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目的:通过研究清热凉血中药对人永生化表皮细胞(Ha Ca T细胞)人β防御素(HBD)-2的表达及信号通路的调控作用,探讨其治疗银屑病的可能机制。方法:用白细胞介素(IL)-1β和肿瘤坏死因子(TNF)-α的混合物刺激Ha Ca T细胞建立信号通路活化及HBD-2高表达的Ha Ca T细胞模型;分别用清热凉血中药、维A酸和生理盐水进行干预,用实时荧光定量核酸扩增法(q PCR)和酶联免疫吸附试验(ELISA)测定清热凉血中药对细胞模型中HBD-2表达水平的影响;用免疫印迹法(westen blot)检测清热凉血中药对细胞模型中HBD-2及丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)和核转录因子kappa B(NF-κB)信号通路的影响。结果:(1)中药血清高剂量组和维A酸血清组均可明显下调Ha Ca T细胞模型中HBD-2m RNA的表达,抑制率分别为66.8%和73.5%,与细胞模型组比较,差异均有统计学意义(均P<0.01),中药血清高剂量组和维A酸血清组比较差异无统计学意义(P>0.05);(2)中药血清高剂量组与维A酸血清组均可抑制细胞培养上清中HBD-2的分泌,与细胞模型组比较,抑制率分别为35.6%和61.6%;(3)100 mg/L的IL-1β和TNF-α混合物作用18 h后可使Ha Ca T细胞中的cJun氨基末端激酶(JNK)2 MAPK信号通路活化;清热凉血中药血清可以明显下调磷酸化c-Jun氨基末端激酶(p-JNK)2蛋白的相对含量,抑制JNK2 MAPK信号通路的活化。结论:清热凉血方药可明显下调HBD-2高表达的Ha Ca T细胞模型及培养上清中HBD-2的表达。清热凉血方药可以抑制JNK2 MAPK信号通路,并阻断IL-1β和TNF-α对HBD-2的激活。
OBJECTIVE: To explore the possible mechanism of heat-and-cool-blood Chinese medicine on the regulation of human β-defensin (HBD) -2 expression and signal pathway in human immortalized epidermal cells (HaCa T cells) Methods: HaCa T cells were stimulated with a mixture of interleukin (IL) -1β and tumor necrosis factor (TNF) -α to establish a signaling pathway-activated and HaCa T cell model with high expression of HBD-2. , Vitamin A acid and physiological saline. The effect of Qingre Liangxue Chinese medicine on HBD-2 expression in the cell model was determined by qPCR and ELISA. Western blotting was used to detect the effect of Qingre Liangxue Chinese herbs on HBD-2 and mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling in the cell model. Results: (1) The HBD-2mRNA expression in HaCa T cell model was significantly decreased by Chinese medicine serum high dose group and Victoria A acid serum group with the inhibition rates of 66.8% and 73.5%, respectively. Compared with the model group, the difference (P <0.01). There was no significant difference between the high-dose Chinese medicine serum group and the Tretinoin group (P> 0.05). (2) Both the high-dose Chinese serum group and the Tretinoin group The inhibitory rates of HBD-2 in cell culture supernatant were 35.6% and 61.6%, respectively. (3) After treated with 100 mg / L IL-1β and TNF-α for 18 h Activating JNK 2 MAPK signal pathway in HaCa T cells; Qingre Liangxue serum can significantly reduce the relative content of phosphorylated c-Jun N-terminal kinase (p-JNK) 2 protein and inhibit JNK2 MAPK Activation of signaling pathways. Conclusion: Qingre Liangxue Recipe can significantly reduce HBD-2-overexpressing HaCa T cell model and HBD-2 expression in the culture supernatant. Qingre Cooling Herbs can inhibit JNK2 MAPK signaling pathway and block the activation of HBD-2 by IL-1β and TNF-α.