Mechanism Of LJP Attenuate The Radiation Induced Submandibular Gland Injury By Regulating Gadd45α Pathway

Objective1 To detect the expression of Gadd45α proteins in the submandibular gland tissue after radiation and explore the effect of Gadd45α proteinson p38 and JNK signaling pathways.2 To observe the effects of Laminaria japonica polysaccharides(LJP)on the expression of JNK,p-JNK,MCP-1 and MIP-2,and explore the role of LJP in the prevention and treatment of sumandibular gland injury.Methods The first part:48 female Kunming mice were randomly divided into the radiation group and the control group.The model of radiation-induced sumandibular gland injury of mice was established by 60 Co γ ray with a dose of 15 Gy.The Control group underwent simulated radiation.Eight mice were randomly taken at 1d,7d and 30 d after radiation for the following tests:1 Measurement of salivary flow rate: The amount of salivary secretion in the mouse was collected within 30 min,and the salivary flow rate(SFR)of the mouse was calculated to observe the effect of radiation on the function of the submandibular gland of the mouse,and it was confirmed whether the modeling was successful;2 HE staining to observe the histological morphology before and after radiation and to confirm the success of modeling;3 The expression of Gadd45α,p38,p-p38,JNK and p-JNK proteins were detected by immunohistochemistry to determine the effect of radiation on the expression of Gadd45α protein,and the phosphorylation of p38 and JNK signaling pathways was also observed.The second part:120 female Kunming mice were randomly divided into control group,LJP group,radiation group and radiation + LJP group.The model of radiation-induced sumandibular gland injury of mice was established by 60 Co γ ray with a dose of15 Gy.The control group and the LJP group underwent simulated radiation after anesthesia.The LJP group and the Rad+LJP group were intraperitoneally injected with LJP for 4 consecutive days from 3 days before the radiation,and then injected every 3 days,100mg/ Kg.The control group and the radiation group were given an equal volume of physiological saline.Six mice were taken randomly at 1d,3d,7d,14 d and 30 d after radiation for the following tests:1 Measurement of salivary flow rate to observe the effect of LJP intervention on submandibular gland function in mice;2 The expression differences of Gadd45α,JNK,p-JNK and MCP-1proteins in submandibular gland of each group were observed by immunohistochemistry.The optical density values of immunohistochemistry were determined by Image Pro Plus to determine The relative expression levels of Gadd45α,JNK,p-JNK and MCP-1 proteins in each group.The effect of Laminaria polysaccharide intervention on up-regulated Gadd45α protein was observed.3 The relative expression levels of Gadd45α,JNK and p-JNK proteins in the submandibular gland of each group were further determined by Western bolt to determine the effect of LJP intervention on JNK phosphorylation.4 The protein concentrations of MCP-1 and MIP-2 in the submandibular gland of each group were quantified by ELISA to confirm the regulation of the downstream molecules MCP-1 and MIP-2 by the JNK signaling pathway activated by radiation,and observe the regulation of LJP intervention on related proteins.Result1 The salivary flow rate of the radiation group was significantly lower than that of the control group,and the saliva flow rate continued to decrease with the increase of the time after the radiation,and the dose of 15 GY was successfully established.In the experiment of Laminaria japonica polysaccharides intervention,the significant effect of Laminaria japonica polysaccharides was not observed in the 1d and 3d groups after radiation(P>0.05).One week after the radiation,the efficacy of Laminaria japonica polysaccharides was significant(P<0.05).2 The results of histomorphology showed that acinar cells vacuolization,nuclear necrosis and inflammatory cell infiltration were observed in the submandibular gland tissue after irradiation,and increased with the increase of time after irradiation.significantly higher than that in the normal control group.The intervention of Laminaria polysaccharide could significantly down-regulate the expression of Gadd45α.The results of p-p38/p38 and p-JNK/JNK are positively correlated with the expression level of Gadd45α,which can up-regulate Gadd45α and activate the phosphorylation of p38 and JNK.The intervention of Laminaria polysaccharide can inhibit the activation of JNK phosphorylation by radiation process.4 The results of ELISA showed that the activated JNK signaling pathway may up-regulate the expression of downstream inflammatory factors MCP-1 and MIP-2,while the intervention of Laminaria japonica polysaccharides may down-regulate MCP-1 and MIP-2 expression.3 Immunohistochemistry and WB assay showed that the expression level of Gadd45α protein in the submandibular gland tissue in the radiation group wasConclusion1 Gadd45α-p38 signaling pathway and Gadd45α-JNK signaling pathway may be involved in the occurrence of radioactive submandibular gland injury;2 The activated JNK signaling pathway may participate in the inflammatory response by up-regulating the expression of MCP-1 and MIP-2,thereby causing radioactive damage of the submandibular gland;3 Laminaria polysaccharide may inhibit the phosphorylation of p38 and JNK and the expression of inflammatory cytokines(MCP-1,MIP-2)by down-regulating the expression of Gadd45α,and reduce the inflammatory response of the submandibular gland caused by radiation.Thereby exerting its protective effect on the radiation of the submandibular gland.