3.1 Akebia saponin D ameliorates depressive-like behaviors of CMS mice
We first evaluated the anti-depressant efficacy of ASD in mice (Fig. 1A). CMS reduced sucrose preference, which was reversed by 3-week treatment with ASD or IMI (Fig. 1B and 1C). Similarly, CMS shortened latency and prolonged immobility in the FST, which ASD reversed as well as IMI (Fig. 1D). ASD also reversed the CMS-induced decrease in body weight (Fig. S1). In contrast, neither ASD nor IMI affected the distance travelled or immobility time in the OFT (Fig. 1E and Fig. S1). These results suggest that ASD ameliorates depressive-like behaviors in CMS mice.
3.2 Akebia saponin D rescues CMS-induced deficits in hippocampal neurogenesis
NSPCs in the dentate gyrus (DG) of the hippocampus proliferate and differentiate into neurons even in adulthood, and this neurogenesis is negatively associated with depression and positively associated with the efficacy of anti-depressants [9, 20]. Consistent with this, CMS strikingly reduced the number of newborn neurons (DCX+) in the hippocampus of our mice (Fig. 2A). In fact, CMS reduced numbers of Ki67+ cells and DCX+-BrdU+ cells in the subgranular zone (SGZ) of the hippocampus, suggesting inhibition of NSPC proliferation and neuronal differentiation (Fig. 2B and 2C). Conversely, CMS strongly increased the number of GFAP+-BrdU+ cells in hippocampus, suggesting the induction of NSPC differentiation into astrocytes (Fig. 2D). ASD reversed these effects of CMS (Fig. 2A-2D).
To examine the effects of ASD on the survival and maturation of proliferative cells, BrdU was injected into mice before CMS exposure (Fig. 2E). CMS reduced the numbers of surviving newborn neurons (BrdU+-NeuN+) in the DG, which ASD reversed (Fig. 2E and 2F). CMS slowed rates of neuronal differentiation and maturation, while accelerating NSPC differentiation into astrocytes, and ASD reversed these effects (Fig. 2G). These results indicate that ASD improves NSPC proliferation, survival, as well as neuronal differentiation and maturation in the hippocampus of CMS mice.
To investigate the role of neurogenesis in antidepressant effects of ASD, we used the temozolomide (TMZ) to ablate neurogenesis in ASD-treated CMS mice (Fig. 2H and 2I). TMZ treatment abolished the anti-depressant effects of ASD in the SPT and FST (Fig. 2J-2L). These results suggest that the anti-depressant effects of ASD depend in part on promoting hippocampal neurogenesis.
3.3 Akebia saponin D reprogrammes a pro-neurogenic microglia in dentate gyrus of CMS mice
Microglia control the neurogenic microenvironment, and the Arg-1+ microglia contributes to hippocampal neurogenesis [9]. Therefore we examined the effects of ASD on Arg-1+ microglia in dentate gyrus of CMS-exposed mice. The results showed that ASD significantly increased the percentage of Arg-1+ microglia in dentate gyrus of CMS-exposed mice (Fig. 3A and 3B). The immobility time in forced swimming test was negatively correlated with Arg-1+ microglia in dentate gyrus of mice (Fig. 3C). ASD also reversed the CMS-induced increases in the pro-inflammatory factors TNF-α, iNOS and IL-1β and decrease in the anti-inflammatory factors IL-4 and Arg-1 in dentate gyrus of mice (Fig. 3D). Analogously, CMS substantially reduced the levels of IGF-1, TGF-β and BDNF, while ASD significantly increased BDNF in dentate gyrus of mice (Fig. 3D). The BDNF levels were positively correlated with Arg-1 levels in dentate gyrus of mice (Fig. 3E). The results from immunofluorescent staining showed that ASD upregulated the BDNF in Arg-1+ microglia of in dentate gyrus (Fig. 3F). Considering that ASD increases microglial secretion of BDNF, which in turn promotes neurogenesis from NSPCs, we examined the levels of phosphorylation of the BDNF-specific receptor TrkB in hippocampus of mice. The results showed that CMS reduced the levels of p-TrkB in the SGZ of hippocampus, which ASD reversed (Fig. 3G).
To confirm that ASD directly regulates microglial function, we examined the effects of ASD on primary cultures of microglia that were treated with LPS as a model of neuroinflammation (Fig. S2). LPS shifts microglia toward a pro-inflammatory phenotype that inhibits NSPC proliferation, survival and differentiation [20]. Pretreatment with ASD at 50 or 100 μM, but not 10 μM, prevented LPS from upregulating iNOS and TNF-α, and increased the expression of IL-10, Arg-1 and BDNF at 24 and 48 h (Fig. S2 and S3).
To confirm that the ASD-induced changes in microglia in turn influence NSPCs, we treated primary cultures of microglia in different ways, then transferred the culture medium to NSPC cultures and observed their proliferation, survival and neuronal differentiation (Fig. 3H and Fig. S4). We first examined levels of p-TrkB in primary NSPCs cultured in conditioned medium from microglia. The conditioned medium from microglia treated with 50 μM ASD + LPS (ASD-M-CM) increase the pTrkB in NSPCs (Fig. 3I). Compared with conditioned medium from PBS-treated microglia (PBS-M-CM), the conditioned medium from LPS-treated microglia (LPS-M-CM) decreased the size of NSPC neurospheres. ASD-M-CM increased the size of NSPC neurospheres when compared with PBS-M-CM or LPS-M-CM. (Fig. 3J). LPS-M-CM inhibited NSPC differentiation into neurons (DCX+ cells), but ASD-M-CM promoted such differentiation (Fig. 3K). Overall, the effects of ASD were similar to those of pioglitazone, an agonist of PPAR-γ pathway, which reprogrammes a pro-neurogenic microglial phenotype.
3.4 The PPAR-γ plays a critical role in reprogramming of pro-neurogenic microglia by akebia saponin D in dentate gyrus of CMS mice
Since mammalian target of rapamycin (mTOR) / PPAR-γ signaling plays a key role in induction of anti-inflammatory microglial phenotypes [37], we asked whether ASD acts via such signaling to exert its “microglial reprogramming” effect. Indeed, CMS reduced expression of mTOR and PPAR-γ as well as their phosphorylated levels in dentate gyrus (Fig. 4A). After treatment with ASD, p-mTOR and p-PPAR-γ were significantly increased in CMS-exposed mice (Fig. 4A). Activation of PPAR-γ with ASD reversed the CMS-induced decrease in BDNF level in dentate gyrus of mice (Fig. 4A). The results from immunofluorescent staining showed that PPAR-γ localized in cytoplasm and nucleus of Arg-1+ microglia in the dentate gyrus of mice that were exposed to CMS and then treated with ASD (Fig. 4B).
To confirm the role of PPAR-γ in induction of the pro-neurogenic microglia in dentate gyrus of ASD-treated mice, we repeated the above experiments in the presence of the PPAR-γ antagonist GW9662 (Fig. 4C), which effectively blocked the PPAR-γ pathway in dentate gyrus (Fig. 4D). Such blockade abolished the ability of ASD to increase numbers of Arg-1+ microglia in the dentate gyrus of CMS mice (Fig. 4E). Blockade of PPAR-γ signaling in ASD-treated primary microglia also abolished the ability of ASD-M-CM to stimulate NSPC proliferation and neuronal differentiation (Fig. 4F-4I). GW9662 treatment abolished the ability of ASD to increase the BNDF and pTrkB levels in dentate gyrus of CMS mice (Fig. 4D). These results suggest that soluble microglial factors such as BDNF activate the TrkB of NSPC to promote NSPC proliferation, survival and neurogenesis. Consistent with this, the TrkB inhibitor K252a prevented ASD-M-CM from stimulating NSPC proliferation and neuronal differentiation (Fig. S5).
Blockade of PPAR-γ or TrkB signaling abolished the ability of ASD to promote hippocampal neurogenesis in CMS mice (Fig. 5A and 5B). Either GW9662 or K252a also blocked the anti-depressant effects of ASD in the sucrose preference test and forced swimming test (Fig. 5C and 5D). These results suggest that ASD reprogrammes the pro-neurogenic microglia in dentate gyrus of CMS mice via the PPAR-γ signaling pathway.