黑布药膏对慢性皮肤溃疡伴有异常纤维化的治疗作用及机制  

Therapeutic efficacy and mechanisms of HeiBu Ointment in the treatment of chronic skin ulcers with abnormal fibrosis

作　　者：姚文涛 彭冰[1] 马慧可 王正春 曲剑华[1] 李萍[1] 何秀娟[1] YAO Wentao;PENG Bing;MA Huike;WANG Zhengchun;QU Jianhua;LI Ping;HE Xiujuan(Beijing Institute of Chinese Medicine,Beijing Hospital of Traditional Chinese Medicine of Capital Medical University,Bejing100010,China)

机构地区：[1]首都医科大学附属北京中医医院北京市中医药研究所,北京100010

出　　处：《北京中医药大学学报》2023年第12期1684-1693,共10页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.82274524);北京市自然科学基金项目(No.7212164)。

摘　　要：目的 研究黑布药膏对转化生长因子-β1(TGF-β1)诱导的人永生化表皮细胞(HaCaT)上皮间质转化(EMT)的影响,探讨黑布药膏治疗慢性皮肤溃疡伴有异常纤维化的作用机制。方法 使用TGF-β1诱导人HaCa细胞EMT模型。将HaCaT细胞分为空白对照组,模型组,以及黑布药膏高、中、低剂量组。各组均加入DMEM高糖培养基(4.00 mmol/L左旋谷酰胺、4.5 g/L葡萄糖)外,模型组加入TGF-β1(0.01 mg/L),黑布药膏高、中、低剂量组加入TGF-β1(0.01 mg/L)与不同剂量黑布药膏提取物(0.5、0.25、0.125 mg/L)。使用CCK-8法检测黑布药膏对HaCaT细胞活性的影响;划痕实验检测黑布药膏对HaCaT细胞迁移能力的影响;免疫荧光法检测EMT上皮标志物上皮钙黏素(E-cadherin),间质标志物神经钙黏素(N-cadherin)和波形蛋白(vimentin)的表达;蛋白质印迹法检测Sma和Mad相关蛋白(Smad)和胞外信号调节激酶(ERK)信号通路相关蛋白的表达。结果 黑布药膏提取物浓度在0.5 mg/L以下对HaCaT细胞活性无影响。与模型组相比,黑布药膏各剂量组可抑制TGF-β1刺激后的HaCaT细胞迁移,促进E-cadherin表达,抑制N-cadherin、vimentin表达,抑制TGF-β1诱导的HaCaT细胞Smad2、Smad3和ERK1/2蛋白的磷酸化(P<0.05)。结论 黑布药膏可能通过抑制Smad和ERK信号通路,抑制表皮细胞向间质细胞过度转化,从而促进创面周围异常纤维化的慢性皮肤溃疡愈合。Objective We aim to investigate the impact of HeiBu Ointment on transforming growth factor-β1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in human immortalized epidermal cells(HaCaT)and elucidate the mechanism underlying HeiBu Ointment's efficacy in treating chronic skin ulcers with abnormal fibrosis.Methods We employed a TGF-β1-induced EMT model using HaCaT.HaCaT cells were categorized into five groups:the blank control group,the model group,and the HeiBu Ointment high-,medium-,and low-dose groups.All the groups were cultured in DMEM high-glucose medium(4.00 mmol/L L-glutamine,4.5 g/L glucose).The model group was treated with TGF-β1(0.01 mg/L),while the HeiBu Ointment high-,medium-,and low-dose groups received TGF-β1(0.01 mg/L)in combination with varying doses of HeiBu Ointment extract(0.5 mg/L,0.25 mg/L,and 0.125 mg/L).The CCK-8 method was used to detect the effect of HeiBu Ointment on HaCaT cell activity;the effect of HeiBu Ointment on the migration ability of HaCaT cells was determined by scratch test;the effects of HeiBu Ointment on the expression of the epithelial marker protein E-cadherin and the mesenchymal marker proteins N-cadherin and vimentin in HaCaT cells were observed by immuno fluorescence method;the expression of Smad and ERK signaling pathway-related proteins was detected by Western blotting.Results HeiBu Ointment below 0.5 mg/L had no effect on HaCaT cell activity.Compared with the model group,HeiBu Ointment significantly inhibited the migration of HaCaT cells after TGF-β1 stimulation(P<0.05),promoted the expression of the epithelial marker protein E-cadherin,inhibited the expression of the mesenchymal marker proteins N-cadherin and vimentin,and inhibited TGF-β1-induced phosphorylation of Smad2,Smad3,and ERK1/2 in HaCaT cells(P<0.05).Conclusion HeiBu Ointment may promote healing of chronic skin ulcers with abnormal fibrosis by inhibiting excessive trausition of epidermal cells to mesenchymal cells through inhibition of the Smad and ERK signaling pathways.

关 键 词：黑布药膏 人永生化表皮细胞 上皮间质转化 慢性皮肤溃疡 皮肤纤维化 

分 类 号：R285.5[医药卫生—中药学]