目的 探讨异氟醚通过抑制细胞间黏附分子（ICAM-1）表达参与减轻肝脏缺血-再灌注（IR）损伤的可能调节机制。 方法 32只雌性SD大鼠分为4组。A组大鼠行腹腔注射1%戊巴比妥钠40 mg/kg麻醉，进行手术但不阻断入肝血流；B组1%戊巴比妥钠麻醉后行部分肝脏IR；C组大鼠仅接受1.0 MAC异氟醚吸入麻醉，不阻断血流；D组采用1.0 MAC异氟醚麻醉，建立肝脏IR模型。肝脏缺血60 min，再灌注3 h后取肝组织和血液标本，检测血清丙氨酸转氨酶（ALT）和天冬门氨酸转氨酶（AST）、肝组织ICAM-1和肝组织还原型谷胱甘肽（GSH）、脂质过氧化物丙二醛（MDA）和超氧化物歧化酶（SOD）含量。 结果 与戊巴比妥钠麻醉比较，采用异氟醚处理后明显降低血清ALT和AST的水平，再灌注肝组织内GSH、SOD含量明显高于而MDA含量降低，同时抑制肝组织ICAM-1的表达。 结论 异氟醚麻醉能够有效减轻肝脏IR损伤，抑制氧自由基的生成和释放，具体机制可能与抑制ICAM-1表达致使细胞内GSH含量增加密切相关。
ObjectiveTo evaluate the effect of ZnPP Ⅸ on the expressions of heme oxygenase-1 (HO-1) and glutathione-Stransferase-π （GST-π） and the chemosensitivity of drug-resistant hepatic carcinoma cell line Bel/Fu, and explore it’s possibility to reverse drug-resistance and the relevant regulating mechanism. Methods①MTT assay was adopted to detect the drug sensitivity for adriamycin, mitomycin, and 5-fluorouracil of Bel/Fu cell after ZnPP Ⅸ being induced for 24 h. ②RTPCR was carried out to detect the expressions of HO-1 and GST-π mRNA after Bel/FU cells being treated with different concentrations ZnPP Ⅸ for 24 h. ResultsAfter Bel/Fu cells being treated with ZnPP Ⅸ for 24 h, the 50% inhibiting concentration (IC50) for drugs was decreased dramatically （Plt;0.05）. Meanwhile, the expressions of HO-1 and GST-π mRNA in the treated cells also decreased dose-dependently (Plt;0.01). ConclusionsZnPP Ⅸ can increase the chemosensitivity of Bel/FU cells by down-regulation of HO-1 and GST-π expression. ZnPP Ⅸ is a potential agent to reverse multidrug resistance of hepatic carcinoma cells.
目的:研究大豆异黄酮对D半乳糖致衰老大鼠抗氧化能力的影响。方法:用D半乳糖注射Wistar雄性大鼠5个月,建立衰老模型。对致衰老模型组、大豆异黄酮组肝脏、心脏和前列腺丙二醛(MDA)含量、超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSHPx)活性进行测定及比较。结果:低、中、高不同剂量大豆异黄酮灌喂组与模型组大鼠相比,各脏器MDA含量(μmol/L)(心脏:695±093,562±112,435±112比802±111;肝脏:815±085,647±120,515±112比935±135;前列腺:715±092,558±115,423±125比833±124)均有降低,差异有统计学意义(Plt;005),而SOD酶活性(nmol/L)(心脏:4732±308,5518±428,6120±368比3225±370;肝脏:18121±506,19015±706,19720±570比17213±512;前列腺:4156±301,4607±421,5015±335比3374±305)和GSHPx酶活性(nmol/L)(心脏:905±096,1111±245,1313±146比713±151;肝脏:902±105,1150±223,1362±192比698±160;前列腺:435±085,613±102,747±155比312±106)有升高,差异同样具有统计学意义(Plt;005);大豆异黄酮摄入量越高,MDA含量越低,而SOD、GSHPx酶活性越高。结论:摄入适量大豆异黄酮可有效增强大鼠机体抗氧化能力,从而延缓D半乳糖诱发的大鼠衰老。
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.
ObjectiveTo establish 16HBE cell lines stably expressing glutathione S-transferase mu 5 (GSTM5) gene, and explore the mechanism of GSTM5 nuclear translocation. MethodsRecombinant lentiviral expression vector containing GSTM5 gene was constructed and lentivirus was produced. After lentivirus infection of 16HBE cells, 16HBE-GSTM5 cell lines were obtained by screening with puromycin. Expression of GSTM5 in different cells was examined by RT-qPCR and Western blot. The nuclear translocation of GSTM5 was observed by confocal laser scanning microscope, after the 16HBE-GSTM5 cell lines were treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) for 0.5 hour. ResultsLentiviral expression plasmids, PLVX-puro-3*flag-SBP-GSTM5-C and PLVX-puro-GSTM5-SBP-3*flag-N, were constructed and lentiviral particles were successfully packed. After infected with lentivirus and screened by puromycin, two cell lines, 16HBE-GSTM5-SBP-3*flag-N and 16HBE-3*flag-SBP-GSTM5-C, were obtained. GSTM5 expression in these two cell lines was significantly higher compared with the control group and parental cells. After treated with TNF-α for 0.5 hour, the nuclear translocation of GSTM5 in 16HBE-GSTM5-SBP-3*flag-N was much more obviously than that in 16HBE-3*flag-SBP-GSTM5-C. ConclusionThe N-terminal region of GSTM5 is critical for nuclear translocation induced by TNF-α, which is mediated by a novel and non-classical nuclear localization signal.
The aim of this research is to investigate the influence of microencapsulation on the expression of the oxidative stress genes and exogenous regulation of HepG2 cells. We compared the expression of hemeoxygenase-1 (HO-1) and glutathione S-transferases-A1 (GST-A1) in HepG2 cells under different culture conditions through real-time PCR. The effects of exogenous antioxidants on cell viability and albumin levels were also evaluated through MTT assay and ELISA assay. The results showed that after culturing for 6 and 16 days, the expression levels of HO-1 in encapsulated cells were approximately 4.9 and 3.1 times higher than that of monolayer cells at the same culture period; As for the expression levels of GST-A1, they were elevated to 11.2 and 33 times of monolayer cells (P<0.05). Accordingly, we found that NAC at 5-10 mmol/L significantly increased the viability by 40%-70% and the biosynthetic function by 20%-30% in microencapsulated HepG2 cells (P<0.05). GSH increased the viability of the encapsulated cells by 20%-55% and the biosynthetic function by 15% (P<0.05). In conclusion, oxidative stress exists in the microcapsules and affects genes expression. Exogenous antioxidants can prevent the inhibition effects of oxidative stress on cellular growth.
Objective To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs). Methods The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall methodin vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×105 cells/mL); group B, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot. Results The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups (P<0.05); but there was no significant difference between groups D and E (P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance (A) values had significant differences between groups (P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E (P<0.05), but there was no significant difference among groups A, D, and E (P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups (P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups (P<0.05). Conclusions GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.
Objective To investigate the expression of presenilin-2(PS2) and glutathione S transferase π(GSTπ) and their role in the prognosis and therapy of infiltrating ductal breast carcinoma. Methods The expression of PS2 and GSTπ in tumor tissues from 210 patients with infiltrating ductal breast carcinoma confirmed by pathologic examination and treated with modified radical mastectomy was examined by using LSAB immunohistochemical method. Results The expression rate of PS2 was 49.5%(104/210) and the expression rate of GSTπ was 48.1%(101/210). The grade of the postoperative 5-year survival rate and 10-year survival rate in four groups of 210 patients, from high to low, was the group 1 (PS2 positive expression/GSTπ negative expression), the group 2 (PS2 positive expression/GSTπ positive expression), the group 3 (PS2 negative expression/GSTπ negative expression) and the group 4 (PS2 negative expression/GSTπ positive expression). Conclusion The prognosis of the group 1 is the best, the group 2 better, the group 3 good and the group 4 the worst. The results suggest that reasonable use of endocrinotherapy and chemotherapy in infiltrating ductal breast carcinoma is necessary.
Objective To study the protective effect of glutamine on the intestinal mucosal antioxidation in endotoxemic rats. Methods Twenty-eight male Wistar rats were randomly divided into three groups, group A:parenteral nutrition supplemented with glutamine, group B:TPN without glutamine,and group C:normal control. Endotoxemia was induced by continous intravenous infusion of lipopolysaccharide(LPS) at a dose of 2 mg/kg per day throughout the 5-day study period. The mucosal protein、DNA、ATP、SOD、MDA、GSH、sIgA were determined. Results The mucosal protein、DNA、ATP、SOD、GSH and sIgA content in endotoxic rats were markedly decreased, MDA was increased as compared with normal control(P＜0.05). The former indices in group A were improved and MDA content was decreased as compared with group B(P＜0.05). Conclusion Glutamine can improve gut energy metabolism, decrease the extent of mucosal injury of free radicals, and give an protective effect on the mucosal probably by increasing GSH.
ObjectiveTo investigate the clinical values of serum histidine decarboxylase (HDC), D-lactate, and alpha-glutathione S-transferase (α-GST) for diagnosing intestinal mucosal injury of patients with intestinal obstruction. MethodsThe expression levels of serum HDC, D-lactate, and α-GST in 28 patients with strangulated intestinal obstruction, 19 patients with simple intestinal obstruction, 17 patients with acute simple appendicitis, and 20 healthy volunteers were determined by enzyme linked immunosorbent assay (ELISA) before the treatment, and then the area under receiver operating characteristic curve (AUC) of these diagnostic indices were compared. In addition, the occurrence rates of systemic inflammatory response syndrome (SIRS) and infectious complications (abdominal cavity infection and pulmonary infection) were closely observed. The relevances of SIRS and infectious complications and the expression levels of these three diagnostic indices were analyzed. ResultsThe expression levels of serum HDC, D-lactate, and α-GST of the patients with strangulated intestinal obstruction were the highest among all the patients (Plt;0.01), and the expression levels of these three indices in the patients with simple intestinal obstruction were higher than those of the patients with acute simple appendicitis (Plt;0.05). The AUC of HDC (0.913) was larger than that of D-lactate (0.872) and α-GST (0.836) (P=0.000, P=0.000, respectively). When the cut off value of HDC was 31.00 μg/L, the sensitivity, specificity, false negative rate, and false positive rate of HDC were 74.5%, 94.6%, 25.5%, and 5.4%, respectively, which were all better than those of D-lactate and αGST. The occurrence rates of SIRS and abdominal cavity infection of the patients with strangulated intestinal obstruction were significantly higher than those of patients with simple intestinal obstruction (P=0.046) and acute simple appendicitis (P=0.027); while there was not significantly different of pulmonary infection among all the patients (P=0.728). The expression level of serum HDC in patients with strangulated intestinal obstruction suffered from SIRS (P=0.000) or abdominal cavity infection (P=0.002) was significantly higher than that of not-suffered from SIRS or uninfected patients. Meanwhile, the expression levels of serum D-lactate and α-GST in the patients with strangulated intestinal obstruction suffered from SIRS were higher than those of notsuffered from SIRS patients (P=0.032, P=0.021, respectively). The expression levels of HDC, D-lactate, and α-GST were significantly correlated with SIRS and abdominal cavity infection (Plt;0.05), among which the level of HDC and the incidence of SIRS had the highest correlation (r=0.608, P=0.001). ConclusionHDC may be a more effective index for diagnosing intestinal mucosal injury of patients with intestinal obstruction.