基于MITF与PI3K/AKT信号通路探讨多种单味中药对黄褐斑的作用机制  

Use of MITF and PI3K/AKT signaling pathways to explore potential mechanisms of a variety of single traditional Chinese medicine on melasma

作　　者：倪晨宁 王小勇 石海兰 沈肖奋 NI Chen-ning;WANG Xiao-yong;SHI Hai-lan;SHEN Xiao-fen(Department of Dermatology,The Second Affiliated Hospital of Zhejiang Chinese Medicine University,Hangzhou,Zhejiang 310005,China)

机构地区：[1]浙江中医药大学附属第二医院皮肤科,杭州310005

出　　处：《浙江中西医结合杂志》2023年第9期788-796,共9页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省中医药科技计划项目(No.2023ZL444)。

摘　　要：目的基于小眼畸形相关转录因子(MITF)与磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路探究多种单味中药对黄褐斑的作用机制。方法水煎法制备甘草、当归、赤芍药液。黑素细胞分为0、50、100、500、1000μg/mL及1000μg/mL+LY294002组,培养基中加入相应浓度的中药药液与25 nmol LY294002,MTT法检测不同剂量药物细胞毒性,测定酪氨酸酶活性与黑色素含量,Western blot检测MITF、PI3K/AKT信号通路相关分子水平。30只5周龄豚鼠按照随机数字表法分为对照组、模型组、甘草组、当归组、赤芍组,每组6只。中波紫外线照射建立黄褐斑模型,甘草组、当归组、赤芍组分别接受0.3 mg/kg甘草、当归、赤芍药液灌胃,对照组、模型组接受等体积生理盐水灌胃,持续4周。切取豚鼠背部造模处皮肤,测定黑色素含量,HE、Fontana-Masson染色观察皮肤组织形态,Western blot、免疫组化检测MITF、PI3K/AKT信号通路相关分子水平。结果与各0μg/mL组比较,甘草、当归、赤芍1000μg/mL组细胞活力、酪氨酸酶活性、黑色素含量均明显降低[甘草:(66.82±9.65)%比100.00%,(65.07±5.13)%比100.00%,(55.56±3.17)%比100.00%;当归:(56.62±15.99)%比100.00%,(52.69±5.52)%比100.00%,(54.69±4.05)%比100.00%;赤芍:(77.51±7.33)%比100.00%,(67.60±10.66)%比100.00%,(61.80±12.89)%比100.00%;P<0.05],因此后续各中药均选用1000μg/mL剂量进行实验。Western blot检测结果显示,与各0μg/mL组比较,甘草、当归、赤芍1000μg/mL组MITF表达水平降低[(0.28±0.04)比(0.99±0.08),(0.28±0.04)比(1.14±0.08),(0.43±0.09)比(0.84±0.13);P<0.05],p-PI3K、p-AKT、p-GSK3β表达水平升高[甘草:(1.08±0.07)比(0.61±0.07),(0.79±0.07)比(0.33±0.08),(0.78±0.06)比(0.41±0.06);当归:(1.38±0.07)比(1.01±0.07),(1.27±0.05)比(0.83±0.08),(0.78±0.06)比(0.39±0.06);赤芍:(0.83±0.12)比(0.41±0.07),(0.79±0.07)比(0.29±0.08),(0.82±0.04)比(0.31±0.06);P<0.05]。与对照组比较,模型组豚鼠背部皮肤�Objective To explore the molecular mechanism of a variety of single traditional Chinese medicine on melasma based on microphthalmia-associated transcription factor(MITF)and phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)signal pathway.Methods Licorice,Angelica sinensis,and Radix paeoniae rubra were prepared as the water decocting.Melanocytes were divided into 0,50,100,500,1000μg/mL and 1000μg/mL+LY294002 groups.The corresponding concentration of traditional Chinese medicine solution and 25 nmol LY294002 were added to the culture medium.MTT assay was used to detect cytotoxicity of different doses of drugs.After that,tyrosinase activity and melanin content were also assayed accordingly,while levels of the MITF and PI3K/AKT signaling pathway-related proteins were analyzed using Western blot.In addition,30 guinea pigs of 5 weeks old were randomly divided into control,model,licorice,Angelica sinensis,and Radix paeoniae rubra groups(n=6 per group).The melasma model group of animals was established by using the ultraviolet B irradiation and then treated with licorice,Angelica sinensis,or Radix paeoniae rubra(0.3 mg/kg individually),while the control and model groups of animals received an equal volume of normal saline for 4 weeks.The skin on the back of guinea pig was biopsied for measurement of the melanin content and H&E and Fontana-Masson staining for observation of skin morphology.Levels of MITF and PI3K/AKT signaling pathway-related proteins were detected by using Western blot and immunohistochemistry in skin tissues.Results Compared with control,the cell viability,tyrosinase activity,and melanin content were significantly decreased after treatment with 1000μg/mL of licorice(66.82±9.65 vs.100.00%,65.07±5.13 vs.100.00%,and 55.56±3.17 vs.100.00%,respectively;P<0.05),Angelica sinensis(56.62±15.99 vs.100.00%,52.69±5.52 vs.100.00%,and 54.69±4.05 vs.100.00%,respectively;P<0.05),and Radix paeoniae rubra(77.51±7.33 vs.100.00%,67.60±10.66 vs.100.00%,and 61.80±12.89 vs.100.00%,respectively;P<0.05).Based

关 键 词：豚鼠 黄褐斑 小眼畸形相关转录因子 磷脂酰肌醇3激酶 蛋白激酶B 甘草 当归 赤芍 

分 类 号：R285.5[医药卫生—中药学]