化瘀祛痰方药及其拆方含药血清对脂多糖诱导的人脐静脉内皮细胞炎症因子表达的影响

Effect of Huayu Qutan Recipe and Its Separated Recipes on Inflammation Factors in EA.hy926 Cells Induced by Lipopolysaccharide

摘要:

目的: 观察化瘀祛痰方药及其拆方对脂多糖(lipopolysaccharide,LPS)诱导人脐静脉内皮细胞EA.hy926炎症因子表达的影响。 方法: SD大鼠随机分为空白对照组、全方组、补气组、化瘀组、祛痰组,生理盐水和相应中药予以ig,第9天腹主动脉采血,分离血清。体外培养EA.hy926细胞,随机分为①正常对照组、②LPS刺激组、③全方组、④补气组、⑤化瘀组、⑥祛痰组、⑦空白血清对照组。其中②组加入终浓度为10mg ·L^-1 LPS,③~⑦组用相应含药血清(浓度为10%)预处理24 h后加入终浓度为10mg ·L^-1 LPS,各组细胞培养24 h后进行各项指标测定。RT-PCR法检测核因子-κB(NF-κB)和肿瘤坏死因子-α(TNF-α) mRNA表达;ELISA法检测TNF-α蛋白的表达;免疫荧光细胞化学法检测NF-κB蛋白的表达。 结果: 经LPS刺激后NF-κB和TNF-α的表达与正常组比较均明显升高(P<0.01)。全方组NF-κB和TNF-α的表达与刺激组相比明显受抑制(P<0.01);拆方各组中,化瘀组NF-κB和TNF-α的表达显著减少。补气、祛痰、空白血清对照组NF-κB和TNF-α的表达显著高于全方组;而化瘀组NF-κB和TNF-α的表达与全方组相比较无统计学差异。 结论: 化瘀祛痰方药及化瘀拆方可显著抑制内皮细胞炎症因子NF-κB和TNF-α的表达,这可能是其抗动脉粥样硬化的作用机制之一。

Abstract:

Objective: To observe the influence of Huayu Qutan recipe and its separated recipes on the expression of inflammatory factors in cultured human umbilical veins endothelial cells EA.hy 926 induced by LPS. Method: SD rats were equally divided into five groups in random: the control group, Huayu Qutan recipe group, Buqi recipe group, Huayu recipe group and Qutan recipe group. They were ig given daily with normal saline and recipes respectively. Blood drawn from abdominal aorta of the rats 2 h after ending of the treatments on the 9^th day, and the serum was takenfor testing. EA.hy926 cells were cultured and equally divided into seven groups randomly: the normal control group, LPS stimulating group, Huayu Qutan recipe group, Buqi recipe group, Huayu recipe group, Qutan recipe group as well as normal serum control group. Then the mRNA levels of NF-κB and TNF-α were tested by RT-PCR. The protein levels of TNF-α and NF-κB were detected by ELISA technique and fluorescent immunocytochemistry method, respectively. Result: The expression of NF-κB and TNF-α were both increased compared to normal group after stimulation of LPS(P<0.01). The expression of NF-κB and TNF-α in Huayu Qutan recipe group and Huayu recipe group were obviously inhibited compared to that of LPS stimulating group (P<0.01). Conclusion: The molecular mechanism of Huayu Qutan recipe and Huayu recipe against atherosclerosis is probably related with the inhibition of expression of NF-κB and TNF-α.