Evaluation Of Prognostic Markers For Sepsis And The Effect Of Berberine In Combination With Yohimbine

AIM:To analyze the relationship between lymphocyte apoptosis,inflammatory cytokine levels, immune function status and the severity and prognosis in patients with sepsis in order to explore the feasibility of them as prognostic predictor for sepsis,and further observe the effect of berberine in combination with yohimbine(Ber+Y) on the prognostic predictors in a rat model of CLP-induced sepsis.METHODS:Part 1:Clinical signifieanc of lymphocyte apoptosis,inflammatory cytokine levels,immune function status in patients with sepsis.From June 2007 to December 2007,a total of 26 patients with sepsis at general intensive care unit(GICU) in Guangdong general hospital were enrolled and devided into sepsis and multiple organ dysfunction syndrome(MODS) due to sepsis groups according to organ function state.In addition,15 healthy persons were included as control.Peripheral venous blood samples were drawed to detect apoptosis rate of lymphocytes,CD4⁺/CD8⁺ rate,Th1/Th2 rate and CD14⁺ monocyte human leukocyte antigen-DR(HLA-DR) expression rate by flow cytometry and plasma levels of cytochrome C(Cyt-c),interleukin-6(IL-6),interleukin-18(IL-18).interleukin-27 (IL-27),γinterferon(IFNγ),high mobility group protein 1(HMGB-1) by Enzyme Linked Immunosorbent Assay(ELISA).Furthermore.sepsis-related organ failure assessment score (SOFA) and acute physiology and chronic health evaluationâ…¡(APACHEâ…¡) were recorded.The differences in these indexes were assessed among the sepsis,MODS due to sepsis and control groups. Partâ…¡:Evaluation of lymphocyte apoptosis,inflammatory cytokine levels and immune function status as prognostic predictors for sepsis.The patients with sepsis were enrolled according to the inclusion Criteria based on the latest diagnosis Criteria at international sepsis conference in 2001,then,the patients who fulfilled the elimination Criteria and had MODS within 3 days were excluded.All patients were provided conventional therapy,and the progression of sepsis and outcome in 28 days was recorded. According to the progression within 7 days,patients were divided into two groups:sepsis and MODS due to sepsis;According to outcome on the 28th day after enrollment,the patients were further divided into survival and non-survival group.The severity of sepsis was classified by APACHEâ…¡and SOFA once a day for 7 days;Plasma Cyt-c,IL-6,IL-8,IL-18,IL-27,IFNγand high mobility group B1(HMGBI) levels were measured by commercial enzyme-linked immunosorbent assay(ELISA) kits according to the manufacturer's recommendations on day 0,1, 3,7 after enrollment(entry into the study was defined as day 0);Apoptosis of lymphocytes,CD4⁺/ CD8⁺ rate,Th1/Th2 rate and CD14⁺ monocyte human leukocyte antigen-DR(HLA-DR) expression were also detected by flow cytometry on day 0,1,3,7 after enrollment.Furthermore,the correlation analysis between these indexes was made.Receiver operating characteristic(ROC) curves,made by plotting sensitivity versus(1 - specificity),were used to evaluate the diagnostic performance for these indexes,which have a difference by mon-factor analysis at various cutoff points,and the disCriminant equation for prediction by multiplicity was established.Partâ…¢:The effect of septic severity on lymphocyte apoptosis in spleen,serum IL-18 and prognosis in a CLP mouse model.Mice subjected to cecal ligation and puncture(CLP) were used in the study,the experiment was performed in three parts:â‘ 32 Kunming mice were randomly divided into the sham group(n= 10),the mild sepsis(mild CLP) group(n = 11) and severe sepsis(severe CLP) group(n = 11), and 7-day mortality of mice among three groups was compared;â‘¡26 Kunming mice were randomly divided into the sham group(n = 8),the mild sepsis(mild CLP) group(n = 10) and severe sepsis(severe CLP) group(n =8),the parameters of functional disturbance for liver,lung, kidney after 20h after CLP were determined;â‘¢30 Kunming mice were randomly divided into the sham group(n = 10),the mild sepsis(mild CLP) group(n = 10) and severe sepsis(severe CLP) group(n = 10),serum Cyt-c,IL-18 levels,caspase-3 activity of splenocytes and lymphocyte apoptosis rate 20 h after CLP were detected.Partâ…£:the effect of berberine in combination with yohimbine(Bet+Y) on lymphocyte apoptosis in spleen,serum IL-18 and survival in mice.A CLP mouse model was established,The experiment was carryed out in two parts:â‘ 30 Kunming mice were randomly divided into sham group(n = 10),CLP group(n = 10) and CLP + Ber + Y group(n = 10),mortality of mice in three groups were recorded once a day for 14 days;â‘¡In another set of experiments,serum Cyt-c,IL-18 levels,caspase-3 activity of splenocytes and lymphocyte apoptosis rate in spleen at 20 h after CLP were examined.Statistical analyses were performed with SPSS for Windows 13.0(SPSS,Chicago,IL). Values for continuous variables are expressed as mean±SE throughout the article,and in case of a skewed distribution as the median and range.The Student's two-tailed/-test was used to compare continuous variables exhibiting a normal distribution between two groups.Nonparametric continuous variables were compared using Wilcoxon's rank-sum(Mann Whitney U) test.To examine linear correlations between two variables,the Pearson or Spearman correlation method was used as appropriate;Survival time compared by using Kaplan-Meier method,and the survival curve was drawn;Data of Cross-table were analyzed by usingχ2 test,multiple comparisonusing partitions ofχ2 method;Multiple comparison in non-parametric test of k-independent samples by using tested Nemenyi test(manual calculation).Significance was assumed when P＜0.05.RESULTS:Partâ… :Changes of lymphocyte apoptosis,inflammatory cytokine levels,immune function status in patients with sepsis.1.The rate of lymphocyte apoptosis and plasma level of Cyt-c were significantly increased in patients with sepsis compared with healthy controls,both of which were higher in patients with MODS due to sepsis than those without MODS;HLA-DR expression rate were decreased in patients with sepsis,and CD4⁺ / CD8⁺ raten were lower in patients with MODS due to sepsis than those without MODS;Plasma levels of IL-18 were significantly higher in patients with MODS due to sepsis than control group;IL-27 levels was also significantly higher in patients with MODS due to sepsis and sepsis than controls;IFNγlevels was significantly higher in patients with MODS due to sepsis than the other two groups.2.There was a positive correlation between the rate of lymphocyte apoptosis,plasma Cyt-c, IL-18,IL-27 and IFNγ,levels and APACHEâ…¡or SOFA score.A negative correlation between HLA-DR%,Th1/Th2 and APACHEâ…¡score or SOFA score also occurred.Furthermore, lymphocyte apoptosis rate and plasma Cyt-c levels were assotiated with HLA-DR%,IL-18,IL-27, IFNγ(P all＜0.05).Partâ…¡:Heart rate,Glasgow Coma Scale(GCS) score,blood platlet count,lymphocyte count,IL-8,Il-18 levels in 24 h after enrollment played an important role in prediction for MODS in 7 days,and blood lymphocyte apoptosis rate,HLA-DR in 24 h after enrollment predicted survival within 28 days.1.IL-8,heart rate,blood glucose,Na⁺ and GCS score on day 0 was significantly inCreased, and lymphocyte count deCreased in the patients with MODS due to sepsis compared to those with only sepsis(P all＜0.05).Compared with sepsis group,IL-18,IFNγ,and Cr on day 1,IL-6 and IFNγon day 3 were significantly higher(P all＜0.05),PaO₂/FiO₂ on day 2,HLA-DR%on day 3 significantly lower(P all＜0.05) and APACHEâ…¡score and SOFA score in 0-3 days were significantly higher in patients with MODS due to sepsis(P all＜0.05);Receiver operating characteristics(ROC) analysis demonstrated the following indexes were useful in the prediction for MODS in 7 days:IL-8(0 d),heart rate,blood glucose level,blood Na⁺,GCS score, lymphocyte count,IL-18(ld),IFNγ(1d),PaO₂/FiO₂(2d),IL-6(3d),IFNγ(3d),HLA-DR%(3d), APACHEâ…¡score(0-3d),SOFA score(0-3d)(P＜0.05);The areas under the ROC curves for each parameter were as follow from high to low:APACHEâ…¡(3d),APACHEâ…¡(2d),SOFA(1d), SOFA(3d),APACHEâ…¡(0d),IL-6(3d),APACHEâ…¡(1d ),IFNγ(3d),SOFA(0d),SOFA (2d),IL-18(1d),IFNγ(1d),IL-8(0d),HLA-DR%(3d),PaO₂/FiO₂(2d):At the optimum cut-off points,Youden index(YI) from high to low was as follow::APACHEâ…¡(3d),APACHEâ…¡(2d),SOFA(1d),IL-18(1d),IL-6(3d),IFNγ(1d),IFNγ(3d),SOFA(3d),SOFA (2d),APACHEâ…¡(0d),SOFA(0d),APACHEâ…¡(1d),IL-8(0d),PaO₂/FiO₂(2d), HLA-DR%(3d).A disCriminant equation,based on PC,heart rate,GCS,serum IL- 18,serum IL-8 and blood lymphocyte count detected within 24 h after enrollment,was established:Y(0) = 1.100×10²x₁+0.451x₂+1.910x₃+5.489×10^-2x₄-0.166x₅+2.322x₆-38.541;Y(1)=-4.635×10^-3x₁+ 0.542x₂+1.574x₃+5.675×10^-2x₄-0.155x₅+1.162x₆-37.148[Y(0) indicates that MODS do not occur,Y(1) presents MODS,x₁-PC(0d),x₂-HR(0d),x₃-GCS(0d),x₄-IL-18(1d),x₅-IL-8 (0d),x₆-lymphocyte count(0d)].DisCriminant analysis:the correct rate=82.7%,sensitivity = 86%,specificity = 79%,Youden index = 65%.The model of prediction for MODS within 7 days has a higher accuracy than any of the above indexes,APACHEâ…¡score and SOFA score in single-factor analysis.2.Cyt-c on day 0 was significantly inCreased,lymphocyte count,and HLA-DR%Th1/Th2 were deCreased significantly(P all＜0.05),lymphocyte apoptosis rate and IL-18 on day 1 and HMGB1 on day 3 were significantly higher(P all＜0.05),and HLA-DR%lower Th1/Th2 on day 3 were deCreased significantly(P all＜0.05),APACHEâ…¡score on day 0-7 and SOFA score on day 1-7 were significantly higher in non-survival group than survival group(P all＜0.05);Cyt-c, lymphocyte count,HLA-DR%and Th1/Th2 on day 0,the rate of lymphocyte apoptosis and IL-18 on day 1,HMGB1,HLA-DR%and Th1/Th2 on day 3 and APACHEâ…¡score on day 0-7 and SOFA score on day 1-7 could predict survival in 28 days(P all＜0.05);ROC analysis demonstrated the following indexes were useful in the prediction for survival in 28 days:Cyt-c(0 d),lymphocyte count(0 d),HLA-DR%(0 d),Th1/Th2(0 d),lymphocyte apoptosis rate(1 d), PaO₂/FiO₂(1 d),IL-18(1 d),HMGB-1(3d),HLA-DR%(3d),Th1/Th2(3d),APACHEâ…¡score (0-7d),SOFA score(0-7d).The areas under the ROC curves for each parameter were as follow from high to low:lymphocyte apoptosis rate(1d),SOFA(1d),APACHEâ…¡(3d),APACHEâ…¡(2d),APACHEâ…¡(4d),APACHEâ…¡(7d),APACHEâ…¡(ld),APACHEâ…¡(5d),HLA-DR% (0d),APACHEâ…¡(6d),SOFA(4d),HLA-DR%(3d),SOFA(2d),Cyt-c(0d),SOFA (3d),SOFA(5d),IL-18(1d),HMGB1(3d),APACHEâ…¡(0d),Th1/Th2(0d);At the optimum cut-off points,Youden index(YI) from high to low was as follow:lymphocyte apoptosis rate(1d),APACHEâ…¡(1d),APACHEâ…¡(Td),SOFA(1d),HLA-DR%(0d),IL-18(1d), APACHEâ…¡(3d),APACHEâ…¡(2d),HLA-DR%(3d),SOFA(4d),APACHEâ…¡(4d),APACHEâ…¡(5d),HMGB1(3d),SOFA(3d),APACHEâ…¡(0d),Cyt-c(0d),APACHEâ…¡(6d),SOFA (5d),SOFA(2d),Th1/Th2(Od).A disCriminant equation,based on lymphocyte apoptosis rate and HLA-DR detected within 24 h after enrollment,was established: Y(0)=0.456x₁+0.315x₂-24.587;Y(1) = 0.392x₁+0.431x₂-22.430[Y(0) indicates survival,Y(1) represents death,x₁- HLA-DR(0 d),x₂- lymphocyte apoptosis rate(1d)].DisCriminant analysis: the correct rate = 78.8%,sensitivity = 77%,specificity = 80%,Youden index = 57%.The model of prediction for survival within 28 days has a higher accuracy than any of the above indexes. APACHEâ…¡score and SOFA score in single-factor analysis. Partâ…¢:The effect of septic severity on lymphocyte apoptosis in spleen and prognosis in a CLP mouse model.1.No mouse died in control group at 168 h,the average survival time＞168h,the average survival time of mice in mild CLP group was 121.09±19.54 h,and 89.46±19.78 h in severe CLP group,there was a significant difference among the three groups in survival time(P＜0.01); There was no significant difference in 7-day mortality between mild and severe CLP groups.2.Serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) at 20 h after CLP inCreased significantly in two model groups than control(P＜0.05);there was a significant difference among the three group in lung wet-dry gravity rate(P＜0.05),and the lung wet-dry gravity rate in severe CLP group was higher than mild CLP group(P＜0.05);there was no significant difference among the three groups in blood urea nitrogen(BUN),Creatinine(CR) and total bilirubin(TB)(P＞0.05).3.Apoptosis rate of spleen lymphocytes,T lymphocytes,CD4⁺ T lymphocyte significantly inCreased,and blood lymphocyte count deCreased in CLP groups compared to control,but there was no difference between mild and severe CLP groups.There was a significant negatively correlation between blood lymphocyte count and apoptosis rate of lymphocytes,T lymphocytes and CD4⁺ T lymphocyte in spleen(P all＜0.05).4.There was a significant positive correlation between liver function enzymes and apoptosis rate of lymphocytes,T lymphocytes,CD4⁺ T lymphocyte and caspase 3 activity in the spleen(P＜0.05);blood lymphocyte count was negatively correlated with liver function enzymes(P＜0.05). No correlation occurred between apoptosis rate of lymphocytes in spleen and lung wet-dry gravity rate.Partâ…£:the effect of berberine in combination with yohimbine(Ber+Y) on lymphocyte apoptosis in spleen and survival in mice.1.The survival rate of mice in CLP+Ber+Y group was higher than that of CLP group.2.T lymphocyte apoptosis rate in the spleen was lower in CLP+Ber+Y group than that in CLP group,but the difference was not statistically significant,but CD4⁺ T lymphocyte apoptosis rate was significantly deCreased in CLP+Ber+Y group compared to CLP group(P＜0.05).CONCLUSIONS:1.The rate of blood lymphocyte apoptosis,plasma Cyt-c,IL-18,IL-27 and IFN-γlevels Th1/Th2 rate,HLA-DR expression,CD4⁺ / CD8⁺ rate may evalute the disease severity and prognosis in sepsis patients to a certain extent;2.The disCriminant equation,which was established based on platelet count,heart rate,GCS, serum IL-18,IL-8 levels and blood lymphocyte count detected within 24 h after enrollment,is useful in the prediction for MODS in 7 days,and its prediction accuracy is up to 82.7%.The other one,which was based on lymphocyte apoptosis rate and HLA-DR detected within 24 h after enrollment,is able to predict survival in 28 days,and its prediction accuracy is 78.8%;3.There was a significant positive correlation between liver function enzymes and apoptosis rate of spleen lymphocytes,but no correlation occurred between apoptosisrate of lymphocytes in spleen and lung wet-dry gravity rate in mice.There was a significant negative correlation between blood lymphocyte count and apoptosis rate of lymphocytes,T lymphocytes and CD4⁺ T lymphocyte in spleen.There was no difference in spleen lymphocyte and its subgroup apoptosis rate at 20 h after CLP and 7-day mortality between mild and severe CLP groups.4.Berberine in combination with yohimbine could suppress lymphocyte apoptosis,especially CD4⁺ T lymphocyte,and then improve survival in septic mice.These data suggest that lymphocyte apoptosis may be a valuable marker in the prediction of outcome and evaluation of theraoeutic intervention in patients with sepsis.