白及胶/微米三七膏含药血清对脂多糖诱导的巨噬细胞核因子-κB信号通路的影响

Study on Effect of Hyacinth Bletilla/Micron Pseudo-ginseng Salve Drug-containing Serum on LPS-induced Inflammatory Injury Model of Macrophages by NF-κB Pathway

目的:研究白及胶/微米三七膏含药血清通过核因子(NF)-κB信号通路对脂多糖(LPS)诱导的巨噬细胞炎症损伤模型的影响。方法:取对数生长期的RAW264.7细胞,用不同浓度(5、10、15、20、25μg/mL)的LPS处理,采用CCK-8法检测细胞增殖率,确定LPS的最佳浓度,并诱导建立RAW264.7细胞炎症损伤模型;将细胞分为正常对照组、LPS组和不同浓度梯度(50、100、150、200、250、300μL/mL)的白及胶/微米三七膏含药血清+LPS组,用CCK-8法检测各组光密度(OD)值,确定含药血清最佳使用浓度;将细胞分为正常对照组、空白血清组、含药血清组、LPS组、LPS+空白血清组和LPS+含药血清组,用Western blotting法测定细胞浆/细胞核NF-κB p65蛋白表达量和细胞浆p-IκBα蛋白表达量。结果:LPS最佳浓度为10μg/mL;白及胶/微米三七膏含药血清最佳浓度为250μL/mL;与正常对照组相比,LPS组细胞浆NF-κB p65蛋白表达量显著降低、细胞核NF-κB p65和细胞浆p-IκBα蛋白表达量显著增高,差异有统计学意义(P<0.01);与LPS组相比,LPS+含药血清组细胞浆NF-κB p65蛋白表达量显著增高、细胞核NF-κB p65和细胞浆p-IκBα蛋白表达量显著降低,差异有统计学意义(P<0.01)。结论:通过NF-κB信号通路,白及胶/微米三七膏含药血清通过抑制NF-κB p65进入细胞核和抑制I-κBα的磷酸化,减少巨噬细胞分泌炎性因子,从而改善LPS诱导的炎症反应。

Objective To explore the effect of Hyacinth Bletilla/Micron Pseudo-ginseng salve drug-containing serum on LPS-induced inflammatory injury model of macrophages by NF-κB pathway.Methods The RAW264.7 cells at logarithmic growth stage are processed with lipopolysaccharide(LPS)at different concentrations(5,10,15,20,25μg/mL).Its proliferation rate is detected by CCK-8 method.The optimal LPS concentration is determined and the RAW264.7 model of inflammatory injury is established.The cells are divided into normal control group,LPS group and Bletilla/Micron Pseudo-ginseng drug-containing serum+LPS group with different concentration gradient(50,100,150,200,250,300μL/mL).The OD value of each group is detected by CCK-8 method to determine the optimal concentration of drug-containing serum.The cells are divided into normal control group,blank serum group,drug-containing serum group,LPS group,LPS+blank serum group and LPS+drug-containing serum group.The protein expression levels of NF-κB p65/p-IκBαin cytoplasm and nuclear are determined by Western blotting.Results The optimal concentration of LPS is 10μg/mL.The optimum concentration of drug-containing serum is 250μL/mL.Compared with the normal control group,the protein expression levels of NF-κB p65 in cytoplasm are significantly decreased in LPS group.The protein expression levels of NF-κB p 65 in nuclear and p-IκBαin cytoplasm are significantly increased in LPS group(P<0.01).Compared with the LPS group,the protein levels of NF-κB p65 in nuclear are significantly increased,NF-κB p65 in nuclear and p-IκBαin cytoplasm are significantly decreased in LPS+containing serum group(P<0.01).Conclusion Bletilla/Micron Pseudo-ginseng drug-containing serum inhibits NF-κB p65 entry into the nucleus and IκBαphosphorylation and reduce macrophage secretion of inflammatory cytokines,thereby improving LPS-induced inflammatory reaction by NF-κB signaling pathway.