| ObjectiveTurtle plate tincture containing sterone S9,which is the active ingredient of turtle plate,was prepared to treat androgenetic alopecia model mice,and the effect of turtle plate tincture on hair growth of androgenetic alopecia model mice was observed.In addition,the mechanism of action was explored from the perspective of Wnt/β-catenin signaling pathway and hair follicle stem cells.This study provides ideas for the research of new drugs for the treatment of androgen alopecia.Methods1.Androgenetic alopecia model mice were made by using DHT 5mg/kg/d subcutaneous injection on the back and divided into five groups:blank group(B),model group(M),matrix group(MA),positive control group(PC),and experimental group(E).Intervention:Hair removal was performed in all five groups.The blank group did not intervene;the model group was injected with DHT daily;the matrix group was injected with DHT+twice a day external matrix solution;the positive control group was injected with DHT+once a day with 5%minoxidil solution;the experimental group was injected with DHT+twice a day with Turtle plate tincture.Observe the hair growth of the mice,including the comparison of the time when the skin in the epilation area begins to darken and the time when the hair grows to the skin surface.2.On the 21st day of the experiment,the skin tissues in the hair removal area of each group of mice were stained with HE,and the effects of the drug on the hair follicles were observed,including the number of hair follicles and the comparison of the ratio of terminal hair to vellus hair.3.On the 21st day of the experiment,the skin tissues of the hair removal area of each group of mice were detected by WB and IF,the quantitative expression of Wnt10b、GSK-3β、DKK1、CD34 and Integrin β1 proteins was detected,and the qualitative expression of Wnt10b,β-catenin,LEF1,c-Myc,CK15,and Integrin β1 proteins was detected.Results1.Effects on hair growth:The hair growth rate of the model group and the matrix group lagged behind the other groups.The time when the skin of the model group started to darken was behind the blank group,P<0.01.The time when the hair of mice in the model group was visible in the skin surface was behind the blank group,P<0.01.2.Effects on hair follicles:In the blank group,model group,and matrix group,the hair shaft pigment is light or even colorless,and there were more vellus hair;the positive control group,terminal hair with deep pigmentation in the hair shaft was seen,but the hair was mainly vellus hair;the experimental group was dominated by terminal hair with deep pigmentation in the hair shaft.The number of hair follicles in the model group was less than that in the blank group,P<0.01;there was no difference in the number of hair follicles between the model group and the matrix group,P>0.05;the number of hair follicles in the positive control group and the experimental group was significantly more than that in the model group,P<0.001;the number of hair follicles in the experimental group was more than that in the positive control group,P<0.01.There was no difference in the ratio of terminal hair/vellus hair between the model group and the matrix group,P>0.05;in the positive control group and the experimental group,the ratio of terminal hair/vellus hair was higher than that in the model group,P<0.01,P<0.001.3.Western blot results showed that compared with the blank group(B),the expression of the Wnt10b protein in the modle group(M)reduced,P<0.01;the expression of the Wnt10b protein in the experimental group(E)was more than that in the group M,P<0.01.The expression of the GSK-3β and DKK1 protein in the group E were less than that in the group M,<0.01,P<0.001.The expression of the CD34 protein in the group E was more than that in the group M,P<0.05.The expression of the Integrin β1 protein in the group E was more than that in the group M,P<0.001;the expression of the Integrin β1 protein in the group E was more than that in the positive control group(PC),P<0.001.4.Immunofluorescence results showed that Wnt10b was mainly expressed in the inner root sheath of the hair follicle,both in the positive control group and the experimental group,especially in the experimental group.β-catenin is mainly expressed in the inner root sheath and basal part of the hair follicle,and the basal part is most strongly expressed.The blank group and the model group are expressed only at the basal part,while the experimental group is expressed in the inner root sheath and basal part of the hair follicle,and the strongest expression.LEF1 was mainly expressed in the inner root sheath of the hair follicle,and only a few hair follicles were expressed in the experimental group.c-Myc was mainly expressed in the outer root sheath and inner root sheath of the hair follicle,and only weakly expressed in the experimental group.CK15 is slightly expressed in the outer root sheath and epidermis of the hair follicle,and is more expressed in the experimental group.Integrin β1 is mainly expressed in the inner root sheath of the hair follicle,and is more expressed in the experimental group.Conclusion1.The use of DHT 5mg/kg/d subcutaneous injection to make a mouse model of androgenic alopecia was successful.2.Turtle plate tincture has the effect of increasing the number of hair follicles,promoting the growth of terminal hair,reducing the number of vellus hair,and increasing the ratio of terminal hairs to vellus hairs.3.Turtle plate tincture activates the Wnt/β-catenin signaling pathway in hair follicle stem cells by enhanced expression of Wnt10b,and inhibits the expression of the negative regulators GSK-3β and DKK1 activated by DHT,thereby making signal transmission smooth and starting the hair cycle.Turtle plate tincture activated hair follicle stem cells,promoted hair follicle stem cell proliferation,thus promoting hair and hair follicles growth in androgenetic alopecia model mice. |
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