补益肝肾法对小鼠皮肤α-MSH表达及黑素合成影响研究
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摘要
目的：以圣愈汤为基础,加菟丝子、泽泻而成的祛斑汤具有补益肝肾、益气养血的功效,临床用于治疗黄褐斑,疗效显著。
观察不同剂量祛斑汤对雌性C57BL/6J小鼠皮肤中α-黑素细胞刺激素(α-MSH)表达和黑素分布的影响,了解该方剂是否通过降低α-MSH表达来抑制黑素合成；观察不同剂量祛斑汤对雌性C57BL/6J小鼠皮肤中两种黑素即优黑素和褐黑素含量的影响,了解该方剂在抑制黑素合成的过程中对两种黑素作用的差异。为深入探索该方剂治疗黄褐斑机制提供实验依据。
方法：选择对体外培养的A375人黑素瘤细胞具有抑制细胞增殖、降低酪氨酸酶活性的方剂祛斑汤,配成低、中、高不同浓度。以雌性C57BL/6J小鼠为实验动物,随机分成5组,分别为祛斑汤低、中、高剂量组、阳性对照组和空白对照组。低、中、高剂量组每只小鼠按0.2ml/10g于每日灌服相应浓度中药1次,阳性对照和空白对照组为等量维生素C液和蒸馏水。30日后皮肤取材。采用免疫组织化学染色方法观察皮肤α-MSH表达和黑色素分布,并用x2检验和单因素方差分析进行统计学分析；采用酶联免疫吸附试验测量皮肤优黑素和褐黑素含量,并用单因素方差分析进行统计学分析。
结果：
1.祛斑汤对C57BL/6J小鼠皮肤α-MSH表达影响：免疫组织化学染色结果显示：光学显微镜下α-MSH表达定位于小鼠表皮角质形成细胞和毛囊黑素细胞胞浆或细胞外基质,为黄色或棕黄色或深棕色颗粒。空白对照组α-MSH阳性表达率为70%,祛斑汤高、中、低剂量组、阳性对照组α-MSH阳性表达率分别为20%、50%、60%、40%。祛斑汤高剂量组与空白对照组β-MSH表达比较,差别有统计学意义(P<0.01)。
2.祛斑汤对C57BL/6J小鼠皮肤黑素分布影响：免疫组织化学染色结果显示：光学显微镜下各组小鼠毛囊基底细胞团中均可见黑素颗粒。黑素颗粒阳性反应的黑素细胞为阳性细胞。空白对照组阳性细胞平均光密度0.35,高于其他组。祛斑汤高、中剂量组阳性细胞平均光密度分别为0.26、0.28,与空白对照组比较,差别有统计学意义(P<0.01)。
3.祛斑汤对C57BL/6J小鼠皮肤优黑素和褐黑素含量影响：酶联免疫吸附试验结果显示：各组优黑素含量比较,祛斑汤各剂量组与阳性对照组优黑素含量均低于空白对照组。祛斑汤高剂量组与空白对照组间差别有统计学意义(P<0.01)。各组褐黑素含量比较,祛斑汤各剂量组与阳性对照组褐黑素含量均高于空白对照组。祛斑汤高、中、低剂量组与空白对照组比较,差别有统计学意义(P<0.01)。
结论：
1.祛斑汤能降低雌性C57BL/6J小鼠皮肤中α-MSH表达、抑制黑素合成,α-MSH是影响皮肤和毛发颜色的重要因素。
2.祛斑汤在抑制黑素合成过程中主要减少优黑素的生成,并促进优黑素向褐黑素转化。
Objective:Quban Decoction that adds cuscuta and alisma orientate to Shengyu Decoction that nourishes liver and kidney and replenishes qi and blood is effective significantly on treatment of melasma.
Observe the effect of different doses Quban Decoction on expression of α-MSH and melanin distribution in the female C57BL/6J mouse skin in order to understand Whether the prescription inhibited melanin synthesis by reducing the expression of α-MSH; Observe the effect of different doses Quban Decoction on the two kinds of melanins such as eumelanin and Pheomelanin in the female C57BL/6J mouse skin in order to understand Whether the prescription influenced the two kinds of melanins differently in the inhibition of melanin synthesis process. Provide the experimental basis for exploring deeply the mechanism of treating melasma by the prescription
Methods:Select Quban Decoction that inhibited proliferation of the A375human melanoma cells in vitro and reduced the activity of tyrosinase and bubbed Quban Decoction into low concentration, middle concentration and high concentration. Female C57BL/6J mice as experimental animals, Were randomly divided into five groups:low dose group, middle dose group, high dose group, the positive control group and blank control group. Each animal was fed with the corresponding concentration of traditional Chinese medicine0.2ml/10g daily in low dose group, middle dose group, high dose group. The animals were fed with the same amount of vitamin C and distilled water in Positive control and blank control group. Take the skin from mice30days later. Use immunohistochemical techniques to observe the expression of a-MSH and melanin distribution and the result was analyzed using the x2test and One-way ANOVA statistically. Use the enzyme-linked immunosorbent assay to measure the content of eumelanin and Pheomelanin in the skin and the result was analyzed using One-way ANOVA statistically.
Results:
1. Effect of Quban Decoction on expression of a-MSH in C57BL/6J mouse skin:the results of immunohistochemical staining showed that: The expression of α-MSH was located in the cytoplasmic or extracellular matrix of epidermal keratinocytes and hair follicle melanocytes by light microscope, yellow or brown or dark brown particles. The positive expression rate of α-MSH in the blank control group is70%. The positive expression rates of high dose group, middle dose group,low dose group, the positive control group were20%,50%,60%,40%. The difference in a-MSH expression between the high dose group and the blank control group was significant statistically (P<0.01)
2. Effect of Quban Decoction on melanocyte distribution in C57BL/6J mouse skin:the results of immunohistochemical staining showed that: There are melanin granules in hair follicles basal cell mass in each group by the light microscope. The melanocytes that contained the melanin granules were named positive cells. The average optical density of positive cells of blank control group was0.35, that was higher than that of other groups. The average optical densities of positive cells of the high dose group and the middle dose group were0.26,0.28. The difference between the high dose group, the middle dose group and the blank control group was significant statistically (P<0.01)
3. Effect of Quban Decoction on content of the eumelanin and Pheomelanin in C57BL/6J mouse skin:ELISA results show that:the eumelanin content of all of the Quban Decoction groups and positive control group was lower than the blank control group. There was significant difference between the high dose group and the blank control group (P<0.01).The Pheomelanin content of all of the Quban Decoction groups and the positive control group was higher than the blank control group.There was significant difference between the high, middle, low dose groups and the blank control group (P<0.01)
Conclusion:
1. The Quban Decoction can reduce the expression of a-MSH in female C57BL/6J mouse skin and inhibit melanin synthesis. a-MSH is an important factor that affects the color of skin and hair.
2. The Quban Decoction can reduce the generation of the eumelanin chiefly in the inhibition of melanin synthesis process and promote the eumelanin to convert to the pheomelanin.
观察不同剂量祛斑汤对雌性C57BL/6J小鼠皮肤中α-黑素细胞刺激素(α-MSH)表达和黑素分布的影响,了解该方剂是否通过降低α-MSH表达来抑制黑素合成；观察不同剂量祛斑汤对雌性C57BL/6J小鼠皮肤中两种黑素即优黑素和褐黑素含量的影响,了解该方剂在抑制黑素合成的过程中对两种黑素作用的差异。为深入探索该方剂治疗黄褐斑机制提供实验依据。
方法：选择对体外培养的A375人黑素瘤细胞具有抑制细胞增殖、降低酪氨酸酶活性的方剂祛斑汤,配成低、中、高不同浓度。以雌性C57BL/6J小鼠为实验动物,随机分成5组,分别为祛斑汤低、中、高剂量组、阳性对照组和空白对照组。低、中、高剂量组每只小鼠按0.2ml/10g于每日灌服相应浓度中药1次,阳性对照和空白对照组为等量维生素C液和蒸馏水。30日后皮肤取材。采用免疫组织化学染色方法观察皮肤α-MSH表达和黑色素分布,并用x2检验和单因素方差分析进行统计学分析；采用酶联免疫吸附试验测量皮肤优黑素和褐黑素含量,并用单因素方差分析进行统计学分析。
结果：
1.祛斑汤对C57BL/6J小鼠皮肤α-MSH表达影响：免疫组织化学染色结果显示：光学显微镜下α-MSH表达定位于小鼠表皮角质形成细胞和毛囊黑素细胞胞浆或细胞外基质,为黄色或棕黄色或深棕色颗粒。空白对照组α-MSH阳性表达率为70%,祛斑汤高、中、低剂量组、阳性对照组α-MSH阳性表达率分别为20%、50%、60%、40%。祛斑汤高剂量组与空白对照组β-MSH表达比较,差别有统计学意义(P<0.01)。
2.祛斑汤对C57BL/6J小鼠皮肤黑素分布影响：免疫组织化学染色结果显示：光学显微镜下各组小鼠毛囊基底细胞团中均可见黑素颗粒。黑素颗粒阳性反应的黑素细胞为阳性细胞。空白对照组阳性细胞平均光密度0.35,高于其他组。祛斑汤高、中剂量组阳性细胞平均光密度分别为0.26、0.28,与空白对照组比较,差别有统计学意义(P<0.01)。
3.祛斑汤对C57BL/6J小鼠皮肤优黑素和褐黑素含量影响：酶联免疫吸附试验结果显示：各组优黑素含量比较,祛斑汤各剂量组与阳性对照组优黑素含量均低于空白对照组。祛斑汤高剂量组与空白对照组间差别有统计学意义(P<0.01)。各组褐黑素含量比较,祛斑汤各剂量组与阳性对照组褐黑素含量均高于空白对照组。祛斑汤高、中、低剂量组与空白对照组比较,差别有统计学意义(P<0.01)。
结论：
1.祛斑汤能降低雌性C57BL/6J小鼠皮肤中α-MSH表达、抑制黑素合成,α-MSH是影响皮肤和毛发颜色的重要因素。
2.祛斑汤在抑制黑素合成过程中主要减少优黑素的生成,并促进优黑素向褐黑素转化。
Objective:Quban Decoction that adds cuscuta and alisma orientate to Shengyu Decoction that nourishes liver and kidney and replenishes qi and blood is effective significantly on treatment of melasma.
Observe the effect of different doses Quban Decoction on expression of α-MSH and melanin distribution in the female C57BL/6J mouse skin in order to understand Whether the prescription inhibited melanin synthesis by reducing the expression of α-MSH; Observe the effect of different doses Quban Decoction on the two kinds of melanins such as eumelanin and Pheomelanin in the female C57BL/6J mouse skin in order to understand Whether the prescription influenced the two kinds of melanins differently in the inhibition of melanin synthesis process. Provide the experimental basis for exploring deeply the mechanism of treating melasma by the prescription
Methods:Select Quban Decoction that inhibited proliferation of the A375human melanoma cells in vitro and reduced the activity of tyrosinase and bubbed Quban Decoction into low concentration, middle concentration and high concentration. Female C57BL/6J mice as experimental animals, Were randomly divided into five groups:low dose group, middle dose group, high dose group, the positive control group and blank control group. Each animal was fed with the corresponding concentration of traditional Chinese medicine0.2ml/10g daily in low dose group, middle dose group, high dose group. The animals were fed with the same amount of vitamin C and distilled water in Positive control and blank control group. Take the skin from mice30days later. Use immunohistochemical techniques to observe the expression of a-MSH and melanin distribution and the result was analyzed using the x2test and One-way ANOVA statistically. Use the enzyme-linked immunosorbent assay to measure the content of eumelanin and Pheomelanin in the skin and the result was analyzed using One-way ANOVA statistically.
Results:
1. Effect of Quban Decoction on expression of a-MSH in C57BL/6J mouse skin:the results of immunohistochemical staining showed that: The expression of α-MSH was located in the cytoplasmic or extracellular matrix of epidermal keratinocytes and hair follicle melanocytes by light microscope, yellow or brown or dark brown particles. The positive expression rate of α-MSH in the blank control group is70%. The positive expression rates of high dose group, middle dose group,low dose group, the positive control group were20%,50%,60%,40%. The difference in a-MSH expression between the high dose group and the blank control group was significant statistically (P<0.01)
2. Effect of Quban Decoction on melanocyte distribution in C57BL/6J mouse skin:the results of immunohistochemical staining showed that: There are melanin granules in hair follicles basal cell mass in each group by the light microscope. The melanocytes that contained the melanin granules were named positive cells. The average optical density of positive cells of blank control group was0.35, that was higher than that of other groups. The average optical densities of positive cells of the high dose group and the middle dose group were0.26,0.28. The difference between the high dose group, the middle dose group and the blank control group was significant statistically (P<0.01)
3. Effect of Quban Decoction on content of the eumelanin and Pheomelanin in C57BL/6J mouse skin:ELISA results show that:the eumelanin content of all of the Quban Decoction groups and positive control group was lower than the blank control group. There was significant difference between the high dose group and the blank control group (P<0.01).The Pheomelanin content of all of the Quban Decoction groups and the positive control group was higher than the blank control group.There was significant difference between the high, middle, low dose groups and the blank control group (P<0.01)
Conclusion:
1. The Quban Decoction can reduce the expression of a-MSH in female C57BL/6J mouse skin and inhibit melanin synthesis. a-MSH is an important factor that affects the color of skin and hair.
2. The Quban Decoction can reduce the generation of the eumelanin chiefly in the inhibition of melanin synthesis process and promote the eumelanin to convert to the pheomelanin.
引文
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[44]邓湘庆,龚盛昭,揭向阳.川芎提取物抑制酪氨酸酶活性的研究[J].中药材,2007,30(4)：469-471.
[45]李洪武,朱文元,夏明玉,等.白术及茯苓提取物对豚鼠皮肤酪氨酸酶mRNA基因表达水平的影响[J].中华皮肤科杂志,2001,34(2)：134-135.
[46]谭城,朱文元,鲁严,等.芦荟素、肉桂酸、苦参碱对酪氨酸酶的抑制作用[J].中华皮肤科杂志,2002,35(2)：134-136.
[47]解士强,陈志强,卜令,等.丹皮酚在体外对人黑素细胞酪氨酸酶活性及黑素生成的影响[J].中华皮肤科杂志,2006,39(11)：639-641.
[48]李洪武,朱文元.治疗黄褐斑的中药复方对酪氨酸酶活性的影响[J].中华皮肤科杂志,2000,33(2)：93-95.
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[50]王正辉,杨壮群.黑素及影响黑素生成的因素.中国美容医学,2002,11(3)：278-280.
[51]陆洪光.黄种人与白种人正常皮肤痣和黑素瘤组织中优黑素和褐黑素含量比较.中国皮肤性病学杂志,2000,14(3)：149-151.
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[55]薛春雨,邢新,李蠡,等.α-MSH和MC-1R对表皮黑素细胞的调控作用.实用美容整形外科杂志,2003,14(5)：263-265.
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[2]Thody AJ, Ridley K, Penny RJ, et al. MSH are present in mammalian skin[J].Peptides,1983,4(6):813-816.
[3]Schiller M, Raghunath M, Kubitscheck U, et al. Human dermal fibroblasts express prohormone convertases land 2 and produce proopiomelanocortin derived peptides[J]. JInvest Dermatol,2001,117(2):227-235.
[4]薛春雨,李蠡.α-黑素细胞刺激素对皮肤合成黑素的调控作用.医学研究生学报,2004,17(9)：823-828.
[5]Tsatmali M, Ancans J, Thody AJ. Melanocyte function and its control by melanocortin peptides[J]. J Histochem Cytochem,2002,50(2):125.
[6]Schioth HB, Phillips SR, Rudzish R, et al. Loss of function mu-tations of the human melanocortion-1 receptor are common and are associated with red hair[J]. Biochem Biophys Res Commun,1999,260(2):488.
[7]闵彦,田野苹.α-黑素细胞刺激素作用的信号传导机制.生命的化学,2011,31(5)：671-673.
[8]Schaffer JV, Bolognia JL. The melanocortin-1 receptor:red hair and beyond[J]. Arch Dermatol,2001,137(11):1477.
[9]Abdel-Malek ZA, Scott MC, Furumura M, et al. Themelanocortin 1 receptor is the principal mediator of the effects of agoutin signaling protein on mammalian mela-nocytes[J].Cell Sci,2001,114(5):1019-1024.
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[11]TsatmaliM, Ancans J, Thody AJ. Melanocyte Function and Its Control by Melanocortin Peptides[J]. J Histochem Cytochem,2002,50(2):125-134.
[12]王秋枫.酪氨酸酶相关蛋白家族.中国美容医学,2003,12(1)：100-103.
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[1]BrzoskaT. α-melanocyte-stimulating hormone and related tripeptides: biochemistry, anti-inflammatory and protective effects in vitro and in vivo, and future perspectives for the treatment of immune-mediated inflammatory diseases. Endocr Rev,2008,29:581-602.
[2]Thody AJ, Ridley K, Penny RJ, et al. MSH are present in mammalian skin[J].Peptides,1983,4(6):813-816.
[3]Schiller M, Raghunath M, Kubitscheck U, et al. Human dermal fibroblasts express prohormone convertases land 2 and produce proopiomelanocortin derived peptides[J]. JInvest Dermatol,2001,117(2):227-235.
[4]薛春雨,李蠡.α-黑素细胞刺激素对皮肤合成黑素的调控作用.医学研究生学报,2004,17(9)：823-828.
[5]Tsatmali M, Ancans J, Thody AJ. Melanocyte function and its control by melanocortin peptides[J]. J Histochem Cytochem,2002,50(2):125.
[6]Schioth HB, Phillips SR, Rudzish R, et al. Loss of function mu-tations of the human melanocortion-1 receptor are common and are associated with red hair[J]. Biochem Biophys Res Commun,1999,260(2):488.
[7]闵彦,田野苹.α-黑素细胞刺激素作用的信号传导机制.生命的化学,2011,31(5)：671-673.
[8]Schaffer JV, Bolognia JL. The melanocortin-1 receptor:red hair and beyond[J]. Arch Dermatol,2001,137(11):1477.
[9]Abdel-Malek ZA, Scott MC, Furumura M, et al. Themelanocortin 1 receptor is the principal mediator of the effects of agoutin signaling protein on mammalian mela-nocytes[J].Cell Sci,2001,114(5):1019-1024.
[10]Dong Y et al. Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to a-melanocyte-stimulating hormone by up-regulating melanocortin-1 receptor. Biochem Biophys Res Commun,2010,396:849-853.
[11]TsatmaliM, Ancans J, Thody AJ. Melanocyte Function and Its Control by Melanocortin Peptides[J]. J Histochem Cytochem,2002,50(2):125-134.
[12]王秋枫.酪氨酸酶相关蛋白家族.中国美容医学,2003,12(1)：100-103.
[13]Schallreuter KU,KothariS,ChavanB,tal.Regulationofmelanogenesis-controversies and new concepts[J]. Exp Derm atol,2007,16 (12):1023-1031.
[14]Sturm RA, Teasdale RD, Box NF. Human pigmentation genes:identification, structure and consequences of polymorphic variation[J]. Gene,2001,277(1-2):49.
[15]Abdel-Malek ZA. Melanocortin receptors:their functions and regulation by physiological agonists and antagonists[J]. Cell Mol Life Sci,2001,58(3):434.
[16]Cardinali G, Ceccarelli S, Kovacs D, et al. Keratinocyte growth factor promotes melanosometransfer to keratinocytes[J]JlnvestDermatol,2005,125(6):1190-1199.
[17]Kadekaro A L, Kavanagh R, Kanto H,et al. Alpha-Mel-anocortin and endothelin-1 activate antiapoptotic pathwaysand reduce DNA damage inhuman melanocytes. Cancer Res,2005,65(10):4292-4299.