Effects And Mechanism Of Dizhi Pill On Delaying Skin Aging

Objective Through the theoretical study,clinical application and experimental study of traditional Chinese medicine,the effect and mechanism of Dizhi Pill on delaying skin aging were discussed.The theoretical research is based on the understanding and pathogenesis of skin aging in ancient and modern times;the clinical research is based on the prescription,pharmaco dynamics and clinical observation of Dizhi pill;the experimental study is based on mice as experimental animals to prepare the aging mice model induced by D-galactose,and to explore the effect of Dizhi Pill on the skin histomorphological structure of model mice.The causes of aging of epidermis and dermis,as well as the effects on cytokines,skin immune function and sex hormones were discussed at the cellular and molecular level.To study the specific target of Dizhi Pill in delaying skin aging,and to provide theoretical,clinical and experimental basis for the mechanism study of Chinese medicine in preventing and treating skin aging.Method1.Theoretical research: By consulting ancient and modern literature,this paper discusses the understanding of skin aging in modern medicine and ancient medicine,and the etiology and pathogenesis of skin aging.2.Clinical application research : The composition analysis,pharmacodynamics research and clinical effect of Dizhi pill were discussed.Sixty female patients were randomly divided into treatment group and control group.The treatment group was treated with Dizhi pill combined with intense pulsed light(IPL),while the control group was treated with intense pulsed light(IPL).After 8 weeks,12 weeks,VISIA skin test analyzer and CK skin moisture analyzer were used to observe the improvement of five indicators: pore,wrinkle,red area,pigments and skin moisture content.The clinical symptoms were observed by using the skin aging scale to quantify the score.The total curative effect was determined by the improvement of the instrument measurement index and the clinical symptoms.The total effective rate was =(significant number + effective number / total treatmen number t)*100%.3.Experimental study:SPF 2-month-old female mice,weighing l8-22 g,were randomly divided into six groups: normal control group,model group,VE control group,high-dose group of Dizhi pill,medium-dose group of Dizhi pill and low-dose group of Dizhi pill.Feeding in cage,constant temperature and humidity in SPF laboratory.After 2 weeks of adaptive feeding,except the normal group,5% D-galactose saline solution(125 mg/kg.d)was injected subcutaneously into the neck and back of mice every day for 42 consecutive days to prepare aging mice model induced by D-galactose.In the fourth week of model establishment,the high,middle and low dose groups of Dizhi Pill were given 6.825 g/kg d,13.65 g/kg D and 27.3 g/kg D respectively.The VE control group was given vitamin E 45.5 mg/kg d by gavage for 3 weeks.The blank group and the model group were given the same amount of distilled water.At the end of the experiment,mice were killed,biopsy samples were taken and HE staining was performed to observe the changes of skin histomorphology and structure under optical microscope;the expression of keratin K19 in epidermis of aging mice induced by D-galactose was detected by immunofluorescence method;the expression of aquaporin AQP3,hyaluronic acid HYP,hyaluronic acid HA and dermal collagen were detected by enzyme-linked immunosorbent assay(ELASA).The expression of matrix metalloproteinase MMP-1,MMP-3 and cytokines TGF-beta 1,and vascular endothelial growth factor in dermis were detected by immunohistochemical method(S-P method);the morphological and quantitative changes of epidermal S100 + Langerhans cells(S100 + LCs)were detected by immunohistochemical method(S-P method);and the levels of serum estradiol E2,luteinizing hormone LH and follicle-forming hormone(FSH)were detected by ELISA method.Data were collected for statistical analysis.Result1.At 8 weeks,the four indexes of pore,red area,pigments and skin water content in the treatment group were improved,but the wrinkle index was not improved.At 12 weeks,the water content of the pores,wrinkles,red area,pigments and skin in the treatment group were improved.The comparison between groups showed that the improvement of the treatment group was better than that of the control group,and the total effective rate of the treatment group was higher than that of the control group,P < 0.05,with statistical significance.2.The effect of Dizhi Pill on histomorphological structure of aging mice induced by D-galactose: Histopathology: In the blank group,the epidermis of mice had complete structure,uniform thickness and close connection.Wave-like fibrous tissue was found in dermis,which was arranged orderly,evenly distributed,dense and abundant,and dermal papillae were clear.The distribution of hair follicle and sebaceous gland was normal.The structure of acinar cells is regular and the ratio of nucleus to plasma is normal.In the model group,the epidermis was uneven in thickness,and the structure was incomplete.The boundary between the epidermis and the dermis was clear.The dermis became thinner,the number of cell layers decreased,and the collagen fibers arranged disorderly and unevenly.The demarcation of subcutaneous tissue is not clear.The acinar cells of sebaceous glands are slightly irregular in structure and normal in nuclear-cytoplasmic ratio.The epidermis structure of mice in VE group and Dizhi pill high,middle and low dose group was more complete,the cell layer was clearer,the epidermal processes and dermal papillae were more obvious,the collagen layer was significantly thicker in the dermis,and the fibrous tissue was arranged in waves,orderly and evenly,the structure of sebaceous acinar cells existed,the morphology was regular,and the nuclear-plasma ratio was normal.The results showed that VE group and Dizhi pill could improve the histomorphological structure of skin.Quantitative analysis showed that the thickness of epidermis and dermis in model group was significantly thinner than that in blank group(P < 0.01).Compared with model group,the thickness of epidermis and dermis in VE group and high dose group of Dizhi pill was significantly thicker(P < 0.01).The thickness of epidermis and dermis in middle and low dose group of Dizhi pill was significantly thicker(P < 0.01).Meaning(P < 0.05);Compared with VE group,the thickness of epidermis and dermis in high dose group of Dizhi pill increased significantly(P < 0.05).3.The effects of Dizhi Pill on keratin K19 and aquaporin AQP3 in epidermis of aging mice induced by D-galactose:Immunofluorescence detection of K19 showed that under fluorescence microscope,K19 in cytoplasm was stained in red and nucleus in blue.The expression of keratin K19 in the basal epidermis of mice in blank group was red fluorescent,while the expression of keratin K19 in model group was decreased.In VE group,the expression of K19 in low,medium and high dose groups of Dizhi Pill was increased to varying degrees.Quantitative analysis showed that the expression of K19 in the model group was significantly lower than that in the blank group(P < 0.01).Compared with the model group,the expression of K19 in the high dose group of Dizhi Pill was significantly increased(P < 0.01);the expression of K19 in the VE group and the medium dose group was significantly increased(P < 0.05).Compared with VE group,the expression of K19 in high dose group of Dizhi pill was increased,and the difference was statistically significant(P < 0.05).ELISA showed that the content of AQP3 in the model group was significantly lower than that in the blank group,and there was a significant difference between the two groups(P < 0.01).Compared with model group,AQP3 content in VE group and low dose group of Dizhi pill increased significantly(P < 0.05);AQP3 content in middle and high dose group of Dizhi pill increased significantly(P < 0.01).Compared with VE group,the content of AQP3 in high dose group of Dizhi pill increased,with statistical significance(P < 0.05).4.The effects of Dizhi Pill on the contents of HYP,HA,collagen and MMP-1,MMP-3 in dermis of aging mice induced by D-galactose:The content of HYP,HA and collagen in dermis of mice detected by ELISA showed that compared with the blank group,the content of HYP,HA and collagen in model group was significantly lower(P < 0.01).Compared with the model group,the contents of HYP,HA and collagen in VE group,Dizhi pill high and middle dose group increased significantly(P < 0.01),and the contents of HYP,HA and collagen in low dose group of Dizhi pill increased significantly(P < 0.05).Compared with VE group,the contents of HYP,HA and collagen in the high and middle dose groups of Dizhi Pill increased,and the difference was statistically significant(P < 0.05).Immunohistochemical staining showed that MMP-1 and MMP-3 were positive in brown-yellow granules.The results showed that the collagen fibers in the blank group were arranged regularly and neatly,and the positive expressions of MMP-1 and MMP-3 were very few.Compared with the normal group,the expression of MMP-1 and MMP-3 increased in the model group,which was dark brown and yellow,with sparse collagen fibers arranged irregularly or scattered.Compared with the model group,the collagen fibers in VE group and Dizhi pill groups were arranged neatly,the number of collagen fibers increased,and the expression of brown and yellow granules decreased in varying degrees,indicating that the expression of MMP-1 and MMP-3 decreased.Quantitative analysis showed that the contents of MMP-1 and MMP-3 in the model group were significantly higher than those in the blank group(P<0.01).Compared with the model group,the contents of MMP-1 and MMP-3 in the VE group,the middle-dose group and the low-dose group were significantly lower(P<0.05),and the contents of MMP-1 and MMP-3 in the high-dose group were significantly lower(P<0.05).Compared with VE group,the contents of MMP-1 and MMP-3 in the high dose group of Dizhi pill decreased significantly(P < 0.05).5.Effects of 5 Dizhi Pills on Growth Factor TGF-beta 1 and VEGF in Aging Mice Induced by D-galactose:Immunohistochemical staining showed that the expression of cytokines TGF-beta 1 and VEGF in the skin epidermis of mice in blank group was weakly positive,showing brown-yellow granules in the cytoplasm.Compared with the blank group,the expression of TGF-beta 1 in the model group was very low,and the expression of VEGF brown granules in the model group was not significantly changed compared with the blank group.Compared with the model group,the expression of TGF-beta 1 was increased in VE group,Dizhi pill high and medium dose group,and the expression of VEGF was not changed significantly.Quantitative analysis showed that compared with the blank group,the expression of TGF-beta-1 in the model group was decreased and the difference was statistically significant(P<0.05);compared with the model group,the expression of TGF-beta-1 in the high and middle dose groups of Dizhi pill was increased(P<0.05),while the expression of TGF-beta-1 in the low dose group and the VE group had no significant change(P>0.05);compared with the VE group,the expression of TGF-beta-1 in the high and middle dose groups of Dizhi pill was increased and worse.The difference was statistically significant(P < 0.05).Compared with the blank group,the expression of vascular endothelial growth factor in the model group was decreased(P < 0.05);compared with the model group,the expression of vascular endothelial growth factor in the VE group and the high,medium and low dose groups of Dizhi pill had no significant change(P > 0.05);compared with the VE group,the expression of vascular endothelial growth factor in the high,middle and low dose groups of Dizhi pill had no significant change(P > 0.05).6.The effects of Dizhi pills on S100 protein-positive Langerhans cells(S100+LC)in aging mice induced by D-galactose:Immunohistochemical detection of S100+LC showed that the cytoplasm and membrane of the blank group were yellow-brown S100+LCs.The cell body was small,round or elliptic,with few and short dendrites,clear cell contour,and typical dendritic cells were not easily seen.The cells were scattered in the epidermis and dermis.In the model group,the cell bodies of S100 + LCs became larger and scattered in the middle and upper spinous layer.LCs were not visible in part of the field of vision.Dendritic processes decreased,shortened or disappeared significantly.Some structures were incomplete.Dendrites were few and short.Typical LCs were rare.Compared with the model group,the morphology and distribution of S100 + LCs in VE group and Dizhi pill low dose group did not change significantly.LCs were distributed widely and evenly in the middle and high dose groups of Dizhi Pills,and partially concentrated in hair follicles and sebaceous glands.LCs were round,quasi-circular and irregular in shape.Dendritic processes increased significantly,and were long and thin.Some processes could extend to several adjacent spinous cells.Compared with the blank group,the number of S100 + LCs cells in the model group decreased significantly(P < 0.05);compared with the model group,the number of S100 + LCs cells in the high-dose group increased significantly(P < 0.01),the number of S100 + LCs cells in the middle-dose group increased(P < 0.05),and the number of S100 + LCs cells in the VE group and the low-dose group did not change significantly(P > 0.05);compared with the VE group,the number of S100 + LCs cells in the high-dose group was higher(P > 0.05).The number of S100 + LCs cells increased significantly in the dose group(P < 0.01).The number of S100 + LCs cells increased in the middle dose group(P < 0.05).The number of S100 + LCs cells did not change significantly in the low dose group(P > 0.05).7.Effects of Dizhi Pill on Serum E2,LH and FSH Levels in Aging Mice Induced by D-galactose:ELISA showed that compared with the blank group,the level of E2 in the model group decreased significantly(P < 0.01),the level of LH and FSH increased significantly(P < 0.01);compared with the model group,the level of E2 in the high dose group increased significantly(p < 0.01),the level of LH decreased(P < 0.05),the level of FSH decreased significantly(P < 0.01).Compared with VE group,the level of E2 in high dose group of Dizhi pill increased significantly(P < 0.01),the level of LH decreased(P < 0.05),the level of FSH decreased(P < 0.01),and the level of E2 in middle dose group of Dizhi pill increased(P < 0.05).