Berberine Inhibits Macrophages Inflammatory Depending On SIRT1

Chronic inflammation of insulin target tissues is the main cause that gives rise to the decrease in the insulin sensitivity, and macrophage is the main cell type that induces this chronic inflammation status. Coptis chinensis is a commonly used Chinese medicine for treating type 2 diabetes mellitus, its effective component berberine has been shown to inhibit macrophage inflammation, but the mechanism is stillunclear. As a type of deacetylase, SIRT1 can prolong life span, regulate apoptosis, improve metabolism and inflammatory reactions. Because the anti-inflammatory properties of berberine are basically consistent with the targets and biological effects of SIRT1, the further study on their relationship will help to provide clues forthe mechanism ofpharmacological effects of berberine as well as novel ideas for the TCM treatment of type 2 diabetes mellitus.Objective:To observe the effect of berberine on the expression of inflammatory factors and SIRT1 in macrophages as well as the influence of SIRT1 on the anti-inflammatory effects of berberine and to preliminarily investigate the mechanism of anti-inflammatory effects of berberine by methodfor culturing RAW264.7 macrophagesin vitro.Methods:1) The MTT method was used to detect the cytotoxicity of different concentrations of berberine (1,5,10,50 and 100 umol/L) on the RAW264.7 macrophages.2) Pretreat the RAW264.7 macrophages with different concentrations (1 and 5 umol/L) of berberine for 24 hours and then replacethe drug maintenance mediumthe one containing 100 ng/ml LPS.Real-time PCR method was utilized to detect the gene expression ofTNF-α, IL-6 and MCP-1 in macrophages.3) The RAW264.7 macrophages were divided into four groups: control group, BBR group, LPS group and BBR+LPS group; the berberine concentration was 5 umol/L; at 24 hours after the pretreatment, the drug maintenance medium were abandoned and replaced by new culture; the LPS working concentration was 100 ng/ml;RNA and protein samples were extracted6 hours after, and Real-time PCR and Western blot method were used to detect the SIRT1 expression in samples.4) The RAW264.7 macrophages were pre-treated with the SIRT1 specific inhibitor EX527 and siRNA interference technology; with the corresponding control groups set, they were randomly divided into blank group and LPS group and LPS+BBR group; the berberine concentration was 5 umol/L; at 24 hours after the pretreatment, the drug maintenance medium were abandoned and replaced by new medium which containedthe 100 ng/ml LPS; 6 hours after, RNA samples were extracted, and Real-time PCR method was used to detect the gene expression of TNF-a, IL-6 and MCP-1 in the samples.Results:1) Berberine at lower concentration (1 and 5 umol/L) had little cytotoxicity on RAW264.7 macrophages (P>0.05), and berberine at a middle and high concentration (10,50 and 100 umol/L) had a significant inhibition effects on theactivity of macrophages (P<0.05);2) Compared with 1 umol/L berberine,5 umol/L berberine could significantly inhibit the expression of proinflammatory factors TNF-a, IL-6 and MCP-1 in macrophages induced by LPS (P<0.05);3) At the gene level, berberine could both up-regulate the gene expression of SERT1 in macrophages at the basic state and the inflammatory conditions (P< 0.05); at the protein level, berberine had no significant effect on the protein expression of SIRTlin macrophages at the basic state (P>0.05); berberine could obviously up-regulate the protein expression of SIRT1 in macrophages at the inflammatory state (P<0.05); in addition, the study also revealed that, LPS inhibited the expression of SIRT1 in macrophages (P<0.05).4) SIRT1 inhibitor EX527 increased the gene expression of pro-inflammatory factors in RAW264.7 macrophages induced by LPS, while attenuatedthe anti-inflammatory effects of berberine; SIRT1 knockdown RAW264.7 macrophages showed an increaseof the gene expression of pro-inflammatory factors at the inflammatory condition, and weakened the anti-inflammatory effects of berberine.Conclusion:1) 5 umol/L berberine has little cytotoxicity on RAW264.7 macrophages, and can significantly inhibit the expression of pro-inflammatory cytokinesin macrophages induced by LPS;2) Berberine can up-regulate the gene expression of SIRT1 in macrophages at the basic state and had no significant effect on protein; berberine can significantly up-regulate the gene and protein expression of SIRT1 in macrophages under the inflammatory condition;3) SIRT1 deletion increases the gene expression of pro-inflammatory factors in macrophages induced by LPS, whileattenuatesthe anti-inflammatory effects of berberine, indicating that SIRT1 is an important part of the anti-inflammatory effects of berberine in macrophages.