青石止痒软膏对TNF-α/IFN-γ诱导角质形成细胞炎症模型的影响及机制  

Effect and mechanism of Qingshi Zhiyang Ointment on TNF-α/IFN-γ induced Keratinocyte model

作　　者：赵欣楠 蔡玲玲[2] 任雪雯 邓宇童 姜晓媛[3] 李雪[1] 胡博 杭小涵 于心荟 李元文[2] ZHAO Xin-nan;CAI Ling-ling;REN Xue-wen;DENG Yu-tong;JIANG Xiao-yuan;LI Xue;HU Bo;HANG Xiao-han;YU Xin-hui;LI Yuan-wen(Beijing University of Chinese Medicine,Beijing 100029,China;Dermatology and Venereal Disease Department,Dongfang Hospital,Bejing University of Chinese Medicine Beijing 100078;Dermatology Department,Fangshan Hospital,Beijing University of Chinese Medicine,Beijing 102400)

机构地区：[1]北京中医药大学,北京100029 [2]北京中医药大学东方医院皮肤性病科,北京100078 [3]北京中医药大学房山医院皮肤科,北京102400

出　　处：《北京中医药》2022年第11期1239-1244,共6页Beijing Journal of Traditional Chinese Medicine

基　　金：北京市自然科学基金面上项目(7192116);北京中医药大学李元文教学名师工作坊(MSGZF-201906);中医药继续教育导航工程-继教专委会建设及精品课程制作。

摘　　要：目的 探讨青石止痒软膏对肿瘤坏死因子α (TNF-α)/γ干扰素(IFN-γ)诱导的人永生化角质形成细胞(HaCaT)和人表皮角质形成细胞炎症模型的影响及机制。方法 将HaCaT细胞和人表皮角质形成细胞分别分为空白组、模型组及实验组。空白组不予任何干预措施;模型组用TNF-α (10 ng/mL)+IFN-γ (10 ng/mL)刺激6 h诱导炎症模型;实验组在造模的基础上分别用0.000 1、0.001、0.01、0.1 g/mL的青石止痒软膏含药培养基培养,并根据以上浓度分为4个亚组。以细胞计数试剂盒-8法测定青石止痒软膏对细胞活力的影响,以实时反转录聚合酶链反应法定量microRNA-145(miRNA-145),免疫印迹法检测信号素3A (Sema3A)蛋白表达,酶联免疫吸附测定法测定细胞培养上清中白细胞介素31(IL-31)、神经生长因子(NGF)、神经营养因子4 (NT-4)水平。结果 不同浓度青石止痒软膏对细胞增殖无明显抑制。HaCaT细胞:与空白组比较,模型组miRNA-145表达低,Sema3A蛋白表达高,细胞培养上清中IL-31、NGF、NT-4水平高(P<0.05)。与模型组比较,0.001 g/mL组、0.01 g/mL组、0.1 g/mL组miRNA-145表达均升高(P<0.05);Sema3A蛋白表达均降低(P<0.05);0.000 1 g/mL组、0.001 g/mL组、0.01 g/mL组、0.1 g/mL组细胞培养上清中IL-31、NGF、NT-4水平均逐渐降低,且后3组差异有统计学意义(P<0.05)。人表皮角质形成细胞:与空白组比较,模型组miRNA-145表达低,Sema3A蛋白表达高,细胞培养上清中IL-31、NGF、NT-4水平高(P<0.05);与模型组比较,0.01 g/mL组、0.1 g/mL组miRNA-145表达高(P<0.05);0.001 g/mL组、0.01g/mL和0.1 g/mL组Sema3A蛋白表达低(P<0.05);0.01 g/mL组、0.1 g/mL组细胞培养上清中IL-31、NGF、NT-4水平低(P<0.05),0.000 1g/mL组、0.001 g/mL组细胞培养上清中IL-31、NGF水平低(P<0.05)。结论 青石止痒软膏可以明显改善HaCaT细胞和人表皮角质形成细胞炎症,其机制可能是通过调控Sema3A/NGF及相关分子水平。Objective To explore the effect and mechanism of Qingshi Zhiyang Ointment on inflammation model of human immortalized keratinocytes(HaCaT) and epidermal keratinocyte induced by tumor necrosis factor-α(TNF-α)/interferon-γ(IFN-γ). Methods HaCaT cells and human epidermal keratinocytes were divided into blank group, model group and experimental group respectively. The blank group received no intervention measures. The model group was stimulated with TNF-α(10 ng/mL)+IFN-γ(10 ng/mL) for 6 h to induce inflammation model. On the basis of modeling, the experimental group was cultured with 0. 000 1, 0. 001, 0. 01 and 0. 1 g/mL of Qingshi Zhiyang ointment, and divided into four subgroups according to the above concentrations. The effect of Qingshi Zhiyang ointment on cell viability was determined by cell counting kit-8 method. Quantify microRNA-145(miRNA-145) was detected by real-time reverse transcriptase polymerase chain reaction(real-time RT-PCR)method,Western Blot(WB) method to detect the expression of Sema3A protein,enzyme linked immunosorbent assay(ELISA)method to determine the expression level of interleukin-31(IL-31), NGF, nerve growth factor 4(NT-4). Results HaCaT cells: Qingshi Zhiyang Ointment with different concentrations has no obvious inhibition on cell proliferation. Compared with the blank group, the levels of miRNA-145 was decreased and Sema3A protein increased, and the levels of IL-31, NGF, and NT-4increased(P<0. 05). Compared with the model group, the expression of miRNA-145 in 0. 001 g/mL group, 0. 01 g/mL group and 0. 1 g/mL group all increased(P<0. 05). Sema3A protein expression decreased(P<0. 05). The levels of IL-31, NGF and NT-4 in cell culture supernatants of 0. 001 g/mL, 0. 001 g/ml, 0. 01 g/mL and 0. 1 g/mL groups decreased gradually, and the differences among the latter three groups were statistically significant(P<0. 05). Human epidermal keratinocytes : compared with the blank group, the expression of miRNA-145 in the model group was lowered, the expression of Sema3A protein was increased

关 键 词：炎症性皮肤病 青石止痒软膏 角质形成细胞 肿瘤坏死因子α Γ干扰素 信号素3A 神经生长因子 

分 类 号：R285[医药卫生—中药学]