| Objective:To investigate the effects of subcutaneous injection of astaxanthin（AST）on skin structure,oxidative stress damage and on matrix metalloproteinase expression in photodamaged mice.Methods:Sixty-four 8-week-old female healthy SPF-grade BALB/c mice were divided into youth group（youth group）,blank control group（control group）,photoaging model group（model group,UV）,solvent group（solvent group,UV+soybean oil for injection）,astaxanthin low-dose group（AST-L,AST10 mg/kg+soybean oil for injection）,astaxanthin medium-dose group（AST-M,AST50 mg/kg+soybean oil for injection）,astaxanthin high-dose group（AST-H,AST100 mg/kg+soybean oil for injection）,and astaxanthin medium-dose group（AST50 mg/kg+soybean oil for injection）.AST10mg/kg+soybean oil for injection),astaxanthin medium dose group（AST-M,AST50mg/kg+soybean oil for injection）,astaxanthin high dose group（AST-H,AST100mg/kg+soybean oil for injection）,and minimal erythema group（MED group）,8mice in each group.The minimum erythema amount of mice in the MED group was 2.16J/cm~2（UVA）and 0.50 J/cm~2（UVB）measured in the pre-experiment.Except for the CONTROL group and YOUTH group,the remaining five groups were irradiated with combined UVA+UVB for 9 weeks without interruption,and the cumulative radiation exposure was 234.36 J/cm~2（UVA）and 54.68 J/cm~2（UVB）to prepare the mouse photoaging model.Thereafter,the mice were irradiated continuously on a daily basis,and the agents were injected subcutaneously in groups 30 min before irradiation on the 7th day,once every 7 days,for a total of 9 times.After the last irradiation,the skin images on the back of mice were recorded and graded using the photoaging Glogau typing method.Then the mice were executed by sodium pentobarbital overdose anesthesia,and the skin of the irradiated area on the back of the mice was cut（YOUTH group was cut before the photoaging model was made）.1/4 of the skin tissues were fixed in 4%paraformaldehyde solution and then HE staining,Masson staining and Weigert staining were performed to observe the changes of skin tissues,collagen fibers and elastic fibers in mice,and 3/4 of the skin tissues were homogenized by The expression levels of MDA,SOD,GSH-Px,Hyp and MMP-1 were measured by ELISA.Body weight,elastin and collagen fiber percentage,MDA,SOD,GSH-Px,Hyp,MMP-1 expression were expressed as mean±standard deviation（X±s）,and ANOVA was used for comparison between groups,rank sum test was used for comparison between groups for rank data（Glogau typing）,and Bonferroni method was used for two-way comparison,and experimental data were analyzed using SPSS 26.0 and Graph Pad Prism 5.0 software for statistical processingResults:1.General situation:All groups of mice survived during the experimental cycle without any changes in living habits such as eating and sleeping after injection,and no behavioral changes such as laziness and abnormal restlessness.The MODEL and SOLVENT groups showed typical photoaging skin tissue appearance,while the AST-L group showed obvious skin folds,a small amount of pigmentation and hemorrhagic spots,and obvious keratinization,and the AST-M and AST-H groups were difficult to distinguish with the naked eye.The body weight of mice in all groups increased during the experimental period,but only the CONTROL group,AST-M group and AST-H group significantly increased their body weight before and after the experiment（P<0.05）.Mouse skin Glogau photoaging typing results:CONTROL group was the same as YOUTH group（P>0.05）.MODEL group,SOLVENT group and AST-L group were all higher than CONTROL group（P<0.05）,AST-M group and AST-H group were the same as CONTROL group（P>0.05）.SOLVENT group,AST-L group and AST-M group were all the same as MODEL group（P<0.05）.AST-L group,AST-M group,and AST-H group were all typed the same as MODEL group（P>0.05）,and AST-H group was typed lower than MODEL group（P<0.05）.AST-L group,AST-M group,and AST-H group were typed the same as SOLVENT group（P>0.05）.AST-L group,AST-M group,and AST-H group were all typed the same between groups（P>0.05）2.Histological changes in the skinHE staining:The epidermal ridges were gentle in the CONTROL group.Compared with the CONTROL and YOUTH groups,the epidermis and dermis of each group receiving UV irradiation were thickened to different degrees,and some areas showed papillary-like hyperplasia and individual epidermal cells showed heterotypy,and the thickening of the epidermis and dermis of AST-H was the lowest.Masson staining and collagen fiber area:The collagen fibers in the CONTROL group were arranged in parallel,and the collagen fibers in each group receiving UV irradiation were disordered and partially clumped.the YOUTH group,MODEL group,SOLVENT group,AST-L group,AST-M group,and AST-H group were lower than the CONTROL group（P<0.05）.the SOLVENT group and AST-M group were lower than the MODEL group（P<0.05）,and the AST-L and AST-H groups were the same as the MODEL group（P>0.05）.the AST-L,AST-M,and AST-H groups were the same as the SOLVENT group（P>0.05）.the AST-L,AST-M,and AST-H groups were the same between groups（P>0.05）.Weigert staining and elastic fiber area:The elastic fibers in the CONTROL group were elongated and scattered among the collagen fibers,and the elastic fibers in the UV-irradiated groups were thickened and broken to different degrees.Except for MODEL group,YOUTH group,SOLVENT group,AST-L group,AST-M group,AST-H group were the same as CONTROL group（P>0.05）.AST-L group and AST-M group were lower than MODEL group（P<0.05）,SOLVENT group and AST-H group were the same as MODEL group（P>0.05）.AST-L group,AST-M group and AST-H group were the same as SOLVENT group（P>0.05）.AST-L group,AST-M group and AST-H group were the same between groups（P>0.05）.3.Skin tissue MDA,SOD,Hyp,GSH-Px,MMP-1 expressionMDA expression:The CONTROL group was lower than the YOUTH group（P<0.05）.Except for the AST-H group,which was the same as the CONTROL group（P>0.05）,the MODEL,SOLVENT,AST-L,and AST-M groups all had higher MDA expression than the CONTROL group（P<0.05）.The SOLVENT,AST-L,AST-M group and AST-H group were significantly lower than MODEL group（P<0.001）.AST-M group and AST-H group were lower than SOLVENT group（P<0.05）;AST-L group was the same as SOLVENT group（P>0.05）.AST-L group was the same as AST-M group（P>0.05）.AST-H group was lower than AST-L group and AST-M group（P<0.05）.SOD expression:The CONTROL group was higher than the YOUTH group（P<0.05）.SOD in the MODEL,SOLVENT,AST-L,AST-M,and AST-H groups was lower than that in the CONTROL group（P<0.05）.The SOLVENT,AST-L,AST-M,and AST-H groups were significantly higher than that in the MODEL group（P<0.001）.AST-M group and AST-H group were higher than SOLVENT group（P<0.05）,AST-L group was the same as SOLVENT group（P>0.05）.AST-M group was the same as AST-L group（P>0.05）,AST-H group was higher than AST-L group（P<0.05）.AST-H group was the same as AST-M group（P>0.05）.GSH-Px expression:The CONTROL group was the same as the YOUTH group（P>0.05）.GSH-Px was higher in the MODEL,SOLVENT,AST-L,and AST-M groups than in the CONTROL group（P<0.05）,and the AST-H group was the same as the CONTROL group（P>0.05）.The SOLVENT,AST-L,AST-M group,and AST-H group were significantly lower than MODEL group（P<0.001）.AST-L group,AST-M group,and AST-H group were significantly lower than SOLVENT group（P<0.001）.AST-L group was the same as AST-M group（P>0.05）.AST-H group was lower than AST-L group and AST-M group（P<0.05）.Hyp expression:The CONTROL group was significantly lower than YOUTH group（P<0.001）.Hyp was higher than CONTROL group in MODEL group,SOLVENT group,AST-L group（P<0.001）,AST-M group and AST-H group were the same as CONTROL group（P>0.05）.SOLVENT group,AST-L group,AST-M group and AST-H group were significantly lower than MODEL group（P<0.001）.AST-M group and AST-H group were significantly lower than SOLVENT group（P<0.001）,and AST-L group was the same as SOLVENT group（P>0.05）.AST-M group and AST-H group were lower than AST-L group（P<0.05）.AST-H group was the same as AST-M group（P>0.05）.MMP-1 expression:The CONTROL group was lower than the YOUTH group（P<0.05）.MMP-1 was higher in the MODEL,SOLVENT,and AST-L groups than in the CONTROL group（P<0.05）,and the AST-M and AST-H groups were the same as the CONTROL group（P>0.05）.The SOLVENT,AST-L,and AST-M group,AST-H group were significantly lower than MODEL group（P<0.001）.AST-L group,AST-M group,AST-H group were lower than SOLVENT group（P≤0.001）.AST-M group,AST-H group were lower than AST-L group（P<0.05）.AST-M group was the same as AST-H group（P>0.05）.Conclusion:AST subcutaneous injection decreases the expression of MDA,Hyp,GSH-Px and MMP-1,increases the expression of SOD and reduces the breakage of collagen and elastic fibers in photoaged skin in a concentration-dependent manner,thus reducing the appearance and histological changes of skin photoaging. |
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