The Role Of Oxidative Stress And TRP Channel In X-ray-induced Pain

Objective:To investigate the role of oxidative stress and TRP channel in X-ray-induced pain.Methods:The X-ray-induced pain model was established with irradiation at a single dose of 20Gy X-ray to the hind limbs and tails of mice.The mechanical thresholds was measured by the Von Frey filament test.The heat thresholds was analyzed by hot-plate test and tail-flick test.The cold thresholds was assessed by the acetone stimulation test.The dorsal root ganglion of lumbar vertebrae 2-5 and foot skin tissue were separated from mice in diffirent time after irradiation.The levels of nitric oxide synthase(iNOS)mRNA,catalase(CAT)mRNA,superoxide dismutase(SOD)mRNA,transient receptor potential vanilloid 1(TRPV1)mRNA and transient receptor potential ankyrin 1(TRPA1)mRNA in the skin tissue or dorsal root ganglion were detected by real-time PCR method.Western blot was used to measure the protein levels of TRPV1 and TRPA1.Immunofluorescent double staining was used to observe the colocalization of TRPA1 and TRPV1 with the lectin B4(IB4),respectively.Results:Compared with the control group,the mechanical pain threshold remarkably decreased at day 1(0.33±0.04 g vs.0.21±0.03 g,p=0.043),and lasted 28 days after X-ray radiation.The heat pain threshold significantly reduced at day 3(5.59±0.21 s vs.4.78±0.24 s,p=0.02),and continued until day 11.The reduced cold pain threshold was observed at day 3(3.79±0.45 vs.1.93±0.33,p<0.001),and lasted 7 days.At 14 days after a single dose of 20Gy radiation,the mRNA level of iNOS expression increased(1.00±0.14 vs.4.35±1.17,p<0.05),while the mRNA levels of SOD and CAT markedly decreased(SOD,1.00±0.12 vs.0.50±0.06,p<0.01;CAT,1.00±0.03 vs.0.54±0.07,p<0.001),the mRNA levels of TRPV1 and TRPA1 respectively increased(TRPV1,1.00±0.25 vs.2.1 ± 1.05,p=0.01;TRPA1,1.00±0.12 vs.1.4 ± 1.02,p<0.05)at day 3 after X-ray radiation in DRG,and lasted 14 days.Western blot analysis showed that the protein level of TRPV1 in DRG increased at day 7 after X-ray radiation(p<0.01),The protein level of TRPA1 also significantly increased at day 3 after X-ray radiation(p<0.01).Immunofluorescent double staining revealed that the proportion of TRPA1+/IB4+cells or TRPV1+/IB4+ cells in IB4 positive neurons was significantly increased at 7 day after X-ray radiation in DRG.Conclusions:The radiation-induced skin injury model can be established by a single dose of 20Gy X-ray to the hind limbs and tails of mice.Oxidative stress induced by X-ray in the foot skin tissue and DRG may activate TRPV1 and TRPA1 channels,thus causing the pain.Oxidative stress and TRP channel may play an important role in X-ray-induced pain,and antioxidants may provide effective methods and strategies to prevent and treat the pain after radiotherapy.