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目的:观察葛根素预处理对脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7活化和分泌细胞因子的影响,探讨其抗炎机制。方法:取对数期生长良好的RAW264.7细胞,随机分为空白对照组、LPS组、葛根素预处理+LPS组。CCK-8法检测葛根素对RAW264.7的细胞毒性作用,Giemsa染色法观察细胞形态学变化,酶联免疫吸附试验(ELISA)检测细胞上清中肿瘤坏死因子-α(TNF-α)、巨噬细胞炎性蛋白-2(MIP-2)的变化,实时荧光定量PCR(qRT-PCR)动态测定NF-κB p65 mRNA的表达。结果:当葛根素的浓度为100,200,400μmol·L-1时,与1 mg·L-1LPS共培养均未显示出细胞毒性作用(P<0.05);与空白对照组相比,LPS组可明显改变RAW264.7细胞的形态(胞体增大,形态不规则,伪足大量伸出),葛根素100μmol·L-1组干预后可明显抑制LPS导致的细胞形态学变化,葛根素200,400μmol·L-1干预组的抑制效果更显著,但2组之间无明显差异;葛根素预处理可使细胞上清液中TNF-α,MIP-2以及细胞内NF-κB p65 mRNA的表达受到抑制(P<0.05),并随着葛根素浓度的增加,抑制效果逐渐增加(P<0.05),但未达到对照组水平。结论:葛根素是一种安全有效的天然抗炎药物,可显著下调炎症细胞因子(如TNF-α,MIP-2)的表达,其作用机制可能与下调NF-κB p65 mRNA的表达有关。
OBJECTIVE: To observe the effect of puerarin on the activation and secretion of cytokines in macrophages RAW264.7 induced by lipopolysaccharide (LPS), and to explore its anti-inflammatory mechanism. Methods: RAW264.7 cells with good logarithmic growth phase were randomly divided into blank control group, LPS group and puerarin pretreatment + LPS group. Cytotoxicity of puerarin on RAW264.7 cells was detected by CCK-8 assay. Cell morphology was observed by Giemsa staining. Tumor necrosis factor-α (TNF-α) was measured by enzyme linked immunosorbent assay (ELISA) The changes of MIP-2 and qRT-PCR were used to detect the expression of NF-κB p65 mRNA. Results: When puerarin was 100, 200 and 400μmol·L-1, the cytotoxicity was not observed in co-culture with 1 mg · L-1 LPS (P <0.05). Compared with the blank control group, LPS group could obviously change The morphological changes of RAW264.7 cells (cell bodies enlarged, irregular shape and large numbers of pseudopodia) were inhibited by 100μmol·L-1 puerarin. The changes of cell morphology induced by LPS were obviously inhibited by puerarin 200 and 400μmol·L- 1 intervention group was more significant, but there was no significant difference between the two groups; puerarin pretreatment inhibited the expression of TNF-α, MIP-2 and NF-κB p65 mRNA in the cell supernatant (P <0.05). With the increase of puerarin concentration, the inhibitory effect increased gradually (P <0.05), but did not reach the level of control group. CONCLUSION: Puerarin is a safe and effective natural anti-inflammatory drug which can down-regulate the expression of inflammatory cytokines (such as TNF-α and MIP-2). The mechanism may be related to the downregulation of NF-κB p65 mRNA expression.