| Objective:In this study, we will investigate the effects of the anti-infla-mmatory of berberine on LPS induced macrophage RAW264.7 in vitro. On the other hand, its moleculear mechanism in the NF-κB, MAPK pathway and the cascades mediated by COX-2 could be evaluated by the express-ion of gene and protein. This research will offer a new strategy for the development of new anti-inflammatory drug.Methods:1 Murine macrophage cells RAW264.7 were stimulated by LPS with 0.1μg/mL. After 18 h, the expression of TNF-α, IL-6 inflammatory cy-tokines in the cell supernatant were detected.2 The cytotoxicity of berberine at the concentrations used (5,10, 15 μg/mL) on murine macrophage RAW264.7 cell was determined by MTT.3 After 3 h, LPS was added to the mixture that berberine were pr-eintervention on murine macrophages RAW264.7 with a final concentrat-ion of 0.1μg/mL and cultured 18 h. The expressions of Mincle, TNF-a, IL-1β,IL-6, and IL-10 in cells supernatant was evaluated by ELISA, quantitateve PCR in protein and gene level.4 Western blot method was used to detect the protein expression of phosphorylation p65(p-p65), p65, phosphorylation IκB-α (p-κB-α), IκB-αin the NF-kappa B(NF-κB) signaling pathway and phosphorylat-ion ERK1/2(p-ERK1/2), ERK1/2, phosphorylation JNK1/2(p-JNK1/2), JNK1/2, phosphorylation p38(p-p38), p38 in the MAPK signaling pathway.5 The expression of NO, PGE2 was evaluated by ELISA method and the protein expression of COX-2 was measured by western blot.Results:1 After murine macrophage cells RAW264.7 Stimulated by 0.1u g/mL LPS, the expression of TNF-α, IL-6 was significantly higher than the normal group(P＜0.01), which showed that cell inflammation model was successfully established.2 Berberine at the concentrations used (5,10,15 μg/mL) had no cytotoxicity to murine macrophage cells RAW264.7. Compared to normal group relative viability of cells was greater than 90%.3 Compared to normal group, different concentrations of berberine was able to significantly reduce the expression of TNF-α, IL-1β, IL-6 and improved the expression of anti-inflammatory cytokine IL-10 (P＜0.05); In addition, berberine significantly reduced the expression of Mincle mRNA(P＜0.05)4 Berberine was able to suppress the protein expression of phosp-horylation p65, phosphorylation I κ B-α, and increase IκB-α total protein in the NF-κB signalingpathway. And it also inhibited the pr-otein expression of phosphorylation ERK1/2, phosphorylation JNK1/2, and increased the total protein of ERK1/2, JNK1/2. But western blot analysis showed that there had no effect on the protein of p38 in the MAPK signaling pathway.5 Berberine significantly decreased the expression of NP、PGE2、 COX-2 protein (P＜0.01).Conclusion:In this paper, we had successfully constructed the cell inflamma-tory response model with 0.1μg/mL LPS. Further more, these results have confirmed that different concentrations of berine could signify-cantly inhibit the expression of proinflammatory cytokines and incre-ase the expression of anti-inflammatory cytokines. These results show that Berberine have good effects on anti-inflammatory in vitro, which can reduce the protein expression belonging to NF-κB and MAPK pathw-ay and inhibite COX-2 mediating cascades in LPS-induced RAW264.7 cell s. In summary, berberine could be a potential reagent for anti-inflam-matory. |
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