不同用量刺蒺藜对小鼠毛囊黑皮素1受体表达的影响
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摘要
研究背景与目的
色素代谢障碍性皮肤疾病(如黄褐斑、白癜风),发病率高,严重影响患者的生活质量,临床缺乏理想疗效的关键是有关色素调节的影响因素、作用机理与规律未能得到深入的科学阐述。黑皮素(melanocortin)是来源于前体阿黑皮素(POMC)的神经肽类激素,包括a-MSH、ACTH等,主要在脑垂体中产生。皮肤源性黑皮素神经肽激素主要是参与调控皮肤的色素沉着过程,而这种作用又主要是通过MC-1R介导。MC-1R是黑素细胞合成优黑素、暗黑素的关键调节物,是调控皮肤色素沉着的重要基因。在这过程中,酪氨酸酶又是色素合成的关键限速酶。MC-1R的激活或者高表达会使酪氨酸酶的合成增加,从而使黑素合成增加。中医对色素病发病机制的认识多是责之气血失调,而提出调和气血的治则,主要是祛风与活血、补肾法则有机结合的方法。近年有关中医药与色素代谢障碍性疾病研究较多的是局限于观察单味中药对酪氨酸酶活性或黑素细胞增殖的影响,鲜有从MC-1R的角度对其色素代谢机制进行阐明,因而也较少具有实际应用意义。我们以往的初步研究结果表明刺蒺藜具有双向调节黑素细胞增殖和酪氨酸酶活性的作用,基于此,我们根据中医临床使用频率高,对黑素代谢有调节作用的刺蒺藜进行了有关色素代谢的一些研究,观察不同用量刺蒺藜水提物对C57BL/6J小鼠毛囊MC-1R表达的影响,初步探讨刺蒺藜在皮肤表皮黑素细胞活化和增殖过程中的作用;为临床治疗色素代谢障碍性皮肤病提供有效的用药参考和实验依据。
方法与内容
48只C57BL/6J小鼠,随机分成4组,每组12只,分别为给予刺蒺藜水提液的3组实验组和给予蒸馏水的1组对照组,实验组小鼠预养2天后,于第3天开始,刺蒺藜生药材分上午9:00和下午16:00两次连续给实验组小鼠灌服刺蒺藜水提液,两次给小鼠灌胃各0.5ml,对照组为等量蒸馏水。于第30日上午给药1小时后将4组小鼠3%戊巴比妥45mg/Kg腹腔麻醉,常规固定,剃除背毛,用组织剪剪取2cm×2cm大小皮肤,10%中性甲醛固定备用。
随机在每组选择相同数量的标本,脱蜡至水,抗原修复后,用链霉素抗生物素蛋白-过氧化物酶连接(S-P)常规染色法检测MC-1R在表皮的阳性表达情况,用缓冲液替代第一抗体做阴性对照,用已知阳性切片作为阳性对照。然后将标本均用200倍显微镜下随机选择20个视野观察表皮MC-1R阳性细胞分布情况,并由JVC彩色摄像头将细胞图像输入Mias系统拍照。
最后采用二级计分方法,对表达MC-1R的阳性细胞进行统计计分,以正常小黑鼠皮肤为对照,应用SPSS13.0统计软件,各组标本之间阳性率采用秩和检验的统计学分析。对实验结果加以分析,均以α=0.05,P＜0.05时判为有显著性差异;
结果
MC-1的表达定位于表皮基底部黑素细胞、角质形成细胞胞浆,为黄色或棕黄色颗粒,MC-1R在正常组小黑鼠皮肤毛囊中呈散在局灶性分布,在试验组呈局灶性或弥漫性分布,在表皮基底层形成棕褐色条带。MC-1在不同用量刺蒺藜组和正常的小鼠毛囊皮片中均有阳性表达,但MC-1R在试验组皮片中的表达与正常对照组皮肤比较,差异有统计学意义(P=0.004＜0.01;X~2=13.515)且MC-1R在试验组较正常对照组表达明显增强,试验组刺蒺藜用量越高表达越明显,高剂量即刺蒺藜用量达225g时候表达最强(平均秩=34.21)。
全文结论
1、本实验所设计低中高剂量刺蒺藜都能够促进MC-1R的表达水平。
2、中高低剂量刺蒺藜未发现能双向调节黑皮素1受体的表达。
3、MC-1R在刺蒺藜作用下的表达可能是通过扩增生物学效应的方式使得黑色素合成增强,从而起到调节黑素代谢的作用。
Study Background and Purpose
Dysbolism pigment skin diseases(such as chloasma,vitiligo),high incidence of serious impact on quality of life of patients,clinical efficacy of the lack of ideals are related to pigment regulation of the key influencing factors,mechanism and laws of science have not been thoroughly expounded.Melanocortin(melanocortin) are derived from precursor pro-opiomelanocortin(POMC) neuropeptide hormones, including a-MSH,ACTH,etc.,produced mainly in the pituitary.Skin-derived melanocortin neuropeptide hormone is mainly involved in regulation of skin pigmentation process,and this role mainly through the MC-1R-mediated.MC-1R are excellent synthetic melanocyte melanocytes,pigment Diablo key mediator, regulation and control are the important skin pigmentation gene.In the process, tyrosinase is the key rate-limiting pigment synthesis enzyme.MC-1R activation,or high expression of tyrosinase synthesis would increase,thus increasing melanin synthesis.TCM Pathogenesis of pigment awareness are responsible for many disorders of the blood,while the proposed rule is to reconcile qi and blood,mainly with Huoxue Qufeng,the organic combination of Bushen Ways law.In recent years, Chinese medicine and pigment-related metabolic disorders studied are limited to observation of single herbs on tyrosinase activity or melanin cells,rarely from the perspective of MC-1R pigment metabolism mechanism of its stated,and thus have a practical application of less significance.Our previous preliminary study results show that the thorn Tribulus terrestris has a bi-directional regulation of melanocyte proliferation and tyrosinase activity in the role,based on this,according to our clinical use of high frequency of melanocyte metabolic regulation has had a thorn Tribulus terrestris on the pigment metabolism in some studies to observe the different amount of water thorn Tribulus terrestris extract on C57BL/6J mice hair follicles MC-1R expression,preliminary study thorn Tribulus terrestris epidermal melanocytes in the skin cell activation and proliferation process;for clinical treatment of pigment metabolism skin barrier provides an effective drug reference and experimental evidence.
Methods and contents
48 C57BL/6J mice were randomly divided into 4 groups,each 12,were given water extract of Tribulus terrestris stabbed the 3 test groups and one group given distilled water control group,experimental group of mice 2 days after pre-dependent, beginning in the first three days,gill medicinal herbs Tribulus terrestris Health at 9:00 am and 16:00 pm on two consecutive give the experimental group of mice fed with aqueous extract of Tribulus terrestris stabbed twice to mice orally each 0.5ml,the control group contour and distilled water.The 30th day in the morning 1 hour after administration of 4 groups of mice 3%pentobarbital 45mg/Kg intraperitoneal anesthesia,conventional fixed,scraped back hair,with scissors cutted Organize size 2cm×2cm skin,10%neutral formaldehyde fixed standby.
Random selection at each of the same number of specimens,dewaxing to water, antigen retrieval,the use of streptomycin avidin - peroxidase Connect(SP) staining of conventional MC-1R expression in the epidermis,the buffer with the first alternative to do the negative control antibody,using known positive biopsy as a positive control. And the specimens were 200 times with random selection under the microscope observation field of vision 20 epidermal MC-1R-positive cells distributed by the JVC color camera image input cells photographed Mias system.
Finally the use of secondary method of calculation of the expression of MC-1R positive cells scoring statistics to the normal black mouse skin as a control,the application of statistical software SPSS13.0,specimens in each group between the positive rate of adoption of W ilcoxon rank sum test statistical analysis.The experimental results of the analysis,areα=0.05,P＜0.05,when the contractor for the difference was significant;
Results
MC-1 expression is positioned at the bottom of Table Picchi melanocyte, keratinocyte cytoplasm,yellow or brown granules,MC-1R in normal mouse skin black hair follicles were scattered in the focal distribution,in the experimental group was focal or diffuse distribution of epidermal basal layer in the formation of brown stripe.MC-1 at different dosage thorn Tribulus terrestris group and normal skin of mice hair follicles have a positive expression of the film,MC-1R in the test group the expression of skin tablets and skin compared to normal control group,the difference has statistical significance(P = 0.004＜0.01;X2 = 13.515) and MC-1R in the test group than in the normal control group was significantly increased,and the test group thorn Tribulus terrestris dosage,the more obvious the higher the expression,that is, high doses thorn Tribulus terrestris when used to express the strongest capacity of 225g(average rank = 34.21 ).
The full text of the conclusions
1,different doses thorn Tribulus terrestris can effectively increase both MC-1R expression level.
2,MC-1R in the thorn Tribulus terrestris expression under possible biological effects through the amplification of the way makes Melanin synthesis enhanced,in order to play a regulating rode of melanin metabolism.
3,in high and low doses should not barbed bi-directional regulation of Tribulus terrestris melanocortin 1 receptor expression.
色素代谢障碍性皮肤疾病(如黄褐斑、白癜风),发病率高,严重影响患者的生活质量,临床缺乏理想疗效的关键是有关色素调节的影响因素、作用机理与规律未能得到深入的科学阐述。黑皮素(melanocortin)是来源于前体阿黑皮素(POMC)的神经肽类激素,包括a-MSH、ACTH等,主要在脑垂体中产生。皮肤源性黑皮素神经肽激素主要是参与调控皮肤的色素沉着过程,而这种作用又主要是通过MC-1R介导。MC-1R是黑素细胞合成优黑素、暗黑素的关键调节物,是调控皮肤色素沉着的重要基因。在这过程中,酪氨酸酶又是色素合成的关键限速酶。MC-1R的激活或者高表达会使酪氨酸酶的合成增加,从而使黑素合成增加。中医对色素病发病机制的认识多是责之气血失调,而提出调和气血的治则,主要是祛风与活血、补肾法则有机结合的方法。近年有关中医药与色素代谢障碍性疾病研究较多的是局限于观察单味中药对酪氨酸酶活性或黑素细胞增殖的影响,鲜有从MC-1R的角度对其色素代谢机制进行阐明,因而也较少具有实际应用意义。我们以往的初步研究结果表明刺蒺藜具有双向调节黑素细胞增殖和酪氨酸酶活性的作用,基于此,我们根据中医临床使用频率高,对黑素代谢有调节作用的刺蒺藜进行了有关色素代谢的一些研究,观察不同用量刺蒺藜水提物对C57BL/6J小鼠毛囊MC-1R表达的影响,初步探讨刺蒺藜在皮肤表皮黑素细胞活化和增殖过程中的作用;为临床治疗色素代谢障碍性皮肤病提供有效的用药参考和实验依据。
方法与内容
48只C57BL/6J小鼠,随机分成4组,每组12只,分别为给予刺蒺藜水提液的3组实验组和给予蒸馏水的1组对照组,实验组小鼠预养2天后,于第3天开始,刺蒺藜生药材分上午9:00和下午16:00两次连续给实验组小鼠灌服刺蒺藜水提液,两次给小鼠灌胃各0.5ml,对照组为等量蒸馏水。于第30日上午给药1小时后将4组小鼠3%戊巴比妥45mg/Kg腹腔麻醉,常规固定,剃除背毛,用组织剪剪取2cm×2cm大小皮肤,10%中性甲醛固定备用。
随机在每组选择相同数量的标本,脱蜡至水,抗原修复后,用链霉素抗生物素蛋白-过氧化物酶连接(S-P)常规染色法检测MC-1R在表皮的阳性表达情况,用缓冲液替代第一抗体做阴性对照,用已知阳性切片作为阳性对照。然后将标本均用200倍显微镜下随机选择20个视野观察表皮MC-1R阳性细胞分布情况,并由JVC彩色摄像头将细胞图像输入Mias系统拍照。
最后采用二级计分方法,对表达MC-1R的阳性细胞进行统计计分,以正常小黑鼠皮肤为对照,应用SPSS13.0统计软件,各组标本之间阳性率采用秩和检验的统计学分析。对实验结果加以分析,均以α=0.05,P＜0.05时判为有显著性差异;
结果
MC-1的表达定位于表皮基底部黑素细胞、角质形成细胞胞浆,为黄色或棕黄色颗粒,MC-1R在正常组小黑鼠皮肤毛囊中呈散在局灶性分布,在试验组呈局灶性或弥漫性分布,在表皮基底层形成棕褐色条带。MC-1在不同用量刺蒺藜组和正常的小鼠毛囊皮片中均有阳性表达,但MC-1R在试验组皮片中的表达与正常对照组皮肤比较,差异有统计学意义(P=0.004＜0.01;X~2=13.515)且MC-1R在试验组较正常对照组表达明显增强,试验组刺蒺藜用量越高表达越明显,高剂量即刺蒺藜用量达225g时候表达最强(平均秩=34.21)。
全文结论
1、本实验所设计低中高剂量刺蒺藜都能够促进MC-1R的表达水平。
2、中高低剂量刺蒺藜未发现能双向调节黑皮素1受体的表达。
3、MC-1R在刺蒺藜作用下的表达可能是通过扩增生物学效应的方式使得黑色素合成增强,从而起到调节黑素代谢的作用。
Study Background and Purpose
Dysbolism pigment skin diseases(such as chloasma,vitiligo),high incidence of serious impact on quality of life of patients,clinical efficacy of the lack of ideals are related to pigment regulation of the key influencing factors,mechanism and laws of science have not been thoroughly expounded.Melanocortin(melanocortin) are derived from precursor pro-opiomelanocortin(POMC) neuropeptide hormones, including a-MSH,ACTH,etc.,produced mainly in the pituitary.Skin-derived melanocortin neuropeptide hormone is mainly involved in regulation of skin pigmentation process,and this role mainly through the MC-1R-mediated.MC-1R are excellent synthetic melanocyte melanocytes,pigment Diablo key mediator, regulation and control are the important skin pigmentation gene.In the process, tyrosinase is the key rate-limiting pigment synthesis enzyme.MC-1R activation,or high expression of tyrosinase synthesis would increase,thus increasing melanin synthesis.TCM Pathogenesis of pigment awareness are responsible for many disorders of the blood,while the proposed rule is to reconcile qi and blood,mainly with Huoxue Qufeng,the organic combination of Bushen Ways law.In recent years, Chinese medicine and pigment-related metabolic disorders studied are limited to observation of single herbs on tyrosinase activity or melanin cells,rarely from the perspective of MC-1R pigment metabolism mechanism of its stated,and thus have a practical application of less significance.Our previous preliminary study results show that the thorn Tribulus terrestris has a bi-directional regulation of melanocyte proliferation and tyrosinase activity in the role,based on this,according to our clinical use of high frequency of melanocyte metabolic regulation has had a thorn Tribulus terrestris on the pigment metabolism in some studies to observe the different amount of water thorn Tribulus terrestris extract on C57BL/6J mice hair follicles MC-1R expression,preliminary study thorn Tribulus terrestris epidermal melanocytes in the skin cell activation and proliferation process;for clinical treatment of pigment metabolism skin barrier provides an effective drug reference and experimental evidence.
Methods and contents
48 C57BL/6J mice were randomly divided into 4 groups,each 12,were given water extract of Tribulus terrestris stabbed the 3 test groups and one group given distilled water control group,experimental group of mice 2 days after pre-dependent, beginning in the first three days,gill medicinal herbs Tribulus terrestris Health at 9:00 am and 16:00 pm on two consecutive give the experimental group of mice fed with aqueous extract of Tribulus terrestris stabbed twice to mice orally each 0.5ml,the control group contour and distilled water.The 30th day in the morning 1 hour after administration of 4 groups of mice 3%pentobarbital 45mg/Kg intraperitoneal anesthesia,conventional fixed,scraped back hair,with scissors cutted Organize size 2cm×2cm skin,10%neutral formaldehyde fixed standby.
Random selection at each of the same number of specimens,dewaxing to water, antigen retrieval,the use of streptomycin avidin - peroxidase Connect(SP) staining of conventional MC-1R expression in the epidermis,the buffer with the first alternative to do the negative control antibody,using known positive biopsy as a positive control. And the specimens were 200 times with random selection under the microscope observation field of vision 20 epidermal MC-1R-positive cells distributed by the JVC color camera image input cells photographed Mias system.
Finally the use of secondary method of calculation of the expression of MC-1R positive cells scoring statistics to the normal black mouse skin as a control,the application of statistical software SPSS13.0,specimens in each group between the positive rate of adoption of W ilcoxon rank sum test statistical analysis.The experimental results of the analysis,areα=0.05,P＜0.05,when the contractor for the difference was significant;
Results
MC-1 expression is positioned at the bottom of Table Picchi melanocyte, keratinocyte cytoplasm,yellow or brown granules,MC-1R in normal mouse skin black hair follicles were scattered in the focal distribution,in the experimental group was focal or diffuse distribution of epidermal basal layer in the formation of brown stripe.MC-1 at different dosage thorn Tribulus terrestris group and normal skin of mice hair follicles have a positive expression of the film,MC-1R in the test group the expression of skin tablets and skin compared to normal control group,the difference has statistical significance(P = 0.004＜0.01;X2 = 13.515) and MC-1R in the test group than in the normal control group was significantly increased,and the test group thorn Tribulus terrestris dosage,the more obvious the higher the expression,that is, high doses thorn Tribulus terrestris when used to express the strongest capacity of 225g(average rank = 34.21 ).
The full text of the conclusions
1,different doses thorn Tribulus terrestris can effectively increase both MC-1R expression level.
2,MC-1R in the thorn Tribulus terrestris expression under possible biological effects through the amplification of the way makes Melanin synthesis enhanced,in order to play a regulating rode of melanin metabolism.
3,in high and low doses should not barbed bi-directional regulation of Tribulus terrestris melanocortin 1 receptor expression.
引文
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[45]A daikan PG,Gauthaman K,P rasad RNV,et al.P roerectilepharmaco logical effects of T ribulus terrestris extract on the rabbit co rpus caverno sal smoo th muscle in vitro.Annals A 2cademy ofM edicine,Singapo re,2000,29(1):22-26.
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[47]孙斌,瞿伟菁,柏忠江.蒺藜皂甙对乳腺癌细胞Bcap-37的体外抑制作用.中药材,2003,26(2):104-106.
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