Study On The Effect And Mechanism Of SDF-1/CXCR4 Pathway Inhibitor AMD3100 On Radiation-induced Skin Fibrosis

Objective: Radiation-induced skin fibrosis is a common complication of chronic cancer radiation therapy.For a long time the study of the pathogenesis of this disease is unknown.Radioactive skin fibrosis in the clinical manifestations of the skin shape changes.Skin contracture and loss of elasticity,difficult to pinch the skin crease,and may be associated with telangiectasia,pain and itching.At present,modern clinical medicine for radiation-induced skin fibrosis lack of effective means of prevention is also no standard treatment options.Treatment methods mostly drug-based,the main drug is divided into three categories,namely anti-inflammatory drugs,the role of drugs in the blood and interferon.These drugs have a certain effect in preventing the formation of fibrosis,but the treatment of fibrosis has been formed is very limited.SDF-1 / CXCR4 biological axis plays an important role in mobilization,migration and homing of hematopoietic stem cells.Studies have shown that SDF-1 / CXCR4 bio-axis plays an important role in the transformation of fibroblasts into myofibroblasts.AMD3100 is a nonpeptide blocker of CXCR4.AMD3100 can effectively bind to CXCR4 to block signal transduction between SDF-1 and CXCR4,but it does not have an agonistic effect on CXCR4.At present,AMD3100 is mainly used to mobilize hematopoietic stem / progenitor cells,inhibit tumor cell proliferation and migration,inflammatory response,autoimmune diseases and asthma and other aspects of the therapeutic effect.We hypothesized that the use of AMD3100 to inhibit the SDF-1 / CXCR4 pathway indirectly affects the TGF-? / Smad pathway signaling,which play a role in reducing the damage caused by ionizing radiation and delay the progress of fibrosis.Methods: At the cellular level,an MTT assay was used to detect the effect of different concentrations of AMD3100 on the survival of human normal skin cells.Human skin fibroblasts(WS1)were treated with different doses of X-ray irradiation together with different concentrations of AMD3100.The expression of TGF-?,Smad2 and p-Smad2 in TGF-? / Smad pathway was detected by Western Blot assay.Immunofluorescence assay was used to detect the changes of Smad2 protein in WS1 cells after the addition of AMD3100 to X-ray irradiation and the addition of exogenous TGF-? growth factor.The luciferase reporter assay was used to measure the activity of Smad2/3 after different concentrations of AMD3100.At the tissue level,the expression of CXCR4 in human skin tissue after irradiation was detected by immunohistochemistry.A model of radion-induced skin injury in SD rats was established(30 Gy).Rats were subcutaneous injected with AMD3100 or PBS group,immediately after irradiation or two weeks after radiation.The semi-quantitative method was used to evaluate skin lesions.HE staining was used to observe the changes of skin tissue structure between different treatment groups.Result: The results of immunohistochemistry showed that the expression of CXCR4 in human skin after irradiation was increased.Results from MTT assay showed that AMD3100 in the concentration range of 50-500 ?mol / L had no significant effect on normal human skin cell viability.The results from Western Blot showed that the expression of TGF-?,Smad2 and p-Smad2 protein in TGF-? / Smad pathway was decreased with the increase of AMD3100 drug concentration.Immunofluorescence assay showed that AMD3100 inhibited the translocation of Smad2 protein into the nucleus of irradiated WS1 cells.The results of luciferase reporter assay showed that AMD3100 decreased the luciferase reporter activity of Smad2/3 response.At the animal level,skin damaged after subcutaneous injection of AMD3100 immediately following radiation was less severe,compared with PBS-injected rat skin.HE staining showed that the skin tissue structure of the rat skin was injected with the AMD3100 group in the skin layer and the number of the skin appendages was the closest to the normal skin tissue.Comparatively,subcutaneous injection of AMD3100 two weeks after radiation showed no curative effect on radiation-induced skin injury.Conclusion: 1.The expression of CXCR4 was significantly higher in normal fibrous tissues than in normal skin tissues,suggesting that CXCR4 molecules play an important role in the development of skin fibrosis.2.AMD3100 plays an indirect inhibitory role in the TGF-? / Smad pathway,an important signaling pathway for fibrosis.This study revealed the potential role of SD3-CXCR4 pathway inhibitor AMD3100 in reducing the damage caused by ionizing radiation and delaying the development of radioactive skin fibrosis.This study provided a novel strategy for the treatment of radiation-induced skin injury and skin fibrosis.