| Objective:By constructing an EAAG animal model,the potential role and impact of RAGE and its ligand S100 B in the pathogenesis of EAAG was investigated to provide a new therapeutic target for autoimmune autonomic ganglioneuropathy.Methods1.EAAG models were constructed using α3g ACh R antigen immunized C57BL/6mice and grouped: 10 healthy control 1-week group（HC-1W group）,10 healthy control2-week group（HC-2W group）,10 EAAG 1-week group（EAAG-1W group）,10 EAAG2-week group（EAAG-2W group）,10 anti-S100 B treatment group（T-S100 B group）,10anti-RAGE treatment group（T-RAGE group）.10 mice in the anti-S100 B treatment group（T-S100 B group）and 10 mice in the anti-RAGE treatment group（T-RAGE group）were treated with RAGE and S100 B specific antagonists respectively 1 week after successful moulding,and their body weight and time spent searching for food were monitored and recorded to evaluate olfactory function.2.The expression of α3g ACh R in the frontal lobe,hypothalamus,brainstem,heart muscle and intestine of each group of mice was detected by immunohistochemistry.3.Western Blotting was applied to detect the expression of α3g ACh R,RAGE and S100 B in frontal lobe,hypothalamus,brainstem,myocardium and intestine of each group of mice and quantitative greyscale analysis were performed.4.Detection of CD4+ T lymphocytes,Th1,Th2,Th17 and Treg cell subsets and their ratios in the spleen lymphoid tissue of mice by flow cytometry5.The expression of s RAGE and inflammatory factors IL-1β,IL-6,IL-10 and TNF-α in the peripheral serum of each group of mice was measured by enzyme-linked immunosorbent assay.6.Welch’s ANOVA test and paired/unpaired t-test were used to compare the differences between groups,p<0.05 between groups was statistically significant.ResultsI.Behavioural tests of autonomic function1.The differences in weight loss and time needed to search for food were statistically significant in the EAAG-1W group compared to the HC-1W group and in the EAAG-2W group compared to the HC-2W group,and in the EAAG-2W group compared to the EAAG-1W group（p<0.05）,while the differences in weight between the EAAG-2W and EAAG-1W groups were not statistically significant（p= 0.5680）.2.The T-RAGE and T-S100 B groups gained weight and spent less time searching for food than the EAAG-2W group;the T-RAGE group spent more time searching for food than the T-S100 B group,and the differences were all statistically significant（p <0.05）.II.Comparison of IHC results1.In the EAAG-1W and EAAG-2W groups,α3g ACh R antigen expression was detected in the frontal lobe,hypothalamus,brainstem and small intestine glandular epithelial cells,and α3g ACh R antigen expression was more significant in the EAAG-2W group.α3g ACh R antigen expression was not detected in cardiac myocytes in the EAAG-1W group,and a small amount of α3g ACh R antigen expression was detected in the EAAG-2W group;α3g ACh R antigen expression was not detected in the HC-1W and HC-2W groups.2.Compared to the EAAG-2W group,the expression of α3g ACh R antigen was reduced in both the T-RAGE and T-S100 B groups in the above-mentioned areas,with the reduction being more significant in the T-RAGE group.III.Comparison of Western Blotting results1.The expression of α3g ACh R protein in the frontal lobe,hypothalamus,brainstem,myocardium and small intestine was higher in the EAAG-1W and EAAG-2W groups than in the HC-1W and HC-2W groups,respectively;the expression ofα3g ACh R protein was lower in the T-RAGE and T-S100 B groups than in the EAAG-2W group,and the differences were statistically significant（p<0.05）.hypothalamus,myocardium and small intestine α3g ACh R protein expression in the T-RAGE group was lower than that in the T-S100 B group（p<0.05）,and the difference in brainstemα3g ACh R protein expression between the two groups was not statistically significant（p=0.8873）.2.Among the above areas,RAGE protein expression in the EAAG-1W and E AAG-2W groups was higher than that in the HC-1W and HC-2W groups,respectively;RAGE protein expression in the T-RAGE and T-S100 B groups was lower than that in the EAAG-2W group,and the differences were statistically significant（p<0.05）.protein expression in the T-RAGE group was lower than that in the T-S100 B group,and the difference was statistically significant（p<0.05）.The difference in RAGE protein expression in the small intestine between the two groups was not statistically significant（p=0.7047）.3.Among the above sites,S100 B protein expression was higher in the EAAG-1W and EAAG-2W groups than in the HC-1W and HC-2W groups,respectively;S100B protein expression was lower in both the T-RAGE and T-S100 B groups than in the EAAG-2W group,and the decrease was more pronounced in the T-S100 B group than in the T-RAGE group,and the differences were all statistically significant（p < 0.05）.IV.Comparison of ELISA results1.IL-1β in the serum of mice in the EAAG-1W group was higher than that in the HC-1W group,and IL-1β in the T-RAGE and T-S100 B groups was higher than that in the EAAG-2W group,and the difference was higher in the T-RAGE group,all of which were statistically significant（p<0.05）,but the difference between IL-1β groups in the EAAG-2W and HC-2W groups was not statistically significant（p= 0.1138）.2.IL-6 in the serum of mice in the EAAG-1W group was higher than that in the HC-1W group,IL-6 in the EAAG-2W group was higher than that in the HC-2W group,IL-6 in both the T-RAGE and T-S100 B groups was lower than that in the EAAG-2W group,and the T-RAGE group had lower IL-6 values than the T-S100 B group,all differences were statistically significant（p < 0.05）.3.IL-10 in the serum of mice in the EAAG-1W group was higher than that in the HC-1W group,and IL-10 in the T-RAGE group was higher than that in the EAAG-2W group,and the differences were statistically significant（p < 0.05）;however,the differences between groups were not statistically significant in the EAAG-2W group compared to the HC-2W group and the T-S100 B group compared to the EAAG-2W group（p = 0.9166,0.1314）.4.TNF-α in the serum of mice in the EAAG-1W group was higher than that in the HC-1W group,TNF-α in the EAAG-2W group was higher than that in the HC-2W group,TNF-α in the T-RAGE and T-S100 B groups was higher than that in the EAAG-2W group,and it was higher in the T-RAGE group,and the differences between the groups were statistically significant（p < 0.05）.5.The s RAGE in the serum of mice in the EAAG-1W group was higher than that in the HC-1W group,and the s RAGE in the EAAG-2W group was higher than that in the HC-2W group.The s RAGE in the T-RAGE and T-S100 B groups was lower than that in the EAAG-2W group,but the s RAGE value in the T-RAGE group was higher than that in the T-S100 B group,and the differences were all statistically significant（p< 0.05）.V.Comparison of flow cytometry results1.CD4+T/T cells,Th1/CD4+T cells,Th2/CD4+T cells,Th17/CD4+T cells,Treg/CD4+T cells,Th1/Th2,Th17/Treg in spleen were higher in the EAAG-1W group than in the HC-1W group,and the differences were all statistically significant（p < 0.05）.2.Th1/CD4+T cells,Th2/CD4+T cells,Th17/CD4+T cells and Th17/Treg were higher in the EAAG-2W group than in the HC-2W group,and Th1/Th2 was lower than in the HC-2W group,all with statistically significant differences（p<0.05）;the differences between Treg/CD4+T cell groups were not statistically significant（p=0.0663）.3.CD4+T/T cells,Th1/CD4+T cells,Th17/CD4+T cells,Th1/Th2 and Th17/Treg were lower in the T-RAGE group than in the EAAG-2W group,and Treg/CD4+T cells were higher than in the EAAG-2W group,all with statistically significant differences（p<0.05）;the differences between the Th2/CD4+T cell groups were not statistically significant（p=0.1383）.4.Th2/CD4+T cells,Th17/CD4+T cells and Treg/CD4+T cells were higher in the T-S100 B group than in the EAAG-2W group,and CD4+T/T cells,Th1/Th2 and Th17/Treg were lower than in the EAAG-2W group,all with statistically significant differences（p<0.05）,while the differences between the Th1/CD4+T cell groups were not statistically significant（p=0.3641）.5.CD4+T/T cells,Th1/CD4+T cells,Th2/CD4+T cells,Th17/CD4+T cells,Treg/CD4+T cells,Th1/Th2 and Th17/Treg were higher in the T-RAGE group than in the T-S100 B group,all with statistically significant differences（p < 0.05）.Conclusion1.Immunization of C57BL/6 mice with α3g ACh R was effective in establishing the EAAG model.1 week of immunization caused weight and olfactory loss in EAAG and 2 weeks after immunization,weight remained stable while olfactory loss persisted.2.Significant expression of α3g ACh R was detected on EAAG frontal,hypothalamic,brainstem and small intestinal glandular epithelial cells,and small amounts of α3g ACh R could be detected next to cardiac myocytes at 2 weeks of immunization.3.RAGE and S100 B protein expression levels were elevated in EAAG-involved tissues,and antagonistic RAGE and S100 B treatment both reduced RAGE and S100 B protein,with antagonistic RAGE treatment being more effective.4.In the early inflammatory response of EAAG,the proportion of Th1 and Th17 is significantly increased,which plays a role in promoting the inflammatory response through the release of IL-6 and TNF-α.Antagonistic RAGE and S100 B treatment both increase the proportion of Treg,and anti-RAGE treatment increases the level of IL-10,suppressing the inflammatory response and promoting the recovery of EAAG. |
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