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

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黑色素含量升高[(3060.21±431.62)%比100.00%,P<0.05],皮肤组织结构紊乱,黑色素沉积明显增多;与模型组比较,甘草组、当归组、赤芍组豚鼠背部皮肤黑色素含量降低[(1820.34±385.08)%、(1181.43±347.24)%、(2229.87±554.25)%比(3060.21±431.62)%,P<0.05],皮肤组织结构较为完整,黑色素沉积减少。Western blot、免疫组化检测结果显示,与对照组比较,模型组MITF表达水平升高[(1.47±0.18)比(0.41±0.07),(2.71±0.17)比(1.00±0.05);P<0.05],p-PI3K、p-AKT、p-GSK3β表达水平降低[p-PI3K:(0.15±0.03)比(0.59±0.06),(0.64±0.06)比(1.00±0.05);p-AKT:(0.25±0.04)比(0.82±0.06),(0.73±0.09)比(1.00±0.05);p-GSK3β:(0.23±0.05)比(0.79±0.07),(0.56±0.09)比(1.00±0.05);P<0.05]。与模型组比较,甘草组、当归组、赤芍组MITF表达水平降低[(0.85±0.07)、(0.67±0.05)、(0.92±0.06)比(1.47±0.18),(1.79±0.15)、(1.51±0.13)、(1.94±0.12)比(2.71±0.17);P<0.05],p-PI3K、p-AKT、p-GSK3β表达水平升高[p-PI3K:(1.18±0.06)、(1.30±0.09)、(1.13±0.08)比(0.59±0.06),(1.57±0.12)、(1.70±0.12)、(1.50±0.08)比(1.00±0.05);p-AKT:(1.12±0.06)、(1.15±0.04)、(1.08±0.06)比(0.82±0.06),(1.68±0.14)、(1.79±0.10)、(1.49±0.12)比(1.00±0.05);p-GSK3β:(1.04±0.12)、(1.09±0.12)、(1.00±0.11)比(0.79±0.07),(1.81±0.14)、(1.96±0.09)、(1.67±0.15)比(1.00±0.05);P<0.05]。结论甘草、当归、赤芍可能通过激活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。

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 on these data,1000μg/mL dose of each agent was selected for subsequent experiments.Western blot results showed that compared with control,levels of MITF protein were also reduced after treatment with 1000μg/mL of each agent(0.28±0.04 vs.0.99±0.08,0.28±0.04 vs.1.14±0.08,and 0.43±0.09 vs.0.84±0.13,respectively;P<0.05),whereas levels of p-PI3K,p-AKT,and p-GSK3βproteins were increased(licorice,1.08±0.07 vs.0.61±0.07,0.79±0.07 vs.0.33±0.08,and 0.78±0.06 vs.0.41±0.06;Angelica sinensis,1.38±0.07 vs.1.01±0.07,1.27±0.05 vs.0.83±0.08,and 0.78±0.06 vs.0.39±0.06;and Radix paeoniae rubra,0.83±0.12 vs.0.41±0.07,0.79±0.07 vs.0.29±0.08,and 0.82±0.04 vs.0.31±0.06,respectively;P<0.05).Moreover,compared with that of controls,the skin melanin content in model animals was also increased(3060.21±431.62 vs.100%;P<0.05),while the skin tissue structure was aberrant and the melanin deposition was significantly increased in model animals.In contrast,compared with the model group,the skin melanin content after treatment with licorice,Angelica sinensis,and Radix paeoniae rubra was all reduced(1820.34±385.08,1181.43±347.24,and 2229.87±554.25 vs.3060.21±431.62%,respectively;P<0.05),while their skin structures were improved and the melanin deposition was reduced after each of the agent treatment.In addition,Western blot and immunohistochemistry confirmed our in vitro data,i.e.,compared with the control animals,level of MITF protein in the model group was increased(1.47±0.18 vs.0.41±0.07 and 2.71±0.17 vs.1.00±0.05,respectively;P<0.05)but levels of p-PI3K(0.15±0.03 vs.0.59±0.06 and 0.64±0.06 vs.1.00±0.05,respectively;P<0.05),p-AKT(0.25±0.04 vs.0.82±0.06 and 0.73±0.09 vs.1.00±0.05,respectively;P<0.05),and p-GSK3β(0.23±0.05 vs.0.79±0.07 and 0.56±0.09 vs.1.00±0.05,respectively;P<0.05)were all reduced in model animals.In contrast,compared with the model group,level of MITF protein was decreased after these three treatments(WB,0.85±0.07,0.67±0.05,and 0.92±0.06 vs.1.47±0.18;IHC,1.79±0.15,1.51±0.13,and 1.94±0.12 vs.2.71±0.17,respectively;P<0.05),whereas levels of p-PI3K(WB,1.18±0.06,1.30±0.07,and 1.13±0.08 vs.0.59±0.06;IHC,1.57±0.12,1.70±0.12,and 1.50±0.08 vs.1.00±0.05 respectively;P<0.05),p-AKT(WB,1.12±0.06,1.15±0.04,and 1.08±0.06 vs.0.82±0.06;IHC,1.68±0.14,1.79±0.10,and 1.49±0.12 vs.1.00±0.05,respectively;P<0.05),and p-GSK3β(WB,1.04±0.12,1.09±0.12,and 1.00±0.11 vs.0.79±0.07;IHC,1.81±0.14,1.96±0.09,and 1.67±0.15 vs.1.00±0.05,respectively;P<0.05)were all increased after licorice,Angelica sinensis,and Radix paeoniae rubra treatment.Conclusion Treatment of animal model of melasma with licorice,Angelica sinensis,or Radix paeoniae rubra was able to reduce expression of MITF protein and melanin deposition,but relieve chloasma symptoms in skin biopsies by activation of the PI3K/AKT signal pathway.