| Backgrounds and Objectives:Port wine stains,also known as nevus flammeus or nevus telangiectaticus,is a common congenital dermal superficial capillary network expansion deformity.The pathogenesis is very complicated and still not clear.The current research suggests that its pathogenesis may include five aspects of endothelial progenitor cells differentiate and develop abnormally,genetic mutation,abnormal expression of vascular growth-related factors,abnormal proportion of vessels and nerves,and abnormal activation of protein kinase in the vasculature.Based on the principle of selective photothermal action,the pulsed dye laser(PDL)has become the gold standard for the treatment of port wine stains.Nevertheless,the rate of complete resolution of the lesions is still less than 25%,and a large number of PWS patients may develop resistance to PDL after a prolonged period of treatment,also the recurrence rate is very high after many years.Therefore,we need for a new treatment that is more effective and safer in clinical practice.Photodynamic therapy(PDT)is a new technology for the diagnosis and treatment of diseases using the principle of photodynamic effects.The basic principle is to use a photosensitizer or a non-toxic dye substance to undergo a series of biochemical reactions with oxygen molecules in surrounding tissues after irradiation with light or laser light of a specific wavelength.It can produce reactive oxygen species and singlet oxygen.Eventually lead to apoptosis,autophagy and even necrosis,vascular occlusion,injury,and inflammation and immune response in surrounding tissues.Hemoporfin is a new type of second-generation photosensitizer.Compared with first generation PDT drugs such as hematoporphyrin derivative,Hemoporfin has a stable structure,stronger photodynamic efficiency,higher photoactivity,faster clearance rate and lower toxicity.Hemoporfin mediated photodynamic therapy has been successfully used in the treatment of port wine stains in China.In recent years,there have been reports on the efficacy,safety and side effects of photodynamic therapy for the treatment of port wine stains,but the specific treatment mechanism has not been fully clarified.Therefore,our study will further investigate the partial mechanism of photodynamic therapy for port wine stains by studying the inhibition of growth,induction of apoptosis and regulation of apoptosis by HMME-PDT.Methods:1.The HUVECs were used as in vitro studies to explore the phototoxic effects of HMME-PDT.The effects of different concentrations of photosensitizer and different doses of light intensity on the activity of cells were examined by CCK-8 assay.And screen the processing parameters used in subsequent experiments.2.The experiment was divided into non-PDT groups and HMME-PDT groups.Flow cytometry,DAPI staining,WB and qRT-PCR were used to further clarify whether the HMME-PDT group showed signs of apoptosis and the effects on Bcl-2 and Bax in HUCEV cells.3.The experiment was divided into control group,low dose group and high dose group.The secretion of VEGF-A in each group was detected by ELISA and immunofluorescence.Western Blot was used to detect the expression levels of AKT,p-AKT,mTOR,p-mTOR,P70S6 and p-P70S6 in VEGF-A/AKT/mTOR pathway.Results:1.In the absence of illumination,cells treated with different concentrations of photosensitizer showed no significant difference in cell viability,and cell viability was almost 100%.At the same time,the light-treated groups showed different degrees of survival decline.Under the same illumination condition,the cell survival rate decreased with the increase of photosensitizer concentration.Moreover,under the same photosensitizer concentration,the cell survival rate decreased with the increase of light intensity.Microscopically,it was observed that the photodynamically treated HUVEC cells began to form cell membrane vesicles with poor refractive index,reduced cell volume,disappearance of cell-to-cell connections,and even cells floating on the medium.This phenomenon is more obvious with the increase in light metering.2.Significant signs of apoptosis were found in the cells 24 hours after HMME-PDT.Separate illumination or incubation of the photosensitizer alone caused almost no obvious apoptosis of the cells,but there was a very obvious apoptosis after photodynamic treatment.After photodynamic treatment,the cells have nuclear pyknosis,chromatin condensation,and even nuclear rupture to form fragments and nuclear disintegration.Compared with the control group,the ratio of Bax/Bcl-2 protein in the PDT groups were significantly increased,and the trend of increasing the ratio of Bax/Bcl-2 mRNA was consistent.3.The fluorescence intensity of VEGF-A and the secretion of VEGF-A in HMME-PDT cells were significantly lower than those in the control group,and it decreased more with the increase of irradiation dose.Compared with the control group,the expression of p-AKT,mTOR,p-mTOR,P70S6 and p-P70S6 showed a PDT concentration-dependent decrease.The expression of phosphorylation and activation of each histone in the three groups of AKT,mTOR and P70S6 proteins were also significantly inhibited.Conclusion1.Hemoporfin mediated photodynamic therapy also has a very effective inhibitory effect on HUVEC cells at low concentrations.The cytotoxic effect of HMME-PDT on HUVEC cells is closely related to the concentration of photosensitizer and the intensity of illumination.At the same time,in the HUVEC cell viability study,cell viability was observed to be almost unaffected in the absence of light,and the dark toxicity of Hemoporfin at concentrations below 25 mg/ml was neglected.2.HMME-PDT showed obvious signs of apoptosis in the cells 24 hours later,indicating that apoptosis plays a crucial role in the therapeutic effect of HMME-PDT.Separate illumination or incubation of the photosensitizer alone caused almost no obvious apoptosis of the cells,but there was a very obvious apoptosis after photodynamic treatment.The corresponding anti-apoptotic protein Bcl-2 was decreased in HUVEC cells after photodynamic treatment,and the pro-apoptotic protein Bax was significantly increased.3.HMME-PDT can inhibit the expression of VEGF-A and inhibit the activation of AKT,mTOR and P70S6.Inhibition of vascular endothelial cell proliferation and vascular remodeling by inhibiting VEGF-A mediated protein activation of the AKT/mTOR pathway. |
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