| Objective:(1)To observe the clinical efficacy and safety of Picoway picosecond laser alone and Picoway picosecond laser laser combined with Sibai ointment in the treatment of melasma,compare the therapeutic effects of the two schemes,and explore the comprehensive treatment method of integrated Chinese and Western medicine for melasma with significant effect and safe mode.(2)Explore the possible mechanism of Action of Sibai ointment in the treatment of melasma based on network pharmacology method.(3)To study the effect of local and external application of Sibai ointment on melasma model rats and its mechanism.Methods:(1)Clinical trials study:A total of 72 patients with melasma who visited dermatology Department of Beijing Friendship Hospital of Capital Medical University from September2019 to December 2022 were selected as the subjects of this study.They were divided into 2groups by random number table method,namely treatment group and control group,with 36patients in each group.The control group was treated with Picoway picosecond laser only,using 1064nm Zoom mode,light spot size 7-8mm,energy density 0.65-0.75J/cm~2,frequency8Hz,once every 4 weeks,continuous 12 weeks.The treatment group was treated with Picoway picosecond laser combined with Sibai ointment.The Picoway picosecond laser treatment was the same as the control group,treated once every 4 weeks,Sibai ointment 1time in the morning and evening,with massage for 3 to 5 minutes,continuous treatment for12 weeks.The observation indexes included modified m MASI score for melasma lesions area and severity,physician’subjective evaluation(PGA),melasma quality of life scale(MELASQo L)score,skin disease Quality of Life Index(DLQI)score,satisfaction evaluation and safety observation.In the statistical part,SPSS26.0 statistical software was used for data processing and analysis,and adverse manifestations and reactions of patients in the treatment stage and follow-up were observed and recorded carefully to evaluate the treatment effect of the two therapeutic schedules.(2)Network pharmacology study:Using TCMSP database and drug name as key words,the active components and corresponding targets were obtained according to ADME,OB≥30%and DL≥0.18,and the disease targets were retrieved by OMIM,Dis Ge NET and Gene Cards databases,and the intersection genes of The ointment and melasma were obtained.PPI network was constructed by importing the intersection targets into STRING database.Cytoscape3.9.1 software was used for network topology analysis to screen out the core targets in the protein interaction network.Cluster Profiler package was called by R 3.6.3 software to obtain the GO and KEGG enrichment analysis results of core intersection genes.(3)Animal experimental study:36 healthy female SD rats were randomly selected and divided into normal group(n=6)and model group(n=30).The animal model of SD rats with melasma was established by referring to the methods of domestic and foreign literatures.After the modeling,6 SD rats in the normal group and 6 SD rats in the model group were selected to detect the amount of MDA and TYR and the liveness of SOD in serum,liver and skin tissues,and pathological examination of skin tissue to observe the successful modeling of SD rat melasma animal model.The rest of 24 rats were randomly grouping 3 parts,every group had 8 rats.Group A was treatment group with Sibai ointment,group B was positive control group with vitamin C aqueous solution,and group C was model control group.After 4weeks of continuous treatment,rats were sacrificed,and the contents of MDA and TYR and the activity of SOD in liver and skin tissues of SD rats were compared.The liver and skin tissues of SD rats were stained by HE and stained by Masson-Fontana for histopathological observation.Results:1.Clinical trials study(1)m MASI score:1)Fitzpatric type:There was no statistical significance in m MASI score of typeⅢand TypeⅣpatients before treatment,at the 4th and 8th week of treatment(P>0.05);At the 12th week of treatment between the two groups,there was statistically significant difference for typeⅢpatients of m MASI score(P<0.05),but there was no statistically significant difference for typeⅣpatients of m MASI score(P>0.05).m MASI score of typeⅢand typeⅣpatients was compared before and after treatment,and the difference was statistically significant(P<0.05).2)Treatment effect:there was no statistically significant difference in m MASI score between 2 groups before treatment,at the 4th and 8th week of treatment(P>0.05).At the 12th week of treatment,there was a statistically significant difference for the two groups of m MASI score(P<0.05).After 12 weeks of treatment,and the difference was statistically significant for the decline score of m MASI in the two groups(P<0.05).(2)MELASQo L score:After 12 weeks of treatment,the difference was no statistically significant for MELASQo L score of 2 groups(P>0.05),while the difference was statistically significant for the decline score of MELASQo L of two groups(P<0.05).(3)DLQI score:After 12 weeks of treatment,the difference was no statistically significant for DLQI score between 2 groups(P>0.05),and the difference was no statistically significant for the decline score of DLQI of 2 groups(P>0.05).(4)Clinical efficacy:There was statistical significance in clinical efficacy between the two groups(P<0.05).(5)PGA score:After 12 weeks of treatment,the difference was statistically significant for the score of PGA of 2 groups(P<0.05).(6)Satisfaction evaluation:After 12 weeks of treatment,the difference was statistically significant in satisfaction for the two groups(P>0.05).2.Network pharmacology studyIn the study,171 active components,274 corresponding targets and 173 targets of melasma disease were obtained.26 genes of intersection of Sibai ointment and melasma were obtained,which were imported into STRING database to construct PPI network,and Cytoscape3.9.1 software was used for network topology analysis.Three core intersecting genes were screened out from the protein interaction network.GO enrichment analysis results include the regulation of DNA-binding transcription factor activity,reactive oxygen metabolism,response to steroid hormones,cell response to drugs and other pathways.The results of KEGG enrichment analysis suggested that the intersecting genes have related effects on various cancer diseases and infectious diseases,as well as the pathways of cellular senescence,melanoma and human skin and visceral kala-azar.3.Animal experimental study(1)Before treatment:1)Direct observation:After 4 weeks of modeling,the skin of SD rats in model group formed clear and stable red-brown patches.2)Biochemical observation:After modeling,SOD activity in liver and skin tissues of SD rats in model group was significantly lower than that in normal group,while MDA and TYR contents in liver and skin tissues were significantly higher than that in normal group,with statistical significance(P<0.05).Consistent with the changes of biochemical indicators of melasma.3)Pathological observation:HE staining showed that the normal group had thinner epidermis without hyperplasia,with a small amount of keratin,reticulated collagen fibers in the dermis,and almost no inflammatory cells and microvascular dilatation.The epidermis of the model group showed different degrees of obvious hyperplasia,keratinosis,pigment granules obviously distributed in the basal layer,the increase of inflammatory cells and the proliferation and expansion of small blood vessels.Masson-fontana staining showed that there was almost no deposition of melanin particles in the epidermis of normal rats.In the model group,there were a large number of melanin particles in the epidermis of rats.It is consistent with pathological changes of melasma.(2)After treatment:1)Direct observation:In group A,it was directly observed that the skin lesions at the molding site became pale,and no obvious pigmentation was observed.The skin lesions were similar to the normal.In group B,it was directly observed that the skin lesions at the molding site became lighter,and the local skin lesions still had pigmentation,and the skin lesions were close to that of Group A.In group C,the skin color,desiccation and desquamation recovered with time change and without any intervention,and the color and area of the spots were still significantly higher than those in groups A and B.2)Biochemical observation:The activity of SOD in group A and group B was increased compared with the group C,the content of MDA and TYR were decreased compared with the group C,the difference was statistically significant(P<0.05).For group A and group B,there was no significant difference(P>0.05).3)Pathological observation:HE staining showed that the degree of epidermal hyperplasia was significantly reduced in group A,the dilatation of small blood vessels was reduced in the dermis,and there were few inflammatory cells.The degree of epidermal hyperplasia in group B was relatively reduced,pigment particles were distributed in the basal layer,and small blood vessels were significantly dilated in the dermis with inflammatory cells.The epidermis of the rats in group C showed obvious hyperplasia and incomplete keratosis,pigment granules were obviously distributed in the basal layer,microvascular dilatation and hyperplasia were observed in the dermis,and inflammatory cells were abundant.Masson-fontana staining showed that melanin particles in the epidermal layer of rats in group A were significantly reduced and scattered.In group B,melanin particles in the epidermis of rats were relatively few and concentrated.The number of melanin granules in the epidermis of rats in group C decreased compared with before treatment.Conclusion:1.Clinical trials study(1)The clinical efficacy of Picoway picosecond laser combined with Sibai ointment in the treatment of melasma is better than that of Picoway picosecond laser alone.(2)Picoway picosecond laser combined with Sibai ointment is a safe and effective treatment for melasma,which is worthy of further study and application in clinical dermatology.2.Network pharmacology studySibai ointment plays a therapeutic effect on melasma through multiple components,multiple targets and multiple pathways.It is speculated that Sibai ointment may play a therapeutic role in melasma by regulating and controlling cell cycle and apoptosis,and then alleviating cell senescence through anti-oxidation.3.Animal experimental study(1)The rat model of melasma induced by UVB combined with progesterone was established successfully,which was consistent with the pathological and biochemical characteristics of melasma.(2)Sibai ointment is effective in the treatment of melasma,and can desalinize melanosis,reduce MDA and TYR content of melasma model rats,and increase SOD activity.It has antioxidant ability,improves the antioxidant capacity of the body,and enhances the therapeutic effect of melasma disease.In terms of skin histopathology,Sibai ointment can reduce the number of melanin particles in the epidermal layer of melasma model rats. |
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