Prevention Effects Of Radiation-induced Submandibular Gland Dysfunction Utilizing Free Radical Scavenger, Tempol, And Its Possible Mechanism In Mouse

ObjectiveRadiation-induced salivary gland injury is one of the serious complications for patients with head and neck malignancies after radiotherapy, without effective preventive measures. Here we investigate the prevention effects of radiation-induced submandibular gland dysfunction utilizing the antioxidant Tempol (TPL), in a C57/6mice model, and its potential radioprotective mechanisms.Method1.1. Model Building:Eighty male C57bl/6mice were randomly divided into four groups:vehicle control group; radiation group;200mg/kg TPL group; and200mg/kg TPL+radiation group. The head and neck regions were then locally irradiated with a single-dose exposure to15Gy electron beam from a medical linear accelerator.2. Drug Administration:For200mg/kg TPL group and200mg/kg TPL+radiation group, SIM (200mg/kg body weight, qd alt) was administered intraperitoneally10min prior to IR. Equal solvent was given to vehicle control group and radiation only group.3. Survival rate and weight change of the mice were observed30days after radiation. Salivary flow rate were test3day, and30day after localized IR.4. Submandibular glands were collected for total superoxide dismutase activity, total glutathione and malondialdehyde content3day after IR.5. Histomorphological observation was performed3day, and30day after IR.6. Terminal deoxynucleotidyl transterase (TdT)-mediated dUTP-digoxigenin nick end labeling method (TUNEL) was used to examine apoptosis of the submandibular glands cells3day after IR.7. Bcl-2, Bax, and cleaved caspase-3protein expression were determined by Western Blot3day after IR.8. Immunohistochemical technique was used to detect Bcl-2and Bax protein distribution3day after IR. Result1. Salivary flow rates of the mice at3and30days in the group of radiation only were separately5.67±1.05,5.27±1.34, compared with irradiated mice pretreated with200mg/kg TPL (13.12±1.17,13.09±2.71), those were significantly smaller, F=226.4,215.3, P<0.05.2. For radiation group, the most obvious change was vaculization of acinarcells at3d postirrdaition, but the most remarkable change was focal fibrosis. In contrast,200mg/kg TPL+radiation group demonstrated clearer lobular structures, fewer vacuoles than radiation group at3d and30d after IR.200mg/kg TPL alone treatment did not alter the histology of submandibular gland (SMG) when compared to the control group.3. TPL could significant decrease MDA content, increase SOD activity and GSH content on day3and day30after irradiation compared with radiation only group (P<0.05).4. Apoptosis indes in the cells of submandibular glands were significant increase on days3and days30after irradiation compared with radiation only group (P<0.05);5. Demonstrated by Western Blot and Immunohistochemical technique, irradiated mice pretreated with200mg/kg TPL could upregulate Bcl-2protein level, and downregulate the level of Bax and cleaved caspase3protein.Conclusion200mg/kg TPL could protect C57bl/6mice salivary glands from radiation injury, and its mechanism maybe via alleviate oxidative stress injury, and regulate the mitochondrial pathway of apoptosis.