TCM, with a reliable amount of clinical practice and unique curative effect in depression treatment, is gaining increasing attention [41]. LBRD standard decoction, which is a classical TCM formula with consisting of lily bulb and fresh rehmannia juice, is extensively implicated in treatment of affective disorder [8, 9, 42]. Each single herb of LBRD standard decoction is beneficial for central nervous system disorders and widely used in an Asian-medicated diet. Although the results show that LBRD is a safe and effective treatment for depression in clinical trials, it is still not clear the effective constituents, and what the underlying possible mechanisms are during the intervention and how it plays a role in this process.
In this work, there are 32 chemical constituents in LBRD standard decoction including catalpol, vidarabine, aminoadipic acid, myristic acid, verbascoside and etc. Among them, the contents of verbascoside and catalpol in LBRD standard decoction met with the standard qualities of lily bulb and raw rehmannia root in the Pharmacopoeia of People's Republic of China (2020 Edition). Furthermore, both of them have been reported to exert antidepressant, antioxidative, anti-inflammatory, immunoregulatory and neuroprotective effect. Thus, verbascoside and catalpol may be served as quality control marker and effective ingredients of LBRD standard decoction, which could benefit the modernization, standardization and internationalization of LBRD decoction as a compound drug.
Our previous reports demonstrated that CUMS for 4 weeks in mice could produce obvious depression-like behavior [39]. Thus, we established a 4-week CUMS mice model to observe the antidepressant effect and possible mechanism of LBRD standard decoction in CUMS-induced depression-like behavior mice. Behavioral results showed LBRD standard decoction could produce antidepressant like effect and its effect was similarly to fluoxetine, which was commonly used for depression treatment in clinic. In addition to anti-depressive effect, LBRD standard decoction was also found to effectively improve the severity of anxiety symptoms. Meanwhile, our current results revealed the protection of LBRD standard decoction on cortex neurons may be related to reduction of glutamate and recover the balance of pro/anti-inflammatory cytokines (Figure. 1). Evidences have showed that the abnormalities in the synthesis, metabolism, and reuptake into neurons of glutamate and inflammatory factors in the brains was contributed to the pathophysiology of depression [12, 43–45]. Further preclinical and animal studies also demonstrated that acute and chronic stress procedures lead to the increased extracellular glutamate in the cortex, and this has led to glutamate-mediated exitotoxicity via actions at extra synaptic N-methyl-D-aspartate receptors (NMDARs) or GluR2-Parkin pathway mediated mitophagy, which are responsible for the loss of neurons in these brain regions [46–48].
GABAergic interneuron have a critical role in orchestrating the balance of excitation and inhibition within the mPFC tissue, as well as excitatory outputs to projection areas by targeting specific subcellular domains of glutamatergic pyramidal neurons, and controlling cortical firing rate, bursting, timing, synchrony and rhythms [49]. In current study, LBRD standard decoction intervention can achieve the balance between the Glu and GABA in mFPC tissues by increasing Gad-67, VGAT and GAT-3 genes expression which are responsible for GABA synthesis, release and uptake. From the treatment perspective, our findings supported the latest hypothesis of depression whereby deficient GABAergic inhibition of the cortical glutamatergic system results in its over-activity [50]. Elevated Glu in depression may result from GABAergic deficit (lower GABA levels or GABAergic interneuron numbers) resulting in glutamatergic disinhibition [12]. Moreover, NMDAR antagonists including ketamine and D-cycloserine (DCS), and GABA agonists targeting Glu and GABA homeostasis have antidepressant benefit in both MDD and bipolar depression [51, 52].
GABAergic interneurons are now recognized to be highly heterogeneous in terms of multiple morphological, electrophysiological, and molecular properties. Studies have also begun to investigate the role of three major largely non-overlapping GABA interneuron subtypes (SST, PV and VIP) in the pathophysiology and treatment of depression [35]. However, there is less evidence of consistent alterations of specific GABAergic cell-type markers [37]. The reason for these discrepancies is unclear, but owing to the length of stress exposure, the type of stressor, and the method of analysis of interneuron markers. Fortunately, the consensus generating from these cellular and molecular studies reveal SST interneuron is a preferentially vulnerable GABAergic subtype to biological adversity [12, 53]. Similarly, our results revealed the number of SST interneuron and the level of SST mRNA and protein expression were decreased in stress induced depression. Reduced SST interneuron cell function alters information processing by cortical microcircuits in result of reduction of temporal and spatial signal to noise ratios of excitatory input onto pyramidal neurons [54]. Furthermore, LBRD standard decoction medications can modulate the balance of GABAergic interneuron subpopulations and reverse SST interneurons deficit in depression by improving GABA related genes expression within it (Figure. 2). Our results revealed deficits in SST interneuron represent a contributing cellular pathology, and therefore becoming a novel target for LBRD standard decoction therapeutic approaches by normalizing altered inhibitory function in depression with reduced SST cell and GABA functions.
Epigenetic modifications, i.e., DNA methylation, post-translational histone modification and interference of microRNA (miRNA) or long non-coding RNA (lncRNA), are able to influence the severity of the depression and the outcome of the therapy [55]. In this study, we successfully constructed a GABA-related ceRNA regulatory network that LncRNA Neat1 and Malat1 acts as a miRNA-144-3p and miRNA-15b-5p sponge to regulate GABA synthesis, release and uptake by mediating Gad-67, VGAT and GAT-3 (Figure. 3). LBRD standard decoction administration can remarkably enhance the levels of Neat1 and Malat1 expression and decrease the miRNA-144-3p level in SST-positive neurons (Figure. 4). LncRNA NEAT1 performs a scaffolding function in the nucleus that releases regulatory proteins after nerve cell activation, fine-tuning the excitatory response that is important for pathological seizure states. In addition, a decrease in lncRNA NEAT1 leads to altered expression of multiple gene transcripts involved in ion channel function following neuronal activation [56–58]. Notably, another lncRNA, MALAT1, which displays considerable expression in neurons, can be linked thanks to rodent models of psychiatry disease to the set of molecular alterations contributing to disease onset [59, 60]. miRNA-144-3p targets a number of genes implicated in the response to stress, aging diseases and mood stabilizer treatment including Pten, Spred1, EGFR, Nrf2, AQP1, NGF, Brg1 and Notch1 [61–64]. Unfortunately, there are few reports on the lncRNA Neat1 and malat1 directed miRNAs involved in the development and treatment of depression. To our knowledge this is the first time to elaborate dysfunctional lncRNA-directed regulatory network contributes to GABA deficits in depression and LBRD standard decoction can reverse reduced SST neuron activity by regulating the miRNA-144-3p mediated GABA synthesis, release (Figure. 5).
Our study has some limitations. First, LBRD standard decoction contains multiple ingredients; we have only demonstrated its antidepressant effects. The activity and synergies between the components are not yet clear and further research is needed to elucidate one or more components responsible for these effects. Second, the results of our study suggest that deregulated ceRNA network contributes to GABA deficits in SST interneuron, but how this alteration influences other GABAergic neurons subtype information processing in neuronal cortical microcircuit, has not been addressed. More importantly, whether changing the GABA-related miRNA network in SST neurons reverses the therapeutic effect of LBRD standard decoction. Therefore, in future work, we will adopt small RNA interference technology, optogenetic technology, as well as single cell sequencing to study the antidepressant effects of LBRD standard decoction based on neuroepigenetics mechanism of GABA deficits.