Gypenoside XVII, an Active Ingredient from Gynostemma Pentaphyllum, Inhibits C3aR-Associated Synaptic Pruning in Stressed Mice

Abstract

Gynostemma pentaphyllum is a herbal medicine widely used in Asian countries, and its saponin extracts have been shown to possess potent anti-inflammatory effects. Gypenoside XVII, an active ingredient isolated from Gynostemma pentaphyllum, has been found to alleviate the inflammation induced by LPS in the BV2 microglia, according to our preliminary study. This study aims to evaluate whether Gypenoside XVII could attenuate depression-like symptoms in vivo and tries to demonstrate the involvement of the complement regulation in its antidepressant-like effect. The results showed that Gypenoside XVII significantly attenuated depression-like behaviors in the forced swimming test, tail suspension test and sucrose preference test. It also alleviated the acute stress-induced hyperactivity of serum corticosterone levels. Additionally, Gypenoside XVII significantly inhibited the activation of microglia and the expression of C3 in mice exposed to chronic unpredictable mild stress (CUMS). Meanwhile, the activation of C3aR/STAT3 signaling and the expression of proinflammatory cytokines was reversed by Gypenoside XVII. Moreover, CUMS induced excessive synaptic pruning by activating microglia, while Gypenoside XVII restored it in the prefrontal cortex. Our data demonstrated that Gypenoside XVII, the active ingredient of Gynostemma pentaphyllum, produced the antidepressant-like effects in mice, which was mediated by the inhibition of complement C3/C3aR/STAT3/cytokine signaling in the prefrontal cortex.

Keywords: Gypenoside XVII; antidepressant; complement C3; microglia; synaptic pruning.

Figure 1

Acute administration with Gypenoside XVII decreased the immobility time but did not affect locomotor activity in the behavioral tests (n = 8). The structure of Gypenoside XVII and the experimental timeline (A). Open-field test (B,C). Tail suspension test (D). Forced swimming test (E). Serum corticosterone levels (F). * p < 0.05 and ** p < 0.01 versus the vehicle group.

Figure 2

Gypenoside XVII reversed depression-like symptoms in depression-like mice (n = 10). Diagram for the experimental protocol (A). Sucrose preference during an 8-week CUMS procedure (B). Body weight during an 8-week CUMS procedure (C). Immobility time at the end of CUMS procedure (D). # p < 0.05 and ## p < 0.01 versus the Normal-vehicle group. * p < 0.05 and ** p < 0.01 versus the CUMS-vehicle group.

Figure 3

The number of microglia was decreased and the levels of C3 were decreased by Gypenoside XVII in the prefrontal cortex (n = 4). The number of microglia (A,B) and the levels of C3 (C,D) were detected by immunofluorescence. ## p < 0.01 versus the Normal-vehicle group. * p < 0.05 versus the CUMS-vehicle group.

Figure 4

Gypenoside XVII inhibits C3aR-STAT3 cytokines signaling in the prefrontal cortex (n = 4–6). C3aR levels were detected by both immunofluorescence (A,B) and Western blot (C). pSTAT3/STAT3 was detected by Western blot (D). The mRNA expression of IL-1β (E), IL-6 (F) and TNF-α (G) was detected by PCR. # p < 0.05 and ## p < 0.01 versus the Normal-vehicle group. * p < 0.05 and ** p < 0.01 versus the CUMS-vehicle group.

Figure 5

Gypenoside XVII prevented synaptic pruning in the prefrontal cortex (n = 4). Presynaptic protein vGluT1 in microglia (A,B). Postsynaptic protein PSD95 in microglia (C,D). ## p < 0.01 versus the Normal-vehicle group. * p < 0.05 and ** p < 0.01versus the CUMS-vehicle group.

Figure 6

Schematic diagram of the antidepressant-like Gypenoside XVII in CUMS-induced mice. Chronic stress activates microglia and enhances C3 release. C3 targets neuron and recruit microglia to eliminate targeted synapses. Meanwhile, C3 binds with C3aR to activate STAT3. Subsequently, STAT3 induces the activation of the pro-inflammatory cytokine to aggravate the inflammatory response.