| Objective: This experiment mainly studies the splenic immunotoxicity induced by PFOA,and studies the protective effect of vitamin C(Ascorbic acid)on splenic immunotoxicity induced by PFOA,and preliminarily discusses its possible molecular mechanism,so as to provide scientific ideas for the prevention and treatment of splenic immune-related diseases caused by PFOA environmental pollutants.Methods:(1)Forty adult male ICR mice were randomly divided into four groups(n =10): normal control group,PFOA model group,Vitamin C low-dose group and Vitamin C high-dose group.(2)After intragastric administration of PFOA for 10 hours,Vitamin C was injected intraperitoneally.Except for the normal control group,all the mice in the other groups were gavaged with the concentration of PFOA of 10mg/kg.At the same time,the mice in the normal control group were given the same volume of PFOA solvent.Vitamin C low-dose group and high-dose group were diluted with sterile saline to the concentration of 100mg/kg and 200mg/kg respectively,while the normal control group and PFOA model group were injected intraperitoneally with the same volume of sterile saline.(3)The experimental period was 21 days,weighing and observing the physiological state of mice at a fixed point every day,and the samples were taken after 21 days of the experiment.The spleen of mice was weighed and the spleen index was calculated.(4)Eyeballs take blood and centrifuge to absorb the upper serum.The contents of IL-1β,TNF-α,IL-6 and IL-8 cytokines in serum were detected by ELISA kit,and the contents of LDH and MDA in serum were detected by biochemical kit.(5)The histopathological sections of spleen were made,and the morphological changes of spleen cells were observed by hematoxylin-eosin staining(HE).(6)The expressions of CD4,CD8,Bcl-2,PCNA,Ik B-β and NF-κBp65 immunoreactive cells in spleen tissue were observed by immunofluorescence(IF).(7)In order to verify the results of IF,Western blotting(Wb)was used to further detect the changes of CD4,CD8,Bcl-2,PCNA,Ik B-β and NF-κBp65 protein expressions in mouse spleen.Results:(1)Compared with the normal control group,the body weight and spleen weight and index of the PFOA model group were significantly increased(P<0.05),and the spleen weight and index of the Vitamin C intervention group were significantly lower than those of the PFOA model group(P<0.05).(2)HE results showed that spleen cells in the normal control group were evenly distributed and a large number of spleen cells were counted,while spleen cells exposed to PFOA showed different degrees of pathological damage such as necrosis,watery degeneration,vacuolation and inflammatory infiltration.After Vitamin C intervention,spleen injury of mice was improved to varying degrees,showing reduction in the number of necrotic cells,degeneration,vacuolization and inflammatory infiltration.(3)Compared with the normal control group,the expressions of IL-1β,TNF-α,IL-6,IL-8,LDH and MDA in serum of PFOA model group increased significantly(P<0.05),while the expressions of IL-1β,TNF-α,IL-6,IL-8,LDH and MDA decreased after Vitamin C intervention(P<0.05).(4)Immunofluorescence showed that compared with the normal control group,the expressions of CD4,CD8,Bcl-2 and PCNA positive cells in spleen tissue of PFOA model group decreased,while Ik B-β and NF-κBp65immunoreactive cells increased(P<0.05);after Vitamin C intervention,the expressions of CD4,CD8,Bcl-2 and PCNA positive cells in spleen cells increased,while the expressions of Ik B-β and NF-κBp65 positive cells decreased(P<0.05).(5)It was further verified by Wb that compared with the normal control group,the expressions of CD4,CD8,Bcl-2 and PCNA protein in spleen tissue of PFOA model group were down-regulated,while the expressions of Ik B-β and NF-κBp65 protein were up-regulated(P<0.05);but the expressions of CD4,CD8,Bcl-2 and PCNA protein in spleen tissue of Vitamin C intervention group were up-regulated,while the expressions of Ik B-β and NF-κBp65 protein were down-regulated(P<0.05).(6)RT-PCR results showed that CD4 and CD8 m RNA expressions were decreased but Ik B-β and NF-κBp65m RNA expressions were increased in the PFOA model group compared with the normal control group(P< 0.05).After Vitamin C intervention,CD4 and CD8 m RNA levels were increased,and Ik B-β and NF-κBp65 m RNA levels were decreased compared with those in the PFOA model group(P <0.05).Conclusion: Vitamin C has a protective effect on splenic immunotoxicity induced by environmental pollutant PFOA in mice,and its mechanism may be related to improving splenic cellular immune function,promoting cell proliferation and inhibiting inflammatory stress. |
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