| Objective:Astaxanthin,a xanthophyll carotenoid,has been extensively studied for its various health benefits in the area of the skin,and several studies have shown astaxanthin to be an effective compound for accelerating wound healing.In recent years,the anti-fibrosis effect of astaxanthin has been confirmed in various organs and tissues,but whether it can improve the fibrotic lesions formed after skin wound healing,that is,scar formation,has rarely been reported,and further research is needed.Therefore,this study is based on applying astaxanthin nano-emulsion to in vivo skin defect and scar models to observe its intervention effects,and to initially investigate its mechanism of action,with a view to providing a new tool for clinical scar treatment applications.Methods:1.A classical rabbit ears scar model was made and divided into two main subject groups(astaxanthin nano-emulsion group and centella triterpenes cream group),each group was controlled by itself.Astaxanthin nano-emulsion and centella triterpenes cream containing equal amounts of the active ingredients were applied evenly to the corresponding area of the wounds 14 days after the modeling procedure,and the rabbits were braked until the drugs were basically absorbed and then were returned to the original environment and kept in a single cage,and the development and regression of the scar were strictly recorded.Specimens of scar tissue from each group were cut after 28 days of medication,half of which were immediately fixed in paraformaldehyde,while the other half were immediately stored in a-80℃refrigerator.Subsequently,HE and Masson staining were performed to observe the arrangement of collagen fibers,distribution of capillaries and infiltration of inflammatory cells in skin.2.SD rats were divided into three groups(blank control group,model group,astaxanthin nano-emulsion group)using a random number table,except for the blank group,models of full skin defect were made in the other two groups.The astaxanthin group applied emulsion on the wound twice a day.The trauma or scar areas were measured on days 1,3,6,7,9,11,14,18,21 and 28 after surgery.Whole skin layers were taken at the 2nd,3rd and 4th week,and the morphology,distribution and content of fibroblasts and collagen fibers in the skin tissues at different periods were observed using HE and Masson staining respectively;The expression of TGF-β1,Col-I and Col-III proteins at week 4 were detected by immunohistochemistry.Protein immunoblotting to detect α-SMA,IL-6 in skin tissues at week 4.3.4D-Fast DIA proteomics study of protein solution samples at week 4 from rats in the astaxanthin group and model group to screen out their differential proteins,and then performed functional annotation,etc.,so as to analyze the key proteins and signaling pathways of astaxanthin to promote wound healing and improve scarring;Protein immunoblotting to detect apoptosis and autophagy related factors and verify the expression of PI3K/Akt/m TOR pathway proteins in skin tissues at week 4.The expression of apoptosis and autophagy related factors at week 4 was detected by immunofluorescence.Results:1.The thickness of the scar tissue was thinned and the collagen fibers were significantly reduced and aligned after the intervention with astaxanthin nano-emulsion in both rectangular and round scars.The scar tissues treated with astaxanthin nano-emulsion and cumene cream showed a reduction in vascular proliferation and a reduction in the number of fibroblasts,and some of the results suggested that the dermal papillae had been reconstructed,indicating the transformation of the scar tissues into normal skin.2.The wounds of the rats in the astaxanthin group and model group gradually shrunk over time.At the 4th week,the quality of skin healing in the astaxanthin group was better than that of the model group.The wound healing rates of the model group and the administration group both increased logarithmically with the healing time,and the astaxanthin group showed faster healing and the higher healing rate,with the most significant difference at day 7 and 9(P<0.01).On post-operative day 21,the scar transformation rates were 1.0 in the astaxanthin group.At the 4th week,the HI was evidently higher in the model group compared to the astaxanthin group(P<0.05).At week 2,there was no significant difference in NA between the two groups.At week 3,NA was lower in the astaxanthin group compared to the model group(P<0.01).At week 4,NA was more significantly lower in the astaxanthin group(P<0.01).At week2,AA was higher in the astaxanthin group compared to the model group(P<0.05);while at weeks 3 and 4,it was significantly lower in the astaxanthin group compared to the model group(P<0.01).At the 4th week,the expression of Col-I,Col-III and TGF-β1 in the astaxanthin group were lower than that in the model group(P<0.01);After 4 weeks of treatment,the expression of α-SMA protein and IL-6 inflammatory factor was lower in the astaxanthin group compared to the model group(P<0.01).3.Proteomics: Fold change 1.5 was selected for bioinformatics analysis,i.e.199up-regulated proteins and 241 down-regulated proteins.Among them,the expression of Atg12 protein and IL-33 were down-regulated(P<0.05).The results of protein function annotation showed that the number of proteins annotated by GO was 4917 and the number of proteins annotated by KEGG pathway was 3230.And then KEGG functional classification found that the differential proteins associated with signal transduction function included Akt,Atg12,Smads,MAPK,etc.Among them,Akt2 isoform had a P-value of <0.01 for A/M,a fold change of 1.2 and an elevated expression level.PI3K/Akt signaling pathway P<0.01,and the enrichment fold >2.0,indicating that the pathway was significantly different in the two comparison groups.SPP1 was down-regulated in this pathway.It was newly found that the NF-κB signaling pathway was also significantly different between the two comparison groups.Among them,PLAU and LBP proteins were down-regulated.GO enrichment analysis showed that the proteins expression of the two comparison groups was significantly different in the regulation of wound healing,response to inflammatory response and differentiation of keratin-forming cells into fibroblasts,macroscopically manifesting as differences in wound healing as well as scar formation.Western Blotting: After 4 weeks of treatment,compared with the model group,apoptotic protein Bax and autophagy-related factor p62 were significantly increased in the astaxanthin group(P<0.05).The expression of m TOR and Akt protein in the astaxanthin group were both higher than that in the model group(P<0.05).Immunofluorescence: After 4 weeks of treatment,the mean optical density of Caspase3 in the astaxanthin group was higher than that of the model group(P<0.01),and the difference with the blank control group was not significant(P>0.05).The mean optical density of Bcl-2 factor was lower in the astaxanthin group than in the model group(P<0.01);The mean optical density of p62 was increased by the astaxanthin intervention compared to the model group(P<0.05).Conclusions:1.Astaxanthin nano-emulsion has an improving effect on hyperplastic scar of rabbit ears.2.Astaxanthin nano-emulsion can promote the healing of full-thickness skin defects in rats,and can reduce the proliferation of fibroblasts and collagen production,and decrease the ECM content,and reduce inflammation,inhibit wound contraction and scar contracture,and then improve the occurrence and development of hyperplastic scar after healing.3.The mechanism of the anti-dermal fibrosis effect of astaxanthin nano-emulsion is related to its induction of apoptosis. |
PDF Full Text Request