🧬 BDNF Extraction Viewer

Yueju pill (YJ), a classical Traditional Chinese Medicine formula for "six stagnations", has long been used for mood disorders. We have previously demonstrated that YJ exerts rapid-onset antidepressant effects. However, the long-lasting antidepressant effects and its underlying neurobiological mechanisms remain elusive. To evaluate the sustained antidepressant efficacy of YJ in a chronic restraint stress model and elucidate its underlying molecular mechanisms through the integration of transcriptomic, pharmacological, and molecular biological analyses. We first assessed quality consistency of YJ via HPLC quantification. YJ's long-lasting antidepressant actions were conducted using behavioral paradigms (NSF, TST, FST, SPT, OFT) from 30 min 5 day in normal or chronic restraint stress model (CRS) mice after acute administration. Hippocampal key targets in mice affecting the therapeutic onset and long-lasting antidepressant efficacy of YJ were anchored through RNA-sequencing. The expression alterations of these identified targets in mouse hippocampus following YJ treatment were further confirmed by Western blot and PCR. Bidirectional causal validation was achieved by region-specific pharmacological antagonism (PACAP6-38) and RNA interference (AAV-PACAP-shRNA) in the dentate gyrus (DG), elucidating the necessity of this pathway for enduring antidepressant responses to YJ. Elisa was utilized to quantify hippocampal synaptic protein expressions in response to YJ and to assess its association with PACAP. Multi-component analysis via simultaneous identification and quantification of four marker constituents established the inter-batch homogeneity of YJ, with determined mean levels of shanzhiside methylester (0.2594 mg/kg), geniposide (44.2805 mg/kg), ferulic acid (0.1031 mg/kg), and gentiobioside (0.6720 mg/kg). In dose-response testing (1.0-2.5 g/kg), YJ at 1.0 g/kg exhibited the optimal antidepressant-like profile, characterized by rapid onset (reduced feeding latency in NSF at 30 min), short-term efficacy (decreased TST immobility at 3 h), and prolonged therapeutic effects (reduced immobility persisting up to 5 days). In the CRS model, acute YJ administration rapidly and robustly reversed stress-induced behavioral deficits, as evidenced by improved performance in NSF at 30 min, TST at 2 h, and SPT at day 1, with sustained antidepressant-like effects observed in FST at day 3. Notably, these behavioral changes occurred without alterations in locomotor activity or center time in OFT. Hippocampal transcriptomic analysis revealed distinct time-dependent molecular signatures following YJ administration. At 30 min, YJ induced a unique transcriptional shift characterized by qPCR-confirmed upregulation of ADCYAP1 (encoding PACAP). Conversely, at 3 days, a separate signature emerged with CSPG4 (NG2) identified and validated as upregulated. Furthermore, YJ treatment increased hippocampal PACAP levels at 30 min and NG2 expression at 3 days in CRS-exposed mice. Intra-dentate gyrus infusion of PACAP6-38 eliminated YJ's rapid antidepressant-like effects (NSF at 30 min; TST at Day 1) but left Day 3 FST efficacy and NG2 upregulation partially intact. However, AAV-shRNA-mediated PACAP knockdown in the dentate gyrus completely blocked both rapid and sustained behavioral benefits and abolished NG2 induction at 3 days and also blocked the acute YJ-induced enhancement of hippocampal synaptic proteins (synapsin 1 and PSD95) and BDNF expression at both 30 min and 3 days post-administration. Our study demonstrates that YJ achieves sustained antidepressant effects through a time-dependent hippocampal mechanism involving sequential PACAP and NG2 activation, ultimately converging on synaptic protein enhancement and BDNF signaling. This multi-component, multi-target, and multi-temporal mode of action embodies the holistic essence of TCM and offers a compelling alternative to current monoamine-based therapies with limited efficacy and delayed onset.