Laminaria Polysaccharide Protects The Submandibular Gland Oxidative Stress By Regulating The Nrf2 Pathway

Part I The protective effect of laminaria polysaccharide onradiation-induced submandibular gland oxidative damageObjective:To explore the effects of laminaria polysaccharides on radiation-induced submandibular gland oxidative damage by observing the salivary flow,submandibular gland index,submandibular gland gross and pathological morphology and oxidative stress index ROS,MDA,inflammatory factor TNF-α,IL-6 and TGF-β.Methods:Totally 96 healthy mice of 2-month-old from Kunming were randomly divided into 4 groups: control group,LJP group,radiation group(IR group)and radiation + LJP group(IR+LJP group),24 in each group.The body weight,water and food intake of the mice were measured every 3 days.The mice in the IR group and the IR+LJP group were given 60 Co γ-ray to establish mice model of radiation-induced submandibular gland injury.LJP(100mg/kg/d)was intraperitoneally injected at 3 days before radiation until 4 days after radiation.The total amount of saliva in 30 min was induced by pilocarpine at 1d,7d,28 d and 56 d after radiation.The weight of submandibular gland and body weight of the mice were also weighed,the submandibular gland index was calculated,and histopathological changes were observed through HE staining.The levels of ROS and MDA in the submandibular gland were detected.The content of TNF-α,IL-6 and TGF-β in mice serum were measured by enzyme-linked immunosorbent assay(ELISA).Results:1.The general condition of mice: the mice from IR group were easy to get anxious,the fur is easy to fall off,while the above abnormal conditions were not obvious in other groups.Compared with the control group,the water intake of mice from the IR group were increased significantly after irradiation(P <0.05)and reached the peak at 7d after irradiation,while the body weight and food intake decreased significantly(P <0.05).Compared with the IR group,the water consumption of the mice decreased significantly after radiation in the LJP+IR group(P <0.05),while body weight and food intake were elevated significantly(P <0.05).2.The changes of salivary flow in each group: There was no significant difference in saliva flow between the LJP and control group(P>0.05).Compared with the control group,the salivary flow of mice from the IR group decreased significantly after radiation,and with the radiation time,the saliva flow showed a gradual decline(P <0.05);the salivary flow of the irradiated mice with treatment of LJP increased gradually 7 days after irradiation(P <0.05)3.The morphology and index of the submandibular gland: Compared with the control group,the submandibular gland from the IR group showed hyperemia and swelling,and the submandibular gland index increased significantly(P <0.05).The swelling submandibular glands from the irradiatted mice with treatment of LJP were improved,and the submandibular gland index was lower than that of the IR group(P <0.05).4.HE staining showed that: the submandibular gland tissue in the IR group showed obvious vacuolization,nuclear pyknosis,ductal dilation and inflammatory cell infiltration.The degree of vacuolization and inflammatory cell infiltration of the submandibular gland in the LJP+IR group were significantly lower than that of the IR group.5.Oxidative stress indicators ROS and MDA showed that: compared with the control group,the levels of ROS and MDA in the submandibular gland of the IR group were significantly increased after radiation(P <0.05);the content of ROS and MDA in the submandibular gland of irradiated mice with treatment of LJP decreased(P <0.05),and returned to the level of the control 56 days after radiation.6.ELISA showed that: Compared with the control group,the levels of TNF-α,IL-6 and TGF-β1 in the IR group were significantly increased(P < 0.05).The levels of TNF-α,IL-6 and TGF-β in the LJP+IR group were significantly lower than that of the IR group(P < 0.05).Conclusions:1.γ-rays can induce oxidative stress damage and inflammatory response in the submandibular gland.2.The protective effect of laminaria polysaccharide on radiation-induced submandibular gland injury may be related to the improvement of oxidative damage and inflammatory response.Part II Molecular mechanism of protective effect of LJP onradiation-induced submandibular gland injury based on Nrf2pathwayObjective:By further observing the changes of Nrf2/HO-1,NQO1,MnSOD and mitochondrial protein SIRT3 expression to evaluate the molecular mechanism of the protective effect of laminaria polysaccharide on radiation-induced submandibular gland oxidative damage.Methods:Immunofluorescence staining,immunohistochemical staining,real-time PCR and Western blotting were used to verify the expression of Nrf2/HO-1,NQO1,MnSOD and mitochondrial protein SIRT3 in submandibular gland.Results:1.Immunofluorescence staining: Nrf2 in the control group and LJP group were expressed in the cytoplasm of acinar cells,while Nrf2 expression in the submandibular gland in the IR group and IR+LJP group was translocated from cytoplasm to nucleus.Compared with control group,MnSOD in submandibular gland in the LJP group show not statistically significant(P >0.05),while the expression of MnSOD in the IR group and LJP+IR group was significantly increased after radiation(P <0.05).Compared with the IR group,the expression of MnSOD in the LJP+IR group was significantly increased(P <0.05).2.Immunohistochemical staining: Compared with the control group,the expression of HO-1 and NQO1 in the LJP group was not significantly different(P >0.05),and the expression of HO-1 and NQO1 in the IR group and IR+LJP group increased significantly after radiation(P <0.05),while expression of Sirt3 in the IR group was significantly lower(P <0.05).The expression of HO-1,NQO1 and Sirt3 in the IR+LJP group was significantly higher than that of the IR group(P <0.05).3.Real-time PCR: Compared with the control group,there was no significant difference in the LJP group(P >0.05),and the mRNA levels of Nrf2,HO-1,NQO1 and MnSOD in the IR group were significantly elevated after radiation(P <0.05),while the mRNA level of Sirt3 was significantly decreased(P <0.05).The level of Keap1 and Nrf2,HO-1,NQO1,MnSOD and Sirt3 in the LJP+IR group was significantly higher than that of the IR group(P <0.05).4.Western blot: Compared with the control group,the levels of Nrf2,HO-1 and NQO1 protein in the IR group and the LJP+IR group were significantly increased(P <0.01),while the levels of Nrf2,HO-1 and NQO1 in the LJP+IR group were more than that of the IR group(P <0.01).Conclusions:1.LJP may improve radiation-induced oxidative stress injury and inflammatory response by activating Nrf2 signaling pathway.2.LJP may improve oxidative stress injury and inflammatory response by up-regulating mitochondrial protein Sirt3 expression.