槲皮素的抗炎免疫及部分机制研究

Research on anti-inflammatory and immune effects of quercetin and its partial mechanism

目的探讨槲皮素抗炎免疫作用及部分机制。方法采用不同浓度的槲皮素对RAW 264.7细胞进行干预,观察其对RAW 264.7细胞活力的影响,选择对细胞活力无影响浓度的槲皮素以进行下一步研究。以无处理的RAW 264.7细胞为空白组,采用1μg/ml脂多糖(LPS)刺激RAW 264.7细胞建立炎症细胞模型,将其分为模型组,槲皮素0.98、1.95、3.90、7.80μg/ml剂量组,观察其细胞活力,活性氧(ROS)、单核细胞趋化蛋白1(MCP1)水平。采用环氧合酶(COX)-1、COX-2抑制剂筛选试剂盒检测槲皮素对COX-1、COX-2的抑制作用。另选取SPF级BALB/C雄性小鼠20只(6~8周龄,体重18~22 g)制备其脾淋巴细胞悬液,采用不同浓度的槲皮素对小鼠脾淋巴细胞进行干预,观察其对细胞增殖情况的影响。以无处理的小鼠脾淋巴细胞为对照组,将经LPS(10μg/ml)或伴刀豆球蛋白A(Con A)(8μg/ml)处理的小鼠脾淋巴细胞分为LPS组、Con A组,以及槲皮素0.98、1.95、3.90、7.80μg/ml剂量组。观察不同浓度槲皮素对小鼠脾淋巴细胞、Con A诱导的T细胞和LPS诱导的B细胞增殖的影响,观察T细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、γ干扰素(INF-γ)的水平。结果槲皮素<7.8μg/ml时RAW 264.7细胞活力与未处理时比较,差异无统计学意义(P>0.05)。槲皮素0.98、1.95、3.90、7.80μg/ml剂量组ROS、MCP1含量低于模型组,差异有高度统计学意义(P<0.01)。槲皮素对COX-1、COX-2的IC50分别为12.73、0.44μg/mL,COX-2与COX-1的IC50比值为0.034。槲皮素浓度为0.98、1.95、3.90、7.80μg/ml时小鼠脾淋巴细胞增殖均高于未处理时,差异有统计学意义(P<0.05或P<0.01)。槲皮素1.95、3.90、7.80μg/ml剂量组T细胞低于Con A组,槲皮素3.90、7.80μg/ml剂量组B细胞低于LPS组,差异有统计学意义(P<0.05)。槲皮素1.95、3.90、7.80μg/ml剂量组TNF-α水平均低于Con A组,槲皮素各剂量组IL-6和INF-γ水平均低于Con A组,差异有高度统计学意义(P<0.01)。结论槲皮素是选择性的COX-2抑制剂,具有良好的抗炎活性,能促进小鼠脾淋巴细胞增殖,抑制T、B细胞增殖和细胞因子的分泌。

Objective To investigate the anti-inflammatory and immune effects of quercetin and its partial mechanism.Methods Different concentrations of quercetin were used to intervene RAW 264.7 cells to observe the effects of quercetin on the viability of RAW 264.7 cells.Quercetin with no influence on the viability of RAW 264.7 cells was selected for further study.Untreated RAW 264.7 cells were used as blank group,and the inflammatory cell model was established by stimulating RAW 264.7 cells with 1μg/ml lipopolysaccharide(LPS).The cells were divided into model group,quercetin 0.98,1.95,3.90,and 7.80μg/ml dose groups.Cell viability,reactive oxygen species(ROS),and monocyte chemoattractant protein 1(MCP1)levels were observed.The cyclooxygenase(COX)-1 and COX-2 inhibitor screening kit was used to detect the inhibition of quercetin on COX-1 and COX-2.In addition,20 SPF BALB/C male mice(6-8 weeks old,18-22 g body weight)were selected to prepare splenic lymphocyte suspension,and different concentrations of quercetin were used to interfere with the splenic lymphocytes of mice,and the effects of quercetin on cell proliferation were observed.Mice spleen lymphocytes were respectively treated with LPS(10μg/ml)or concanavalin A(Con A)(8μg/ml)were divided into LPS group,Con A group,and quercetin 0.98,1.95,3.90,7.80μg/ml groups.The effects of different concentrations of quercetin on the proliferation of spleen lymphocytes,T cells induced by Con A and B cells induced by LPS,and the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interferonγ(INF-γ)in T cells were observed.Results When quercetin<7.8μg/ml,there was no significant difference in the activity of RAW 264.7 cells compared with untreated cells(P>0.05).The levels of ROS and MCP1 in quercetin 0.98,1.95,3.90,and 7.80μg/ml groups were lower than those in model group,and the difference was highly statistically significant(P<0.01).The IC50 of quercetin to COX-1,COX-2 were 12.73 and 0.44μg/ml,respectively,and the IC50 ratio of COX-2 to COX-1 was 0.034.The proliferation of spleen lymphocytes of mice with quercetin concentration of 0.98,1.95,3.90,and 7.80μg/ml were higher than those without quercetin,and the differences were statistically significant(P<0.05 or P<0.01).T cells in quercetin 1.95,3.90,and 7.80μg/ml dose groups were lower than those in Con A group,and B cells in quercetin 3.90 and 7.80μg/ml dose groups were lower than those in LPS group,with statistical significance(P<0.05).The levels of TNF-αin quercetin 1.95,3.90,and 7.80μg/ml groups were lower than those in Con A group,the leves of IL-6 and INF-γin quercetin dose groups were lower than those in Con A group,and the differences were highly statistically significant(P<0.01).Conclusion Quercetin is a selective COX-2 inhibitor with good anti-inflammatory activity,which can promote the proliferation of splenic lymphocytes in mice,inhibit the proliferation of T and B cells and the secretion of cytokines.