| BackgroundSkin photoaging is a series of damage to skin tissues and functions caused by long-term repeated exposure to ultraviolet(UV)light,manifested as erythema,roughness,thickening,wrinkle formation and pigmentation at the exposed parts of the skin,and severe damage may appear as benign or malignant tumors.UV exposure-induced skin photoaging is associated with oxidative stress,inflammatory responses,and degradation of collagen fibers mediated by matrix metalloproteinases(MMPs).Dendrobium officinale polysaccharides(DOP)is the main component of D.officinale,and DOP has great potential for application in skin diseases due to its moisturizing,antioxidant,immune enhancing and inflammation suppressing properties.However,there is still a lack of in vivo experiments to validate the bioactivity of DOP,and few reports focus on the preventive and/or therapeutic effects of DOP on skin photoaging.DOP is limited in transdermal absorption due to its molecular properties.The W/O/W multilayer emulsion(ME)is a mature drug delivery system,which have the advantages of enhancing drug absorption,promoting slow release and improving drug stability,and have been widely used in clinical practice.Therefore,coating DOP into multilayer emulsions may facilitate the transdermal absorption of DOP and thus further exert its protective effect on the skin.In this study,a mouse model of photoaging was established using combined UVA+UVB simulated sunlight irradiation for 16 weeks,and the protective effect of DOP on skin photoaging was initially explored;and on this basis,the difference between DOP solution and DOP multilayer emulsion in improving skin photoaging in mice was evaluated and compared;in addition,the effect of DOP on proliferation,apoptosis and autophagy of Ha Ca T cells after UV radiation was further explored by the Ha Ca T photoaging model,to provide a new idea for anti-aging research,open up a new field for the clinical application of Dendrobium officinale and its related traditional Chinese medicine preparations,and further develop the application of traditional Chinese medicine in dermatology.Materials and Methods:Part I.Preliminary investigation on the protective effect of DOP on skin photoaging in mice1.Experimental grouping:Female BALB/C mice were randomly divided into 6groups,including normal control group(Control),simple light irradiation group(UVA+UVB),positive control group(VE),low-dose DOP solution group(5 mg/m L,DOP-L),medium-dose DOP solution group(10 mg/m L,DOP-M)and high-dose DOP solution group(50 mg/m L,DOP-H).2.Mouse photoaging model establishment:Mice were irradiated with UV for 16weeks,with a cumulative exposure of 148.35 J/cm~2of UVA and 17.2 J/cm~2of UVB.3.Mouse physical appearance evaluation:Mice skin was photographed every 4weeks.In addition,the thickness of double-layered skin and elasticity recovery time of mice were measured by skin fold thickness measurement and skin lift test.4.Histomorphometry measurements:The structural changes of mouse skin were observed by HE,Masson and EVG staining of pathological sections.5.Antioxidant indexes assay:The changes of SOD,GSH and MDA expression in skin tissues were measured to reflect the changes of antioxidant enzymes.6.Inflammatory factors assay:The changes of TNF-α,IL-1β,IL-6 and NF-κB content in the homogenate of mouse skin tissue were detected by ELISA.7.Matrix metalloproteinase assay:The differences of MMP-1 and MMP-3expression in the homogenate of mouse skin tissue were detected by ELISA.Part II Effect of DOP multilayer emulsion on improving UV induced skin photoaging in mice1.DOP multilayer emulsion preparation:W/O/W type multilayer emulsions containing different concentrations of DOP were prepared by a two-step emulsification method,and the structure of DOP multilayer emulsions was observed under a microscope.2.Experimental grouping and model building:Female BALB/C mice were randomly divided into 9 groups,the Control,UVA+UVB,VE,DOP-L,DOP-M and DOP-H groups were the same as part I,low-dose DOP multilayer emulsion group(0.5%),medium-dose DOP multilayer emulsion group(1%)and high-dose DOP multilayer emulsion group(5%).Mice were irradiated with UVA+UVB for 16 weeks to establish a photoaging model.3.Determination of the properties of DOP multilayer emulsion:The thermal stability of DOP multilayer emulsion,the emulsion loading,encapsulation rate and transdermal release of DOP multilayer emulsion were tested.4.The methods of gross appearance evaluation,histomorphology observation,antioxidant indexes detection,inflammatory cytokines and matrix metalloproteinases detection were the same as those in the first part.Part III Photoprotective effects of DOP through inhibition of oxidative stress and enhancement of autophagy1.Cell proliferation activity assay:Explore the effects of different UV irradiation doses and different concentrations of DOP on the proliferation activity of Ha Ca T cells and the proliferation-promoting effect of DOP on photoaged Ha Ca T cells by CCK8 assay.2.Reactive oxygen assay:Detect the intracellular ROS levels of Ha Ca T cells via flow cytometry.3.Cell cycle assay:Detect the effect of DOP on the cell cycle of photoaged Ha Ca T cells via flow cytometry.4.DNA damage assay:Detect the DNA damage markerγH2AX via flow cytometry.5.Apoptosis assay:Detect the effect of DOP on apoptosis of photoaged Ha Ca T cells via flow cytometry.6.Autophagy level assay:Detect cellular autophagic flow using autophagic double-labeled adenovirus(m RFP-GFP-LC3)transfection under confocal microscopy;Use western blot to detect autophagic expression levels in photoaged mice.Results:Part I.Preliminary investigation on the protective effect of DOP on skin photoaging in mice1.After 16 weeks of combined UVA+UVB irradiation,the skin of mice showed wide transverse wrinkles,roughness and hardness on the back,accompanied by severe skin hyperplasia and local erythema and erosion,showing typical photoaging phenomena;in addition,the double-layer thickness and elasticity recovery time of the skin of mice in the UVA+UVB group increased significantly(P<0.0001);while DOP solutions(especially at high doses)could effectively improve the skin lesions of mice and reduce the skin thickness and recovery time(P<0.001).2.Pathological sections manifested that the epidermis of mice in the UVA+UVB group showed obvious irregular thickening,consisting of 6-10 layers of keratin-forming cells,disorganized collagen fiber structure,and some fibers were thickened,broken and homogenized,and elastic fibers were degenerated and reduced in number;high concentration of DOP could effectively improve the structural damage of skin tissue caused by UV.3.Compared with mice in the UVA+UVB group,the skin tissues of mice in the DOP solution(especially at high doses)group showed significantly higher SOD and GSH activity,reduced MDA content,significantly lower expression of inflammatory factors including TNF-α,IL-1βand IL-6,reduced NF-κB expression,and lower MMP-1 and MMP-3 content(P<0.05).Part II Effect of DOP multilayer emulsion on improving UV induced skin photoaging in mice1.Smooth and delicate multilayer emulsion with good continuity was produced by a two-step emulsification method.Microscopic observation showed that DOP emulsion droplets were multilayer concentric vesicles similar to onion structure,which confirmed its multilayer composite structure.2.The structure of DOP multilayer emulsion was stable at 4℃and 25℃;While the normal structure was destroyed at 70℃,and the emulsion showed delamination and liquid precipitation;A large number of broken structural fragments could be observed under microscope;The encapsulation rates of DOP multilayer emulsions at low,medium and high doses were 93.58%,92.03%and 93.91%,respectively;Transdermal release assay showed that 6-hour cumulative DOP release:DOP-H(ME):1166.06μg/cm~2>DOP-H:624.17μg/cm~2.3.The results of animal experiments showed that DOP multilayer emulsion improved the skin lesions of photoaged mice better than DOP solution,and the skin of mice in the high-dose DOP multilayer emulsion group was in the best condition among all UV-exposed mice,with overall smoothness and elasticity,no skin hyperplasia and deep and wide transverse wrinkles.4.Compared with the UVA+UVB group,the skin condition of the mice in the medium-and high-dose DOP multilayer emulsion group was improved significantly.The epidermal thickness of mice was significantly reduced and only consisted of 2-3layers of keratin-forming cells,the dermal fiber bundles were neatly arranged with lax interstices,and the elastic fibers were normal in shape and evenly distributed.5.The antioxidant index measurement showed that high-dose DOP solution and medium-and high-dose DOP multilayer emulsion could stably and effectively enhance SOD and GSH activities and reduce MDA content in the skin of photoaged mice,so as to resist the interference of long-term UV radiation,among which the high-dose multilayer emulsion group had the best effect(P<0.05).6.The expression of TNF-α,IL-1β,IL-6,NF-κB,MMP-1 and MMP-3 in the DOP multilayer emulsion group was significantly reduced compared with that in the UVA+UVB group(P<0.05),and the levels of inflammatory factors and matrix metalloproteinases in the high-dose DOP multilayer emulsion group were lower than those in the high-dose DOP solution group.Part III Photoprotective effects of DOP through inhibition of oxidative stress and enhancement of autophagy1.The proliferative activity of Ha Ca T cells was lowest when they were irradiated with UVA(5 J/cm~2)+UVB(19.2 m J/cm~2)(P<0.0001);DOP at 80 mg/L significantly increased the proliferative activity of Ha Ca T cells(P<0.0001);Compared with the UVA+UVB group,the medium and high doses of DOP treatment could effectively enhance the proliferative activity of photoaged Ha Ca T cells(P<0.05).2.UV exposure significantly increased ROS expression level and enhanced mean fluorescence intensity of ROS in Ha Ca T cells,and 80 mg/L DOP pretreatment significantly reduced ROS expression(P<0.05).3.UV radiation caused S-phase block in Ha Ca T cells,while the proportion of G1phase was increased and S-phase block was significantly reduced in photoaged Ha Ca T cells pretreated with 40 and 80 mg/L DOP(P<0.05).4.The expression levels ofγH2AX in G1 phase,S phase,G2/M phase and total cell cycle were significantly increased in photoaged Ha Ca T cells,and DOP pretreatment significantly reduced the expression levels ofγH2AX(P<0.05).5.Combined UV radiation increased the apoptosis of Ha Ca T cells,while DOP pretreatment at 40,80 and 160 mg/L were all effective in reducing the total apoptosis level of photoaged Ha Ca T cells(P<0.05).6.DOP up-regulated the autophagy level of photoaged Ha Ca T cells,enhanced the expression ratio of LC3B II/I and decreased the expression level of P62 in skin tissues of photoaged mice.Conclusion:The skin photoaging model of mice was successfully established by UVA+UVB irradiation.Topical DOP solution,especially high-dose DOP solution,could improve photoaging manifestations such as rough and thickened skin and wide wrinkles by enhancing the body’s antioxidant system,reducing inflammatory response,and inhibiting the expression of MMPs to counteract the damage of epidermal and dermal collagen fibers and elastic fibers by long-term UV radiation.A more stable DOP multilayer emulsion was produced by a two-step emulsification method,which showed concentric multi-capsular structure under microscopy.Compared with the DOP solution,the DOP multilayer emulsion promoted the transdermal absorption of DOP,thus improving the photoaging performance caused by long-term UV radiation more effectively.DOP could enhance the proliferation activity of photoaged Ha Ca T cells,reduce the level of oxidative stress,apoptosis and cell cycle arrest,and improve autophagy to play a protective role in photoaging.DOP multilayer emulsion can effectively enhance the level of autophagy in the skin tissue of photoaged mice to achieve photoaging protection. |
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