| 倪晨宁.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研究[J].浙江中西医结合杂志,2023,33(9): |
| 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研究 |
| Based on MITF and PI3K/AKT signal pathway to explore the mechanism of single traditional Chinese medicine on melasma |
| 投稿时间：2023-04-14 修订日期：2023-06-19 |
| DOI： |
| 中文关键词: 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 |
| 英文关键词:Melasma Microphthalmia-associated transcription factor Phosphatidylinositol 3 kinase Protein kinase B Licorice Angelica sinensis Radix paeoniae rubra |
| 基金项目:浙江省中医药科技计划项目（项目编号：2023ZL444 ） |
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| 中文摘要: |
| 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 μg/ml、50 μg/ml、100 μg/ml、500 μg/ml、1000 μg/ml、1000 μg/ml+LY294002组,培养基中加入相应浓度的中药药液与25 nM 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,WB检测MITF、PI3K/Akt信号通路相关分子水平。30只豚鼠随机分为对照组、模型组、甘草组、当归组、赤芍组,每组6只。中波紫外线(UVB)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,WB、免疫组化检测MITF、PI3K/Akt信号通路相关分子水平。结果：与各组0 μg/ml组相比,甘草、当归、赤芍500、1000 μg/ml组细胞活力降低(P＜0.05)；甘草500、1000 μg/ml组、当归100、500、1000 μg/ml组、赤芍1000 μg/ml组酪氨酸酶活性降低(P＜0.05)；甘草500、1000 μg/ml组、当归50、100、500、1000 μg/ml组、赤芍100、500、1000 μg/ml组黑色素含量降低(P＜0.05)。WB检测结果显示,与各组0 μg/ml组相比,1000 μg/ml组MITF表达水平降低(P＜0.05),p-PI3K、p-AKT、p-GSK3β表达水平升高(P＜0.05)。与对照组相比,模型组、甘草组、当归组、赤芍组背部皮肤黑色素含量升高(P＜0.05),皮肤组织结构紊乱,黑色素沉积明显增多；与模型组相比,甘草组、当归组、赤芍组背部皮肤黑色素含量降低(P＜0.05),皮肤组织结构较为完整,黑色素沉积减少。WB、免疫组化检测结果显示,与对照组相比,模型组MITF表达水平升高(P＜0.05),p-PI3K、p-AKT、p-GSK3β表达水平降低(P＜0.05),甘草组、当归组、赤芍组MITF、p-PI3K、p-AKT、p-GSK3β表达水平升高(P＜0.05)；与模型组相比,甘草组、当归组、赤芍组MITF表达水平降低(P＜0.05)。结论：甘草、当归、赤芍可通过激活PI3K/AKT信号通路,抑制MITF表达,减少皮肤黑色素沉积,进而缓解黄褐斑症状。 |
| 英文摘要: |
| Objective: Based on microphthalmia-associated transcription factor (MITF) and phosphatidylinositol 3 kinase / protein kinase B (PI3K/AKT) signal pathway, to explore the mechanism of action of many kinds of traditional Chinese medicine on melasma. Methods: Preparation of licorice, Angelica sinensis and Radix Paeoniae Radix by water decocting. Melanocytes were divided into 0 μg/ml, 50 μg/ml, 100 μg/ml, 500 μg/ml, 1000 μg/ml, 1000 μg/ml+LY294002 groups. The corresponding concentration of traditional Chinese medicine solution and 25 nM LY294002 were added to the culture medium. MTT assay was used to detect cytotoxicity of different doses of drugs. Tyrosinase activity and melanin content were determined. The levels of related molecules in MITF and PI3K/Akt signaling pathway were detected by WB. 30 guinea pigs were randomly divided into control group, model group, licorice group, Angelica sinensis group and Radix Paeoniae Radix group, with 6 in each group. The model of melasma was established by ultraviolet B (UVB) irradiation. Licorice group, Angelica sinensis group and Radix Paeoniae Radix group received 0.3 mg/kg licorice, Angelica sinensis and Radix Paeoniae Radix liquid, respectively, while control group and model group received equal volume of normal saline for 4 weeks. The skin on the back of guinea pig was cut and the content of melanin was measured. HE and Fontana-Masson staining were used to observe the skin morphology. The levels of MITF and PI3K/Akt signaling pathways were detected by WB and immunohistochemistry. Results: Compared with 0 μg/ml groups, the cell viability of licorice, Angelica sinensis and Radix Paeoniae Radix in 500 and 1000 μg/ml groups decreased (P＜0.05). Tyrosinase activity of licorice 500, 1000 μg/ml groups, Angelica sinensis 100, 500, 1000 μg/ml groups and Radix Paeoniae Radix 1000 μg/ml groups decreased (P＜0.05). The content of melanin in licorice 500, 1000 μg/ml groups, Angelica sinensis 50, 100, 500, 1000 μg/ml groups and Radix Paeoniae Radix 100, 500, 1000 μg/ml groups decreased (P＜0.05). WB detection results showed that compared with 0 μg/ml group, the expression levels of MITF in 1000 μg/ml group were decreased (P＜0.05), while the expression levels of p-PI3K, p-AKT and p-GSK3β were increased (P＜0.05). Compared with the control group, the melanin content in the back skin of model group, licorice group, Angelica sinensis group and Radix Paeoniae Radix group was increased (P＜0.05), the skin tissue structure was disordered, and the melanin deposition was significantly increased. Compared with model group, the content of melanin in back skin of licorice group, Angelica sinensis group and Radix Paeoniae Radix group was decreased (P＜0.05), the skin structure was more complete, and the deposition of melanin was reduced. WB and immunohistochemical test results showed that compared with the control group, the expression level of MITF in the model group was increased (P＜0.05), and the expression levels of p-PI3K, p-AKT and p-GSK3β were decreased (P＜0.05), while the expression levels of MITF, p-PI3K, p-AKT and p-GSK3β in the licorice group, Angelica sinensis group and Radix Paeoniae Radix group were increased (P＜0.05). Compared with model group, the expression level of MITF in licorice group, Angelica sinensis group and Radix Paeoniae Radix group decreased (P＜0.05). Conclusion: Licorice, Angelica sinensis and Radix Paeoniae Rubra can inhibit the expression of MITF, reduce melanin deposition and relieve chloasma symptoms by activating PI3K/AKT signal pathway. |
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