MicroRNA-204 may participate in the pathogenesis of hypoxic-ischemic encephalopathy through targeting KLLN.

Hypoxic-ischemic encephalopathy (HIE) is a common neonatal disease that can lead to high neonatal mortality rates. Previous studies have indicated that microRNAs (miRs) may be involved in the pathogenesis of HIE; however, the specific mechanisms underlying their involvement require further investigation. The aim of the present study was to investigate the roles of miR-204 and its target gene killin p53 regulated DNA replication inhibitor (KLLN) in HIE using rat HIE models. Brain injury was induced by surgery and incubation of hypoxic incubator brain using 10-day-old pup rats. On day 3, rats were sacrificed, and the infarct size of the brain was determined using a tetrazolium chloride assay. Terminal deoxynucleotidyl transferase UTP nick-end labeling staining was performed to detect the cell death rate in the brain tissue. Following this, the brain tissues were collected, and reverse transcription-quantitative polymerase chain reaction, western blot analysis and immunohistochemistry assays were performed to examine the expression levels of miR-204 and KLLN. Furthermore, neurons were cultured and transfected with miR-204 inhibitors or mimics, and the effect of miR-204 on the proliferation and apoptosis of neurons was examined using MTT and flow cytometric assays. Finally, a dual-luciferase reporter assay was performed to confirm whether KLLN is a direct target of miR-204. The expression of miR-204 was significantly downregulated and the expression of KLLN was significantly increased in the brain tissue of HIE rats (P<0.001). In addition, the transfection with miR-204 inhibitors significantly decreased the proliferation rates and significantly increased the apoptosis rate of neurons; however, transfection with miR-204 mimics prompted the opposite results. The dual-luciferase reporter assay also confirmed that KLLN is a direct target of miR-204. Taken together, the results of the present study demonstrated that miR-204 was downregulated in HIE and that miR-204 may serve important roles in the pathogenesis of HIE through targeting KLLN.

NF­κB regulates HSF1 and c­Jun activation in heat stress­induced intestinal epithelial cell apoptosis.

Heat stress may induce intestinal epithelial cell apoptosis; however, the molecular mechanisms have not yet been identified. The present study used IEC­6 rat small intestinal epithelial cells to investigate heat stress­induced production of reactive oxygen species (ROS), which may be involved in nuclear factor (NF)­κB activation during heat stress. IEC­6 cells were transfected with NF­κB p65­specific small interfering RNA (siRNA), and observed a significant increase in cell apoptosis and caspase­3 cleavage; however, in cells transfected with adenovirus that constitutively overexpressed p65, the opposite results were obtained. Furthermore, p65 knockdown increased the heat stress­induced expression and activity of heat shock transcription factor 1 (HSF1); conversely, p65 overexpression slightly decreased HSF1 activity. The levels of heat stress­induced c­Jun phosphorylation were also examined: Knockdown of p65 resulted in a reduction of c­Jun phosphorylation, whereas p65 overexpression resulted in increased phosphorylation. Furthermore, siRNA­mediated knockdown of HSF1 in IEC­6 cells significantly increased heat stress­induced apoptosis. Cells pretreated with c­Jun peptide, an inhibitor of c­Jun activation, exhibited a significant reduction in apoptosis. These findings indicated that heat stress stimulation in IEC­6 cells induced the pro­apoptotic role of NF­κB by regulating HSF1 and c­Jun activation.

Diagnosis of overt disseminated intravascular coagulation in critically Ill adults by Sonoclot coagulation analysis.

Disseminated intravascular coagulation (DIC) diagnosis is hampered by the limited availability of reliable clinical or laboratory tests. Currently available tests are time consuming and expensive. We investigated whether coagulation and platelet function analyses using the Sonoclot system were suitable for overt DIC diagnosis in critically ill adults. This was an observational diagnostic study performed in 498 patients presenting with an underlying disorder associated with DIC. Overt DIC patients were identified according to an International Society on Thrombosis and Hemostasis (ISTH) score of >5. Coagulation and platelet parameters were analyzed using the Sonoclot system, and compared with ISTH as the gold standard. Receiver operating characteristic curves and area under the curves were used to evaluate the value of the Sonoclot parameters. There were no differences for age or gender between the groups. Significant correlations were observed between activated clotting time (ACT) and ISTH score (r = 0.7; P < 0.001), clot rate (CR) and ISTH score (r = 0.5; P < 0.001), platelet function (PF) and ISTH score (r = -0.6; P < 0.001), and PF and platelet count (r = 0.5; P < 0.001). An ACT cut-off value of 213.5 s alone or combined with CR presented good sensitivity (76.7 and 86.8 %, respectively) and specificity (96.2 and 93.3 %, respectively). Sonoclot analysis can be performed using a point-of-care device that effectively discriminates low and high ISTH scores, and that effectively predicts coagulation dysfunction in patients with overt DIC.

Vascular endothelial cell injury partly induced by mesenteric lymph in heat stroke.

Animal models have shown that mesenteric lymph plays important roles in the pathogenesis of endothelium injury in many critical ill states. Gut-derived septicemia and endothelium injury are the two key pathogenesis of heat stroke (HS); however, it is unclear whether mesenteric lymph is cytotoxic to endothelium in HS. HS rat models were prepared in a prewarmed incubator. Mesenteric lymph, collected pre-, during, and post-HS, was analyzed for biological activity on human umbilical vein endothelial cell (HUVEC) in vitro. The effect of HS lymph on the production of von Willebrand factor (vWF), thrombomodulin (TM), and IL-6 by HUVEC was investigated. In vivo, vascular endothelium injury biomarkers, circulating endothelial cell (CEC), as well as serum soluble vWF and TM were tested in rats of HS and HS with mesenteric lymph duct ligation (HS-LDL). HS but not heat stroke sham mesenteric lymph-injured endothelial cells showed significantly increased HUVEC cytotoxicity and enhanced HUVEC monolayer permeability as well as elevated levels of vWF and TM production by HUVEC. IL-6 production by HUVEC was augmented by HS lymph in vitro. The effects of HS lymph on IL-6 production had a time course resembling that of the toxic effects of HS lymph on HUVEC. In vivo, when compared with HS rats, decreased CEC counts as well as lower serum vWF and TM concentrations were detected in HS-LDL rats. HS mesenteric lymph is probably harmful to vascular endothelium, which indicates that the modulation of mesenteric lymph may have some potential benefits to HS.

Xuebijing injection alleviates liver injury by inhibiting secretory function of Kupffer cells in heat stroke rats.

OBJECTIVE: To evaluate the effects of Xuebijing (XBJ) injection in heat stroke (HS) rats and to investigate the mechanisms underlying these effects. METHODS: Sixty anesthetized rats were randomized into three groups and intravenously injected twice daily for 3 days with 4 mL XBJ (XBJ group) or phosphate buffered saine (HS and Sham groups) per kg body weight. HS was initiated in the HS and XBJ groups by placing rats in a simulated climate chamber (ambient temperature 400C, humidity 60% ). Rectal temperature, aterial pressure, and heart rate were monitored and recorded. Time to HS onset and survival were determined, and serum concentrations of tumor necrosis factor (TNF)-alpha interleukin (IL)-1beta, IL-6, alanine-aminotransferase (ALT), and aspartate-aminotransferase (AST) were measured. Hepatic tissue was harvested for pathological examination and electron microscopic examination. Kupffer cells (KCs) were separated from liver at HS initiation, and the concentrations of secreted TNF-a, IL-beta and IL-6 were measured. RESULTS: Time to HS onset and survival were significantly longer in the XBJ than in the HS group. Moreover, the concentrations of TNF-alpha, IL-1beta, IL-6, ALT and AST were lower and liver injury was milder in the XBJ than in the HS group. Heat-stress induced structural changes in KCs and hepatic cells were more severe in the HS than in the XBJ group and the concentrations of TNF-alpha, IL-beta and IL-6 secreted by KCs were lower in the XBJ than in the HS group. CONCLUSION: XBJ can alleviate HS-induced systemic inflammatory response syndrome and liver injury in rats, and improve outcomes. These protective effects may be due to the ability of XBJ to inhibit cytokine secretion by KCs.

[Effects of early supplemental parenteral nutrition on nutrition intakes and clinical outcomes in trauma patients].

OBJECTIVE: To evaluate energy and protein intake changes in early supplemental parenteral nutrition (PN) in trauma patients, and to assess its impact on clinical outcomes. METHODS: Clinical results of patients receiving or not receiving additional PN during the first 7 days after injury were retrospectively analyzed, with a total of 195 patients classified into two groups: control group (n=105) and mixed nutrition group (n=90). The time of nutrition support, intakes of protein and energy within 14 days after trauma, and clinical outcomes were compared between two groups. RESULTS: The degree of injury was comparable between two groups with no significant differences in acute physiology and chronic health evaluation II score, injury severity score (ISS) and Glasgow coma score (GCS). Compared with the control group, the mixed nutrition group received parenteral nutritional support earlier (40.0±21.0 hours vs. 55.1±23.5 hours, P<0.01), with later beginning of enteral nutrition (EN, 75.2±54.5 hours vs. 55.1±23.5 hours, P<0.01) and lower rate of EN in 48 hours after admission [14.4% (13/90) vs. 43.8% (46/105), P<0.01]. The time of restoring oral diet was not different between the mixed nutrition group and control group (10.8±3.7 days vs. 11.4±3.6 days, P>0.05). The energy intake was significantly higher in the mixed nutrition group than in the control group in 3, 7, 14 days (3 days: 3981.6±2209.3 kJ vs. 2683.2±1414.9 kJ, 7 days: 5477.5±2008.4 kJ vs. 3619.1±1429.9 kJ, 14 days: 6250.2±2533.2 kJ vs. 5199.9±1972.7 kJ, P<0.05 or P<0.01). In both groups the protein intake was insufficient, and it was significantly lower in the mixed nutrition group than in the control group on day 3 (20.6±18.4 g vs. 26.5±13.8 g, P<0.05). The patients in the mixed nutrition group had longer hospital stay time (73.9±62.5 days vs. 50.9±33.3 days, P<0.01). The mortality rate of mixed nutrition group and control group was 4.4% (4/90) and 3.8% (4/105) respectively, the rate of infection and acute respiratory distress syndrome (ARDS) were 8.9% (8/90) and 3.8% (4/105), 5.6% (5/90) and 7.6% (8/105) respectively, duration of mechanical ventilation (days) was 8.3±4.6 and 7.3±4.7, duration of stay in ICU was 17.6±13.2 days and 14.2±11.3 days respectively, and no significant difference was found between two groups (all P>0.05). CONCLUSION: Although early supplemental PN within 7 days after injury increases energy intake, PN without a standard protocol does not improve clinical outcomes and may prolong hospital stay time.

[Analysis of energy balance and risk factors on clinical outcomes in patients with severe traumatic brain injury].

OBJECTIVE: To observe the energy expenditure in severe traumatic brain injury patients, and to assess the impact of cumulative energy balance on clinical outcomes. METHODS: Using prospective self-controlled study, the change in energy expenditure of 42 patients with severe traumatic brain injury was measured by indirect calorimetry (IC). Daily energy intake was recorded. Afterwards, energy balance was calculated. The levels of nutritional biochemical indicators were compared. Logistic regression analysis was used to analyze the correlation of cumulative energy balance with clinical outcomes. RESULTS: Mean practical energy intake of all patients was (6787 ± 1848) kJ/d, and mean negative energy balance was (913 ± 285) kJ/d. The negative energy balance was most crucial in first 3 days after admission. Meanwhile, practical energy intake was significantly lower than target energy intake (kJ: 2859 ± 1370 vs. 6027 ± 899, P < 0.01). The practical energy intake was increased with time, and it was found that the first 14 days were crucial for development of negative energy balance. On 7th day after admission, albumin (g/L) level in plasma was lowest compared with that on 3rd day (29.5 ± 5.0 vs. 35.9 ± 3.8, P < 0.01), and then it was increased gradually returning to normal level on 28 days (34.1 ± 2.8). Three days after admission, prealbumin (mg/L: 122.5 ± 23.3) was obviously lower than normal level, but it rapidly elevated on 7th day (214.3 ± 38.6, P < 0.01) and continued to rise till 28th day (257.7 ± 25.2). On the 3rd day after admission, C-reactive protein (mg/L: 139.5 ± 54.4) was obviously higher than normal level. However, it significantly fell on 7th day (108.4 ± 42.2, P < 0.01), and it continued to fall. Logistic regression analysis showed a strong association of cumulative negative energy balance with infection and upper gastrointestinal bleeding [odds ratio (OR) of infection was 2.130, 95% confidence interval (95%CI) 1.540 to 29.661, P = 0.023; OR of upper gastrointestinal bleeding was 0.083, 95%CI 0.013 to 0.542, P = 0.009]. CONCLUSIONS: Cumulative negative energy balance may be correlated with the occurrence of complications in patients with severe traumatic brain injury. On the basis of the measurements of changes in energy by IC, early supply of sufficient energy may improve the outcome of patients.

[Comparison of measuring energy expenditure with indirect calorimetry and traditional estimation of energy expenditure in patients in intensive care unit].

OBJECTIVE: To compare measurement of energy expenditure (MEE) by indirect calorimetry (IC) with traditional estimation of energy expenditure (EEE), to provide a basis for energy supplementary for critically ill patients. METHODS: Using self-controlled study,the energy expenditure of 57 intensive care unit (ICU) patients was measured by IC. Meanwhile, EEE was also calculated using the following equations : Harris-Benedict (HB), HB×factor , or 104.6 kJ/kg. Body weight were calculated using actual body weight (ABW) or ideal body weight (IBW). If body mass index (BMI)<18.4 kg/m(2) it was considered as underweight , and the IBW was selected from the IBW table. The potential adequacy of estimated energy was assayed by ratio of EEE/MEE. RESULTS: There was significant difference in MEE by IC and EEE by HB, HB×factor and 104.6 kJ/kg [(6 335 ± 1 004) kJ, (9 125 ± 1 795) kJ, (7 188 ± 1 029) kJ vs. (7 753 ± 1 439) kJ ,P<0.05 or P<0.01]. There was significant difference between EEE by HB×factor and 104.6 kJ/kg (P<0.01) , and EEE by 104.6 kJ/kg×ABW , and the latter was closer to MEE. Underfeeding would occur in most ICU patients if HB equation was used [100% (4/4) in underweight patients and 73.59% (39/53) in normal weight (BMI 18.5-23.9 kg/m(2))]. EEE as calculated by 104.6 kJ/kg×IBW was reasonable in the underweight patients 100% ( 4/4 ), but EEE in the patients with normal weight by using HB×factor or 104.6 kJ/kg×ABW resulted in significant underfeeding [39.62% (21/53) and 43.39% (23/53)] or overfeeding [24.53% (13/53) and 13.22% (7/53)]. CONCLUSION: EEE derived from the equations was extremely inaccurate and may result in significant underfeeding or overfeeding in individuals. On the basis of this study we would recommend IC for measuring energy expenditure in ICU patients. Otherwise , the equations of 104.6 kJ/kg×IBW in underweight and 104.6 kJ/kg×ABW in normal weight patients may be reasonable.

[Clinical effects of ulinastatin and thymosin alpha1 on immune-modulation in septic patients].

OBJECTIVE: To analyze clinical effect of immuno-modulatory therapy with ulinastatin and thymosin alpha1 on patients with sepsis. METHODS: Two hundred and forty-two septic patients admitted to Guangzhou General Hospital of Guangzhou Military Command intensive care unit (ICU) during 2004.10-2008.6 were included, and they were randomly divided into treatment group (128 cases) and control group (114 cases). The patients in control group were given regular conventional treatment according to Surviving Sepsis Campaign (SSC) in 2004, including early fluid resuscitation, antibiotic therapy, mechanical ventilation (MV) and blood purification. The treatment group received conventional treatment plus immuno-modulation therapy including ulinastatin (first 200 kU injection intravenous twice a day for 4 days and 100 kU for another 6 days) and thymosin alpha1 (1.6 mg subcutaneous twice a day for 4 days, followed by 1.6 mg per day subcutaneous for another 6 days). The total treatment course was 10 days. General demographics were observed, and acute physiology and chronic health evaluation II (APACHE II) scores were recorded. Serum interleukin-6 (IL-6), IL-10 levels of peripheral blood were detected by enzyme linked immunosorbent assay (ELISA). Peripheral blood CD14(+) monocyte human leucocyte antigen DR (HLA-DR) expression, and ratio of helper T lymphocyte 1 (Th1) cytokines interferon-gamma (CD4(+)IFN-gammaww(+)), and Th2 cytokines (CD4(+) IL-4(+)) were assessed with flow cytometer. Duration of infection and MV, length of ICU stay, rate of development of multiple organ dysfunction syndrome (MODS) and mortality rate on 28 days were observed as end-point. RESULTS: Before treatment, there was no difference in all biomarkers between two groups (all P>0.05). After treatment, peripheral blood CD14ww+ monocyte HLA-DR expression and the ratio of CD4(+)IFN-gamma (+)/CD4(+) IL-4(+) increased significantly in the treatment group (both P<0.05), with serum IL-6, IL-10 levels and APACHE II scores all reduced remarkably (all P<0.05). The values showed significant differences compared with those of control group (all P<0.05). The MODS development rate in the treatment group was much lower than that of control group (21% vs. 47%, P<0.05), and the length of use of MV was significantly reduced [(6.08+/-2.46) days vs. (8.23+/-3.47) days, P<0.05]. There was no difference in the infection duration and length of ICU stay (both P>0.05). The mortality rate on 28 days in the treatment group was much lower than that in control group (20% vs. 33%, P<0.05). CONCLUSION: The immuno-modulation therapy of ulinastatin and thymosin alpha1 can remarkably improve the duration of MV and the development rate of MODS and mortality rate on 28 days in the patients with sepsis, probably due to its effect in ameliorating the immuno-imbalance state of the patients. However, the duration of infection and length of ICU stay are not effected.

[Clinical value of monitoring CD14+ monocyte human leukocyte antigen (locus) DR levels in the early stage of sepsis].

OBJECTIVE: To explore the relationship of monitoring CD14(+) monocyte human leucocyte antigen (locus) DR (HLA-DR) and the outcome in the early stage of sepsis. METHODS: Thirty-six definitely diagnosed septic patients in intensive care unit (ICU) were included. CD14(+) monocyte HLA-DR levels were detected by flow cytometer on the first day of the study, and acute physiology and chronic health evaluation II (APACHE II) scores were evaluated. Their clinical values in predicting the outcome of the disease were assessed through correlation analysis. RESULTS: Among 36 sepsis patients CD14(+) monocyte HLA-DR level<30% was found in 6 patients (16.67%). The average APACHE II score was 24.17+/-4.45 (r=0.212, P=0.687), all of them die, CD14(+) monocyte HLA-DR level <40% was 27.78% (10/36), the scores of APACHE II score was 23.50+/-4.30 (r=-0.0251, P=0.484), and the mortality rate was 80% (8/10). CONCLUSION: CD14(+) monocyte HLA-DR level <30% is an immunosuppressive index. In predicting the outcome of sepsis, it might be better than APACHE II scores. Immunosuppression is primarily found in the early stage of sepsis, suggesting that the classical compensatory anti-inflammatory response syndrome (CARS) hypothesis needs to be revised and improved.