Study On The Efficacy And Mechanism Of Black Hair Silk Moisturizing Wuze Essence Solution Transdermal Administration On White Hair Mice Model

Objective:To clarify the efficacy and part of the mechanism of action of Black Hair Moisturizing and Black Hair essence through the efficacy study of the transdermal administration of Black Hair Moisturizing and Black Hair essence in the treatment of white hair in mice,so as to lay a solid theoretical and experimental foundation for the external treatment of white hair in traditional Chinese medicine.Methods:（1）Systematically sort out the understanding of TCM on the etiology and pathogenesis of white-haired disease,the differentiation of disease and the treatment of white-haired disease,and the progress of TCM on the treatment of white-haired disease,as well as the research progress of western medicine on the etiology and pathogenesis,research methods and treatment of white-haired disease,and grasp the frontier of Chinese and western medicine on white-haired disease;Summarize the research progress and propose the existing problems and solutions.（2）The C57BL/J mouse model of white hair disease was established by using hydrogen peroxide（H₂O₂）and hydroquinone（HQ）modeling methods.The most suitable animal model for this study was selected.The changes of hair color on the back of mice were observed by taking pictures on the 7th day（D7）,D14,D21,D42),the morphological changes of hair follicles were observed by HE staining,and the number of hair follicles without melanin was counted by melanin staining section,The expression of TYR mRNA,TRP-1 mRNA,TRP-2 mRNA and MITF mRNA in mouse skin was detected by qPCR method,and the expression of TYR and MITF protein in mouse skin was detected by Western blot method as the model evaluation standard.（3）To verify the efficacy of Black Hair Silk Moisturizing Black Hair essence in the treatment of white hair disease by transdermal administration of Black Hair Silk Moisturizing Black Hair essence in the treatment of white hair model mice.In this study,C57BL/J male mice were selected as research objects,and were divided into blank group,model group,101 Ruisi Yangyuan Black Hair Solution positive drug control group,low,medium and high dose groups of Black Hair Silk Moisturizing Black Hair essence.Before and after administration,photographs were taken to observe the changes in the back hair color of mice.After 30 consecutive days of administration,HE staining was used to observe the morphological changes of mouse hair follicles,and melanin staining sections were used to count the number of hair follicles without melanin,Detection of TYR expression in mouse skin tissue by immunohistochemistry,qPCR was used to detect the gene expression of TYR mRNA,TRP-1 mRNA,TRP-2mRNA and MITF mRNA in the skin of mice,and Western Blot was used to detect the expression of TYR and MITF protein in the skin tissue of mice,so as to explore the efficacy and mechanism of Black Hair Silk Moisturizing Black Hair essence.Results:（1）TCM has a long history and unique advantages in treating white hair.In recent years,the number of clinical articles on the treatment of white hair by percutaneous administration has gradually increased,but the basic research is very few.One of the important reasons is the lack of stable and reproducible animal models;Although western medicine has done a lot of mechanism research on white hair disease,there is no specific drug for white hair disease.At present,there is an urgent need for a Chinese herbal transdermal drug delivery preparation with significant efficacy,high safety and mature dosage form.Black Hair Silk Moisturizing Black Hair essence has significant clinical efficacy and research value.（2）After modeling,compared with the control group,obvious white hair was found in the back administration area of 4%HQ group mice,and obvious white hair was found in the back administration area throughout the modeling cycle;The overall hair color of mice in 3%H₂O₂group and 5%H₂O₂group turned brown,and there were different degrees of injury and scab on the back.The HE staining results showed that compared with the control group,the hair follicles in the 4%HQ group were complete in shape,deeper in length and thicker in different degrees in the epidermal tissue,and the hair follicles were in the transitional stage from the resting stage to the regressive stage;No mature hair follicles were found in the 3%H₂O₂group and 5%H₂O₂group,basal melanocytes and epidermal melanocytes were produced in large quantities,and the hair follicles were in an active growth stage.The results of melanin staining showed that compared with the control group,a small amount of light-colored melanin dots were found in the pathological sections of 4%HQ group mice;A large number of dark melanin dots were found in the pathological sections of mice in the 3%H₂O₂group and 5%H₂O₂group.The statistical results of the number of hair follicles containing melanin showed that compared with the control group,the number of hair follicles containing melanin in the 4%HQ group decreased（P<0.05）;The number of melanin hair follicles in the 3%H₂O₂group and 5%H₂O₂group increased（P<0.01）.The results of qPCR showed that compared with the control group,the expression levels of TYR mRNA and TRP-2mRNA in the skin tissue of 4%HQ group were significantly lower（P<0.05）,and the expression level of TRP-1 mRNA was significantly lower（P<0.01）.Western blot showed that the relative expression of TYR protein in the skin tissue of mice in the 4%HQ group was significantly lower than that in the control group（P<0.05）.（3）After being treated with the black hair nourishing Wuze essence solution of the successfully modeled mice,compared with the model group,black hair appeared on the back of the mice in each treatment group,and the hair color was black and white.The HE staining results showed that compared with the model group,the mature hair follicle morphology was not found in the mice of each treatment group,and the basal melanocytes and epidermal melanocytes increased significantly.The results of melanin staining showed that compared with the model group,a large number of melanin spots were found in the pathological sections of mice in each treatment group.The statistical results of hair follicles containing melanin showed that compared with the model group,the number of hair follicles containing melanin in mice in each treatment group was significantly increased（P<0.05）.The results of TYR immunohistochemical staining showed that compared with the model group,the area of TYR positive expression in the skin tissue of mice in each treatment group was significantly increased（P<0.05）,and the average optical density of TYR in the black hair treatment group was significantly increased（P<0.05）.The results of qPCR showed that compared with the model group,the expression level of TYR mRNA in the skin tissue of mice in each treatment group was significantly higher（P<0.05）;The expression level of TRP-1 mRNA in black hair treated group was significantly higher（P<0.05）;The expression level of TRP-2 mRNA in black hair treated group was significantly higher（P<0.05）;The expression level of MITF mRNA in black hair treated group was significantly higher（P<0.05）.Western blot showed that compared with the model group,the relative expression of TYR protein in black hair high-dose group and black hair middle-dose group was significantly higher（P<0.05）;The relative expression of MITF protein was significantly increased in each treatment group（P<0.05）.Conclusion:（1）The mouse model of white hair disease can be successfully prepared by 4%HQ method.（2）Black Hair Silk Moisturizing Wuze essence has a significant therapeutic effect on the mouse model of white hair disease through transdermal administration.Its mechanism may be related to stimulating the increase of melanocyte number,improving TYR activity,and increasing melanin production.（3）It enriches the theoretical content of treating white-haired disease by external treatment and disease differentiation.