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Objective:To investigate the effect and mechanism of histone methyltransferase enhancer of zeste homolog 2 (EZH2) on sepsis-induced T cell dysfunction.Methods:Twenty-four male C57BL/6 mice were divided into three groups randomly: sham operated group, sepsis model group [cecum ligation and puncture (CLP)+dimethyl sulfoxide (DMSO) group] and EZH2 selective inhibitor treated group (CLP+GSK126 group), with 8 mice in each group. Sepsis murine model was reproduced by CLP. CLP+DMSO group and CLP+GSK126 group were treated with DMSO or GSK126 (10 mg/kg) respectively right after surgery through intraperitoneal injection. The mice were sacrificed 24 hours after operation, and the mesenteric lymph nodes were collected. The expression of EZH2, apoptosis rates, cell proliferation marker ki-67 antigen positive T lymphocytes (ki-67 + cell), interferon-γ positive T lymphocytes (IFN-γ + cell), programmed death receptor-1 positive T lymphocytes (PD-1 + cell) and programmed death-ligand 1 positive T lymphocytes (PD-L1 + cell) were determined by flow cytometry. Results:Compared with sham operated group, the expression of EZH2 in T lymphocytes was up-regulated on mesenteric lymph nodes of CLP+DMSO group. Compared with CLP+DMSO group, the ratio of CD3 + T lymphocytes in CLP+GSK126 group was up-regulated (0.70±0.02 vs. 0.50±0.07, P < 0.01), indicating that the EZH2 inhibitor could increase the number of T lymphocytes in lymph nodes of septic mice; the ratio of ki-67 + cells in CD4 + and CD8 + T lymphocytes in CLP+GSK126 group was increased (CD4 +: 0.74±0.05 vs. 0.63±0.04, CD8 +: 0.82±0.06 vs. 0.70±0.04, both P < 0.05), indicating that the EZH2 inhibitor could increase the ratio of T lymphocytes with high proliferative activity in lymph nodes of septic mice. However, no significant difference was found on both CD4 + and CD8 + T lymphocytes apoptosis rates in the mesenteric lymph nodes of mice between CLP+GSK126 group and CLP+DMSO group [CD4 +: (21.53±2.87)% vs. (20.48±3.21)%, CD8 +: (8.34±1.02)% vs. (7.71±1.38)%, both P > 0.05], indicating that no extra T lymphocytes apoptosis was induced by EZH2 inhibitor. Compared with CLP+DMSO group, the ratios of IFN-γ + CD4 + and IFN-γ + CD8 + T lymphocytes were increased in CLP+GSK126 group (IFN-γ +CD4 +: 0.31±0.11 vs. 0.14±0.06, IFN-γ +CD8 +: 0.30±0.10 vs. 0.13±0.06, both P < 0.05), suggesting that secretion of IFN-γ in lymph nodes by sepsis T lymphocytes was augmented after EZH2 inhibitor administration. Furthermore, compared with CLP+DMSO group, the ratio of PD-1 + cell in CD8 + T lymphocyte was down-regulated in CLP+GSK126 group (0.092±0.006 vs. 0.135±0.004, P < 0.01), suggesting that EZH2 inhibitor restrained the PD-1 expression on sepsis lymphoid node CD8 + T lymphocytes, however, it had no significant effect on PD-L1 + cells. Conclusion:EZH2, regulates sepsis-induced T lymphocyte dysfunction, possibly through modulating the expression of PD-1.
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Objective:To investigate the dynamic expression of histone methyltransferase (enhance of zeste homolog 2, EZH2) in peripheral blood B lymphocytes (CD19 +B) and memory B lymphocytes (CD19 +CD27 +B) of septic patients and its value in predicting prognosis in sepsis. Methods:From June 2018 to January 2020, 48 septic patients in the Intensive Care Unit of Shanghai East Hospital were enrolled, and 40 healthy adult volunteers were recruited as healthy controls. Septic patients were divided into the non-survivors (18 cases) and the survivors (30 cases) according to whether the patients survived at 28 days. Blood samples were collected at day 1, 3 and 7, blood routine, IL-6 and blood gas analysis were collected, and SOFA and APACHE Ⅱ scores were counted. Flow cytometry was used to detect the positive rate and the mean fluorescence intensity of EZH2 on CD19 +B lymphocytes, and the positive rate of EZH2 on CD19 +CD27 +B lymphocytes at different time points. In the healthy controls, fasting was taken only once in the morning. ROC curve was drawn and the area under the curve (AUC) was calculated to evaluate the value of expression of EZH2 on CD19 +B lymphocytes and CD19 +CD27 +B lymphocytes in predicting the prognosis of septic patients. Results:(1) Compared with the healthy controls, the positive rate and average fluorescence intensity of EZH2 on CD19 +B lymphocytes and the positive rate of EZH2 expression on CD19 +CD27 +B lymphocytes were significantly increased at day 1, 2 and 3 in septic patients ( P<0.05). Over time, the expression of EZH2 in CD19 +B lymphocytes and CD19 +CD27 +B lymphocytes increased gradually ( P<0.05). (2) Compared with the survivors, the positive rate of EZH2 on CD19 +B lymphocytes of the non-survivors was increased at day 1, but the positive rate of EZH2 on CD19 +CD27 +B lymphocytes of the non-survivors was decreased at day 3 and 7 ( P<0.05). (3) The positive rate of EZH2 on CD19 +B lymphocytes, APACHE Ⅱ score, SOFA score and IL-6 level in septic patients at day 1 were independently associated with 28-day mortality. (4) The AUC of APACHEⅡ score was 0.907 (95% CI: 0.825-0.990), and the sensitivity and the specificity were 88.89% and 76.67%. The AUC of SOFA score was 0.831 (95% CI: 0.706-0.955), and the sensitivity and the specificity was 66.67% and 86.67%; The AUC of EZH2 positive rate on CD19 +B lymphocytes were 0.799 (95% CI: 0.657-0.941), and the sensitivity and specificity were 88.89% and 80.77%, respectively, the sensitivity was better than SOFA score, and the specificity was higher than APACHEⅡ score. Conclusions:The high expression of EZH2 on B lymphocytes in septic patients is associated with poor prognosis. Dynamic monitoring of EZH2 expression on B lymphocytes has certain predictive value for sepsis.
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Objective:To evaluate the early diagnostic value of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein-7 (IGFBP-7) in acute kidney injury induced by sepsis.Methods:A total of 85 sepsis patients admitted to the EICU and GICU in Shanghai East Hospital from September 2017 to June 2019 were divided into theAKI group ( n=37) and the non-AKI group ( n=48) according to KIDGO diagnostic criteria, and 20 healthy volunteers were served as the control group. The clinical data were recorded and samples of urine were collected at 0 h, 6 h, 12 h, 1 d, 3 d and 7 d post sepsis. The levels of TIMP-2 and IGFBP-7 in the urine were analyzed with ELISA at different time points. Based on the receiver operating characteristic curve (ROC) and the area under the curve (AUC), the early diagnostic value of urinary TIMP-2 and IGFBP-7 in sepsis-induced AKI patients was determined. Results:Compared with the control group, the levels of TIMP-2 and IGFBP-7 of the AKI group were significantly higher at the above time points ( P<0.05), while those of the non-AKI group showed no significant differences. The levels of TIMP-2 and IGFBP-7 of the AKI group were significantly higher than the those of the non-AKI group ( P<0.05). ROC analysis showed that when the AUC of urine TIMP-2 peaked at 1 d, the sensitivity and specificity reached 97.5% and 81.2%, separately with the cutoff value of 151.23 ng/mL. Furthermore, when the AUC of urine IGFBP-7 peaked at 12 h, the sensitivity and specificity reached 100% and 72.8%, separately with the cutoff value of 14.91 ng/mL. Interestingly, when the AUC of combined TIMP-2×IGFBP-7 peaked at 12 h, the sensitivity reached 98.0% and specificity reached 91.5% with the cutoff value of 2.09 [(ng/mL) 2/1 000]. There was no significant correlation between the levels of TIMP-2 and IGFBP-7 with SOFA and APACHEⅡ score at 1 d, 3 d and 7 d post sepsis in the AKI group ( P>0.05). Conclusions:Urine TIMP-2 and IGFBP-7 have early diagnostic value in sepsis-induced AKI. Besides, the combination of the two biomarkers have superior predictive value than each single of them.
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Objective? To?investigate?the?effect?of?histone?methyltransferase?(EZH2)?inhibitor?on?the?polarization??of?peritoneal?macrophages?in?septic?mice.? Methods? Thirty-six?healthy?male?C57BL/6J?mice?were?divided?into?three?groups?by?random?number?table?method?(n?=?12):?sham?operated?group?(Sham?group),?sepsis?model?group?(CLP?group)??and?EZH2?inhibitor?treatment?group?(CLP+3-DZNeP?group).?Sepsis?animal?model?was?established?by?cecum?ligation?and?puncture?(CLP);?Sham?group?was?challenged?only?by?cecum?traction?without?ligation.?3-Deazaneplanocin?A?(3-DZNeP)?1?mg/kg?was?intraperitoneal?injected?24?hours?before?and?1?hour?after?CLP?in?CLP+3-DZNeP?group.?Eight?mice?in?each?group?were?sacrificed?at?24?hours?after?surgery.?The?levels?of?proinflammatory?cytokines?interleukin-6??(IL-6)?and?tumor?necrosis?factor-α(TNF-α)?in?peritoneal?lavatory?fluid?were?detected?by?high?throughput?liquid?protein?chip.?The?expression?levels?of?inducible?nitrogenase?(iNOS)?and?macrophage?mannose?receptor?(CD206)?were?analyzed?by?flow?cytometry.?Mouse?peritoneal?macrophages?were?isolated?and?purified?by?adherent?method,?the?protein?expressions?of?EZH2,?peroxisome?proliferator-activated?receptorγ(PPARγ)?were?detected?by?Western?Blot.?The?remaining?4?mice?? were?sacrificed?at?48?hours?after?surgery,?the?histopathological?changes?of?lung?and?kidney?tissue?were?evaluated?by?hematoxylin-eosin?(HE)?staining.? Results? Compared?with?Sham?group,?the?infiltration?of?inflammatory?cells?in?lung?and?kidney?of?the?CLP?group,?the?levels?of?IL-6?and?TNF-α?in?peritoneal?lavatory?fluid?were?significant?increased??[IL-6?(ng/L):?7?794.75±405.56?vs.?78.63±74.09,?TNF-α(ng/L):?147.25±25.19?vs.?18.20±5.03,?both?P?<?0.01],?the?percentage?of?M1?type?macrophages?was?significantly?increased?[iNOS+?F4/80+:?(13.18±8.80)%?vs.?(1.57±0.77)%,?P?<?0.05],?and?the?protein?expression?of?EZH2?was?significantly?increased?(EZH2/GAPDH:?0.84±0.11?vs.?0.11±0.03,?P?<?0.01),?while?the?protein?expression?of?PPARγ?was?significantly?decreased?(PPARγ/GAPDH:?0.09±0.01?vs.?0.27±0.09,?P?<?0.01).?Compared?with?CLP?group,?the?histopathological?changes?of?lung?and?kidney?in?CLP+3-DZNeP?group?were?significantly?alleviated,?the?levels?of?IL-6?and?TNF-α?in?peritoneal?lavatory?fluid?were?significantly?decreased?[IL-6?(ng/L):?4?207.10±876.60?vs.?7?794.75±405.56,?TNF-α(ng/L):?63.00±25.37?vs.?147.25±25.19,?both?P <?0.01?],?the?percentage?of?M1?type?macrophages?was?significantly?decreased?[iNOS+?F4/80+:?(3.64±0.89)%?vs.?(13.18±8.80)%,??P?<?0.05],?while?the?percentage?of?M2?type?macrophages?was?significantly?increased?[CD206+?F4/80+:?(17.68±5.63)%?vs.?(7.60±3.17)%,?P?<?0.01],?the?protein?expression?of?EZH2?was?significantly?decreased?(EZH2/GAPDH:?0.53±0.09?vs.?0.84±0.11,?P?<?0.05),?and?the?protein?expression?of?PPARγ?was?significantly?increased?(PPARγ/GAPDH:?0.39±0.14?vs.?0.09±0.01,?P?<?0.05).? Conclusions? Sepsis?induces?high?expression?of?EZH2?in?peritoneal?macrophages,?and?may?induce?polarization?of?M1?type?macrophages?by?inhibiting?the?expression?of?PPARγ?protein.?EZH2?inhibitor?3-DZNeP?can?lessen?the?inflammatory?cytokines?release?by?inhibiting?the?M1?type?macrophages?polarization.
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Objective To study the superiority of severe multiple trauma treatment model based on damage control strategy. Methods In the intergrated injury first-aid mode, the intensive care unit-guided damage control strategy was used to treat severe multiple trauma. Results A total of 789 severe multiple damage patients were treated with damage control strategies in our hospital from December 2018 to December 2018. Sixty-nine patients died and the survival rate was 91.25％. Conclusions The intensive care unit-guided trauma control strategy has a satisfactory clinical effect in the treatment of patients with severe multiple trauma.
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It is reported that fibrotic changes seemed to be associated with poor prognosis in patients with organ dysfunction, unfortunately no effective treatment existed currently. Recent studies have demonstrated that macrophages can interact with epithelial cells to induce epithelial mesenchymal transdifferentiation (EMT) which is thought to play a key role in the pathogenesis of a variety of fibrotic diseases. Therefore, this review will summarize the potential role of macrophages in the molecular mechanisms of EMT-associated fibrosis. We aimed to provide a possible therapeutic approaches for the fibrotic diseases.
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Objective To explore the predicting value of peripheral blood CD20+ CD24hi CD38hi regulatory B cells (Bregs) on the prognosis of elderly patients with sepsis.Methods A prospective study was conducted. Septic patients aged > 65 years old, compliance with diagnostic criteria for Sepsis-3, admitted to emergency and emergency intensive care unit (ICU) of Shanghai East Hospital of Tongji University from April 2016 to February 2017 were enrolled. Procalcitonin (PCT), C-reaction protein (CRP) and lactate (Lac) were routinely measured. According to the worst clinical index value within 24 hours, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score and sequential organ failure assessment (SOFA) score were recorded. The concentrations of peripheral blood CD20+ CD24hi CD38hi Bregs were measured by flow cytometry at 1, 3 and 7 days after diagnosed in elderly patients. All patients with sepsis were followed up for 28 days and then divided into death group and survival group according to 28-day outcome. The difference of clinic data and Bregs were compared between the two groups. The significant different factors of elderly sepsis patients were analyzed by binary logistic regression analysis. The correlation between Bregs level and other indicators was analyzed by Spearman correlation. The receiver operating characteristic curve (ROC) was used to evaluate the prognosis value of Bregs in elderly patients with sepsis.Results Fifty-eight patients were enrolled in the study, with 38 male and 20 female; age of (79.91±7.97) years; 32 in sepsis group, 26 in septic shock group; 35 deaths, 28-day mortality rate was 60.3%. APACHE Ⅱ score and SOFA score in death group exhibited much higher than that in survival group (APACHE Ⅱ: 18.14±4.52 vs. 14.91±3.56, SOFA: 8.80±4.56 vs. 6.35±3.00, bothP < 0.05), the Bregs was significantly decreased at 1, 3 and 7 days in death group [cells/μL: 0.70 (0.20, 1.40) vs. 1.50 (0.70, 2.20), 0.54 (0.20, 1.00) vs. 1.42 (1.10, 2.12), 0.25 (0.10, 0.50) vs. 0.80 (0.50, 1.00), allP < 0.05]. Correlation analysis showed that the concentrations of peripheral blood Bregs at 1 day in elderly patients with sepsis was negatively correlated with APACHE Ⅱ score (r = -0.351,P = 0.007), and it was not correlated with PCT, CRP, Lac or SOFA score. It was shown by binary logistic regression that Bregs [odds ratio (OR) = 1.865,P = 0.028] and APACHE Ⅱ score (OR = 0.853,P = 0.026) were independent risk factors for elderly sepsis outcome. It was shown by ROC curve analysis that the prognostic value of the levels of Bregs at 1, 3, 7 days and APACHE Ⅱ score were higher in the elderly patients with sepsis, and the area under ROC curve (AUC) and 95% confidence interval (95%CI) were 0.842 (0.647-0.954), 0.770 (0.564-0.911), 0.888 (0.703-0.977), 0.855 (0.661-0.961), respectively, allP < 0.01. The 7-day Bregs was most powerful to predict outcome, when the cut-off value was 0.50 cells/μL, the sensitivity was 72.73% and specificity was 86.67%. Conclusions The level of peripheral blood CD20+ CD24hi CD38hi Bregs could predict the clinical outcome of elderly patients with sepsis.
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Objective To investigate the regulatory role of programmed death ligand-1 (PD-L1) on acute lung injury (ALI), and its molecular mechanism. Methods Twenty C57BL/6 male mice and 20 PD-L1 knock out male mice were collected, and they were divided into two groups by random number table, respectively: namely sham group and ALI group, 10 mice in each group. The model of ALI was reproduced by two-hit of hemorrhagic shock and sepsis, and the mice in sham group were only got bilateral femoral artery exposure and ligation without bleeding, cecal separation without ligation and perforation. The mice were sacrificed 24 hours after model reproduction, and the lung tissue and bronchoalveolar lavage fluid (BALF) were collected. The mRNA and protein expression levels of PD-L1 in the lungs were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot. The pathological changes were observed with microscopy. The protein levels in BALF were determined. The granulocyte differentiation antigen 1 (Gr1) positive cells was determined by cytometry, and myeloperoxidase (MPO) activity in lung tissue was determined. The levels of proinflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and chemotatic factors keratinocyte chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2) in lung homogenates and BALF were determined by enzyme-linked immunosorbent assay (ELISA). Results Compared with sham group, the mRNA and protein levels of PD-L1 in lung tissue of C57BL/6 mice in ALI group were significantly elevated [PD-L1 mRNA (2-ΔΔCt): 3.20±0.76 vs. 1.01±0.03, PD-L1 protein (A value): 0.98±0.16 vs. 0.15±0.04, both P < 0.05]. It was shown by light microscopy that the alveolar wall was thickened, congestive, edema and spot bleeding with a large number of inflammatory cell infiltration in the lung tissue of C57BL/6 mice in ALI group, and an obvious protein leakage was found in BALF (ng/L: 0.18±0.06 vs. 0.05±0.01, P < 0.05). The lung injury degree of PD-L1 knockout ALI mice was significantly less than that of C57BL/6 ALI mice, and the protein leakage was significantly reduced in BALF (ng/L: 0.11±0.02 vs. 0.18±0.06, P < 0.05). Compared with corresponding sham group, the number of Gr1 positive cells, MPO activity in lung tissue as well as the levels proinflammatory factors and chemotatic factors in lung tissue and BALF in ALI group were significantly increased. However, when compared with C57BL/6 ALI mice, above parameters in lung homogenates and BALF were significantly decreased in PD-L1 knockout ALI mice [number of Gr1 positive cells: (39.0±4.0)% vs. (45.0±8.0)%, MPO activity (U·μg-1·min-1): 2.85±0.62 vs. 4.52±1.16; lung IL-6 (ng/g): 461±111 vs. 728±28, TNF-α (ng/g): 1 123±175 vs. 1 500±327, KC (ng/g): 150±34 vs. 250±28, MIP-2 (ng/g): 1 263±468 vs. 1 763±323; BALF IL-6 (ng/L): 134±22 vs. 258±38, TNF-α (ng/L): 598±102 vs. 889±139, KC (ng/L): 934±286 vs. 1 258±336, MIP-2 (ng/L): 650±130 vs. 950±256; all P < 0.05]. Conclusion PD-L1 may play an important protective role in the immunological mechanism of ALI, which may be mediated by decreasing chemotactic factor KC and MIP-2 and mitigating neutrophil chemotaxis in lung tissue.
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Objective To observe the relationship between high-mobility group box-1 protein HMGB-1 andthe prognosis of systemic inflammatory response syndrome (SIRS) patients after multiple trauma. Method Sixtypatients with SIRS after multiple trauma in the emergency trauma center were divided into multiple organ dysfunc-tion syndrome (MODS) group and non-MODS group according to the MODS diagnostic criteria, and were followedup for 28 days and then assigned to survival group and fatal group, respectively. Another 20 healthy people wereera-oiled in the conrtol group. The levels of HMGB-1 were measured by ELISA at 24 hours, 3 days, 5 days and 7days after admission. APACHE Ⅱ scores were calculated. Results The concentrations of HMGB- 1 in the patientswith SIRS after trauma at every time point were higher than those in control group (P<0.01). The concentrationsof HMGB-1 and APACHEⅡ scores in the MODS group and the fatal group were higher than those in non-MODSgroup and the survival group (P<0.05 and P<0.01 separately). There was positive correlation between theconcentrations of HMGB-1 and APACHE Ⅱ scores (r=0.7938, P<0.05). Conclusions As an importantpro-inflammatory cytokine in sepsis, HMGB-1 may play a role in the development of SIRS after multiple trauma andcan be used to assess the severity of illness and prognosis.