| Objective:Septic shock is characterized by infection combined with organ and tissue hypoperfusion or organ dysfunction. The mortality rate of septic shock is as high as 80%. Septic shock is a serious public health problem, consuming huge social and health resources, and in a very long henceforth period of time it will be an important diseases threatening human life and health. The main mechanisms of sepsis are SIRS and compensatory anti-inflammatory response syndrome (compensatory antiinflammatory response syndrome, CARS) fluctuation. SIRS is the body of a variety of non inflammatory and infectious lesions by reaction of infection, to activate inflammatory cells and release lots of inflammatory factor. It is also reported that vascular endothelial injury, increased endothelial permeability, and the disruption of barrier function are involved in sepsis pathogenesis. Continuous veno venous hemofiltration (CVVH) and ulinastatin (ULI) used in treating inflammatory diseases play an important role in alleviating endothelial cell injury. However, the clinical significances of CVVH and ULI in the treatment of patients with severe sepsis is still unclear. Possible reasons for these conflicting results are:the failure of the stochastic approach, the modification of the treatment plan during treatment, combined with the use of different types of renal replacement therapy method, inadequacy and the number of cases. Therefore, it is necessary to address this issue through the multi center clinical study, randomized, controlled clinic research and to further explore the CVVH and ULI effect on the permeability of endothelial cell and its mechanism in endothelial cell model.Methods:Clinical research:1 the research objectMulti center (Zhengzhou City intensive medicine, Third People’s hospital intensive medicine, Genenral Hospital of PLA Guangzhou Military Area of Henan province intensive medicine, infectious disease hospital of PLA 309 Hospital blood purification center), random, study time is from June 1,2013 to December 30,2014.2 inclusion criteria:(1) consistent with septic shock diagnosis standard;(2) aged>18 years,<75 years old;(3) the hospitalized time>24 hours;(4) and cardiac index>3 L/min/m2;(5) patients or family members signed the consent.3 exclusion criteria:(1) the hemodynamics in patients with unstable, cannot tolerate the blood filter;(2) admitted within 24 hours of death;(3) patients or family members refused to participate in clinical trials or refused to sign the informed consent;(4) with acute cardiac injury or stroke patients.4 Research groups:Using a computer random number technology patients were randomly divided into 4 groups, namely control group, continuous hemofiltration group (CVVH), ulinastatin group (ULI) and continuous hemofiltration combined with ulinastatin group (CVVH-ULI). The control group only with traditional treatment, if the patients suffered from renal failure, hemodialysis with conventional scheme can be carried out; CVVH group uses traditional conservative treatment at the same time with polyflux 14S (membrane area of 1.4 square meters) of blood filter; group ULI,2 times daily intravenous infusion of Wu of pentostatin, each 200000 units; CVVH-ULI is using the CVVH combined with ulinastatin.5 General therapy:Each group were diagnosed as sepsis after 2012 sepsis in accordance with the guidelines for early goal-directed therapy in patients with acute renal failure, can be in accordance with the conventional renal replacement for dialysis.6 Hemofiltration configuration:the diagnosis of severe sepsis 2 hours began hemofiltration treatment. Catheter in internal jugular vein or femoral vein indwelling deep venous catheter, three cavity (USA ARROW company). With continuous veno venous hemofiltration (CVVH) therapy. CVVH treatment machine for Baxter production Aquarius blood filter, the application of polyflux 14S (membrane area of 1.4 square meters) and supporting pipeline, every 24 hours to replace the 1 filter, a total of 72 hours of treatment. Replacement amount 40ml/kg.h, using pre dilution mode. The blood flow rate of 200ml/min. The use of heparin, blood filter machine before use with 3000ml normal saline and heparin 20mg pre washed 30min, and then to the initial dose is 3000IU, then the 500IU/h intravenous infusion, blood coagulation index adjustment with heparin dosage, the bleeding tendency in patients using non heparin treatment.7 general record index(6) general:age, sex, body weight, vital signs (body temperature, heart rate, blood pressure, oxygen saturation, breathing), urine volume, the main diagnosis, APACHII score and SOFA score.(7) the evaluation indexes:the change of MAP; norepinephrine dosage; cytokine detection; mark evaluation object detection and organ function injury of endothelial cells:first,2,3 days morning 6:30 blood check four items of blood coagulation, blood routine, liver and kidney function, HLA-DR, BNP, cardiac biochemical, blood gas analysis, vWF and E- selectin etc., and the APACHII score and SOFA score. Prognosis:ICU mortality, in-hospital mortality,28 day and 60 day mortality.Cell research:Serums of patients from groups noted above were incubated with human umbilical vein endothelial cells (HUVECs). Endothelial permeability were determined by detecting the leakage of FITC-albumin, endothelial cytoskeleton was stained withrhodamine-phalloidin. Cell apoptosis were stained with Annexin-PI staining and detected by a flow cytometry. Autophagy in endothelial cell was evaluated by MDC staining. The formation of autophagosome was viewed by electron microscopy. The expression of p38, ERK and JNK protein and their phosphorylation levels were determined by Western-blot.Results:clinical study:(1) lactic acid level before treatment groups were higher than those of normal, but no significant differences between the groups, suggesting the presence of tissue and organ perfusion is obviously insufficient. While the use of ulinastatin, hemofiltration, or hemofiltration combined with ulinastatin treatment group blood lactate level is significantly lower than the control group. (2) Before the treatment of each group were APACHE II score more than 24 points in the SOFA, and the maximum value in the above 14 points, with no significant differences between groups. But by using Ulinastatin, hemofiltration, or hemofiltration combined with ulinastatin in the treatment of APACHE in patients with II and SOFA score were significantly lower than those of control group. But the effect of ulinastatin, hemofiltration, or hemofiltration combined effects between he d treatment group had no significant difference. (3) of patients in each group were due to the presence of hypotension and shock symptoms, use a dose of dopamine and norepinephrine, dopamine dosage which about 6 mu g/kg.min, and norepinephrine dosage at 0.3 g/kg.min. Treatment of 72 small time, groups of dopamine and norepinephrine content all appeared different degree of decline. The effects of ulinastatin, hemofiltration, or hemofiltration combined with ulinastatin group were significantly lower than those in the control group, but in Ulinastatin, hemofiltration, or hemofiltration combined with ulinastatin treatment group between the three groups, no significant differences in the amount of dopamine and norepinephrine. (4), control group ICU mortality rate,28 day mortality and hospital mortality rates were 38%, 39% and 42%. And ulinastatin, hemofiltration, or hemofiltration combined with ulinastatin treatment significantly reduced the mortality rate in patients with ICU,28 day mortality and in-hospital mortality rate. (5) before treatment in patients with sepsis showed central venous pressure and intrathoracic blood volume decreased, and extravascular lung water content is basic and normal, but systemic vascular resistance and cardiac index decreased characteristics. This is consistent with severe sepsis patients with early pre load, after load decreased, heart function damage performance. By 72 hours after treatment, the control group of patients with central venous pressure, intrathoracic blood volume, lung water content and system of extravascular vascular resistance was higher than the normal value of cardiac index increased, compared with those before treatment. Ulinastatin, hemofiltration, or hemofiltration combined effects between statin therapy group and control group of central venous pressure and systemic vascular resistance did not change significantly, intrathoracic blood volume and extravascular lung water in lower than in the control group, and cardiac index improved significantly better than the control group. In addition, the use of hemofiltration in treatment of the 2 groups of extravascular lung water should be lower than that of the pure Ulinastatin treatment group or a control group, which suggested the hemofiltration superior in capacity management of ulinastatin.Cell research:(1) in severe sepsis patients before treatment (Group untreated) of serum stimulation makes albumin leakage significantly increased endothelial cells. Regardless of is the commonly used conventional therapy, or ULI, CVVH or CVVH combined with ULI in the treatment of endothelial cells, leakage of albumin was significantly lower than untreated. Indicates that the ULI or CVVH on endothelial cell permeability has significant protective effect; (2) the control group patients serum stimulated endothelial cell cytoskeleton disorder and rearrangement, and the effects of changes in endothelial cytoskeletal he Ding, hemofiltration or the combination of the two treated patients serum induced significantly weaker than the control group. This suggests that, rearrangement of ulinastatin and hemofiltration treatment can alleviate the endothelial cytoskeleton. (3) using the control group patients serum stimulated endothelial cells occurred apoptosis, and the effects of endothelial cell apoptosis of his Ding, hemofiltration and combination of the two treated patients serum stimulation was significantly decreased. Prompt, ulinastatin and hemofiltration can reduce the apoptosis of endothelial cells. (4) in control group serum stimulation of endothelial cell autophagic activity can influence is not obvious, and the effect of ulinastatin, hemofiltration or the combination of the two treatment significantly increased the autophagic activity. (5) the serum stimulation can on endothelial cell autophagic vesicle formation does not significantly affect the control, and the effect of ulinastatin, hemofiltration or the combination of the two treatment significantly increased the formation of autophagic vesicles. (6) endothelial cell p38 phosphorylation in septic patients untreated serum group or routine treatment group stimulation increased significantly, while the use of serum ULI and CVVH in endothelial cells after treatment stimulated p38 phosphorylation level was significantly lower than the non treatment group and routine treatment group. While ERK and JNK phosphorylation level had no significant differences between the groups in the. (7) in serum of patients with sepsis stimulates endothelial cell permeability increased significantly, while the p38 inhibitor SB203580 significantly inhibits the increase of permeability, but ERK and JNK inhibitors can inhibit the growth of septic serum induced endothelial cell permeability. (8) the endothelial cytoskeleton rearrangement of p38 inhibitor can significantly inhibit the serum of the patients with sepsis induced by.Conclusions:ULI and CVVH can significantly improve the prognosis of patients with septic shock, the mechanisms were related to reduce endothelial cell permeability in septic patients. |
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