavior induced by OVX [46]. Consequently, we contend that the decision to employ E2 as a physiological dose should be based on standard serological assessments. Therefore, in our investigation, a dosage of E2 was administered to obtain a twofold increase in the concentration of E2 in the peripheral blood of mice, which exceeded the physiological range.

Recent research has shown that depressed patients exhibited lower cognitive abilities compared to people who were not depressed [47]. It is generally believed that depression precedes the development of cognitive deficits [48]. Notably, we found that although sE2 aggravated depressive-like behaviors in OVX mice, it did not affect their short-term memory based on the results of the Y-maze test. This observation could be explained from two perspectives. First, the pathogenetic causes of both memory deficits and depression were different, i.e., depression was not necessarily sufficient to impair the memory function of the brain [49]. Second, the brain damage caused by sE2 was not sufficient to affect the neuronal circuits responsible for memory [50, 51].

Furthermore, studies have shown that elevated E2 levels lead to decreased levels of follicle-stimulating hormone (FSH) due to negative feedback from the pituitary gland [52, 53]. Recent studies have shown that elevated FSH levels are an important factor in the development of Alzheimer's disease in menopausal women [66]. On the other hand, the high concentration of E2 could decrease the viability of primary neurons by inducing metabolic disorders, and HT22 cells themselves showed a strong regulatory ability to maintain the metabolic balance. In addition, in vitro models do not fully represent in vivo conditions. Therefore, further studies are still needed to confirm the security and the specific mechanisms in vivo of sE2.

Finally, the untargeted metabolomics results showed that the sE2 caused the imbalance of multiple metabolites involved in glycerophospholipid metabolism, retrograde endocannabinoid signaling, and so on, which are associated with depression. These results are consistent with the findings of Zhang et al. [6], showing that the long-term ERT treatment promotes CB1 ubiquitination in the brain of OVX mice, ultimately causing the fear extinction disorder [67]. Previous studies have shown that gut microbes induce depression by regulating glycerophospholipid metabolism, people with sleep deprivation and depression have abnormal glycerophospholipid metabolism, while abnormal glycerophospholipid metabolism also occurs in the brain of the depression rat model [68,69,70]. However, the molecular mechanisms regulating the participation of these metabolites in the above metabolic pathways remain unclear. Further studies are necessary to confirm whether the metabolic imbalance caused by sE2 is limited to neuronal cells, which also affects the metabolisms of microglia and astrocytes.

In conclusion, the sE2 induced depression in OVX mice in various ways, including the abnormal response of microglia and metabolic imbalance of neuronal cells. This study has revealed the potential risks and the underlying mechanisms of the application of sE2 in ERT in the treatment of menopausal depression.