The Research Of EtNBSe Combined With Photodynamic Therapy On The Impact Of Fibroblast Proliferation And TGF-β1Secretion And On Signaling Pathway

Objectives1. To observe the impact of EtNBSe combined with photodynamic therapy on fibroblast (in vitro proliferation activity and TGF-J31secretion.2. To observe the impact of EtNBSe combined with photodynamic therapy on activity of ERK, INK and P38of MAPK family members in fibroblast (in vitro)and time-based change of signaling proteins, and the impact on levels of phosphorylation expression with the intervention of inhibitors.Method1. Isolating human foreskin fibroblasts cultured normally, identificate keratin, type I and type III collagen by staining fibroblasts.2. using gradient red light irradiation, the gradient EtNBSe concentration and EtNBSe combined with red irradiation influence fibroblasts; in the studies of inhibitor intervention, applicating signaling pathway-specific inhibitors of ERK, JNK and P38(PD98059, of SP600125, SB203580,) on fibroblasts, using tetrazolium bromide (MTT) assay investigate the changes of proliferation and inhibition of each group.3. using EtNBSe combined with red light irradiated on fibroblasts, and then,using enzyme-linked immunosorbent assay (ELISA) detect the changes of TGF-(31secretion in fibroblasts.4. Using Western blot analysis method check the activity and the phosphorylation of ERK, JNK and P38,at the same time, observing the changes of phosphorylation of ERK, JNK, P38with the intervention of inhibitors.Results1. The cells (cultured in vitro) show positive when treated with type Ⅰ and type Ⅲ collagen staining, and keratin staining show negative, meanwhile the cells owns fibroblast morphology.2. Compared with the control group, fibroblast irradiated by gradient red light (10,30,60,90min), haven’t produced significant cell proliferation and cell toxic effects (P＞0.05). using gradient EtNBSe (30,60,120,240pM) for1h and2h respectively,each group of fibroblasts haven’t produced significant cell proliferation and cell toxic effects (P>0.05).In the group with gradient EtNBSe (30,60,120,240the pM) combined with red light treatment, after1h illuminated, the cells haven’t produced significant cytotoxic response and cell proliferation activity compared with the control group(P＞0.05); while the group was illuminated for2h, EtNBSe (30,60,120pM) increased activity on fibroblast proliferation while EtNBSe (60,120pM) produced significant proliferative activity (P＜0.05). In the groups with intervention of inhibitors, compared with the control group, the group of inhibitors (PD98059, SB203580) combined with photodynamic therapy, cell activity was significantly inhibited (P＜0.05);while for SP600125, cell activity had no significant changes.3. Compared with the control group, treaed with60and120pM EtNBSe combined with photodynamic therapy, TGF-β1secretion was increased (P＜0.05), with significant increase in120pM.4. Concentrations groups:compared with the control group,30,60,120pM EtNBSe combined with photodynamic therapy can promote the activation of phosphorylated ERK protein;30,60pM can promote the activation of phosphorylated of JNK and P38protein, and gradually suppress the activity of them with the increasing dose. In Time Groups: ERK, JNK, P38has different levels of activation, compared with the control group, ERK peaked at60minutes; JNK and P38peaked at90minutes. In Intervention groups:compared with the control group, PD98059and SB203580can significantly inhibit the activity of phosphorylation of ERK and P38; while SP600125had no significant inhibition on the phosphorylation of JNK.Conclusions1. EtNBSe combined with photodynamic therapy can promote the proliferation of fibroblasts cultured in vitro and increase the secretion of TGF-β1. 2.The EtNBSe combined with photodynamic therapy can stimulate the activity MAPK family members (ERK, JNK, p38) in fibroblasts cultured in vitro, which have two-way roles on JNK and p38.