Effects Of CpG ODN1826 To Radiation-Induced Lung Injury In Mice

Lung cancer, one of the common malignant tumors in many countries in the world, has become the first tumor death causes in human. Radiation therapy is one of the most common forms of lung cancer treatment, but the current clinical curative effects are not very ideal. The main reasons include low radiosensitivity of lung cancer and radiation-induced lung injury, which are the main restricting factors influencing tumor target dose and local control. CpG ODN (unmcthylated cytosine-phosphateguanine oligodeoxynucleotides, CpG ODN), is CpG dinucleotide with non-specific sequences of DNA methylation, which can specific activate TLR9 (toll like receptor 9, TLR9) to mediate immune system. CpG ODN has specificity immune function to different species, and to mouse the CpG ODN1826 (including sequence GACGTT) is the best. Basic research confirmed that the CpG ODN1826 has significant radiotherapy sensitization effect for lung cancer. Thus, it has the potential to become a new type of lung cancer clinical radiotherapy sensitizer. However, whether CpG ODN1826 improvs the sensitivity to radiation of the lung tissue around the lung tumor or causes radiation-induced lung injury are unknown. There is rare research on this area at home and abroad.We established the radiation-induced lung injury mouse model, with CpG ODN1826 to intervene the radiation-induced lung injury mice to observe the effect of CpG ODN1826 in radiation-induced lung injury mice, and preliminarily studied the mechanism of CpG ODN826 in the process of radioactive lung injury in mice. ODN alone was used as negative control. ICR mice were divided into Control group, Radiation(RT) group, CpG group, Radiation combined with CpG ODN1826(RT+CpG) group, ODN group and Radiation combined with ODN(RT+ODN) group. Each group had 40 mice. Mice in RT group, RT+CpG group and RT+CpG group were exposed to a single left lung 20 Gy. On day 1,3,5,7,9 after exposure, intraperitoneal injection of 0.2 ml saline to Control group and RT group, intraperitoneal injection of 0.1 mg/ml concentration of CpG ODN solution 0.2 ml to CpG group and RT+CpG group, and intraperitoneal injection of 0.1 mg/ml concentration of ODN solution 0.2 ml to ODN group and RT+ODNgroup. Before irradiation, mice were anesthetized by injecting intraperitoneal 0.004 ml/g body mass of 10% chloral hydrate. The mice were immobilized and shielded under a home-made device. Single dose of 20 Gy 6MV X ray was delivered to a 2 cm ×2 cm area in the left lung at a rate of 2.0 Gy/min. Non-irradiated mice underwent the same procedure but were not exposed to radiation. The mice were executed to get the sample on day 1,5,15,30 and 90 after the exposure. The numbers and time of the mice death were recorded, Autopsy to probe the cause of the death were performed. The lung and body weight, the appearance scores, and the lung indexes were recorded. Paraffin section with HE staining and Masson staining of radiation lungs, and scored pneumonia and fibrosis was prepared. ELISA method was used to detect the serum malondialdehyde (MAD), superoxide dismutase (SOD), glutathione (GSH), transforming growth factor betal (TGF-β1) and tumor necrosis factor alpha (TNF-α) concentration, and alkaline hydrolysis method was used to detect lung tissue concentrations of Hyp. Real-time PCR was used to detect the relative expression quantity of MnSOD gene and FoxP3 gene in each group. The results showed irradiated groups had a lower appearance scores, weight gain but a higher lung index than non-irradiated groups. RT+CpG group had a higher appearance score, weight gain but a lower lung index than the other RT group and RT+ODN group. RT+CpG group had lower pneumonia and pulmonary fibrosis scores than RT group and RT+ODN group. Irradiated groups had higher serum MDA, TGF-β1, TNF-α concentration but lower serum SOD and GSH concentration than non-irradiated groups. RT group and RT+ODN group had higher serum MDA, TGF-β1, TNF-α concentration but lower serum SOD and GSH concentration than RT+CpG group. Hyp in the irradiated lung of RT group and RT+ODN group was higher than that of RT+CpG group. The resluts of Real time PCR suggested that the relative expression quantity of MnSOD of RT+CpG group was higher than that of RT group and RT+ODN group and the relative expression quantity of MnSOD of CpG group was higher than that of Control group and ODN group. On the contrary, the relative expression quantity of FoxP3 of RT+CpG group was lower than that in RT group and RT+ODN group and the relative expression quantity of FoxP3 of CpG group was lower than that of Control group and ODN group. We find that CpG ODN 1826 can improve the living state of radiation-induced lung injury mice (improved appearance, reduced weight loss, resulted in lower pneumonia and pulmonary fibrosis scores), increase the expression of anti-oxidant gene MnSOD and the quantity of antioxidants, and reduce injury from radiation-induced reactive oxygen species production. CpG ODN also reduces expression of the FoxP3 gene, which lowers the secretion of TGF-β1, thus alleviating pulmonary fibrosis. In conclusion, CpG ODN1826 exerts protective and therapeutic effects on radioactive lung injuries.