Effects Of ALA-PDT On Skin Cell Growth And Study On The Role Of Nrf2 Correlative Factors

Background and Objective Photodynamic therapy(PDT)was a therapeutic method of the redox reaction produced by a combination of photosensitizer,oxygen,and a specific wavelength of light.In recent years,photodynamic therapy had been widely used in Dermatology and achieved gratifying results.Its indications included a variety of skin diseases,such as acne,actinic keratosis,squamous cell carcinoma,basal cell carcinoma,condyloma acuminatum and bacteria or fungal infections,etc..In the course of treatment,the researchers found a interesting phenomenon that the high-dose PDT was mainly used to kill tumor cells and microorganisms,and the low-doses PDT was different.More and more studies showed that the low-dose PDT treated photoaging,promoted cell proliferation,and accelerated skin healing,even prevented high-risk population of photoaging and skin cancer after transplantation.There were several groups of experiments using a split-face study showed that the skin texture,wrinkles and skin color of the treatment side were significantly improved than that of the control side.In 2016,the clinical consensus guidelines of the American Society for Surgery suggested the application of PDT in the rejuvenation.Researchers studied the mechanism of photodynamic therapy,and were mainly concentrated in high-doses to kill tumor cells,inflammatory cells and microorganisms.But the biological effects and the related mechanism of the low-dose PDT were less and unclear.Singlet oxygen was the core factor of PDT treatment.The previous studies indicated that the different of light source,the different of photosensitizer,the site of action,the cell of classification,the cell of oxygen metabolism,and active oxygen of intensity,produced the different oxidative stress effect.Nuclear factor erythroid2-related factor 2(Nrf2)was a key factor in the Oxidative stress response.It regulated the expression of downstream antioxidant proteins and growth factors,also played an important role in the treatment of skin photoaging and the protection of cell defense.The effection of the PDT on the growth of skin cells and related signaling pathways of the Nrf2 in the treatment of the low-dose PDT were worth further study.Methods At first,the cell morphological changes of keratinocytes and fibroblasts after the treatment of ALA-PDT by optical microscope.The next,ALA-PDT exerted on cell proliferation of keratinocytes and fibroblasts,using the method of MTT.Moreover,the effect of ALA-PDT on the secretion of bFGF and VEGF by fibroblasts was analyzed by ELISA test.For the second time,the activities of SOD in Fibrobalsts was tested after the treatment of ALA-PDT by WST-8 test.Finally,the changes of Nrf2 and TGF-?1 detected by western blot in the fibroblasts after the treatment of ALA-PDT.Results1.Influences of ALA-PDT on the growth of human skin cellsUnder the inverted microscope,a small number of HaCaT cells,keratinocytes and fibroblasts died after the intervention of high-dose ALA-PDT(1mM,48J/cm2)for 48 hours.The MTT result showed that,at 0.1mM ALA concentration,after 24 hours light treatment at the intensity of 21J/cm2 and 42J/cm2,HaCaT cells,Keratinocytes and Fibroblasts showed no significant difference with these cells under the pure red light;after 48 hours,all the three cells showed the tendency of proliferation after low-dose ALA-PDT(0.1mM,21 J/cm2),while in high dose(1mM,48J/cm2)the proliferation was inhibited.2.Effects study of ALA-PDT on the Fibroblasts Nrf2 and the expression of related factorsThe ELISA results showed that,the bFGF concentration presented a rising tendency in both high-dose ALA-PDT group(1mM,48J/cm2)and low-dose ALA-PDT group(0.1mM,21J/cm2),especially on the point of 24 hours;while the concentration of VEGF also increased at both high and low energies light,and in all the three ALA-concentration group that mentioned above.The increasing of VEGF under PDT intervention was much more obvious than that in the pure red light,especially after 48 hours treatment.In 1 hour after ALA-PDT intervention,the activity of SOD in each dosage group showed no obvious different;6 hours later,the activities of SOD was obviously enhanced in the low-dose group,and was significantly declined in the high-dose group.The result of western blotting showed that the expression of Nrf2,TGF-?1 and MMP-9 was increased after 1h low-energy ALA-PDT,and the content of Nrf2 in low-dose group was higher than that in high-dose group.After 6h,the content of TGF-?1 in each dose group still increased,the content of Nrf2 in each group has no obvious different,and the content of MMP-9 in high-dose was decreased.In addition,after 24h,the content of Nrf2 increased again in the two groups of PDT.The levels of MMP-9 in each group was significantly lower than those in the initial state,and two dose groups of PDT had no bands at 48h.TGF-?1 of the three groups were still increasing at 24h and 48h,and the increase of PDT group was more obvious.Conclusion 1)Low-dose of ALA-PDT could promote the growth of keratinocytes and fibroblast to a certain extent.2)Low-dose of ALA-PDT could up-regulate the expression of Nrf2 in fibroblasts.3)Low-dose ALA-PDT could promote the secretion of bFGF and VEGF in fibroblasts,up-regulate the expression of TGF-?1,promote the expression of MMP-9 in short phase,and inhibit MMP-9 in long phase.4)Low-dose ALA-PDT could increase the activity of SOD to play an antioxidant response to protect cells.