Discussion On The Role And Significance Of TIGIT In Stress Rats Based On The Theory Of "Rage Impairing Liver"

Objective:To investigate the expression and role of immune regulatory receptor TIGIT in thymic tissue and peripheral blood T cells of "angry" psychological stress rats,and to clarify the role of this immune regulatory receptor in the occurrence and development of psychological stress.It will provide new research opportunities and more theoretical basis for the pathogenesis of " Rage impairing liver " in Traditional Chinese medicine.Methods:Forty-eight Wistar male rats aged 8 weeks were randomly divided into stress-free control group(A),“anger” model 7-day group(B)and 14-day group(C),21 days group(D),12 in each group.Except for the blank group,the rest of the groups were replicated in "anger" model rats by "social isolation method" and "plantar electric shock method".Then the above stress time node is studied as follows.1.Before and after the experiment,open field test and aggressive behavior test were used to observe the behavioral changes of "angered" rats.2.HE staining was used to observe the thymus structure and cell morphology,and Tunel method was used to observe the apoptosis of thymus cells,in order to clarify the apoptosis of immune cells in rats after receiving "anger" stress.3.Immunohistochemical method(SP method)and western blot(WB)were used to detect the changes of TIGIT / CD155 and PDCD5 in thymus tissue and peripheral blood mononuclear cells(PBMC)of stressed rats from the protein level.4.Analysis of the distribution ratio of CD3 +,CD4 +,and CD8 + T cell subsets in peripheral blood PBMCs by multi-color fluorescence analysis with flow cytometry and calculating the CD4 / CD8 ratio to observe the T cell differentiation selection.5.Elisa method was used to measure the levels of CRH,ACTH,IL-2 and IL-10 in peripheral blood,and judge the stress state of rats from hormone levels and the immune function of rats from the secretion of immune factors.6.The statistical methods of the above experimental data were compared using single factor analysis of variance,and the behavioral score comparison before and after stress was analyzed by paired t test;SP method results of thymus and peripheral blood PBMC Image Pro Plus 6.0 software was used to calculate the average optical density of positive images Value(MD);Correlation analysis was performed on the MD value using Graph Pad Prism6.0 software.Results: 1.Behavior test results:Open field test: One day before the experiment,there was no significant difference in the behavioral parameters of the rats in each group of "Ang" model compared with the control group(P>0.05);after the experiment,the level score(HM)of the 21-day group of "Ang" model,The vertical score(VM)and modification times(MT)scores were significantly increased(P<0.01),the HM of the model 7-day group and the model 14-day group were increased(P<0.05 or P<0.01).Before and after the experiment,the HM and VM of the control group showed a gradual decreasing trend(P<0.05 or P<0.01);the "Ang" model group showed a downward trend and then an upward trend(P<0.05 or P<0.01).Aggressive behavior test: One day before the experiment,the behavioral parameters of the rats in each group of the "Ang" model were not significantly different from those in the control group(P>0.05);Decreased(P<0.01),the number of attacks(NA)and cumulative attack time(CTA)were significantly increased(P<0.01 or P<0.05),and there was no significant difference in the total test score(TSA).2.HE staining results:Under 400 x light microscope,the control group of rats had full thymus cortex,densely distributed primitive T lymphocytes,nuclei stained dark blue purple,neatly arranged,loose spaces,less cytoplasm,and a few macrophages were pale red.The thymus cortex of rats in each group of "ang" model showed different degrees of tissue structure damage,the arrangement of primitive T cells was disordered,the number was reduced compared with the control group,the cell gap was reduced,part of the "stripe" was piled up,and the apoptosis phenomenon increased: the nucleus The staining becomes lighter,and some of the nuclear shrinkage,splitting,or dissolving in "flakes" occur.3.Tunel test results:Immunofluorescence showed positive(apoptotic state)thymus T cell nuclei showed green fluorescence,DAPI negative staining was blue fluorescence;Apoptosis intensity relationship: control group<model 21 day group<model 14 day group<model 7 day group,And the difference is significant(P<0.05 or P<0.01);compared between the model groups,the 7-day group was significantly higher than the 14-day and 21-day groups(P<0.01),and the 14-day group was significantly higher than the 21-day group(P<0.01).4.SP method test results:Thymus: The brown-yellow reaction zone was wrapped around the naive T cell membrane(TIGIT)or nucleus(PDCD5)of the thymus cortex,respectively.The intensity of the MD values of TIGIT and PDCD5 protein expression was consistent,and the order was: control group <model 21 day group< In the model 14-day group <model 7-day group,except that the expression of the two proteins in the model 21-day group did not increase significantly,the difference between the two proteins in the remaining groups was statistically significant(P<0.05 or P<0.01).Peripheral blood PBMC: The dark brown reaction zone was wrapped around the smear cell membrane,and the relationship between the strength of TIGIT protein expression was consistent with the above thymus T cell results.The TIGIT expression activity of the 7-day group of the "anger" model was the most obvious(P<0.01).5.Western-Blot test results:The relationship between thymus TIGIT protein levels is as follows: Control group<model 14 day group<model 21 day group<model 7 day group,the protein content of each model group was significantly higher than the control group(P<0.01);comparison between model groups: model 7 day group was significantly higher than model 14 Days and 21 days group(P<0.01),model 14 days group was significantly lower than model 21 days group(P<0.01).The relationship of thymus CD155 protein levels was as follows: Each model group was significantly higher than the control group(P<0.01),and there was no significant difference between the model groups,with no statistical significance.6.Flow cytometry test results:CD3 +,CD4 + T cell subgroup expression level relationship: Model 7-day group <model 14-day group <model 21-day group <control group.The 7-day and 14-day models of the model were significantly different from the control group(P<0.05 or P<0.01).The CD8+ T cell subsets increased in each model group,with the model 7-day group being the most significant(P<0.05).In addition,the CD4+/CD8+ ratios of all groups in the "Ang" model were reduced,with the model 7-day group being the most significant(P<0.01).7.Measurement results of serum CRH,ATCH,IL-2,IL-10 concentrations:The concentrations of hormones CRH and ACTH in the 7-day and 14-day groups of the "ang" model were significantly increased compared with the control group(P <0.01),and the changes in the 21-day group of the model were not obvious;the level of IL-2 expression in each group of the "ang" model showed a downward trend.The model 7-day group was the most significant(P <0.01).In contrast,the IL-10 expression level showed an upward trend,and the model 14-day group was the most prominent(P <0.01).8.Correlation analysis:TIGIT in thymus was positively correlated with PDCD5 protein expression level(r = 0.6784,P <0.0001);TIGIT protein expression in thymus was negatively correlated with CD4 + / CD8 + ratio(r =-0.7918,P <0.0001);single peripheral blood The expression level of TIGIT protein in nuclear cells was negatively correlated with the ratio of CD4 + / CD8 +(r =-0.7902,P <0.0001).Conclusion: 1.During chronic "angry" stress,the behavioral scores of rats showed significant fluctuations;the peripheral blood CRH and ACTH hormones increased periodically,and the secretion level was highest at 7 days of stress.2.During chronic "angry" stress,the TIGIT and PDCD5 proteins of the rat thymus showed consistent and periodic expression,of which the activity was highest at 7 days of stress;the two together mediated the "failure" of immature thymus gland cells and caused thymus atrophy To achieve an immunosuppressive effect.3.During chronic "angry" stress,the TIGIT protein expression activity of rat peripheral blood mononuclear cells is periodically up-regulated,which is the highest at 7 days of stress,resulting in an imbalanced distribution of peripheral effector T cell subsets and disturbance of immune factor secretion To achieve an immunosuppressive effect.In summary,the inhibitory regulation of the immune system by the "angry" stress response can be achieved through thymus morphological changes and the TIGIT-CD155 signaling pathway mediated by the immunomodulatory receptor TIGIT.TIGIT may be serve as neuro-endocrine-immunity and cell The important interconnected molecules in the apoptosis reaction play an important role in the pathological process of psychological stress.