ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
ObjectiveTo study the effects of the new small molecular oxygen free radical scavenger Tempol on the survival and vasculogenesis of the long random pattern skin flap (LRPSF) and its mechanism. MethodsEighty-four male Sprague Dawley rats were randomly divided into control and Tempol groups (42 rats in each group). LRPSF of 9 cm×3 cm in size were prepared on the backs of rats in two groups based on the Mcfarlane flap. Rats were administered with Tempol (100 mg/kg) in the Tempol group and with normal saline in the control group by intraperitoneal injection at 15 minutes before operation and at 1-7 day after operation. The rat and the skin flap survival conditions were observed after operation; the survival rate of skin flap was measured, and the vascular structure, vascular volume, and total length of blood vessels were analyzed with Micro-CT three-dimensional imaging after 7 days; HE staining was used to observe the structure of the skin flaps and inflammation, immumohistochemical staining to observe vascular endothelial growth factor (VEGF) expression; water-soluble tetrazolium-1 method was used to measure the content of superoxide dismutase (SOD) and malondialdehyde (MDA), and ELISA to detect the expressions of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) after 1, 3, and 7 days. ResultsAll of rats survived after operation, without hemorrhage, edema, and infection. With the extension of time, necrosis occurred in the distal part of the skin flaps in 2 groups, but the necrosis degree of the Tempol group was lower than that of control group; meanwhile, the blood vessel distribution and continuity were better than those of control group. The skin flaps survival rate, vascular volume, and total length of blood vessels of Tempol group were significantly higher than those of control group after 7 days (P<0.05). The clearer skin flaps structure, lighter inflammation reaction and inflammation cell infiltration, and higher VEGF staining intensity were observed in the Tempol group than the control group after 7 days. There was no significant difference in SOD, MDA, and TNF-α, and IL-6 contents between the 2 groups at immediate after operation. SOD significantly increased, but MDA, TNF-α, and IL-6 contents significantly decreased in the Tempol group when compared with control group after 1, 3, and 7 days (P<0.05). ConclusionTempol can significantly promote the LRPSF survival rates, its mechanism is closely related to the promotion of vasculogenesis and reduction of oxidative stress and inflammation.
目的：研究银杏达莫对糖尿病大鼠肾功能的影响，并从抗氧化应激反应的角度探讨其可能的作用机制。方法：50只Wistar大鼠随机分为正常组（10只），糖尿病模型组（20只）及糖尿病模型加银杏达莫组（20只）。采用单次腹腔注射链脲佐菌素（55 mg/kg）诱导糖尿病肾病（DN）大鼠模型，腹腔注射银杏达莫水溶液。生化法测定血糖，血、尿肌酐及尿白蛋白；尾静脉取血ELISA法检测血清血管内皮先长因子（VEGF）水平；肾脏匀浆后测肾脏丙二醛（MDA）、一氧化氮（NO）的含量。结果：糖尿病模型组和银杏达莫组生化指标均高于正常组（Plt;0.05），银杏达莫组MDA、NO及VEGF的表达减少与模型组比较差异有显著性（Plt;0.05）。结论：银杏达莫具有减轻糖尿病大鼠蛋白尿，提高尿肌酐排泄，减轻肾脏损害的作用，其机制可能与提高肾脏抗氧化系统功能有关。
Febrile seizures (FS) are one of the most common neurological disorders in pediatrics, commonly seen in children from three months to five years of age. Most children with FS have a good prognosis, but some febrile convulsions progress to refractory epilepsy (RE). Epilepsy is a common chronic neurological disorder , and refractory epilepsy accounts for approximately one-third of epilepsies. The etiology of refractory epilepsy is currently complex and diverse, and its mechanisms are not fully understood. There are many pathophysiological changes that occur after febrile convulsions, such as inflammatory responses, changes in the blood-brain barrier, and oxidative stress, which can subsequently potentially lead to refractory epilepsy, and inflammation is always in tandem with all physiological changes as the main response. This article focuses on the pathogenesis of refractory epilepsy resulting from post-febrile convulsions.
Objective To observe the effects of cigarette smoke extract ( CSE) on the proliferation and secretion of hydrogen peroxide ( H2O2 ) in human lung fibroblasts ( HLFs) induced by transforming growth factor-β1 ( TGF-β1 ) . Methods Cultured HLFs were divided into a normal group and a model group induced by TGF-β1 ( 5 ng/mL) , then intervened with CSE at different concentrations ( 0% , 2. 5% , 5% ,10% , respectively) . Brdu ELISA assay was used to detect cell proliferation. H2O2 release from cultured cells was assayed using a fluorimetric method. Cellular localization of H2O2 and expression of α-SMA were performed using a fluorescent-labeling strategy. Results TGF-β1 stimulated group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In TGF-β1 stimulated group, 2. 5% and 5% CSE promoted cell proliferation ( P lt; 0. 01 or 0. 05) , while 10% CSE inhibited cell proliferation ( P lt; 0. 01) . In the normal group, both low and high concentration of CSE inhibited cell proliferation ( P lt; 0. 01 or P lt; 0. 05) , and the inhibition effect was dose-dependent. HLF induced by TGF-β1 generated low constitutive levels of extracellular H2O2 that was markedly enhanced by CSE treatment ( P lt; 0. 01) . Pretreatment with DPI, an inhibitor of NADPH oxidase, abolished secretion of H2O2 . Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast. Conclusions Low concentration of CSE can promote myofibroblast proliferation, and markedly increase extracellular secretion of H2O2 . CSE possibly take part in the development and progress of idiopathic pulmonary fibrosis by increasing oxidative stress.
Objective To study the changes of receptor activator of nuclear factor-κB ligand (RANKL, an osteoclastogenesis-promoting factor) and osteoprotegerin (OPG, the decoy receptor for RANKL), oxidative stress and bone turnover markers in obstructive sleep apnea-hypopnea syndrome (OSAHS), in order to understand the potential mechanisms underlying bone loss in OSAHS patients. Methods Ninety-eight male patients with OSAHS, confirmed by polysomnography (PSG) study, were enrolled. The patients were divided into mild-moderate groups and severe groups. Forty-two male subjects who were confirmed as not having OSAHS served as the controls. The subjects’ bone mineral density (BMD) and T-score were assessed in lumbar spine and femoral neck using dual-energy X-ray absorptiometry. Blood samples were collected from all subjects for measurement of RANKL, OPG, the bone formation marker bone-specific alkaline phosphatase (BAP), the bone resorption marker tartrate-resistant acid phosphatase-5b (TRAP-5b), total antioxidant capacity (TAOC). Twenty-eight severe OSAHS patients accepted continuous positive airway pressure (CPAP) treatment voluntarily. After 6 months, PSG was conducted, and serum RANKL, OPG, TAOC, TRAP-5b, BAP was measured after six months treatment. Results The BMD, T-score of the femoral neck and the lumbar spine were significantly lower in OSAHS patients as compared to the control group. The level of BAP was significantly decreased in the OSAHS group as compared to the control group, and there was no significant difference in TRAP-5b level between two groups. As compared with the control group, levels of OPG, TAOC and the OPG/RANKL ratio decreased significantly. None of these parameters (BMD, T-score, RANKL, OPG, TRAP-5b, BAP) showed significant difference between patients with mild-moderate and severe OSAHS group. Correlation analysis showed that the apnea hypopnea index and oxygen desaturation index were correlated with TAOC. BAP level was positively correlated with TAOC and lowest pulse oxygen saturation. The serum level of TAOC was lower in the OSAHS group after CPAP therapy, but the levels of RANKL, OPG, TRAP-5b, BAP were not different. As compared with the OSAHS group before CPAP therapy, the BMD of the femoral neck and the lumbar spine were not significant difference. Conclusions In patients with OSAHS, the oxidative stress response is enhanced, and imbalance of OPG/RANKL is shifted, which participates in the occurrence of osteoporosis. The oxidative stress injury of severe OSAHS patients was relieved after non-invasive ventilation treatment, but the effect of oxidative stress response on bone metabolism still needs further evaluation.
目的 研究同型半胱氨酸转硫途径、维生素B6及内源性硫化氢在慢性阻塞性肺疾病急性加重期（AECOPD）中的作用。 方法 2010年2月－4月间筛选AECOPD患者16例和健康志愿者（对照组）13例，测定AECOPD患者加重期、缓解期及对照组的肺功能、血清硫化氢（H2S）、丙二醛（MDA）、叶酸、维生素B12、C反应蛋白、白介素6、血浆同型半胱氨酸、胱硫醚、半胱氨酸和维生素B6的浓度。计算半胱氨酸转化率（半胱氨酸浓度/胱硫醚浓度）与胱硫醚转化率（胱硫醚浓度/同型半胱氨酸浓度）参与分析。 结果 ① 加重期血清MDA水平[（7.3 ± 5.1）nmol/L ]比缓解期[（3.0 ± 1.4）nmol/L ]和对照组[（3.0 ± 2.2）nmol/L ]均升高（P＜0.01）；血清MDA水平与第1秒用力呼气容积/用力肺活量（FEV1/FVC）、第1秒用力呼气容积占预计值百分比（FEV1%预计值）呈负相关。② 加重期血清H2S水平与血浆维生素B6水平较缓解期与对照组降低（P＜0.01）；缓解期血清H2S水平[（47.2 ±5.1） μmol/L ]高于对照组[（38.8 ± 2.1） μmol/L ]，P＜0.01；血清H2S水平、血浆维生素B6水平均与FEV1%预计值呈正相关（r=0.651、0.680，P＜0.01），均与血清MDA水平呈负相关（r=-0.334、-0.448，P＜0.05）。③ 加重期半胱氨酸转化率（3.97 ± 2.41）低于缓解期（5.92 ± 2.18）与对照组（6.14 ± 3.15）差异有统计学意义（P＜0.05）；而胱硫醚转化率则相反。④ 叶酸与维生素B12水平各组间均无差异。 结论 提高AECOPD患者维生素B6及H2S浓度可能能促使AECOPD患者向稳定状态转归，减轻氧化应激损伤。维生素B6与H2S可能成为AECOPD患者的一个新的治疗点。Objective To study the roles of homocysteine (Hcy) transsulfuration pathway, Vitamin B6 and endogenous hydrogen sulfide in treating patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Methods Sixteen AECOPD patients and 13 healthy controls (Control group) from February to April 2010 were recruited in this study. Lung function, serum hydrogen sulfide (H2S), malondialdehyde (MDA), folate, vitamin B12, C-reactive protein (CRP), interleukin-6 (IL-6), Hcy, cystathionine, cystein (Cys) and vitamin B6 were all measured for all the patients in the acute exacerbation period and alleviation period and healthy controls. The conversion rate of Cys (expressed as Cys/cystathionine) and the conversion rate of cystathionine (expressed as cystathionine/Hcy) were calculated for analysis. Results Serum MDA level for patients in the acute exacerbation period (AE period) [(7.3 ± 5.1) nmol/L] was significantly higher than that in the alleviation period [(3.0 ± 1.4) nmol/L] and in the healthy controls [(3.0 ± 2.2) nmol/L] (P ＜ 0.01). Serum MDA level was negatively correlated with percentage of FEV1 in predicted FEV1 (FEV1% pred) and FEV1/FVC. Serum H2S level and plasma vitamin B6 level for patients in the AE period were significantly lower than those in the alleviation period and in the healthy controls (P ＜ 0.01), and serum H2S level was significantly higher in the alleviation period [(47.2 ± 5.1) μmol/L] than in the controls [(38.8 ± 2.1) μmol/L] (P ＜ 0.01). Both serum H2S and plasma vitamin B6 levels were correlated positively with FEV1% pred for patients in the AE period and healthy controls (r=0.651, 0.680; P ＜ 0.01), but negatively correlated with serum MDA level (r=-0.334, -0.448; P ＜ 0.05). The conversion rate of Cys for patients in the AE period (3.97 ± 2.41) was significantly lower than that in the alleviation period (5.92 ± 2.18) and the control group (6.14 ± 3.15) (P ＜ 0.05), but the conversion rate of cystathionine was just the opposite (P ＜ 0.05). There were no significant differences in the levels of serum folate and vitamin B12 among the three groups. Conclusion Raising the Vitamin B6 and H2S level may facilitate stabilizing of conditions in patients with AECOPD and reduce oxidative stress. Therefore, it may become a new treatment method for AECOPD.
ObjectiveTo establish a cell inflammation model induced by tumor necrosis factor-α (TNF-α) in human bronchus epithelial cells, and investigate the effects of glutathione S-transferase mu 5 (GSTM5) on the inflammation and oxidative stress. Methods16HBE cells were treated with TNF-α (10 ng/mL, 24 h) in the absence or presence of the constructed GSTM5 eukaryotic expression vector (1 μg/mL). The concentration of malondialdehyde (MDA) and total antioxidation capacity (T-AOC) were detected by colorimetric method. The survival rate of cells was assessed by the methyl thiazolyl tetrazolium (MTT) assay. The transcription level of NADPH oxidase-1 (NOX1), NOX2, NOX3, NOX4, NOX5, dual oxidase-1 (DUOX1) and DUOX2 were evaluated by RT-PCR. Western blot was performed to investigate the protein levels of NOX1 and NOX2. ResultsTNF-α simulation significantly increased the level of MDA in cells, and decreased the level of T-AOC and survival rate of 16HBE. When transfected with the GSTM5 eukaryotic expression vector, the concentration of MDA significantly decreased (P < 0.05), and the activation of T-AOC increased dramatically (P < 0.05). Consequently, the survival rate of 16HBE in the GSTM5 group improved (P < 0.05). The 16HBE cells transfected with the constructed GSTM5 eukaryotic expression vector had a lower transcription and protein levels of NOX1 and NOX2 (all P < 0.01). There were no significant changes in the mRNA expressions of NOX3, NOX4, NOX5, DUOX1 or DUOX2. ConclusionGSTM5 may down-regulate the transcription level of NOX1 and NOX2 to reduce the inflammation and oxidative stress induced by TNF-α.
Objective To establish a cell culture model in vitro of acute lung injury and investigate the effects of NF-κB p65 on the inflammation and oxidative stress in TNF-α-activated type Ⅱ alveolar epithelial cells. Methods A549 cells were treated with TNF-α ( 10 ng/mL, 24 h) in the absence or presence of NF-κB p65 siRNA ( 50 nmol /L) . RT-PCR and Western blot were performed to analyze the silence efficiency of RNAi targeting NF-κB p65. The contents of IL-1β, IL-4, and IL-6 in the culture supernatant were measured by ELISA. The concentration of MDA and SOD were detected by colorimetric method. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium ( MTT) assay. Results P65 RNAi significantly decreased the transcription and translation of NF-κB p65 induced by TNF-α( P lt; 0. 05) . The levels of IL-1β, IL-4, and IL-6 were significantly lower in the supernatants of A549 cells pretransfected with NF-κB p65 siRNA ( P lt;0. 05) , while the concentration of MDA markedly decreased ( P lt; 0. 05) , and the activation of SOD increased dramatically ( P lt; 0. 05) . Consequently, the survival rate of A549 in the p65 siRNA group improved( P lt; 0. 05) . Conclusions NF-κB p65 plays a key role in the oxidative stress induced by TNF-α. NF-κB p65 silencing can down-regulate the inflammation and oxidative stress induced by TNF-αand enhance the proliferation of alveolar epithelial cells.
摘要:目的:通过建立高脂血症脂肪肝动物模型,探讨杜仲叶醇提取物(alcohol extractive of Folium Eucommiae,AEFE)对脂肪肝的防治作用。方法:高脂饮食8周加维生素D36×105IU/kg分3d腹腔注射，建立大鼠高脂血症脂肪肝动物模型。各给药组从造模第3周开始分别灌胃给予洛伐他汀(4mg·kg-1·d-1)、 AEFE(70、140和420mg·kg-1·d-1),共给药6周。各组每天灌服等量生理盐水，灌胃容积为0.5mL/100g体重。结果:AEFE用药6周后显示,各剂量组血清胆固醇、甘油三酯和游离脂肪酸、谷丙转氨酶和谷草转氨酶均较模型组明显降低(Plt;005),肝组织丙二醛含量降低和超氧化物歧化酶活性升高,肝脂肪变性和炎症反应减轻,尤以中剂量组作用最明显(Plt;001)。AEFE对肝内肿瘤坏死因子α浓度无明显影响。结论:AEFE改善高脂性大鼠肝脏脂肪变性,此作用可能与其降血脂、抗氧化损伤有关。