Background: The interaction between the gut microbiome and neurological outcomes is increasingly recognized, including in the context of opioid use and its immunomodulatory effects. This study investigates how morphine treatment interacts with the gut-brain axis, examining its impact on gut microbiome alterations and inflammatory gene expression, along with behavioral responses in a rat model.
Materials and Methods: Wistar rats were assigned to a morphine treatment group that was administered a fixed escalating dose of morphine sulfate (30 mg/kg) or a saline control. We further evaluated the expression of the inflammatory genes IL-6, TNF-α, and INF-γ in the intestinal tissue using quantitative real-time polymerase chain reaction. The behavior test used included the elevated plus maze (EPM) to quantify anxiety-like behavior. Gut microbiome alteration was assessed using the quantitative real-time polymerase chain reaction technique. 
Results: Morphine-treated rats showed a significant increase in anxiety-like behaviors in the EPM test compared to controls (P<0.01). Gut microbiome analysis revealed significant alterations, including a decrease in beneficial bacterial species, specifically Akkermansia muciniphila (P=0.0008) and Bifidobacterium longum (P=0.003), alongside a significant increase in potentially pathogenic species, such as Bacteroides fragilis (P<0.001) and Fusobacterium nucleatum (P<0.001). Inflammatory gene expression analysis demonstrated a significant increase in INF-γ (P<0.001) and TNF-α (P=0.03), while IL-6 did not reach statistical significance (P=0.06). 
Conclusion: This study demonstrates considerable changes in the gut microbiome and increased anxiety-like behaviors after morphine administration, corroborating a bidirectional gut-brain relationship during opioid exposure. The results bring to light promising microbiota-targeted therapies as treatment options for opioid-related behavioral dysregulation.