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Objective:To explore the clinical characteristics of patients with colchicine poisoning, and analyze the risk factors affecting the prognosis of colchicine poisoning and its value in the prognostic assessment.Methods:Patients with colchicine poisoning admitted to the Emergency Intensive Care Unit of the First Affiliated Hospital of Wenzhou Medical University from December 2017 to October 2022 were retrospectively included and divided into the survival group and death group according to the 14-d outcome. The general conditions of the two groups of patients were compared, and the clinical characteristics of patients with colchicine poisoning were analyzed. The differences of laboratory indexes, electrocardiogram, cardiac ultrasound and other clinical indexes during the first admission of patients between the two groups were compared, and their value in the prognosis evaluation of patients with colchicine poisoning was explored.Results:There were 41 patients with colchicine poisoning, aged 15-85 years, including 35 males and 6 females. There were 27 patients (65.9%) in the survival group and 14 patients (34.1%) in the death group, including accumulative poisoning (58.7%) and suicide poisoning (41.3%). The main clinical manifestations of patients with colchicine poisoning were gastrointestinal symptoms (82.93%), multiple organ dysfunction (78.05%), infectious fever (73.17%), myocardial damage (48.78%), coagulation dysfunction (46.34%), and bone marrow suppression (41.46%). Intestinal obstruction (19.51%) and rhabdomyolysis (2.44%) occurred in some patients. Multivariate Logistic regression analysis showed that the increase in absolute value of QTc interval ( OR=1.028, 95% CI: 1.000~1.056, P<0.05), lactic acid ( OR=1.599, 95% CI: 1.088~2.350, P<0.05), prothrombin time ( OR=1.205, 95% CI: 1.002~1.450, P<0.05), D-dimer ( OR=1.242, 95% CI: 1.089~1.417, P<0.05), and alkaline phosphatase ( OR=1.013, 95% CI: 1.002~1.024, P<0.05) were the risk factors for the prognosis of patients with colchicine poisoning. The decrease in the absolute value of ADL score ( OR=0.947, 95% CI: 0.909~0.988, P<0.05) and indirect bilirubin ( OR=0.756, 95% CI: 0.572~0.999, P<0.05) were the protective factors for the prognosis of patients with colchicine poisoning. D-dimer (AUC=0.913), lactic acid (AUC= 0.875) and alkaline phosphatase (AUC=0.770) had predictive value for the prognosis of patients with colchicine poisoning, and their cut-off values were 8.965 mg/L, 4.05 mmol/L and 230.5 U/L, respectively. Conclusions:The patients with colchicine poisoning have multiple organ dysfunction on admission, and are in a critical condition. The early levels of D-dimer, lactic acid and alkaline phosphatase could effectively predict the prognosis of patients with colchicine poisoning.
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Objective:To explore the clinical characteristics of patients with paraquat mixed with diquat poisoning.Methods:The clinical data of 145 patients with paraquat mixed with diquat poisoning admitted to the Department of Emergency of the First Affiliated Hospital of Wenzhou Medical University from January 20, 2016 to March 31, 2022 were retrospectively analyzed. According to the detection results of plasma toxicants in patients with poisoning, the patients were divided into the paraquat diquat mixed group (mixed group), paraquat group (PQ group) and diquat group (DQ group). The clinical indexes, organ dysfunction, different poisoning doses and prognosis of the three groups were compared. Patients in the mixed group were divided into the survival group and death group according to their 90-day survival, and the differences of each index between the two groups were compared. Kaplan-Meier survival analysis was conducted for each index. After Log-rank test, multivariate Cox regression was used to analyze the risk factors of death in the mixed group.Results:A total of 31 patients were included in the mixed group, 92 patients in the PQ group, and 22 patients in the DQ group. There were significant differences in age, toxic dose, number of organ dysfunction, PSS score and APACHE II score among the three groups ( P<0.05). The main injured organs of the mixed group were gastrointestinal tract, kidney, liver, lung and nervous system. The proportion of organ damage in the mixed group was higher than that in the PQ group and DQ group. The white blood cell count, neutrophil count, HB, creatinine, AST, lactic acid, PT and APTT were statistically significant among the three groups ( P<0.05). In the mixed group, patients taking oral administration of < 20 mL all survived; 8 patients taking oral administration of 20 -50 mL died; 11 patients took oral administration of 51-100 mL and 8 (72.7%) died; and 10 patients took oral administration of more than 100 mL and 9 patients (90%) died. In the mixed group, patients with the concentration of diquat > 5000 ng/mL died. Among 31 patients with mixed poisoning, 30 patients (96.78%) had significantly higher concentrations of diquat than paraquat. There were no significant differences in sex, age, time from poisoning to hospitalization, ingestion amount, lymphocyte count, Hb, BNU, CK, total bilirubin, PH, and PT between the survival group and the death group ( P>0.05). Multivariate Cox regression analysis showed that the ingestion amount, plasma PQ concentration at admission, plasma DQ concentration at admission, and lactic acid were independent risk factors for death ( P<0.05). Conclusions:Paraquat mixed with diquat can cause multiple organ function damage. The main damaged organs are gastrointestinal tract, kidney, liver, lung and nervous system. Compared with PQ or DQ poisoning, mixed poisoning has a higher incidence of organ damage, a more serious condition, and a higher mortality rate. Ingestion amount, plasma PQ concentration at admission, plasma DQ concentration at admission and lactic acid were independent factors influencing the prognosis of mixed poisoning.
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Objective:To explore the value of vascular endothelial-cadherin (VE-cad) in evaluating the severity of sepsis.Methods:A prospective study was conducted to select 85 patients with sepsis treated in the emergency ward of the First Affiliated Hospital of Wenzhou Medical University from June 1, 2015 to November 1, 2017. The gender, age, medical history, first infection site, number of affected organs, laboratory indexes, acute physiology and chronic health evaluationⅡ(APACHEⅡ), simplified acute physiology score Ⅱ(SAPSⅡ), sequential organ failure assessment (SOFA) and the total length of stay, emergency intensive care unit (EICU) length of stay, 28-day at admission and survival during hospitalization were measured, and the VE-cad level within 24 hours at admission was measured. The patients were divided into sepsis group and septic shock group according to the progress of the disease. The patients were divided into multiple organ dysfunction syndrome (MODS) group and non MODS group according to whether they were accompanied by MODS. The differences of the above indexes in patients with different disease progression, MODS and different prognosis were analyzed and compared. The receiver operator characteristic curve (ROC curve) was drawn to evaluate the value of VE-cad in evaluating the severity of sepsis.Results:A total of 85 patients were included, mainly respiratory tract infection. Among them, 38 cases were sepsis and 47 cases were septic shock, 39 cases had MODS, 46 cases had no MODS, 64 cases survived and 21 cases died within 28 days after admission. Compared with sepsis group, the number of affected organs in septic shock group was greater [3 (2, 4) vs. 1 (0, 2)], APACHE Ⅱscore [13 (10, 21) vs. 7 (5, 12)], SAPS Ⅱscore [35 (31, 55) vs. 7 (5, 12)], SOFA score [7.0 (5.0, 10.0) vs. 3.0 (0, 5.0)], blood lactic acid [Lac (mmol/L): 3.5 (2.4, 6.2) vs. 1.9 (1.2, 2.2)], C-reactive protein [CRP (mg/L): 90.0 (58.1, 90.0) vs. 50.5 (38.0, 90.0)] and VE-cad levels [mg/L: 1.427 (1.141, 2.150) vs. 1.195 (0.901, 1.688)] were significantly increased, while platelet count [PLT (×10 9/L): 113.4±67.2 vs. 202.5±109.5] and hemoglobin (Hb) levels (g/L: 106.3±36.3 vs. 118.6±18.0) were significantly decreased (all P < 0.05). Compared with non MODS group, APACHE Ⅱ score [14 (10, 22) vs. 8 (6, 13)], SAPS Ⅱ score [36 (32, 56) vs. 29 (24, 35)], SOFA score (7.9±3.9 vs. 4.0±3.8), in-hospital mortality [53.8% (21/39) vs. 0% (0/46)], Lac [mmol/L: 3.1 (2.3, 6.3) vs. 2.1 (1.4, 4.6)] and VE-cad levels [mg/L: 1.427 (1.156, 1.937) vs. 1.195 (0.897, 1.776)] in MODS group were significantly higher, the length of stay in EICU was significantly longer [days: 6 (3, 12) vs. 3 (0, 7)], and the PLT level was significantly lower (×10 9/L: 118.2±80.0 vs. 182.5±104.0, all P < 0.05). Compared with the death group, the number of affected organs in the survival group was fewer [2 (1, 3) vs. 3 (1, 5)], APACHE Ⅱ score [9 (6, 13) vs. 21 (13, 25)], SAPS Ⅱ score [31 (25, 36) vs. 55 (35, 63)] and SOFA score (4.7±3.7 vs. 8.9±4.5) were significantly reduced, and the length of stay in EICU [days: 4 (1, 8) vs. 8 (3, 15)] was significantly shorter (all P < 0.05). ROC curve analysis showed that area under the ROC curve (AUC) of VE-cad, SOFA score and VE-cad combined with SOFA score in evaluating the severity of sepsis were 0.632 [95% confidence interval (95% CI) was 0.513-0.750], 0.830 (95% CI was 0.744-0.916) and 0.856 (95% CI was 0.779-0.933), respectively. When the cut-off value of VE-cad was 1.240 mg/L, the sensitivity was 68.1% and the specificity was 55.3%, the sensitivity of VE-cad combined with SOFA score was 85.1%, the specificity was 73.7%. Conclusion:VE-cad has a certain evaluation value for the severity of sepsis, and the evaluation value of VE-cad combined with SOFA score is better than that of VE-cad single index.
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Objective:To explore the relationship between hematocrit, early fluid therapy, and clinical outcomes in patients with septic shock, and to provide evidence for fluid resuscitation therapy and prognosis assessment in these patients.Methods:The clinical information of patients with septic shock who were diagnosed and treated in the Emergency Intensive Care Unit (EICU) of the First Affiliated Hospital of Wenzhou Medical University from January 1, 2018 to December 31, 2020 were collected. Taking the survival or death of patients 28 days after admission as the end point of clinical research, the patients were divided into the survival and death groups. After analyzing the basic data of the two groups, the univariate and multivariate COX regression analyses were used to analyze the evaluation value of Δ Hematocrit (HCT) d2-d1 and ΔHCT d3-d1 on the prognosis of patients with septic shock. At the same time, the Kaplan-Meier survival curve was used to analyze the overall survival rate of patients with septic shock, and the smooth curve fitting graph was used to verify its relationship with net fluid intake and death. Results:There were 241 cases in the survival group and 67 cases in the death group. Univariate COX analysis showed statistically significant differences between the survival and death groups in acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) ( P=0.0006), red cell volume distribution width (RDW) ( P=0.0043), age ( P=0.0184), ΔHCT d2-d1 ( P=0.0136), ΔHCT d3-d1 ( P=0.0204), and white blood cell (WBC) ( P=0.0444). Multivariate COX analysis showed that ΔHCT d2-d1 ( P=0.0115) and ΔHCT d3-d1 ( P=0.0029) were independent risk factors for death in EICU patients with septic shock. ΔHCT d2-d1 and ΔHCT d3-d1 were divided into three groups according to the three-digit method. The Kaplan-Meier survival curve showed no significant difference among the three groups in the overall survival rate related to ΔHCT d2-d1 ( P=0.16), but there was a statistically significant difference in the overall survival rate among the three groups related to ΔHCT d3-d1 ( P=0.025). The smooth fitting curve of ΔHCT d3-d1, net fluid intake, and prognosis showed that ΔHCT d3-d1 was negatively correlated with net fluid intake, and the middle ΔHCT d3-d1 group had the best prognosis. Conclusions:The value of ΔHCT d3-d1 is related to the net fluid intake of patients with septic shock. An appropriate decrease in HCT on the third day can improve the prognosis of patients with septic shock. The dynamic changes of hematocrit can provide a certain basis for fluid resuscitation and prognosis evaluation in patients with septic shock.
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Objective:To explore the predictive value of the serum C-reactive protein (CRP)/albumin (ALB) ratio (CAR) for organ damage in tsutsugamushi disease.Methods:The clinical data of 166 patients with tsutsugamushi disease admitted to the First Affiliated Hospital of Wenzhou Medical University from January 1, 2010 to December 31, 2020 were retrospectively analyzed. The patients were divided into the organ damage group (72 cases) and non-organ damage group (94 cases) according to the organ damage criteria. The general data and laboratory test results of the two groups of patients were compared. The significant indicators of univariate analysis were analyzed by multivariate logistic regression analysis. The receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to analyze the predictive value of CAR for organ damage in patients with tsutsugamushi disease.Results:There were no significant differences in age, sex, days of fever, and admission body temperature between the organ damage group and non-organ damage group ( P>0.05). However, the body mass index, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ), sequential organ failure assessment (SOFA), length of hospital stay, hospitalization expense, percentage of neutrophils (NEUT), lymphocyte count, procalcitonin, CRP, and CAR in the organ damage group were significantly higher than those in the non-organ damage group ( P<0.05), and ALB was significantly lower than that in the non-organ damage group ( P<0.05). Multivariate logistic regression analysis showed that APACHEⅡ( P=0.039), NEUT ( P=0.003), and CAR ( P=0.011) were independent risk factors for tsutsugamushi disease complicated by organ damage. The ROC curve showed that the AUCs of APACHEⅡ, NEUT, and CAR were 0.655, 0.716, and 0.727, respectively. When the cut-off value of CAR was 2.86, the sensitivity was 55.6%, and the specificity was 79.8%. Conclusions:Elevated CAR is an independent risk factor for tsutsugamushi disease complicated with organ damage and can be used as an important indicator to evaluate the presence or absence of organ damage in patients with tsutsugamushi disease.
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Objective:To explore the influencing factors of severity of upper gastrointestinal bleeding (UGIB) and to establish the early warning evaluation model in the form of line chart, so as to provide a feasible basis for emergency nurses' triage.Methods:A total of 680 UGIB patients admitted to the Emergency Department of the First Affiliated Hospital of Wenzhou Medical University from January 2019 to January 2020 were retrospectively analyzed. They were divided into a modeling group ( n=510) and a validation group ( n=170) by random number table method, and were divided into a high-risk group and a low-risk group according to the expert Consensus on Emergency Diagnosis and Treatment Procedures for Acute Upper Gastrointestinal Bleeding in 2020. The differences of various indicators between groups were compared, the factors affecting the severity of the disease were analyzed by Logistic regression, and the nomogram was drawn and validated. Results:Multivariate logistic regression analysis showed that hematemesis ( OR=3.875, 95% CI: 2.212-6.79), diabetes ( OR=2.64, 95% CI: 1.184-5.883), syncope ( OR=10.57, 95% CI: 3.675-30.403), heart rate ( OR=3.262, 95% CI: 1.753-6.068), red blood cell distribution width ( OR=3.904, 95% CI: 2.176-7.007), prothrombin time ( OR=3.665, 95% CI: 1.625-8.269), lactic acid ( OR=3.498, 95% CI: 1.926-6.354) and hemoglobin ( OR=4.984, 95% CI: 2.78-8.938) were the influencing factors of the severity of UGIB patients ( P < 0.05). The nomogram model showed good consistency and differentiation (C-index=0.903, 95% CI: 0.875-0.931), and was verified internally (C-index=0.895) and Hosmer-Lemeshow goodness-of-fit test ( P=0.7936). Externally verified C-index was 0.899 (95% CI: 0.846-0.952). The calibration curve prompt warning evaluation model had good stability and the prediction efficiency was better than the modified early warning score ( P < 0.05). Conclusions:The early warning evaluation model has a reliable predictive value, which can provide a reference for emergency medical staff to screen high-risk patients and formulate targeted nursing interventions.
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Objective:To investigate the role of growth arrest specific protein 6 (Gas6) in regulating macrophage polarization in wound healing.Methods:Clean male B6 mice were randomly(random number) divided into the normal group, skin defect group, skin defect group + normal saline group (PBS group), skin defect + Gas6 (1 μg) group, skin defect + Gas6 (5 μg) group, and skin defect + Gas6 (10 μg) group. Ten mice in each group were used to observe the healing of skin wounds. Macrophages were isolated from the wound tissues of the remaining 6 mice on the fifth day after modeling. The levels of IL-6 and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA), the mRNA expression levels of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) were detected by RT-PCR, and flow cytometry was used to detect the expression of M1 marker CD197, M2 marker CD163 and F4/80. HE staining was used to detect the pathological changes of skin wounds. Masson staining was used to analyze the granulation tissue and collagen deposition.Results:Scab began to form on the surface of the wound on the third day after the skin defect model was established. The wound area of the Gas6 treatment group was smaller than that of the PBS group, and the wound healing was better than that of the PBS group. Compared with the normal group, the proportion of CD197 in macrophages of the skin defect group was significantly increased ( P=0.00 49), the proportion of CD163 and F4/80 double positive was significantly decreased ( P=0.00 86), the level of IL-6 was significantly increased ( P=0.00 13), the level of IL-10 was significantly increased ( P=0.00 14), the level of iNOS mRNA was significantly increased ( P=0.00 8), and Arg-1 was significantly increased in the skin defect group The mRNA level was significantly decreased ( P=0.01 21), and the inflammatory infiltration was aggravated. Compared with the PBS group, the proportion of CD197 in the Gas6 treatment group was significantly decreased ( P=0.00 0), the double positive rates of CD163 and F4/80 were significantly increased ( P = 0.00 0), the level of IL-6 was significantly decreased (P = 0.00 0), the level of IL-10 was significantly increased ( P=0.00 03), the level of iNOS mRNA was significantly decreased ( P=0.00 18), the level of Arg-1 mRNA was significantly increased ( P=0.00 1), and the number of inflammatory cells and the number of collagen fibers were increased. Conclusions:Gas6 can promote the transformation of macrophages from M1 to M2 in mice with skin defect, which is beneficial to the wound healing of skin defect.
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Objective To investigate the protective effect and mechanism of curcumin on acute lung injury in septic mice.Methods Totally 120 clean BALB/c male mice were randomly (random number) divided into 8 groups:sham group,sepsis group,curcumin control group,curcumin intervention group,negative virus-sepsis group,negative virus-curcumin intervention group,Mfn2 interference-sepsis group,and Mfn2 interference-curcumin intervention group,15 rats in each group.Mice in the sepsis and the curcumin groups were given the cecal ligation and puncture (CLP) mice in the curcumin intervention and curcumin control groups were given curcumin 200 mg/(kg·d) for 1 week,and mice in the negative virus-sepsis group and negative virus-curcumin intervention groups were established by injection of a negative adeno-associated virus in the tail vein.The Mfn2 interference-sepsis and Mfn2 interference-curcumin intervention groups were established by injecting an adeno-associated virus carrying the Mfn2 interference sequence through the tail vein.Mice were sacrificed after 24 h in each group.The degree of lung injury was examined by lung wet-to-dry weight ratio and pathological examination.The inflammatory factors of alveolar lavage fluid including TNF-α and IL-6 were detected by ELISA,the activation of caspase-3,a key molecule for apoptosis,was detected by Western blot,and apoptosis was detected by TUNEL.The data were analyzed by SPSS 22.0 software,the count data was analyzed by x2 test,and the comparison of measurement data between groups was analyzed by one-way ANOVA.Results Compared with the sham group,the wet-to-dry weight ratio of lung tissue in the sepsis group was significantly increased (71.11 ± 3.78 vs 31.11 ± 5.61,P=0.002),the histopathological score was significantly higher (P=0.006),the inflammatory factors TNF-α (P=0.001) and IL-6 (P=0.012) were dramatically increased,and the apoptosis of lung tissue and the expression of caspase-3 cleaved were also significantly increased (P=0.001).Compared with the sepsis group,the wet-to-dry weight ratio and the histopathological score of lung tissue in the curcumin-treated group was significantly lower (32.84 ± 6.15 vs 71.11 ± 3.78,P=0.004),and the inflammatory factors TNF-α(P=0.013) and IL-6 (P=0.003) were obviously decreased,and apoptosis and apoptosis-related protein caspase-3 cleaved expression were also dramatically decreased (P=0.012).After Mfn2 was down-regulated,Mfn2 interference-curcumin intervention group interfered with Mfn2.Compared with the sepsis group,the dry-to-wet weight ratio and the histopathological score of the lung tissue of the mice was not significantly decreased.Further studies found that after down-regulating Mfn2,compared with the Mfn2 interfere-sepsis group,Mfn2 interfere-curcumin intervention group had no such performance.The inflammatory factors TNF-α and IL-6 were not significantly decreased,and the apoptosis of lung tissue and the expression of apoptosis-related protein caspase-3 cleaved were not significantly reduced.Conclusion Curcumin may attenuate acute lung injury in sepsis by up-regulating the expression of Mfn2.
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Objective@#To investigate the effect of cycloartenyl ferulate (CF) on lipopolysaccharide (LPS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC), and to explore its relative mechanism.@*Methods@#Human umbilical vein endothelial cells were cultured in vitro, and the experiment was divided into the normal group, CF group, LPS group, and LPS+CF groups at different concentrations. After the corresponding treatment of cells in each group, the cell viability and apoptosis of each group were tested by the cell counter Kit-8 (CCK-8) assay and TdT-mediated dUTP nick end labeling (TUNEL) assay. The protein expression of Bax, Bcl-2, caspase-3 and the effect of CF on the protein expression of Nrf2/HO-1 pathway were determined by Western blot. One-way ANOVA were performed in multigroup mean comparison, LSD-t test was used to compare the mean values of two samples, and P<0.05 was considered statistically significant.@*Results@#Compared with the LPS group, the survival rate of HUVECs cells in the CF group was significantly increased with different doses, and the survival rate increased significantly in the 320 μmol/L CF group [(69.85±1.2)% vs ( 100.2±1.824)%, P< 0.01]. TUNEL staining showed that compared with the LPS group, the number of apoptotic positive cells in the 320 μmol/L CF group was significantly reduced [(27.33 ± 3.40) vs (11.67 ± 2.04), P<0.01]. Compared with the LPS group, Bcl-2 protein expression level was significantly increased in the CF group at different doses (P<0.01), caspase-3 and Bax protein expression were significantly decreased (P<0.01). Nrf2 protein expression level increased significantly (P<0.01), and HO-1 protein level increased significantly (P<0.01).@*Conclusion@#CF can alleviate LPS-induced apoptosis of HUVEC, which may be related to the increase of bcl-2 expression, the decrease of caspase-3 and Bax protein expression and the activation of Nrf2/ HO-1 signaling pathway.
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Objective To investigate the effect of cycloartenyl ferulate (CF) on lipopolysaccharide (LPS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC),and to explore its relative mechanism.Methods Human umbilical vein endothelial cells were culturedin vitro, and the experiment was divided into the normal group, CF group, LPS group, and LPS+CF groups at different concentrations. After the corresponding treatment of cells in each group, the cell viability and apoptosis of each group were tested by the cell counter Kit-8 (CCK-8) assay and TdT-mediated dUTP nick end labeling (TUNEL) assay. The protein expression of Bax, Bcl-2, caspase-3 and the effect of CF on the protein expression of Nrf2/HO-1 pathway were determined by Western blot. One-way ANOVA were performed in multigroup mean comparison, LSD-t test was used to compare the mean values of two samples, andP<0.05 was considered statistically significant.Results Compared with the LPS group, the survival rate of HUVECs cells in the CF group was significantly increased with different doses,and the survival rate increased significantly in the 320 μmol/L CF group [(69.85±1.2)％vs ( 100.2±1.824)％,P< 0.01]. TUNEL staining showed that compared with the LPS group, the number of apoptotic positive cells in the 320 μmol/L CF group was significantly reduced [(27.33 ± 3.40)vs (11.67 ± 2.04),P<0.01]. Compared with the LPS group, Bcl-2 protein expression level was significantly increased in the CF group at different doses (P<0.01), caspase-3 and Bax protein expression were significantly decreased (P<0.01). Nrf2 protein expression level increased significantly (P<0.01), and HO-1 protein level increased significantly (P<0.01).Conclusion CF can alleviate LPS-induced apoptosis of HUVEC, which may be related to the increase of bcl-2 expression, the decrease of caspase-3 and Bax protein expression and the activation of Nrf2/ HO-1 signaling pathway.
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Objective To investigate the protective effect of Baicalin on inflammation induced by lipopolysaccharide in H9C2 cardiomyocytes and its possible mechanism. Methods H9C2 myocardial cells were cultured and pretreated with baicalin at the final concentration of 10, 20, 30 μmol/L for 12 hours, then stimulated with LPS at the final concentration of 1 μg/mL for 6 hours. The control group was treated with the same amount of saline to collect cell samples. CCK-8 (The Cell Counting Kit-8) was used to detect cell activity, enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of interleukin-6 (IL-6), interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), Western blot was used to detect the protein expression levels of NF-κB p65, p-NF-κB p65, p38 MAPK, p-p38 MAPK, IκBα and p-IκBα. SPSS 23.0 statistical software was used. Independent sample t test was used for comparison between two groups, and one-way ANOVA test was used for comparison among multiple groups. Results The survival rate of myocardial cells in the control group was (93.67 +1.453)％. Compared with the control group, the survival rate of H9C2 myocardial cells induced by LPS decreased (P< 0.05). In the control group, the expression of IL-6 in H9C2 myocardial cells was (49.33 +2.42) pg/mL, the expression of TNF-α was (86.33 +1.85) pg/mL, and the expression of IL-1β was (28.67 +4.66) pg/mL. Compared with the control group, the expression levels of IL-6, IL-1β and TNF-α in H9C2 myocardial cells increased after LPS induction (P< 0.05), while the levels of p-NF-κ B p65, p-p38 MAPK and p-I κ B α protein increased (P< 0.05), while the levels of I κ B α protein decreased (P< 0.05), while the expressions of NF-κ B p65 and p38 MAPK protein did not change significantly (P> 0.05). Compared with LPS group, the survival rate of H9C2 myocardial cells in baicalin intervention group increased (P<0.05), the expression levels of IL-6, IL-1β and TNF-a decreased (P < 0.05), the levels of p-NF-κB p65, p-p38 MAPK, p-I κBα protein decreased (P< 0.05), and the level of IκBα protein increased (P< 0.05), while the expression of NF-κB p65 and p38 MAPK did not change significantly. (P>0.05). Conclusions Baicalin may alleviate LPS-induced cardiomyocyte inflammation by inhibiting the activation of NF-kappa B and p38 MAPK, and improve cell survival.
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Objective To explore the antioxidant mechanism ofhistone deacetylase 2 (HDAC2) regulating Nrf 2 acetylation in lipopolysaccharide (LPS)-induced type Ⅱ alveolar epithelial cell injury.Methods The experiment was divided into two parts.The first part was the routine culture of type Ⅱ alveolar epithelial cells of mice.The cells were stimulated with different concentrations of LPS (10 ng/ mL,100 ng/mL and 1 000 ng/mL).CCK-8 was used to detect the cell activity at 0 h,6 h,12 h,24 h and 48 h,respectively.The second part:Alveolar epithelial cells of type Ⅱ were cultured and divided into the normal control group (control group),LPS group,HDAC2 lentivirus interference group (siRNA-HDAC2 group) and HDAC2 lentivirus overexpression group (LV-HDAC2 group).The expression of HDAC2 and Nrf2 were detected by Western blot,the acetylation of Nrf2 was detected by immunoprecipitation,and the stability of nrf2 was detected after actinidone action.The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by chemical colorimetry.SPSS 23.0 statistical software was used.LSD-t test was used for comparison between two groups,and one-way ANOVA test was used for comparison among multiple groups.Results Compared with the control group,the expression of HDAC2 protein in the LPS group increased (t=5.974,P=0.027),the acetylation level of Nrf2 decreased (t=7.223,P=0.002),the Nrf2 protein level increased (t=2.929,P=0.043),the protein stability of Nrf2 increased,the SOD activity decreased (t=121,P＜0.01),and the MDA content increased (t=10.45,P=0.000 5).Compared with the LPS group,Nrf2 acetylation level decreased in the LV-HDAC2 group (t=1 1.29,P=0.000 4),Nrf2 protein expression increased (t=3.194,P=0.033),Nrf2 protein stability increased,SOD activity increased (t=4.678,P=0.009),and MDA content decreased in the LV-HDAC2 group (t=5.417,P=0.005 6).While the opposite trend was observed in the siRNA-HDAC2 group.Conclusion After LPS stimulation,oxidative stress of type Ⅱ alveolar epithelial cells was aggravated.HDAC2 could decrease the level of Nrf2 acetylation,increase the expression of Nrf2 protein,and alleviate LPS-induced oxidative stress.
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Objective To investigate the effects of histone deacetylase inhibitors trichostatin A (TSA) on acute lung injury in septic mice.Methods Septic mice model was induced by cecal ligation and puncture(CLP).Ninty male BALB/c mice of clean grade were randomly(random number) divided into six groups(n=15),namely sham operation group,CLP group,CLP+DMSO group,CLP+TSA 1 mg group,CLP+TSA 5 mg group,and CLP+TSA 10 mg group.TSA(1 mg/kg,5 mg/kg,10 mg/kg) was administrated 12 hours before operation by intraperitoneal injection.And mice in sham group were only treated with laparotomy without CLP,and 24 h later,all survived mice were sacrificed to obtain specimens.ELISA method was employed to detect the concentrations of TNF-α and IL-1β in BALF.The lung wet/dry ratio was calculated.Histopathology changes of lung tissues were observed under light microscope.Lung tissue cell apoptosis was detected by TUNEL method.Caspase-3,Caspase-9 and CytC were assayed by Western blotting.The survival rate of mice in each group was calculated by additional 120 mice.Data were analyzed by SPSS 23.0.Statistical analyses were performed using independent sample t-test to compare between two groups or one-way analysis of variance test to compare among muhiple groups.The survival rate of mice was analyzed by univariate analysis using log-rank test.Results The lung W/D(P=0.021),the concentrations of TNF-α(P=0.000 1)and IL-1β(P=0.000 6)in BALF,puhnonary pathological change(P=0.001 6),lung tissue cell apoptotic index(P=0.000 9),the levels of apoptosis proteins (P＜0.05) in CLP group were higher than those in sham group,while survival rate (P=0.000 1) in CLP groups was lower than that in sham group.Compared with DMSO,the TSA significantly reduced the lung W/D,the levels of TNF-α.IL-1β in BALF,pathologic changes of lung tissue,lung tissue cell apoptotic index and the levels of apoptosis proteins in septic mice(P＜0.05).The increase in survival rate (P=0.007 2) associated with TSA(10 mg/kg)administration.Conclusion TSA exerts protective effects through attenuating pro-inflammatory cytokines and lung tissue cell apoptosis in sepsis induced acute lung injury in mice.
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Objective To investigate the role of miR-10a in CD4+CD25+Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis.Methods Sepsis mouse model was established by cecal ligation and puncture(CLP).Balb/c mice of clean grade were sacrificed 1,3,5,and 7 days after operation.Blood as well as spleen samples were harvested at given intervals.The splenic CD4+CD25+Treg cells and CD4+T cells were isolated by MACS microbeads.Cells were cultured,and phenotypes were analyzed by flow cytometry.The miR-10a expressed in Treg cells were detected by Real-time PCR.After administration of LV-mmu-miR-10a-5p-inhibition,the immunosuppressive function have been detected.Statistical analyses were performed using one-way analysis of variance (SPSS 19.0,Chicago,USA) test followed by Dunnett-t test to compare among three or more groups or by Student's t-test to compare between two groups.Results The percentages of splenic Tregs (CD4+CD25+/CD4+T) was (7.34±1.2)％ in normal group,and the increase in percentage of Tregs in spleen has been observed in septic mice (P＜0.05).The mean fluorescence intensity (MFI) of Foxp3+Treg was increased in septic mice compared with sham group (P＜0.05).The expression of miR-10a was significantly elevated on CLP 1-7 day (P＜0.05).After down-regulation of miR-10a in septic mice,the percentages of Tregs (CD4+CD25+/CD4+T) was significantly increased in septic mice (P＜0.05),the MFI of Foxp3+Treg was increased in septic mice compared with control group (P＜0.05).The CD4+T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group (P＜0.05).After down-regulation of miR-10a in septic mice,the CD4+T cell proliferative activity was significantly suppressed compared with control group (P＜0.05).Conclusions Treg plays a critical role in immunosuppression in septic mice.Inhibition of miR-10a in vivo could enhence immunesuppression of CD4+CD25+Treg.Therefore miR-10a may participate in the regulation of CD4+CD25+Treg immunosuppression in sepsis and become the target for immunotherapy.
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ObjectiveTo investigate the protective effect of paraoxonase 1 (PON1) gene against liver oxidative stress injury in mice due to dichlorvos poisoning.Methods Experiment 1: 12 male Balb/c mice were randomly divided into three groups, with 4 mice in each group: control group, green fluorescent protein lentivirus control group (Lv-GFP group), and recombinant PON1 lentivirus group (Lv-PON1 group). 2×107 TU of Lv-GFP or Lv-PON1 was transfected via tail vein, while normal saline was given to those in control group. Blood was collected on 0, 1, 3, 5, 7, 9 days via fundus venous plexus for the assay of serum PON1 activity. PON1 mRNA and protein expression levels were respectively determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot on the 3rd post-lentivirus transfection day. Experiment 2: according to the random number table method, another 96 male Balb/c mice were divided into four groups of 24 mice in each control group, dichlorvos group, Lv-GFP intervention group, and Lv-PON1 intervention group. Lv-GFP or Lv-PON1 was transfected via tail vein followed by intraperitoneal injection of dichlorvos 9 mg/kg, while those in control group were given normal saline. Six mice in each group were sacrificed respectively at 6, 12, 24, 48 hours, and liver tissue was collected. PON1 mRNA and nuclear factor E2-related factor 2 (Nrf2) mRNA expression levels were determined by RT-PCR, and PON1 protein level was determined by Western Blot. The content of malondialdehyde (MDA) and glutathione (GSH) in the liver tissue were determined by chemical colorimetry. The activity of superoxide dismutase (SOD) and catalase (CAT) were measured by double antibody sandwich enzyme linked immunosorbent assay (ELISA).Results Experiment 1: after Lv-PON1 was transfected to normal mice, PON1 activity in serum gradually increased and maintained a high level on 3rd day, while that of the control group and Lv-GFP group showed a normal low level. On the 3rd post-lentivirus transfection day, PON1 mRNA and PON1 protein expressions in liver were significantly higher than those of control group and Lv-GFP group. Experiment 2: compared with control group, the mice in dichlorvos group showed significant decreases in PON1 mRNA, PON1 protein, Nrf2 mRNA as well as GSH, SOD, CAT levels at 6 hours [PON1 mRNA (gray value):0.237±0.075 vs. 0.674±0.011, PON1 protein (gray value): 0.602±0.086 vs. 0.998±0.124, Nrf2 mRNA (gray value): 0.089±0.012 vs. 0.126±0.010, GSH (mg/g): 3.84±0.33 vs. 5.52±0.40, SOD (μg/g): 0.383±0.040 vs. 0.564±0.052, CAT (ng/g): 7.32±1.28 vs. 12.46±1.54, allP 0.05), and it was higher than that of the control group at 48 hours (0.206±0.028 vs. 0.124±0.010,P< 0.05). Compared with that of the dichlorvos group, Lv-PON1 intervention group showed a significant increase in PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels [PON1 mRNA (gray value): 0.726±0.021 vs. 0.237±0.075, PON1 protein (gray value): 0.739±0.050 vs. 0.602±0.086, Nrf2 mRNA (gray value): 0.158±0.007 vs. 0.089±0.012, GSH (mg/g): 4.30±0.22 vs. 3.84±0.33, SOD (μg/g): 0.454±0.062 vs. 0.383±0.040, CAT (ng/g): 8.98±1.02 vs. 7.32±1.28, allP< 0.05], and a decrease in MDA content (nmol/g: 6.56±0.44 vs. 7.78±0.41,P< 0.05).Conclusion Regulation of PON1 gene can reduce MDA content, enhance SOD and CAT activities, increase GSH content, and it may also up-regulate Nrf2 mRNA expression to play a protective effect against oxidative stress of liver injury induced by dichlorvos poisoning.