| [Objective] To explore whether shikonin can reduce the degree of oxidative stress in fibroblasts,inhibit the development of skin inflammation in burns,and elucidate the mechanism of shikonin improving oxidative stress in fibroblasts and repairing skin wounds caused by burns.[Methods](1)Using a mouse second degree burn model to explore the therapeutic effect of shikonin.Observing the repair of burned and scalded skin in mice treated with shikonin through tissue pathological sections;Using in vivo tracing proliferation cell method,analyze the proliferation of skin cells in burned and scalded skin treated with shikonin;Immunofluorescence staining technique was used to evaluate the distribution of CK14 and CK19 proteins in burned and scalded skin after the action of shikonin,as well as the effect of shikonin on fibroblasts at the wound site;Real time fluorescence quantitative technology is used to detect the expression of inflammatory factors in burned and scalded skin by shikonin;Observe the collagen recovery of burned and scalded skin after the action of shikonin under polarized light with Sirius red staining.(2)Establishing a fibroblast oxidative damage model using hydrogen peroxide stimulation to explore the protective mechanism of shikonin on fibroblasts.Detect the effect of shikonin on fibroblast viability under stress using tetramethylazolamide colorimetry;α-SMA labeled cellular immunofluorescence assay and Transwell chamber assay were used to observe the improvement and recovery ability of shikonin on cytoskeleton and migration of stress fibroblasts;Ed U detection of the effect of shikonin on the proliferation ability of fibroblasts under oxidative stress;Observe the fluorescence intensity of intracellular ROS to preliminarily evaluate whether shikonin can effectively alleviate oxidative stress in fibroblasts.(3)Use a flow cytometry to sort the fibroblasts from the skin of newborn mice and inject the identified fibroblasts into the burn site,and combine them with shikonin to treat the second degree burn and scald skin of mice.The effect of combined treatment was observed by histopathological section,and the effect of combined treatment on JNK,P38 MAPK,P65 NF-κB in burned and scalded skin was investigated by Western blot and apoptosis signal pathway and immunohistochemical experiments were conducted to observe the effect of combined therapy on the immune status of burned and scalded skin.[Results](1)Through routine pathological staining results,it was found that shikonin can accelerate the regeneration of the epidermal layer and tissue repair in the dermis of traumatic skin.The results of immunofluorescence staining showed that in second degree burned skin,shikonin can promote the proliferation of skin cells and the normal distribution of cytokeratin in the damaged area.And the early application of shikonin can effectively prevent the occurrence of excessive inflammatory reactions,increase the number of fibroblasts in the damaged area,and promote the structural reconstruction of skin collagen,playing a positive role in the treatment of scarless healing of burned and scalded skin.(2)After shikonin treatment,the activity of stress fibroblasts was significantly increased,which promoted the cytoskeleton and cell migration,and the proliferation ability was restored,and the percentage of ROS positive cells in shikonin group was significantly reduced(P<0.001).(3)The combination of shikonin and fibroblast therapy can effectively promote the repair of second degree burned skin,inhibit the phosphorylation levels of JNK,P38,P65,and the expression of pro apoptotic protein BAX,increase the expression of anti apoptotic protein BCL2,and reduce the large aggregation of macrophages and neutrophils in burned skin.[Conclusion] Shikonin can effectively promote the healing of skin tissue and collagen structure reconstruction in mice with second degree burns,alleviate cellular oxidative stress and excessive immune inflammation caused by external stimuli.The combination of shikonin and fibroblasts has a certain therapeutic effect on burned and scalded skin,and it has been preliminarily found that this treatment method can inhibit the expression of JNK,P38 MAPK,NF-κB and mitochondrion mediated apoptosis signal pathway target proteins and alleviate the excessive inflammatory response of burned and scalded skin. |
PDF Full Text Request