Evaluation Of EZH2 Expression On Peripheral Blood T Lymphocytes To Predict Survival Of Patients With Sepsis

Objective:The relationship between the expression of histone methyltransferase enhancer zeste homolog 2（EZH2）in Drosophila melanogaster on the immune cells of patients with sepsis and its function as well as mechanism is still unknown.In this study,we explored the expression and dynamic changes of EZH2 on the major subgroups of T lymphocytes in peripheral blood of patients with sepsis（CD4⁺T cells,CD8⁺T cells）,and probed into the predictive value of EZH2 expression on prognosis.Based on its specificity and sensitivity,it may help to determine the timing of intervention and provide a theoretical basis for finding targeted treatment points for sepsis.Method:1.Subjects:according to the definition of sepsis 3.0,patients with sepsis in the emergency ICU and central ICU were selected as the septic patients group and healthy people who were examined at the Shanghai East Hospital Physical Examination Center during the same period were selected as the healthy control group.According to the inclusion and exclusion criteria,a total of 48 patients with sepsis（septic patients group）and 40 healthy people（healthy control group）were selected as the research subjects.2.Collect clinical data of all patients with sepsis included in the experiment:（1）Gender,age,28-day survival rate,ICU admission and transfer time,history of chronic diseases,whether to use mechanical ventilation,whether to use vasoactive drugs and other basic information.（2）Collect the blood routine,blood gas analysis,and infection indicators including C-reactive protein,procalcitonin,and interleukin-6 of the study subjects on the 1st,3rd,and 7th days after diagnosis.（3）Calculate the GCS score,SOFA,and APACHE II scores by combining the indicators on the 1st,3rd,and 7th days.（4）The expression of EZH2 on CD4⁺and CD8⁺T lymphocyte subpopulations was detected by flow cytometry on the 1st,3rd,and 7th days.Collect the basic data of the healthy control group included in the experiment:（1）Gender and age.（2）Collect blood routine,SOFA and APACHE II scores and other indicators.（3）The expression of EZH2 on CD4+and CD8⁺T lymphocyte subsets was detected by the flow cytometry.3.Patients with sepsis were divided into death group and survival group according to the survival or death results of patients admitted to ICU 28 days after diagnosis of sepsis.The relationship between the expression level of EZH2 and the 28-day survival rate in patients with disease course was analyzed.According to the corresponding ROC curve of the prognostic indicators of septic patients,the best cut-off value was found,and the best stage value was used to regroup them being divided into high expression group and low expression group to further predict the efficacy of prognosis model of sepsis.4.SPSS20.0 was used for data analysis.The measurement data were tested for normality,and the normal distribution data were tested by independent sample T test.Non-normally distributed data were compared using the Mann Whitney U test.Qualitative data were tested byχ2.The difference was statistically significant at P<0.05.Single-factor logistic regression analysis was performed to compare the statistically significant indicators.Multi-factor logistic regression analysis was performed on single-factor statistically significant indicators to screen independent risk factors for death in patients with sepsis.Use the ROC curve to find the best cutoff point,and calculate the area,sensitivity and specificity under each index curve.Spearman correlation analysis was used to analyze the correlation between EZH2 and disease severity,SOFA score,APACHEII score,infection-related indicators and inflammatory factor.Results:1.The positive rate of EZH2 expression in CD4⁺T cells,the mean fluorescence intensity of EZH2 in CD4⁺T cells,the positive rate of EZH2 expression in CD8⁺,and the mean fluorescence intensity of EZH2 in CD8⁺T cells were significantly higher in the sepsis group than in the healthy control group.With the exacerbation of sepsis patients,the expression of EZH2 increased.2.The positive rate of EZH2 expression in CD4⁺T cells,the mean fluorescence intensity of EZH2 in CD4⁺T cells,the positive rate of EZH2 expression in CD8⁺,and the mean fluorescence intensity of EZH2 in CD8⁺T cells were higher in the death group than in the survival group.The disease progressed,and the expression of EZH2 on peripheral blood T cells increased.The CD4⁺,CD4⁺to CD8⁺ratio in the death group were lower than those in the survival group.However,the expression of EZH2 on peripheral blood T cells was not significantly different in the death group on the 1^st,3^rdand 7^th days.3.Multivariate analysis of clinical data and flow cytometry data of patients with sepsis on the 1^st and 3^rd day with different prognosis on 28 days.（1）The mean fluorescence intensity of CD8⁺T cell EZH2 on the1^st day of sepsis patients is an independent risk factor for predicting the patients’28-day death.ROC curve analysis:The area under the curve of the mean fluorescence intensity of EZH2 in CD8⁺T cells is 0.775（0.631-0.918）,which can predict the 28-day prognosis.And the mean fluorescence intensity of EZH2 in CD8⁺T cells predicts the sensitivity and specificity of28-day death 75%and 82.1%,respectively.The specificity of predicting 28-day death is better than APACHE II score,and the sensitivity is better than SOFA score,which has certain clinical significance.Combination of patients with sepsis day 1 SOFA score,APACHE II score EZH2+,mean fluorescence intensity of EZH2 in CD8⁺T cells,the area under the ROC curve is 0.973（0.924-1.000）,the sensitivity was 93.8%,the specificity was 96.4%,is better than that of single index prediction,which can rise to the value of early predicting the prognosis of patients with sepsis,judging the prognosis of patients with play an important role.（2）On the 3^rd day of sepsis,the positive rate of EZH2 expression in CD4⁺T cells,the mean fluorescence intensity of EZH2 in CD4⁺T cells,the positive rate of EZH2expression in CD8⁺T cells,and the mean fluorescence intensity of EZH2 in CD8⁺T cells were independent risk factors for predicting the patients’28-day death.ROC curve analysis:the area under the mean fluorescence intensity curve of CD8⁺T cells EZH2 is0.936（0.842-1.000）with sensitivity degree 66.7%and the specificity degree 100%.The specificity degree for predicting 28-day death is better than the SOFA score and APACHEII score.It has certain clinical significance for the long-term prognosis of patients with sepsis.The area under the mean fluorescence intensity curve of CD4⁺T cells EZH2 is 0.829（0.625-0.998）with specificity degree 52.17%and sensitivity degree100%.The sensitivity degree at 28-day death was better than the SOFA score.4.The Spearman correlation between the mean fluorescence intensity of EZH2⁺in CD8⁺T cells and sepsis severity,survival time,and length of stay in the sepsis group showed that the mean fluorescence intensity of EZH2 in CD8⁺T cells on the 1^st day of the sepsis group was positively correlated with APACHEII scores on the 3^rd and 7^thdays,and negatively correlated with survival time.The mean fluorescence intensity of EZH2 in CD8⁺T cell on the 3^rd day in the sepsis group was positively correlated with the APACHEII scores on the 1^st,3^rd,and 7^th days,negatively correlated with the survival time and the number of hospital stays,and positively correlated with SOFA score on the1^st and 3^rd days.In addition,we also found that the positive rate of EZH2 expression in CD4⁺T cell on the 1^st day was positively correlated with the SOFA score on the 1^st day.5.The positive rate of EZH2 expression in CD4⁺T cells,the mean fluorescence intensity of EZH2 in CD4⁺T cells,the positive rate of EZH2 expression in CD8⁺T cells,and the mean fluorescence intensity of EZH2 in CD8⁺T cells s on the 3^rd day of sepsis were positively correlated with the procalcitonin,lactic acid,neutrophil to lymphocyte ratio,and platelet to lymphocyte ratio on the 7^th day.Conclusion:1.Compared with healthy people,the expression of EZH2 in peripheral blood T lymphocytes in sepsis patients increased significantly.And the level of EZH2 expression increased with the worsening of sepsis patients.2.The expression of EZH2 in peripheral blood T lymphocytes in sepsis patients was significantly different on the 1^st,3^rd and 7^th day.The death group was higher than the survival group.As the disease progressed,the increase in EZH2 expression on peripheral blood T cells increased.of.But There were no significant differences of the EZH2expression in the T cells of the patients in the death group among the 1^st,3^rd and 7^th days,which is considered to be related to the patient’s severe illness and no obvious remission.3.The mean fluorescence intensity of EZH2 in CD8⁺T cell on the 1^st day of sepsis patients,the positive rate of EZH2 expression in CD4⁺T cell on the 3^rd day,the mean fluorescence intensity of EZH2 in CD4⁺T cell,the positive rate of EZH2 expression in CD8⁺T cell and the mean fluorescence intensity of EZH2 in CD8⁺T cells are independent risk factors for 28-day prognosis.And its prognostic efficacy may be better than the commonly used sepsis SOFA score and APACHE II score.4.The expression of EZH2 in peripheral blood T lymphocytes in sepsis patients is related to the severity of the disease,and the higher the expression of EZH2,the more severe the disease in patients with sepsis.5.The expression of EZH2 in peripheral blood T cells on the 3^rd day of sepsis patients is related to the inflammation index on the 7^th day.It is considered to have certain guiding significance for clinical prognosis and has considerable predictive power.