The Study On Expression And Function Of CMTM4 In Septic T Lymphocytes And The Predictive Value Of NLR,PLR For Myocardial Iniuries In Sepsis

Part 1 The study on expression and function of CMTM4 in septic T lymphocytesObjectiveAnti-inflammatory/pro-inflammatory imbalance and immune dysfunction are important pathogenesis of sepsis.The persistent adaptive immune system deficiency,immunosuppression,and immune paralysis during the inflammatory process are the main reasons for.the poor prognosis of patients with sepsis,and exploring the immune mechanism of sepsis has become a top priority.The main characteristic of sepsis-adaptive immune system deficiency is damage and exhaustion of T lymphocytes,which are manifested as decreased cell number,increased apoptosis,and weakened function.Studies have shown that CMTM4,a member of the human chemokine-like factor superfamily,interacts with negative regulatory factors in tumor,negatively regulates T cell function,and leads to tumor immune escape.However,there is no relevant study about CMTM4 in sepsis.This study intends to explore the expression level of CMTM4,in septic T lymphocytes and its biologic influence on the immune function of septic T cells,so as to pave the way for future researchers to explore the immune pathogenesis of sepsis and hope to provide noval ideas for the clinical diagnosis and treatment of sepsis.Methods(1)Adopt the latest version of the guidelines Sepsis 3.0 issued in 2016 as the diagnostic criteria to enroll patients with sepsis,collect their peripheral blood,and collect the peripheral blood from healthy volunteers at the same time,use the lymphocyte separation liquid to separate human peripheral blood lymphocytes,Peripheral blood T lymphocytes were sorted by CD3+magnetic beads;the difference of CMTM4 genetic expression between the normal group and the sepsis group was detected by RT-qPCR;the difference in protein level between the two groups was detected by Western Blot.(2)The human acute T-cell leukemia Jurkat cell line was cultured in vitro,and LPS was given to stimulate the septic cellular model.RT-qPCR and Western Blot were used to detect the difference in gene expression between the experimental group and the control group.(3)Use small interfering RNAs(siRNAs)to silence the CMTM4 gene of the Jurkat cell line.After LPS stimulation was given to the silenced group and the control group,CCK-8 test was used to detect the cell proliferation of the two groups;PI was used to detect the effect of CMTM4 on cell cycle;Annexin V-FITC compares the apoptosis ratio of the silent group after LPS treatment and the control group to determine the effect of CMTM4 on the apoptosis of septic T lymphocytes;ELISA was used to detect IL-2,IL-10 to explore the secrection function changes of septic T lymphocytes treated with siRNAs.(4)Cecal ligation and puncture method to establish a mouse model of sepsis.CLP group and sham group were set up and sacrificed at 6h,12h,and 24h.The peripheral immune organs of mice were collected.The expression of CMTM4 in the CLP group and the sham group was detected at the transcription and translation level,and the expression of CMTM4 in the tissues was observed by immunohistochemistry.Results(1)Compared with healthy volunteers,the expression of CMTM4 in peripheral blood T lymphocytes of patients with sepsis was significantly higher at both transcription and translation levels(P<0.05).(2)Compared with the blank control group,the sepsis T cell showed increased expression of CMTM4 after LPS stimulation at the RNA and protein levels(P<0.05).(3)After silencing the CMTM4 gene of Jurkat cells with siRNAs,the two groups of cells in the si-CMTM4 and si-NC groups were given LPS stimulation for 6h,12h,and 24h to explore the effect of CMTM4 on the function of septic T lymphocytes,and found that the cell proliferation function of the si-CMTM4 group was stronger than that of the si-NC group.The proportion of cells in the si-CMTM4 group entering the S phase of the cell cycle was significantly higher than that of the si-NC group,and the proportion of cells stagnating in the G1 phase of the si-NC group was higher than that of the si-CMTM4 group(P<0.05).After LPS stimulation,the proportion of apoptosis in the si-CMTM4 group was significantly lower than that of the si-NC group(P<0.05),and the proportion of survival cell was higher.In addition,the concentrations of IL-2 and IL-10 in the cell supernatant of the si-CMTM4 group were higher than those of the si-NC group(P<0.05).(4)In the CLP mice model,compared with the sham group,the expression of CMTM4 in the peripheral immune organs spleen and abdominal lymph node tissues increased at the RNA and protein levels(P<0.05).Morphologically,the results of immunohistochemistry showed that the expression level of CMTM4 in the tissues was increased.Conclusion(1)CMTM4,a member of the human chemokine-like factor superfamily,has increased expression in septic T lymphocytes.(2)CMTM4 has an immunosuppressive effect on septic T lymphocytes,which is specifically manifested as inhibiting the proliferation of T cells,affecting the cell cycle,promoting the occurrence of apoptosis,and inhibiting the secretion of inflammatory cytokines by T lymphocytes.(3)The expression of CMTM4 is elevated in the peripheral immune organs of septic mice.Therefore,we believe that CMTM4 participates in the occurrence of sepsis immunosuppression by interfering with the biological functions of T lymphocytes,and has a certain impact on the immune pathogenesis of sepsis.The increased expression of CMTM4 in sepsis patients leads to suppression of T cell immune function and poor prognosis.Part 2 The Predictive Value of NLR,PLR for Myocardial Injuries in SepsisObjectiveSepsis 3.0 pointed out that sepsis is a life-threatening organ dysfunction caused by the body’s disorder of infection,and emphasizes the important role of immune system disorders and organ damage in the disease.Among them,acquired immunity is the top priority.Lymphocytes with complex subgroups not only affect the immune state,but also participate in the occurrence of organ damage.The role of lymphocytes in septic organ damage has been concerned by many researchers.The first part of this study is about basic experiments on septic T lymphocyte,and the second part will analyze the role of lymphocytes in septic organ damage through clinical patient data.Lymphocyte-related indicators involved in the study---neutrophil/lymphocyte ratio(NLR)and platelet/lymphocyte ratio(PLR)---combine the important inflammatory cells involved in sepsis,neutrophils,lymphocytes and platelets,and represents the relationship between immunity/immunity and immunity/coagulation in sepsis.Past studys show that these two indicators can reflects the severity of inflammation to a certain extent,and has gradually become a new biomarker of certain inflammatory diseases.This study focuses on septic myocardial injury to explore whether the index of NLR and PLR have predictive value for target organ damage caused by sepsis.It is hoped that it can be used as a reference for clinical identification of septic myocardial injury.MethodsThis study successively and retrospectively recruited 142 patients with sepsis who were admitted to the ICU of Peking University People’s Hospital from January 2017 to August 2019.The demographic and comorbidities were recorded,including neutrophil count,lymphocytes count,and platelet count,were collected when sepsis was diagnosed to calculate NLR and PLR;CRP,myocardial injury indicators(cTnI,BNP,CK-MB,myoglobin)and APACHE Ⅱ score within 24 hours of ICU admission were collected.According to the value of cTnI(cardiac troponin),sepsis cases were divided into two groups-non-myocardial injury group(n=74)and myocardial injury group(n=68);independent sample t-test analysis was used to analyze differences between the two groups;Spearman analysis was used to judge the correlation between NLR,PLR and myocardial injury indicators;Logistic regression analysis was used to detect the independent risk factors of myocardial injury in patients with sepsis;Receiver operating characteristic(ROC)curves was used to evaluate the value of NLR and PLR on predicting myocardial injury in patients with sepsis.ResultsThe NLR value of the myocardial injury group was significantly higher than that of the non-myocardial injury group,and the PLR was lowered(P<0.05).Spearman correlation analysis showed that NLR and CRP were positively correlated in patients with sepsis(r=0.604,P<0.05),and PLR was negatively correlated with BNP(r=-0.884,P<0.05).Logistic regression results showed that NLR,PLR and APACHE Ⅱ scores were independent risk factors of myocardial injury in patients with sepsis(P<0.05).ROC curve results show that NLR and PLR have a certain predictive value for myocardial injury in patients with sepsis(P<0.001).ConclusionNLR and PLR have a certain predictive value for myocardial injury in patients with sepsis,and NLR has a greater predictive value.