The Investigation Of ALA-PDT Induced Keloid Fibroblast Necroptosis

Objective: To investigate whether 5-aminolevulinic acid photodynamic therapy can induce necroptosis of keloid fibroblasts(KFB)and its possible mechanism.Methods:1.Tissue block adherent method was used to cultivate keloid fibroblasts in vitro and immunocytochemistry method was performed to identify fibroblasts.2.After passaged,the 3rd ~ 10 th generation of the logarit-hmic growth phase fibroblasts were choosed for further research.Added different concentrations of ALA,in high glucose with serum-free DMEM,avoid light 4h,optical power meter,light dose of 10J/cm2、20J/cm2 、40J/cm2、80J/cm2、120J/cm2.After incubation for 24 hours,CCK8 method was used to detect fibroblasts activity after the intervention,calculated the proliferation level,found the optimal combination of photosensitizer + energy(ALA concentration was 4.0mmol/L,PDT energy was 80J/cm2).3.The growth of subculture in good condition of KFB were divided into 6 groups,A was blank control group,B was ALA group,C was PDT group,D was ALA +PDT group,E was Necrostatin-1(necroptosis inhibitor)+ ALA + PDT group,F was SB203580(p38 signal pathway blocker)+ ALA + PDT group.Intervened with ALA was 4mmol/L and light dose was 80 J/cm2.Flow cytometryinstrument(Annexin-V/PI staining)was used to test the necrosis rates of KFB.Western blot was used to detect the expression of RIP3 protein and phosphorylated p38 in KFB of each group.Results: 1.Cells climbed out of the tissue blocks after about 5 ~ 7 days.about 30~40 days,cell crawled about 80% of the culture bottle bottom,some prominences stretched out from the edge,vimentin immunocytochemistry staining result was positive,confirmed as fibroblasts.2.The results from the detection of CCK8 showed that the proliferation activity of KFB was restrained after ALA-PDT intervention,and within a certain range,the proliferation activity reduced with the increase of concentration of photosensitizer and light dose.3.The results of KFB necrosis rate from flow cytometry instrument showed that: A: 2.76%,B: 5.89%,C: 6.44%,D: 37.38%,E: 13.81%,F: 15.85%.Compared with A,the necrosis rates of B、C、D、E 、F rised(P < 0.05);compared with B and C,the necrosis rates of D、E 、F rised significantly(P < 0.05);compared with D,the necrosis rates of E、F decreased(P < 0.05);4.Western blot test showed that: the expression of RIP3 protein rised in B、C、D、E、F,compared with A(P < 0.05);the expression of RIP3 protein rised significantly in D,compared with E、F;while the expression of phosphorylated p38 rised in B、C、D、E、F,compared with A(P < 0.05);The level of phosphorylated p38 in D group was significantly higher than that in other groups(P < 0.05).Conclusion: 1.ALA-PDT can induce KFB necroptosis.2.p38 MAPK signaling pathway is involved in ALA-PDT induced programmed necroptosis of KFB.