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Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.

Yueju confers antidepressant effects in a rapid and long-lasting manner, similar to ketamine. CREB (cAMP-response element binding protein) signaling is implicated in depression pathology and antidepressant responses. However, the role of CREB and associated brain derived neurotrophic factor (BDNF) signaling in rapid and long-lasting antidepressant effects remains unclear. Here, we demonstrated that ICR and Kunming strain mice conferred antidepressant responses lasting for 1 and 5 days, respectively, following a single dose of Yueju. One day post Yueju in Kunming but not ICR strain mice, expression of total and phosphorylated CREB, as well as the CREB signaling activator, PKA (protein kinase A) was up-regulated in the hippocampus. Although BDNF gene expression increased at 3 hours in both strains, it remained up-regulated at 1 day only in Kunming mice. Ketamine showed similar strain-dependent behavioral effects. However, blockade of PKA/CREB signaling blunted the antidepressant effects and reversed the up-regulation of BDNF gene expression by Yueju, but not ketamine. Conversely, blockade of mammalian target of rapamycin signaling led to opposite effects. Taken altogether, prolonged transcriptional up-regulation of hippocampal BDNF may account for the stain-dependent enduring antidepressant responses to Yueju and ketamine, but it was mediated via PKA/CREB pathway only for Yueju.

Postpartum depression (PPD) affects over 10% of new mothers and adversely impacts the health of offspring. One of the greatest risk factors for PPD is prepregnancy stress but the underlying biological mechanism is unknown. Here we constructed an animal model which recapitulated prepregnancy stress induced PPD and tested the role of Akt-mTOR signaling in the hippocampus. Female virgin Balb/c mice received chronic restraint stress, followed by co-housing with a normal male mouse. We found that the chronic stress led to a transient depressive-like condition that disappeared within two weeks. However, prepregnantly stressed females developed long-term postpartum depressive-like (PPD-like) symptoms as indicated by deficient performance in tests of sucrose preference, forced swim, and novelty-suppressed feeding. Chronic stress induced transient decrease in Akt-mTOR signaling and altered expressions of glutamate receptor subunits NR1 and GluR1, in contrast to long-term deficits in Akt-mTOR signaling, GluR1/NR1 ratio, and hippocampal neurogenesis in PPD-like mice. Acute ketamine improved the molecular signaling abnormality, and reversed the behavioral deficits in PPD-like mice in a rapid and persistent manner, in contrast to ineffectiveness by chronic fluoxetine treatment. Taken together, we find that chronic prepregnancy stress potentiates a long-term PPD, in which Akt-mTOR signaling may play a crucial role.