| Skin photoaging is accelerated skin aging that results from long-term solar ultraviolet radiation.As a skin disease caused by external factors,it not only undermines people's appearance and mentality,but also has an etiological connection with the occurrence of skin cancer.The occurrence of photoaging has grown particularly in recent years due to increasing ultraviolet radiation from the ozone layer to the ground,excessive use of sunbathing or the use of phototherapy equipment.Therefore,the exploration of natural and effective active substances for the prevention and treatment of skin photoaging has become the research focus in the field of life science,medicine and aesthetic medicine.In this thesis,18? glycyrrhetinic acid(GA),a natural active substance,was studied as a drug against skin photoaging.GA nanocrystals(NGA)were combined with modified amphiphilic chitosan(ACS)to obtain amphiphilic chitosan /18 ? glycyrrhetinic acid nanocomposite(ANGA),and the transdermal and anti-skin photoaging effect of ANGA was researched to reveal its permeability and how it prevented skin photoaging.(1)NGA was prepared by high-pressure homogenization and represented with dynamic light scattering,electrophoretic light scattering,X-ray diffraction(XRD),scanning electron microscopy(SEM),and thermogravimetric analysis.The results showed that the prepared NGA suspension was stable and negatively charged(-45.9 ±1.3),and the particles were about the same size,with an average particle size of 288.6± 7.3 nm.Compared with coarse GA,the crystallinity and thermal stability of NGA was significantly reduced,while the solubility increased markedly.Franz diffusion cells were used to investigate the in vitro transdermal permeation of NGA.The results showed that the capability of NGA to penetrate the skin was 25% higher than that of coarse 18? glycyrrhizic acid(CGA).The TPA-induced mouse ear inflammation model was used to investigate the anti-inflammatory effect of NGA.The results confirmed that compared with CGA and commercial indomethacin,NGA significantly reduced the difference in weight and thickness of the mouse's ear.Furthermore,NGA has inhibited the expression of tumor necrosis factor-?(TNF-?),interleukin 1?(IL-1?)and interleukin 6(IL-6),lowered the myeloperoxidase(MPO)activity,and reduced the infiltration and aggregation of neutrophils in a mouse's ear tissues,showing better antiinflammatory efficacy.(2)Sialic acid-chitosan-18? glycyrrhizic acid(ACS)was prepared by means of EDC/NHS coupling chemical reaction.It was represented with infrared,thermogravimetric,and electrophoretic light scattering analysis.The results showed that,compared with pure chitosan(CS),the thermal stability of CS derivatives was reduced,and the solubility in water and PBS(p H 7.2)changed from water-insoluble to easily water-soluble.In PBS(p H 4.0 and p H 7.2)buffers,the molecule of the ACS was positively charged.Furthermore,the blood compatibility and the cytotoxicity to human immortalized keratinocytes(Ha Ca T)were evaluated by CCK-8.The results showed that ACS had good blood compatibility and was non-toxic to Ha Ca T when the concentration of ACS was under 1 mg/m L.(3)ANGA composites were prepared by electrostatic adsorption,and the ratio of ACS to NGA was examined on the basis of Zeta potential and the average particle size.The results showed that when the mass ratio of ACS to NGA was 1:10,a stable dispersion system could be obtained,and the ANGA composites were positively charged.The changes in particle sizes and Zeta potential of the ANGA complex before and after autoclaving were also investigated.The results showed that after autoclaving,the average particle size and Zeta potential of the ANGA complex did not change significantly compared with those before autoclaving.CCK-8 was used to evaluate the toxicity of ANGA composites to Ha Ca T.The results showed that when the concentration was higher than 40 ?g/m L,the survival rate of Ha Ca T decreased significantly.Below this concentration,there was no significant inhibitory effect on the proliferation of Ha Ca T.Meanwhile,Franz diffusion cells were used to investigate the in vitro transdermal permeability of the NGA and ANGA composites.The results indicated that the cumulative permeability of the ANGA composites reached 75.3%,which was 18.8% higher than that of the NGA with a cumulative permeability of 56.5%.(4)A UV-irradiated photoaging model was constructed to study the anti-skin photoaging efficacy of ANGA composites.The results showed that ANGA composites could significantly improve UV-induced mouse skin photoaging such as coarse and deep wrinkles,laxity and leather-like appearance,increase the water content markedly and keep skin full in UV-induced mouse photoaged skin,significantly improve the degeneration of physiological structures of UV-induced photoaged mouse skin,especially structural changes such as hyperplasia of the epidermis,fracture and caking of collagen and elastic fibers in the dermis,and improve skin elasticity.Furthermore,the morphology,structure and content of collagen fiber,and the expression of MMP-1and MMP-3 were investigated by Masson,Sirius red staining and ELISA.The results showed that the ANGA composites significantly inhibited the abnormal expression of MMP-1 and MMP-3,thus inhibiting the degradation of collagen fibers(especially type I collagen fibers)in the dermis and increasing collagen content,which in turn alleviated structural damage.(5)Western Blot,q PCR,and ELISA were used to investigate NF-?B and Keap1-Nrf2-ARE signaling pathways involved in the anti-photoaging mechanism of ANGA composites.The results showed that the ANGA composites could effectively upregulate the I-?B? gene,down-regulate the nuclear factor p65 gene and the expression of TNF-?,IL-1?,and IL-6 subsequently,and thus inhibited the inflammatory reaction induced by UV.Meanwhile,ANGA composites effectively reduced the anomalous increase of UV-induced ROS and MDA in mouse skin tissues,activated Nrf2 genes and then up-regulated the expression of downstream target genes,such as SOD,GSH-Px,CAT,HO-1 and NQO1.Finally,ANGA composites quenched excessive free radicals and slowed down the oxidative stress induced by UV. |
PDF Full Text Request