Proanthocyanidins alleviate lipopolysaccharide-induced inflammatory response by up-regulating SIRT1 expression and inhibiting NF-κB pathway in mouse RAW264.7 macrophages / 细胞与分子免疫学杂志

Objective To investigate the role of proanthocyanidins (PC) in lipopolysaccharide (LPS)-induced inflammatory response and its possible mechanism in RAW264.7 macrophages. Methods RAW264.7 macrophages were cultured and treated with PBS and different concentrations of PC for 24 hours, followed by 1 μg/mL LPS for 6 hours. Real-time PCR was used to detect the mRNA expression of interleukin1β (IL-1β), IL-6, monocyte chemoattractant protein 1 (MCP-1), tumor necrotic factor α (TNF-α), IL-4 and arginase 1 (Arg1) in RAW264.7 macrophages. Flow cytometry was used to detect the effects of PBS group, LPS group and PC combined with LPS group on M1 and M2 polarization of macrophages. The protein expressions of silenced information regulator 1 (SIRT1), nuclear factor kappa B p65(NF-κB p65) and acetylated NF-κB p65 (Ace-p65) were detected by Western blot analysis after different concentrations of PC treatment. Co-immunoprecipitation assay was used to detect the binding effect of SIRT1 to NF-κB p65 in macrophages treated with PC. Results Compared with PBS group, the mRNA expression of macrophage pro-inflammatory cytokines IL-1β, IL-6, MCP-1 and TNF-α decreased and the mRNA expression of anti-inflammatory factors IL-4 and Arg1 increased in PC group. Compared with LPS group, PC combined with LPS group could significantly inhibit M1 polarization and promote M2 polarization of macrophages. With the increase of PC concentration, the expression of SIRT1 was up-regulated, and NF-κB p65 protein did not change significantly. The expression of Ace-p65 protein decreased significantly when treated with high concentration of PC. Conclusion PC can significantly alleviate the LPS-induced inflammatory response by up-regulating the expression of SIRT1 and inhibiting NF-κB pathway in RAW264.7 macrophages.

Neuropathic Pain Relieving in Rats by the Inhibition of Monomer of Lamiophlomis Rotata on AKT-mTOR Signaling Pathway in Spinal Dorsal Horn / 中国药师

Objective:To observe the effect of intrathecal administration of 8-O-acetyl-SM(8-OaS) on chronic neuropathic pain in rats by inhibiting the expression of protein kinase B(AKT)-mammalian target of rapamycin(mTOR) signaling pathway in spinal dorsal horn after spinal nerve ligation.Methods:The rat model of neuropathic pain was established by lumbar 5 spinal nerve ligation(SNL), and Von Frey filament was used to investigate the mechanical allodynia. Immunofluorescent histochemistry was adopted to investigate the distribution of pAKT and pmTOR in spinal dorsal horn. The protein levels of pAKT and pmTOR in spinal dorsal horn after drug ad-ministration were quantitatively determined by Western blot. Results: Compared with that in the sham group, the paw withdrawal threshold (PWT) significantly decreased(P<0.01),and the intrathecal administration of 8-OaS attenuated mechanical allodynia ob-viously during the first day and the seventh day after the operation(P<0.05). Meanwhile,double immunofluorescent staining showed the co-expression of pAKT and astrocytes marker glial fibrillary acidic protein(GFAP),and positive labeling of pmTOR was expressed in spinal astrocytes and neurons. The results of Western blot revealed that the protein levels of pAKT and pmTOR in spinal dorsal horn were significantly reduced after the treatment of 8-OaS. Conclusion:Intrathecal administration of 8-OaS attenuates the PWT of SNL-in-duced chronic neuropathic pain. The underlying mechanism of the potential anti-allodynia effect of 8-OaS may be related to the suppres-sion of spinal astrocytes via decreasing the phosphorylation of AKT-mTOR signaling pathway resulting in attenuating the development of neuropathic pain.