The Treatment Effect And Mechanisms Of Hypericin Photodynamic Therapy On Port Wine Stains

Port wine stains(PWSs) are a congenital form of vascular malformation, which is characteristic of ectatic capillaries, postcapillary venules and increased vascular density in the diseased tissue. PWSs commonly occur on the face and neck. The clinical presentations are reddish, dark red or purple spots. Over time, the diameters of the vessels increase, resulting in a worsening clinical condition and a progressively disfiguring lesion. Diffuse thickening of the lesion gradually occurs, with approximately two-thirds of patients developing hypertrophic or nodular lesions by age 50. The presence of a PWSs tends to impede the emotional and psychosocial development of affected individuals, as well as their familie. Studies showed that photodynamic therapy(PDT) has been successfully used in treating PWSs. However, hematoporphyrin(HP) and its derivatives, which have been used as conventional photosensitizers(PS), have not yet obtained the satisfactory treatment effect in treating PWSs. The elimination half-life for HP was more than several days, thereby leading to skin photosensitivity to sunshine and toxicity to visible light(i.e., dark toxicity) for long. Thus, it is necessary to find new PS with shorter half-lives but more efficient in therapeutic effect, and thereby relatively reducing dark toxicity and are more good for PDT to treat PWSs in clinical application.Hypericin(HY) is a naturally Hypericum perforatum extract. Importantly, compared with HP, HY were found to be shorter circulation half-life, which might in part solve the drawback of the long-term dark toxicity that causes by HP treatment.In this study, we used Roman chicken cockscomb and human umbilical vein endothelial cells(HUVECs) as model of human PWSs in vivo and in vitro to investigate the effect of HY-PDT in treating PWSs. By comparing with that of HP-PDT, the efficacy of HY-PDT was then evaluatd. Furthermore, its mechanisms underlying HY-PDT have also been investigated. It has presented theoretical basis to exploit HY as a promising PS to substitute HP using in clinical treatment of PWSs. The concrete research content is as follows:The first chapter is background and objective, providing theoretic support for this study.The second chapter of this study, we investigated the therapeutic effect and mechanisms of HY-PDT on Roman chicken cockscombs(animal model of PWSs). HP was used as postived PS. Our results found that HY combined 585 nm PDT indeed damaged the Ectatic capillaries in chicken cockscomb model by damaging dermal capillary endothelial cell mitochondria function and promoting the release of cytochrome c(cyto c). Impotantly, it also showed HY-PDT exhibited a much stronger effect than HP-PDT. All the above results suggested one of the mechanisms of inhibiting angiogenesis in the Ectatic capillaries in chicken cockscomb model is activating the intrinsic mitochondrial-mediated caspase pathway.In the third chapter, the photocytotoxic effects of HY on HUVECs was explored. HP was used as postived PS. The effect of HY combined 585 nm PDT on HUVECs was evaluated by observations of proliferation, migration and lumen formation. First. HY-PDT indeed exhibited a strong effect on inhibition of the growth of HUVECs through MTT assay and showed typical features of apoptosis by morphological observation,. Furthermore, Matrigel experiment and cell detachment test also showed that HY-PDT inhibited cell migration and lumen formation in dose-dependent way. In addition, HY-PDT exhibited a stronger effect than HP-PDT at a lower concentration.In the fourth chapter, the mechanisms of HY-PDT were further investigated. HY-PDT significantly inhibited cell viability and induced apoptosis of HUVECs by detections of relative proteins and RNA using ELISAs, western blot and qRT-PCR. Our findings indicated that HY-PDT promoted reactive oxygen species(ROS) production, leading to necrosis and initiation of the inflammatory responses. In addition, the data also showed that it induced apoptosis of HUVECs via triggerring the mitochondrial-mediated caspase pathway and preventing the VEGF-A-mediated PI3K/Akt(vascular endothelial growth factor-A/ phosphatidylinositol 3 kinase/protein kinase B,VEGF-A/PI3K/Akt) pathway. Altogether, through the above-mentioned pathways, HY-PDT could induce HUVECs apoptosis by activating caspase cascade, indicating induction of apoptosis may be one of the mechanisms of inhibiting angiogenesis.In conclusion, in our investigations, the treatment effect and mechanisms of HY-PDT on PWSs were studied both in vivo and in vitro, using Roman chicken cockscombs and HUVECs, respectively. Firstly, it showed that, in chicken cockscomb model, HY-PDT indeed exhibited more satisfactory therapeutic effect than HP-PDT by damaging dermal capillary endothelial cell mitochondria function and promoting the release of cyto c. Secondly, it aiso showed that, in HUVECs model, HY-PDT indeed inhibited angiogenesis more effectively than HP-PDT by inhibiting HUVEC cell proliferation, migration and lumen formation. Thirdly, in HUVECs, HY-PDT promoted ROS production, leading to necrosis and initiation of the inflammatory responses. In addition, it induced apoptosis of HUVECs via triggerring the mitochondrial-mediated caspase pathway and preventing the VEGF-A/ PI3K/Akt pathway. Altogether, through the above-mentioned pathways, HY-PDT could induce HUVECs apoptosis by activating caspase cascade, which may be one of the mechanisms of inhibiting angiogenesis. Taken together, HY-PDT exhibited satifactory therapeutic effect on PWSs through inducing cell apoptisis to inhibit angiogenesis both in chicken and HUVECs models at a lower concentration, suggesting that HY could be a promising PS to substitute HP owing to its shorter elimination half-life but higher photocytotoxic effects compared to those of HP in treating PWSs in clinical application.