| Objective: Parkinson’s Disease(PD)is a common neurodegenerative disease in middleaged and elderly people.Epidemiological studies show that the occurrence of PD is contrary to the incidence of malignant tumors.The incidence of lung cancer,colon cancer and other cancers in Parkinson’s patients is significantly lower than that in the general population.These findings suggest that individuals with PD tendency have higher cancer resistance.On the other hand,cancer is closely related to PD.Some pathogenic genes of PD,such as Parkin,PINK1 and SNCA,are also closely related to the incidence of ovarian cancer,lung cancer,liver cancer and other malignant tumors.As a result,the incidence of cancer in PD patients has decreased significantly.PD patients are individuals with high inflammatory tendency,and their peripheral immunity is activated for a long time.The secretion of inflammatory factors in patients’ peripheral blood is significantly increased,and the excessive expression of multiple inflammation-related receptors on peripheral immune cells,including the basic antigen presenting protein MHCII,suggests abnormal activation of peripheral immunity in PD patients.On the other hand,many disease-causing genes of PD have been found to be actively involved in inflammatory pathways and homeostasis of inflammation in vivo.In addition,genome sequencing results of PD patients showed that 24 gene loci may lead to the risk of PD,among which 14 loci were confirmed to be involved in immune inflammation regulation.In conclusion,the high inflammatory tendency of PD patients is part of the pathogenesis of Parkinson’s disease.Individuals with high inflammatory tendency can produce excessive inflammatory response under the stimulation of external malignant transformation,which can prevent the occurrence of tumor,but cause neurodegenerative damage in the brain.Compared with normal wild mice(C57BL/6),DBA/1 mice were given malignant stimulation with the carcinogenic agent diethyl nitrosamine(DEN).Compared with normal wild mice(C57BL/6),the incidence of tumor was reduced,but the damage of dopaminergic neurons in brain was significantly increased.Moreover,the administration of CTLA-4 and PD-1 inhibitors enhanced the inflammatory response dominated by T cells,leading to a decrease in tumor incidence and increased dopamine neuron death and neurodegeneration.These results suggest the close relationship between hyperinflammation,tumor and neurodegeneration,and the profound influence of inflammation manipulation on the outcomes of the other two.Methods:The first part of this study: The mice were randomly divided into DBA/1 mice untreated group(DBA group),C57BL/6 mice untreated group(C57 group),DBA/1 mice DEN induced group(DD group)and C57BL/6 mice DEN induced group(CD group).The levels of TNF-α,IL-1β,IL-10 and IL-4 in serum of mice were detected by ELISA.The incidence of tumor in each group was detected by HE staining.M1 microglia were labeled by IBA-1 and i NOS,M2 microglia were labeled by IBA-1 and Arg1.The activation of microglia in the substantia nigra of each group was detected by immunofluorescence staining,and the levels of T cell activation-related factors CD4,CD28,MHCII,CTLA-4 and PD1 in peripheral blood of each group were detected by flow cytometry.The expression levels of T cell activation-related proteins CD4,CD28,MHCII,CTLA-4 and PD1 in the substantia nigra of each group were detected by immunohistochemistry,and the levels of inflammatory factors TNF-α,IL-1β,IL-10 and IL-4 in the brain tissues of each group were detected by ELISA.Western blot and immunofluorescence were used to detect the expression levels of blood-brain barrier permeability related proteins ZO-1,Claudin-5 and Occludin in the substantia nigra of each group of mice,and TUNEL staining was used to observe the neuronal apoptosis level in the substantia nigra of each group of mice.The number of dopamine neurons in each group was detected by TH immunofluorescence staining.The second part of this study: Spleen T lymphocytes were extracted from each group and cultured in vitro for 24 h.The levels of pro-inflammatory cytokines TNF-α,IL-1β,IL-10 and IL-4 in the supernatant of cells were detected by ELISA.Midbrain slices were prepared by brain biopsy of C57BL/6 mice and cultured in the supernatant of T cells for48 h.By TUNEL staining detection between groups on T cells in mice brain slice of dopamine neurons apoptosis of qing cultivation,through TH immunofluorescence staining test groups clear training on T cells in mice brain slice of dopamine neurons number,by immunofluorescence staining to detect each group clear training on T cells in mice brain slice of microglia activation markers and MHCII and Iba-1 level.The levels of inflammatory factors TNF-α,IL-1β,IL-10 and IL-4 were detected by ELISA,and the m RNA expression levels of inflammatory factors TNF-α,IL-1β,IL-10 and IL-4 were detected by q RT-PCR.The expression levels of inflammatory related proteins TLR4,My D88 and NF-κB were detected by Western blot.The third part of this study: CTLA-4 and PD-1 blockers were injected into highly inflammatory individuals after malignant stimulation.Tumor incidence of mice in each group was detected by HE staining,and the effects of CTLA-4 and PD-1 blockers on Th1 and Th2 cell-mediated inflammatory factors in peripheral blood of substantia nigra in highly inflammatory individuals were detected by ELISA.ELISA was used to detect the effects of CTLA-4 and PD-1 blockers on the levels of Th1 and Th2 cell-mediated inflammatory factors in the substantia nigra of individuals with high inflammatory tendency,and immunofluorescence staining was used to detect the effects of CTLA-4and PD-1 blockers on the polarization of microglia in the substantia nigra of individuals with high inflammatory tendency.Flow cytometry was used to detect the effects of ct LA-4 and PD-1 blockers on the levels of T cell activation-related factors in peripheral blood of individuals with high inflammatory tendency.Immunohistochemistry was used to detect the effects of CTLA-4 and PD-1 blockers on the levels of T cell activation-related factors in the substantia nigra of individuals with high inflammatory tendency.Western blot and immunofluorescence were used to detect the expression levels of blood-brain barrier permeability related proteins ZO-1,Claudin-5 and Occludin in the brain tissues of mice in each group.TUNEL staining was used to detect the effects of CTLA-4 and PD-1blockers on neuronal apoptosis in the substantia nigra region of individuals with high inflammatory tendency.TH staining was used to detect the effect of CTLA-4 and PD-1blockers on the number of neurons in the substantia nigra in highly inflammatory individuals.Results:The first part of this study: After carcinogenic induction,the incidence of tumor in highly inflammatory individuals was reduced,the microglia in the substantia nigra area of mouse brain tissue were significantly activated,M1 microglia were dominant,CD4,CD28,MHCII positive cells in peripheral blood lymphocytes were significantly increased,and CTLA-4,PD1 positive cells were significantly decreased.The expression levels of CD4,CD28 and MHCII proteins in the substantia nigra of mouse brain tissue increased over time,while the expression levels of CTLA-4 and PD1 proteins decreased over time.The levels of TNF-α and IL-1β mediated by Th1 cells continued to increase,while the levels of IL-10 and IL-4 mediated by Th2 cells continued to decrease.The expression levels of ZO-1,Claudin-5 and Occludin in the substantia nigra of mouse brain tissue continued to decrease,and the apoptosis rate of neurons in the substantia nigra of mouse brain tissue increased significantly,and the number of neurons decreased significantly.The second part of this study: After induction of carcinogenesis,TNF-α and IL-1β levels in supernatant of T cell culture in spleen of hyperinflammatory individuals were significantly increased,while IL-10 and IL-4 levels were significantly decreased.Apoptosis rate and number of neurons in substantia nigra region of mouse T cell supernatant culture was significantly increased.The number of IBA-1 and MHCII positive cells in the substantia nigra region of mouse T cell supernatant culture brain slices were significantly increased,the levels of TNF-α and IL-1β were significantly increased,and the levels of IL-10 and IL-4 were significantly decreased.The expression levels of TLR4,My D88 and NF-κB protein in mouse T cell supernatant cultured brain slices were significantly increased.The third part of this study: The combination of CTLA-4 and PD-1 blocker reduced the tumorgenesis of highly inflammatory individuals,increased the levels of TNF-α and IL-1β in serum and brain tissues of mice,decreased the levels of IL-10 and IL-4,activated microglia,increased the number of M1 microglia,and decreased the number of M2 microglia.It increased the expression of CD4,CD28 and MHCII in peripheral lymphocytes and substantia nigra,decreased the expression of CTLA-4 and PD-1,significantly decreased the expression of ZO-1,Claudin-5 and Occludin in substantia nigra of mouse brain tissue,and increased neuronal apoptosis in substantia nigra of mouse brain tissue.Decreased the number of neurons in the substantia nigra of mouse brain tissue.Conclusion: In the context of high inflammation,malignant stimulation activates T cell immunity and induces central nervous system inflammation,and the incidence of tumor induced by malignant stimulation decreases.Ctla-4 /PD-1 blocker promotes peripheral and intracranial inflammatory responses of malignant stimulation in the context of high inflammation by regulating T cell immunity. |
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