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目的:研究中药清热活血解毒复方(QHJD)对肿瘤坏死因子-α(TNF-α)诱导的人永生化角质形成细胞株(Ha Cat)增殖和凋亡的影响,为治疗银屑病的可能作用机制提供依据。方法:体外培养Ha Cat细胞,将高、中、低(30,25,20 g·L~(-1))质量浓度的QHJD水提物分别作用于TNF-α诱导的Ha Cat细胞24 h,采用倒置显微镜观察QHJD水提物处理后的Ha Cat细胞形态学改变;采用CCK-8技术检测QHJD对TNF-α诱导的Ha Cat细胞增殖的影响;采用流式细胞术检测QHJD对TNF-α诱导的Ha Cat细胞凋亡的影响;采用Western blot技术检测QHJD对TNF-α诱导的Ha Cat细胞B细胞淋巴瘤/白血病-xl(Bclxl),Bcl-2相关X蛋白(Bax)蛋白表达的影响;采用Real-Time PCR技术检测清热活血解毒复方对TNF-α诱导的Ha Cat细胞Bcl-xl,Bax mRNA表达的影响。结果:与空白组比较,QHJD各组细胞镜下单位面积的细胞均呈现数量减少,细胞体积缩小,细胞间连接变少或是消失,细胞间隙变大;QHJD各组均可抑制Ha Cat细胞的增殖,差异有统计学意义(P<0.05),其中QHJD中、高剂量组极显著(P<0.01);QHJD中、高剂量组可以促进Ha Cat细胞的凋亡,差异有统计学意义(P<0.05),其中高剂量组促进Ha Cat细胞凋亡更明显(P<0.01);QHJD各组Bax蛋白表达均有上升,差异有统计学意义(P<0.05);QHJD各组Bax mRNA表达均有上升,差异有统计学意义(P<0.05)。结论:QHJD可能通过上调Bax蛋白和mRNA的表达,来诱导角质形成细胞的凋亡,进而抑制TNF-α诱导的Ha Cat细胞的增殖,以发挥治疗银屑病的作用。
Objective: To investigate the effect of QHJD on the proliferation and apoptosis of human keratinocyte-derived keratinocytes (HaCAT) induced by tumor necrosis factor-α (TNF-α) and its potential role in the treatment of psoriasis Mechanism to provide the basis. Methods: Ha Cat cells were cultured in vitro. High, medium and low concentrations of QHJD aqueous extracts (30,25 and 20 g · L -1) were respectively treated with TNF-α-induced Ha Cat cells for 24 h. The morphological changes of Ha Cat cells treated with water extract of QHJD were observed by inverted microscope. The effect of QHJD on the proliferation of Ha Cat cells induced by TNF-α was detected by CCK-8. The induction of TNF-α by QHJD was detected by flow cytometry The effects of QHJD on the expression of Bcl-2 and Bcl-2 protein in Ha Cat cells induced by TNF-α were detected by Western blot. Real-Time PCR was used to detect the effect of compound recipe of clearing heat and promoting blood circulation to detoxify on the expression of Bcl-xl, Bax mRNA in HaCa cells induced by TNF-α. Results: Compared with the blank group, the number of cells per unit area in QHJD group decreased, the cell volume decreased, the number of intercellular junctions decreased or disappeared, and the intercellular space became larger. In each group of QHJD, the number of Ha Cat cells (P <0.05). The QHJD medium and high dose groups were significantly (P <0.01). The medium and high dose QHJD groups could promote the apoptosis of Ha Cat cells (P <0.05), the difference was statistically significant (P < <0.05). The apoptosis of Ha Cat cells in high dose group was more significant (P <0.01), while the expression of Bax protein in QHJD group was higher (P <0.05). The expression of Bax mRNA in QHJD group There was a significant difference between the two groups (P <0.05). Conclusion: QHJD may induce the apoptosis of keratinocytes by up-regulating the expression of Bax protein and mRNA, and then inhibit the proliferation of Ha-Cat cells induced by TNF-α, so as to treat psoriasis.