Chaihu-Jia-Longgu-Muli Tang ameliorates chronic restraint stress-induced depressive-like behaviors in association with modulation of the gut microbiota-NLRP3 inflammasome-microglia axis.

Chronic stress-induced neuroinflammation and gut microbiota dysbiosis have been implicated in the pathogenesis of depression. The classic herbal formula Chaihu-Jia-Longgu-Muli Tang (CLM) has been reported to exert antidepressant effects, but the underlying mechanisms remain poorly understood. This study aimed to investigate whether CLM ameliorates depressive-like behavior in chronic restraint stress (CRS)-induced mice and to explore its association with the gut microbiota remodeling, NLRP3 inflammasome signaling, and microglia activation. A mouse model of depression was established through exposure to CRS. Depressive-like behaviors were assessed using the sucrose preference test (SPT), tail suspension test (TST), and open field test (OFT). Hippocampal neuronal damage was evaluated by HE and Nissl staining. Intestinal barrier integrity was examined by HE staining and immunofluorescence staining for ZO-1 and occludin. Colonic and hippocampal levels of pro-inflammatory cytokines were measured by ELISA. Gut microbiota composition was analyzed by 16 S rRNA gene sequencing. Hippocampal M1 microglial polarization and microglial NLRP3 localization were assessed by Iba-1/CD86 and Iba-1/NLRP3 double immunofluorescence staining, and NLRP3 inflammasome-related protein expression was further evaluated by Western blotting. CLM attenuated CRS-induced depressive-like behaviors and hippocampal neuronal damage, restored gut barrier integrity as indicated by increased ZO-1 and occludin expression and reduced colonic IL-6, TNF-α, and IL-1β levels, and reshaped the gut microbiota by increasing SCFA-producing Muribaculaceae, Alloprevotella, and Bacteroides and decreasing Dubosiella and Lactobacillus spp. Exploratory Spearman analyses identified several nominal associations between microbial taxa and behavioral or inflammatory variables; however, these associations did not remain significant after Benjamini-Hochberg FDR correction and should therefore be interpreted as hypothesis-generating. CLM-H also reduced hippocampal CD86⁺/Iba-1⁺ cells, decreased NLRP3+/Iba-1+ co-localization, and reduced NLRP3, caspase-1 p20, and pro-IL-1β expression. CLM-H ameliorates CRS-induced depressive-like behavior and neuronal damage in association with modulation of the gut microbiota composition, NLRP3 inflammasome-related signaling, and microglial activation. These findings support the potential of CLM as a multi-target intervention for depression-related phenotypes, while further causal validation is still required.