LJP Regulates The Radiation Induced Submandibular Gland Injury Events Via Inhibiting Activation Of NF-?B Signaling

Radiation induced salivary gland injury is one of the common complications of radiation therapy for head and neck.At present,the mechanism has not been clearly clarified,and there is no proper treatment in clinical practice.Studies have shown that early inflammatory reactions play an important role in the acute phase of radioactive salivary gland injury.It is significant to investigate the mechanism of radiation-induced early inflammatory reaction of salivary glands and to find effective therapeutic drugs according to the mechanism to prevent and cure the radiation-induced salivary gland injury.Nuclear factor-kappa B?NF-?B?,which regulates many inflammatory signals,such as tumor necrosis factor-??TNF-??,intercellular adhesion molecule-1?Intercellular Adhesion Molecule-1,ICAM-1?,it plays a key role in the inflammatory response,and its inhibition is considered to be an effective anti-inflammatory approach.Laminaria japonica polysaccharides?LJP?,which is a naturally non-toxic polysaccharide purified from Laminaria japonica,has anti-inflammation potential.Little is kown about whether LJP can reduce radiation-induced inflammatory responses by inhibiting NF-?B activation or not.Objective1 To investigate the changes of expression of NF-?B/I?B,TNF-?,and ICAM-1 in the radiation induced submandibular gland injury of mice,and try to clarify the possible mechanism of radiation-induced salivary gland injury.2 To investigate the regulation of NF-?B/I?B,TNF-?and ICAM-1 factors in the submandibular gland by intervention of LJP,and try to clarifythe possible mechanism of LJP in the prevention and treatment of radiation-induced salivary gland injury.Method96 healthy female Kunming mice,weighing 32±2g,were randomly divided into control group,LJP group,radiation group,and radiation+LJP group?n=6/each group?Administration time and dose:The LJP group and the radiation+LJP group were intraperitoneally injected LJP 1 day before irradiation,and then treated with LJP at dose of 100 mg/kg one time per day for 7 days,and then the dose were changed to 1/2d for the next 7 days;the control group and the radiation group were given equal amounts of physiological saline at the same time.Irradiation method:radiation group and radiation+LJP group were given⁶⁰Co?at a dose of 15Gy irradiation,control group and LJP group given 0Gy irradiation.After the radiation,the volume of daily drinking water was observed in each group of mice,and 6 mice were randomly selected from each group on the 1d?3d?7d and 14d after irradiation.Then the experiments were conducted as follows:1 Saliva amount:The total volume of saliva secreted by the mice was collected for 30 minutes,and the saliva volume was calculated at 1 g/ml.2 Morphological observation of submandibular gland:Histopathological changes of acinus,stroma,duct,and blood vessels were observed by HE staining.3 Indirect immunofluorescence was performed to detect NF-?B p65 subunit localization in the submandibular gland.4 Immunohistochemical method was used to detect the expression of I?B?and ICAM-1 protein in submandibular glands.5 ELISA assay was used to quantify mouse serum TNF-?content.Result1 Comparison of drinking water amount in mice from 1 to 14 days in each group:compared with the control group,there was no significant difference in LJP group?P₁>0.05?,and the drinking water in radiation group and radiation+LJP group increased significantly?P₁<0.05?;Compared with radiotherapy group,the amount of drinking water in radiation+LJP group was significantly reduced?P₂<0.05?.2 Comparison of saliva volume of mice in different groups at different time points:Compared with the control group,there was no significant difference in the LJP group?P₁>0.05?,and the saliva volume in the radiation group and the radiation+LJP group was significantly reduced at each time point after irradiation?P₁<0.05?.Compared with the radiotherapy group,the saliva volume of the radiation+LJP group increased significantly?P₂<0.05?.With the increase period after radiation,the amount of saliva in the radiation group decreased progressively,and the saliva volume in the radiation+LJP group returned to the level of the control group as the treatment time increased.3 HE staining results showed that there was no obvious abnormality of glandular morphology in the LJP group compared to the control group;the progressively worsened interstitial edema,inflammatory cell infiltration,nuclear condensation,acinar atrophy in the radiation group.Degeneration and other pathological changes;radiation+LJP group were significantly lighter than the radiation group.4 At each time point,NF-?B p65 in the submandibular glands of mice in the control group and LJP group were all expressed in the cytoplasm.1d?3d?7d and 14d after irradiation,some submandibular glands in the radiation group and the radiation+LJP group showed partially fluorescently labeled NF-?B p65was transferred from the cytoplasm to the nucleus.5 1d?3d?7d and 14d after irradiation,the expression of I?B?in the submandibular gland of mice in the radiation group and the radiation+LJP group was significantly lower than that in the control group?P₁<0.05?,and reached the lowest value at 7d after radiation;radiation+LJP group were significantly higher than those in the radiation group?P₂<0.05?.There was no significant difference between the LJP group and the control group?P₁>0.05?.6 1d?3d?7d and 14d after irradiation,ICAM-1 expression in the submandibular gland of the radiation group and the radiation+LJP group was significantly higher than that of the control group?P₁<0.05?,and the expression of the radiation+LJP group was significantly lower than that of the radiation group?P₂<0.05?;there was no significant difference between the LJP group and the control group?P₁>0.05?..7 1d?3d?7d and 14d after irradiation,the levels of serum TNF-?in radiation group and radiation+LJP group were significantly higher than those in control group?P₁<0.05?.The content of radiation+LJP was lower than that of radiation group.There was significant difference between the 3d?7d and 14d after radiation?P₂<0.05?.There was no significant difference between the LJP group and the control group?P₁>0.05?.Conclusion1 Radiation-induced early damage of salivary glands is related to NF-?B pathway activation via increasing the expression of TNF-?and ICAM-1.2 LJP prevents the radioactive salivary gland damage via inhibiting the NF-?B pathway activation.