Anisodamine hydrobromide alleviates LPS-induced inflammation via the miR-1195/G3bp1/NF-κB axis in RAW264.7 cells

Anisodamine Hydrobromide (Ani) and microRNAs (miRNAs) are essential regulators of immune responses, but the mechanisms of many Ani-related miRNAs remain insufficiently characterized. This study focused on miR-1195 as a potential regulator of Ani and its role in inflammation modulation in Lipopolysaccharide (LPS)-treated RAW264.7 cells.

Ani concentration was optimized using CCK-8. RAW264.7 cells were stimulated with LPS, with or without Ani pre-treatment. Inflammatory cytokines (TNFα and IL-1β) levels were assessed by RT-qPCR and ELISA. MiRNA sequencing was used to identify differentially expressed miRNAs, which were then validated by RT-qPCR. The interaction between miR-1195 and the 3' UTR of G3bp1 was confirmed using luciferase assays in 293 T cells. For functional analysis, RAW264.7 cells were transfected with miR-1195 mimics, inhibitors, and G3bp1 inhibitors, followed by assessment of TNFα and IL-1β expression via RT-qPCR and ELISA. Western blot was performed to detect the protein levels of phosphorylated p65 (p-p65) and P65 to explore the downstream pathway of G3bp1.

Ani reduced TNFα and IL-1β expression at both mRNA and protein levels, and reversed the LPS-induced downregulation of miR-1195. Overexpressing miR-1195 led to a marked reduction in TNFα and IL-1β levels in LPS-treated RAW264.7 cells, particularly at higher LPS concentrations. Besides, miR-1195 directly targeted and downregulated G3bp1 expression. Inhibition of miR-1195 enhanced cytokine release and promoted NF-κB activation, as indicated by increased p-p65 expression; these effects were reversed by G3bp1 knockdown.

Ani reduces LPS-induced inflammation by modulating miR-1195 and regulating G3bp1, partly through the NF-κB pathway, offering novel therapeutic insights for inflammatory control.