| Background:The aim of this study was to investigate the effects of mild hypothermia and minimally invasive evacuation of hematoma on the brain function of patients with cerebral hemorrhage. Seventy-six patients with acute cerebral hemorrhage were divided into the minimally invasive evacuation of hematoma (MIHE) and mild hypothermia and minimally invasive evacuation of hematoma (MHMIHE) groups. National Institutes of Health Stroke Scale (NIHSS) scores on the day of admission of the patient and one, three and seven days after the procedure were recorded. Perihematoma brain tissue morphology was observed using hematoxylin and eosin staining. Nuclear factor-κB (NF-κB) expression was determined by immunohistochemistry. The tumor necrosis factor-a (TNF-a) level was detected by ELISA. NIHSS scores in the MHMIHE group were significantly lower than those in the MIHE group on days three and seven. TNF-α and NF-κB levels peaked on day three, and the MHMIHE group had significantly lower levels of TNF-a and NF-κB than the MIHE group. In conclusion, the present study demonstrated that mild hypothermia and minimally invasive evacuation of hematoma can effectively reduce inflammation and improve the brain function of patients. Objective:Mild hypothermia combined minimally invasive evacuation of hematoma can induce NF-κB inflammatory reaction and improve brain function after cerebral hemorrhage in patients, and it is safe and feasible. Methods:Seventy-six patients with acute cerebral hemorrhage were studied and divided into two groups:minimally invasive evacuation of hematoma group and mild hypothermia combined minimally invasive evacuation of hematoma group with NIHSS scores at admission and on 1 d,3 d,7 d after operation. Minimally invasive evacuation of hematoma was used intracranial hematoma smash puncture needle for minimally invasive treatment of intracranial hematoma and mild hypothermia combined minimally invasive evacuation of hematoma was given a general water circulation type cooling blanket+ice cap cerebral local mild hypothermia sub low temperature treatment. Take the perihematomal brain tissue fragments flushed with the cerebral haematoma during the operation and on 1 d,3 d,7 d after operation in all patients. Fixed in formalin preservation and embedded with the paraffin after slicing. To study the expression of protein by using the slicing of surrounding tissue of cerebral haematoma with HE.At the same time,to research the NF-κB by investigating with the immune-histochemistory. TNF-α was tested by ELISA in the venous blood. Results: The NIHSS scores of mild hypothermia combined minimally invasive evacuation of hematoma group was significantly lower than that of minimally invasive evacuation of hematoma group on 3 d,7 d and the difference between the two groups was statistically significant (p<0.05). The former is better. The expression of TNF-α and NF-κB peaked at 3 days but the expression of them in mild hypothermia combined minimally invasive evacuation of hematoma group was lower than that of minimally invasive evacuation of hematoma group (p<0.05). Conclusion:The operation of mild hypothermia combined minimally invasive evacuation can better relieve the NF-κB inflammation and immunoreaction and reduce brain injury and improve brain function It is safe and feasible.Background:Cerebrovascular disease is a major cause of mortality and disability worldwide. The main cause of cerebrovascular disease is hypertension and atherosclerosis(AS), and the most common risk factor for atherosclerosis is hypertension. Therefore, anti atherosclerosis and antihypertensive treatments are recommended for the prevention of cerebrovascular disease. Atherosclerosis is a chronic inflammatory disease that develops by immune responses to autoantigens or by cross-reactions to foreign antigens. Immune cells initiate and sustain local inflammation. Local immune inflammatory reaction is probably essential for atherosclerosis development and progression. Dendritic cells (DCs) and T lymphocyte might contribute to local immune inflammatory responses and disease progression in the artery. DCs are professional antigen-presenting cells (APCs) that present various exogenous and endogenous antigens to T lymphocytes, providing an important link between the innate and adaptive immune responses. The costimulatory molecules CD80 and CD86, which are known to be involved in immunological synapse formation and activation of T cells, and MHC-Ⅱ of DCs binding to TLR and NLR families receptors are crucial for the ability of DCs to discriminate self and foreign antigens for presentation to T cells. We made an attempt to delineate several DC-mediated processes that are potentially relevant to atherosclerosis; namely presentation of therosclerosis-relevant antigens to T lymphocytes, DCs-T cell interactions, production of chemokines and cytokines to sustain local inflammation.Receptors being binded with DCs, activation precursor cells (ThO) of T helper cells are activated. ThO cells are divided into Th1 cells and Th2 cells. Interferon gamma (IFN-y), the main cytokine secreted by Th1. It mediates cellular immune responses. Intracellular IL-10(IL-10) and intracellular IL-4(IL-4), the main cytokine are secreted by Th2. IL-10 inhibits differentiation of Thl. Usually the cytokines secreted of Thl and the hyperfunction of it are seen as proinflammatory factors. The cytokines secreted of Th2 are seen as antiinflammatory factors. CD4+T cells are the main lymphocytes in AS lesions. According to the local environment is different and T cells are differentiated into Th1 and Th2. The high level of AngⅡ makes Thl hyperfunction and imbalance of Thl/Th2. Hypertension via T lymphocytes promote AS. OLmesartan, Ang Ⅱ receptor antagonist (ARB), both reduces blood pressure, and inhibits the hyperfunction of T lymphocytes.AS is a chronic inflammatory disease, which involves immune system, vascular cells,and several organs. The renin angiotensin system (RAS) contributes to vascular damage associated with hypertension and AS, which are important risk factors underlying cardiovascular, cerebrovascular, renal and metabolic diseases. Circulating kidney-derived renin regulates cardiovascular function through Ang Ⅱ binding to its AT1 and AT2 receptors on target tissues. Ang Ⅱ has been detected also in peripheral tissues (such as aortic tissue), suggesting a possible role of the local renin-angiotensin system in atherosclerosis. AT1 receptor is widely expressed on different cell types involved in atherosclerogenesis. AT1R mediates most of the pathophysiological effects of Ang Ⅱ, including vasoconstriction, inflammation growth, and fibrosis, whereas AT2R may counteract many of the AT1R-mediated actions. Over the recent past, researchers think of Ang Ⅱ have changed from being a simple vasoconstrictor to that of a complex growth factor that mediates effects through diverse signalling pathways involving PLC/PKC/Ca2+ mobilization, PLA2, PLD, MAP kinases, tyrosine kinases, proto-oncogene expression, RhoA/Rho kinase, and oxidative stress. Through increased Nox-derived ROS generation and activation of redoxsensitive transcription factors, Ang Ⅱ promotes expression of cell adhesion molecules and induces synthesis of proinflammatory mediators and growth factors. These molecular and cellular processes facilitate increased vascular permeability, leukocyte recruitment, calcification and vascular fibrosis leading to vascular injury. Targeting some of these molecular events with RAS inhibitors may protect against vascular injury and promote vascular health.Ang Ⅱ evokes inflammatory responses and plays a central role in AS mediated by Ang Ⅱ type 1 (AT1) receptor. AT1 receptor blockers (ARBs) prevent the diverse effects of Ang Ⅱ. Each of ARBs being available for clinical use also has unique molecule-specific effects, although all ARBs have common effects. In nonsignificant coronary stenotic lesions, it is important that we use aggressive medical treatments using ARBs to induce the regression and stabilization of coronary plaque. Although all ARBs have class effects, All ARBs may not have similar depressor effects. The angiotensin receptor blocker (ARBs)-Olmesartan, is an important drug. The specific molecule effects of Olmesartan prevents the increase in coronary atheroma volume. In particular, the Olmesartan also has an anti-inflammatory effect. This article discusses on current evidence regarding the Olmesartan-modified dendritic cells of rat in vitro by down-regulated T cells proliferation.Objective:Research on effects of Olmesartan-modified dendritic cells on immune response of T cells of rat in vitro.Methods:1. Tolerogenic DCs preparation and modificationGet female Lewis rats under aseptic conditions. Fully washed marrow cavity and grinded bone marrow,then prepare the single cell suspension. After breaking the red cell membrane, put the cells fluid was placed in the flasks,adding complete medium at 37℃, under 5% CO2 culture for 3 days. Medium was replaced after 18 hours on seventh days in the flasks and add Olmesartan solution (final concentration 10 μM), while in the control group culture flask was added an equal volume of DMSO solution preparation.Cultured for 2 days suspended cells were collected, recorded as olmesartan modified dendritic cells and without olmesartan modified DCs.2. Flow cytometric analysis of surface CD80, CD86 and MHC-Ⅱ on DCsCounted cells of every flow cytometry assay tube is 1×106 cells/ml. Added lml PBS and wash cells,2000 rpm,5 minutes. After adding 1.5 ml 0.5% BSA and washing cells. Put in 1 μl antibody and put at 4℃ refrigerator incubated for 30 minutes. Washed two times by 1.5 ml 0.5% BSA, Put in 500 μ PBS and blend cells. Suspension of cells again. After being filtered and analyzed by flow cytometry.3. IL-10 and TGF-(3 cytokine of DCs flow cytometry detectionCounted cells is 1×106 cells/ml of every flow cytometry assay tube. Added 2 ml PBS to wash cells 2 times,2000 rpm,5 minutes. Added 2% paraformaldehyde and inserted at 4℃. Being incubated for 20 minutes. Washed cells for 2 times with 2 ml 0.5% BSA. Put in 1 ml the liquild to broke cell membranes and placed at room temperature for 20 minutes. Added IL-10 and TGF-P antibody. Being incubation for 30 minutes at room temperature, avoided light. Washed two times by 1.5 ml 0.5% BSA, Put in 500 μl PBS. Blended the cells and suspensed cells again. After being filtered and analyzed by flow cytometry.4. Establishment of immune rat modelUsing healthy, female Lewis rats, the 50 μg OVA is dissolved in 0.9% NaCl and heat-inactivated Mycobacterium tuberculosis strains H37Ra and incomplete Freund’s adjuvant, the first double after foot pad immunization subcutaneously in rats, the amount of each rat was 200 μl. Injection scheduled for the same day 0,8 days later rats were injected with the same dose as the 1st dose in the back once again with double-blind observation, the daily weight and performance of rats until after the second immunization 5 days. Rat model of immune is established.5. Preparation of lymph node MNCAnesthesia rats which immunized by OVA. Take bilateral inguinal lymph node of rats under sterile conditions, grinded with a sterile grinding rod until the tissue is changed into white. The abrasive liquid fully absorbed in the centrifuge tube, to prepare into lymph nodes in single cell suspension. Use the counting plate to count cells. Adjust the cells concentration of 2×107cells/ml for use.6. CD4+ T cell sortingThe sorting method for CD4+T cells by using the nylon wool column filtration. First, take 150 mg nylon cotton loaded into a 10 ml syringe. Make it as high as 6 centimeters and prepare to be spared after autoclaving. Nylon wool column was washed 2 times by using FBS 1640 culture medium. It was placed at 37℃ for 1 hour. Filtered liquid within the cells to cotton column before release. Count cells for 2 × 107 cells/ml of lymph nodes in single cell suspension. Drop slowly into the cotton inside the column at the rate of 20 drops per minute. The nylon wool column was preincubated for 1 hour and washed nylon cotton column with complete culture medium for 3 times at the normal temperature. Collect the initial effluent. Cells count and adjust the cells concentration of 1 × 106 cells/ml. They were namely for the separation and purification of CD4+T lymphocytes.7. T Cells proliferation assayCFSE method:Take 10 × 106 T lymphocytes within the centrifuge tube. After centrifugation heavy hanging. Add in CFSE. Hatched at 37℃. Washed cells with 1640 for 2 times. After 72 hours of incubation, containing 1 × 106 cells, DCs and T cells suspended in 200 aliquots were cultured in microtitre flat-bottomed plates which had 96-well in the presence. AT the same time, put OVA in them(final concentration 10 μM). After 72 hours of incubation, cells were analyzed by flow cytometry.8. Using ELISA determinate cytokine IFN-y of T cellsT lymphocytes stimulated by OVA in lymph node were co-cultured with DCs for 60 hours. Then took the cell culture supernatant retained. In accordance with IFN-y kit instructions, ELISA operations was performed. The expression of the cytokine IFN-y level of cell culture supernatant was detected.9. Statistical analysisStatistical analysis of data of the experimental results was used SPSS 17.0 software. Between the groups were used independent samples/test, p< 0.05 was considered differences, statistic significant. Results showed by the mean ± standard deviation.Result:1. Olmesartan could not induce tolerogenic DCs in vitroFlow cytometry was used between the two DCs found expression of surface molecules have a tendency. The expression of costimulatory molecules CD80, CD86 and MHC-Ⅱ in the DCs modified by Olmesartan group was lower than those in the DCs without being modifid by Olmesartan group. But there was no statistical difference (p>0.05).2. Level of Intracellular IL-10 in DCs up-regulated by OlmesartanDetected by flow, The expression of IL-10 in the DCs modified by Olmesartan group was lower than that in the DCs non-modified by Olmesartan (p=0.07). However, the level of TGF-β between the two groups has no statistical significance, although that has the trend.3. T cell proliferation inhibited by Olmesartan modified DCsThere are statistically significant differences of T lymphocyte proliferative response between two groups-DCs modified by Olmesartan group and DCs without Olmesartan detected by the CFSE. T lymphocyte proliferation of Olmesartan modified DCs group was significantly lower than that non-Olmesartan modified DCs group (p<0.001).4. Cytokine IFN-γ of T lymphocyte culture supernatant detectedT lymphocytes of rat lymph node-derived which immunized by OVA were co-cultured with DCs. Detection of IFN-γ level of cell culture supernatant by ELISA. The expression of IFN-γ in the DCs modified by Olmesartan group was lower than that in the non-modified DCs group.Conclusions:1. Olmesartan could not induce tolerogenic DCs in vitro, but up-regulated the level of intracellular IL-10 in Olmesartan-DCs.2. The effects of Olmesartan-DCs on T cells inhibited lymphocyte proliferation. Meanwhile it decreased level of IFN-γ cytokine of T cells in cells supernatant. |
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