臭氧化盐水对肝细胞Keap1-Nrf2-ARE通路的激活作用

目的 研究臭氧化盐水对肝组织细胞Keap1-核因子E2相关因子2(Nrf2)-抗氧化元件(ARE)通路中Nrf2的作用.方法 采用成年健康雄性Sprague-Dawley大鼠,随机分为正常对照组(NC组)、模型组、臭氧等渗盐水(OS)组,OS对照组(OSC组).OS组、OSC组分别予5 ml/kg OS,模型组予5 ml/kg氧气盐水每日尾静脉注射,连续15 d,第16天分别予OS组及模型组50%CCl4橄榄油溶液2ml/kg腹腔注射造肝损伤模型.NC组及OSC组予植物油2ml/kg腹腔注射,24 h后,检测大鼠血清ALT、AST、肝组织总抗氧化能力(TAOC)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GPx)、过氧化氢酶(CAT).再提取处死大鼠肝组织的核蛋白,应用Western blot测定其细胞核中Nrf2的含量,免疫荧光组织化学技术检测细胞内Nrf2的分布.结果 与模型组比较,OS组大鼠ALT、AST降低[(1240.4±188.2)U/L、(1245.4±176.9)U/L对比(539.8±175.3)U/L、(546.0±130.2)U/L)],差异有统计学意义(P<0.01),TAOC、GSH,GPx,CAT 活性升高,分别为(0.72±0.24)U/mg、(1.05±0.21)mg/g,(676.9±115.1)U/mg、(45.2±14.3)U/mg对比(1.37±0.19)U/mg、(2.23±0.55)mg/g、(1024.6±162.9)U/mg、(68.2±9.9)U/mg,差异有统计学意义(P<0.01).与NC组比较,OSC组大鼠肝组织TAOC、GSH、GPx,CAT活性升高,差异有统计学意义(P<0.01或P<0.05).Western blot及免疫荧光均显示O3能增强肝细胞核内Nrf2的表达,Keap1-Nrf2-ARE通路的激活在O3抗氧化过程中发挥了重要的作用.结论 臭氧化盐水静脉注射可减轻CCl4所致大鼠肝损伤.其机制可能通过激活Keap1-Nrf2-ARE通路及其下游基因,增强细胞抗氧化和抗自由基的能力.

Abstract：

Objective To study the effect of ozonized saline on the activation of the Keapl-Nrf2ARE signaling pathway in rat liver cells. Methods Twenty maleSprague-Dawley rats were randomly divided into ozonized saline(OS) group, model group, ozonized saline control (OSC) group and normal control (NC)group. The rats in OS group and model group were intravenously administered with OS or oxygen saline (5 ml/kg) respectively, once a day for 15 days, and then intraperitoneally injected with CCU dissolved in Oliver oil. The rats in OSC group were pretreated with OS for 15 days. The rats in NC group were fed normally for 15 days. On the 16th day, the rats in OSC group and NC group were intraperitoneally injected with Oliver oil (2 ml/kg) without CCU. After 24 hours of CCU or olive oil intraperitoneal injection, the serum levels of alanine transaminase (ALT) and aspertate aminotransferase (AST) were measured. The liver tissues were also collected for detection of total anti-oxygen capability (TAOC), glutathione (GSH), catalase (CAT), Glutathione peroxidase (GPx). Western Blot was used to detect Nrf2 and immunofluorescence staining assay to display intracelluar distribution of Nrf2. Results Compared with the rats in model group,the serum ALT and AST levels of rats in OS group were significantly lower (P < 0.01) ,which were (1240.4 ± 188.2) U/L and (1245.4 ± 176.9) U/L vs (539.8 ± 175.3) U/L and (546.0 ± 130.2) U/L, and the TAOC, CAT, GPx and GSH activity of rats in OS group were significantly higher, which were (0.72 ± 0.24) U/mg, (1.05 ±0.21) mg/g, (676.9 ± 115.1) U/mg and (45.2 ± 14.3) U/mg vs (1.37 ± 0.19) U/mg, (2.23 ± 0.55) mg/g,(1024.6 ± 162.9) U/mg and (68.2 ± 9.9) U/mg, respectively. In contrast with NC group, pretreatment of OS in OSC group elevated TAOC, CAT, GPx and GSH activity (P < 0.01 or P < 0.05). Ozonized saline can strengthen the Nrf2 expression in liver cells. Conclusions Preconditioning injection of ozonized saline can reduce rat's liver injury induced by CCl4- The ozonized saline, as a novel Nrf2 activator, can reduce the oxidative damage of radical oxygen species (ROS) and the deleterious substance by activating the KeaplNrf2-ARE signaling pathway and its downstream genes expression.     

Effect of ginkgo biloba extract on livers in aged rats

AIM: To investigate the protective effect of ginkgo biloba extract (GBE) on livers of aged rats and the associated mechanisms. METHODS: Two-mo- and 20-mo-old rats were treated with GBE/saline for 3 mo. Liver tissue samples from 5-mo-old rats treated with saline (group Y) and 23-mo-old rats treated with GBE (group E) or saline (group N) were used for histopathological examinations (hematoxylin-eosin and Masson staining, Lipofuscin staining-Schmorl staining) and determination of expression of tissue inhibitor-1 of metalloproteinase (TIMP-1) and the level of malondialdehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD). Blood samples were collected for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin. RESULTS: Microscopic studies with Masson staining revealed mild liver fibrosis in aged rats (group N), while the livers of aged rats receiving GBE (group E) showed amelioration in fibrosis (2.2±0.1 vs2.8±0.1, P＜0.01) and deposition of lipofuscin (33.7±5.3 vs62.8±5.7, P＜0.01). The expression of TIMP-1 and the level of liver MDA (1.0±0.1 vs1.2±0.2,P＜0.05) also decreased but the activity of GPx (97.1±15.3 vs61.8±14.5, P＜0.01) increased in group E. Compared with group Y, the level of liver MDA (0.8±0.1 vs1.2±0.2, P＜0.01), lipofuscin (32.4±6.0 vs 62.8±5.7, P＜0.01) and TIMP-1 expression were increased, while the activity of GPx (103.2±17.6 vs61.8±14.5, P＜0.01) and SOD (16.7±4.4 vs11.8±3.9, P＜0.05) was decreased in group N. There was no difference in liver function among these three groups. CONCLUSION: GBE has protective effects on aging liver. The possible mechanisms might be its antioxidant activity and inhibition of TIMP-1 expression.     

阿苯达唑联合黄芪对华支睾吸虫感染大鼠肝功能异常的疗效观察

Objective To observe the levels of alanine aminotransferase(ALT), total bilirubin(TBIL), hyaluronic acid (HA), procollagen type Ⅲ aminoterminal peptide (P Ⅲ NP) and larninin (LN) in the sera of rats infected with Clonorchis sinensis (C. sinensis) after treatment of albendazole combined with milkvetch root. Methods Thirty-two healthy adult Wistar rats were randomly divided into four groups with 8 in each based on body mass: control group, non-treatment group, Albendazole group(ALB group) and albendazole combined with milkvetch root group(ALB+MR group). The rats in non-treatmen, ALB and ALB+MR groups were infected orally with metacercariae of C. sinensis 50 per rat. The rats in control group were mock-infected with saline. The rats in ALB group were treated to each rat with 50 mg/kg alhendazole for 5 days, and ALB+MR groups were given to same treat with albendazole, meanwhile each rat injected with 800 mg/kg milkvetch root intraperitoneally for 30 days. All rats were killed after infestation 14 weeks and their sera samples were collected to detect ALT, TBIL, HA, PⅢNP, LN. Results There were statistically significant differences in the levels of ALT and LN in the sera of rats between groups(F=31.40,11.82, P<0.01). Compared with control[ (47.88±4.88)U/L, (51.20±4.12)μg/L], the levels of ALT and LN in rats in non-treatment group [(85.50±9.65)U/L, (64.20±4.18) μg/L] and ALB group [(65.29± 7.78) U/L, (58.23±2.55) μg/L] were significantly increased (P<0.05). Compared with non-treatment group, the levels of ALT and LN in rats in ALB group and ALB+MR groups[(50.25±9.29)U/L, (53.68±5.63)μg/L] were significantly decreased(P<0.05), and they decreased more obviously in ALB+MR group (P<0.05). There were statistically significant differences in the levels of TBIL, PⅢNP and HA in the sera of rats between groups (χ2=15.309,21.418,19.759, P<0.01). Compared with control[(0.700±0.350)μmol/L, (26.085±4.075)μg/L, (81.935±42.550)μg/L], the levels of TBIL, PⅢNP and HA in rats in non-treatment group(2.400 μmol/L, 46.220 μg/L,310.885 μg/L) and ALT group(1.200 μmol/L,36.540 μg/L, 178.010 μg/L) were significantly increased(P<0.05). Compared with non-treatment group, the level of TBIL in ALT+MR group(0.750 μmol/L), the levels of pⅢNP and HA in ALT and ALT+MR group(30.470,100.240 μg/L) were significantly decreased(P< 0.05). The levels of TBIL, PⅢNP and HA decreased more obviously in ALB+MR group(P<0.05). Conclusions The liver function in rats infected with C. sinensis is abnormal. The liver function and fibrosis are improved after treatment with albendazole or albendazole combined with milkvetch root. The treatment of albendazole combined with milkvetch root is more effective.     

Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice

AIM: To investigate the effects of leptin administration on liver fibrosis induced by thioacetamide (TAA). METHODS: Twenty-four male C57Bl/6 mice were randomly allocated into four groups, which were intra-peritoneally given saline (2 mL/kg), leptin (1 mg/kg), TAA (200 mg/kg), TAA (200 mg/kg) plus leptin (1 mg/kg) respectively, thrice a week. All mice were killed after 4 wk. The changes in biochemical markers, such as the levels of alanine aminot-ransferase (ALT) and aspartate aminotransferase (AST) in serum and superoxide dismutase (SOD), malondialdehyde (MDA) in liver were determined. For histological analysis, liver tissues were fixed with 10% buffered formalin, embedded with paraffin. Hematoxylin-eosin (HE) staining and picric acid-Sirius red dyeing were performed. The level of α1(I) procollagen mRNA in liver tissues was analyzed by RT-PCR. RESULTS: Apparent liver fibrosis was found in TAA group and TAA plus leptin group. Compared to saline group, the levels of ALT and AST in serum and MDA in liver increased in TAA group (205.67±27.69 U/L vs50.67±10.46 U/L, 177.50±23.65 U/L vs 76.33±12.27 U/L, 2.60±0.18 nmol/mg pro vs 1.91±0.14 nmol/mg pro, P<0.01) and in TAA plus leptin group (256.17±22.50 U/L vs 50.67±10.46 U/L, 234.17±27.37 U/L vs 76.33±12.27 U/L, 2.97±0.19 nmol/mg pro vs 1.91±0.14 nmol/mg pro,P<0.01). The level of SOD in livers decreased (51.80±8.36 U/mg pro vs 81.52±11.40 U/mg pro, 35.78±6.11 U/mg pro vs 81.52± 11.40 U/mg pro, P<0.01) and the level of α1(I) procollagen mRNA in liver tissues also increased (0.28±0.04 vs 0.11± 0.02, 0.54±0.07 vs 0.11±0.02, P<0.01). But no significant changes were found in leptin group and saline group. Compared to TAA group, ALT, AST, MDA, and α1(I) procollagen mRNA and grade of liver fibrosis in TAA plus leptin group increased (256.17±22.50 U/L vs 205.67± 27.69 U/L, P<0.05; 234.17±27.37 U/L vs 177.50±23.65 U/L, P<0.05; 2.97±0.19 nmol/mg pro vs 2.60±0.18 nmol/mg pro,P<0.05; 0.54±0.07 vs 0.28±0.04, P<0.01; 3.17 vs 2.00, P<0.05), and the level of SOD in liver decreased (35.78±6.11 U/mg pro vs 51.80±8.36 U/mg pro, P<0.05). There were similar changes in the degree of type I collagen deposition confirmed by picric acid-Sirius red dyeing. CONCLUSION: Leptin can exacerbate the degree of TAA-induced liver fibrosis in mice. Leptin may be an important factor in the development of liver fibrosis.     

Gender differences in hepatic ischemic reperfusion injury in rats are associated with endothelial cell nitric oxide synthase-derived nitric oxide

AIM: This study was designed to examine the hypothesis that gender differences in I/R injury are associated with endothelial cell nitric oxide synthase (eNOS)-derived nitric oxide (NO). METHODS: Wistar rats were randomized into seven experimental groups (12 animals per group). Except for the sham operated groups, all rats were subjected to total liver ischemia for 40 min followed by reperfusion. All experimental groups received different treatments 45 min before the laparotomy. For each group, half of the animals (six) were used to investigate the survival; blood samples and liver tissues were obtained in the remaining six animals after 3 h of reperfusion to assess serum NO, alanine aminotransferase (ALT) and TNF-α levels, liver tissue malondialdehyde (MDA) content, and severity of hepatic I/R injury. RESULTS: Basal serum NO levels in female sham operated (FS) group were nearly 1.5-fold of male sham operated (MS) group (66.7±11.0 μmol/L vs45.3μ10.1 μmol/L, P<0.01). Although serum NO levels decreased significantly after hepatic I/R (P<0.01, vs sham operated groups), they were still significantly higher in female rat (F) group than in male rat (M) group (47.8±8.6 μmol/L vs 23.8±4.7 μmol/L, P<0.01). Serum ALT and TNF-α levels, and liver tissue MDA content were significantly lower in F group than in M group (370.5±46.4 U/L, 0.99±0.11 μg/L and 0.57±0.10 μmol/g vs668.7±78.7 U/L, 1.71±0.18μg/L and 0.86±0.11 μmol/g, respectively, P<0.01). I/R induced significant injury to the liver both in M and F groups (P<0.01 vs sham operated groups). But the degree of hepatocyte injury was significantly milder in F group than in M group (P<0.05 and P<0.01). The median survival time was six days in F group and one day in M group. The overall survival rate was significantly higher in F group than in M group (P<0.05). When compared with male rats pretreated with saline (M group), pretreatment of male rats with 17-β-estradiol (E2) (M+E2 group) significantly increased serum NO levels and significantly decreased serum ALT and TNF-α levels, and liver tissue MDA content after I/R (P<0.01). The degree of hepatocyte injury was significantly decreased and the overall survival rate was significantly improved in M+E2 group than in M group (P<0.01 and P<0.05). The NOS inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) treatment could completely abolish the protective effects of estrogen in both male and female rats. CONCLUSION: The protective effects afforded to female rats subjected to hepatic I/R are associated with eNOS-derived NO.     

Anti-hepatoma effect of arsenic trioxide on experimental liver cancer induced by 2-acetamidofluorene in rats

AIM: To study the anti-hepatoma efficiency of arsenic trioxide (As2O3) in the treatment of experimental rat hepatocellular carcinoma (HCC) induced by 2-acetamidofluorene (2-FAA) and to elucidate the possible mechanisms. METHODS: SD rats (2 mo old) had been fed with 2-FAA for 8 wk to induce HCC, and then they were treated with As2O3 or matrine. On d 29, the rats were killed and the liver was weighed and liver tumors were counted. The histological changes of liver tissue were observed under microscope, and the cellular dynamic parameters were studied by flow cytometry. Immunohistochemistry (two-step method) was used to observe the expression of vascular endothelial growth factor (VEGF) and micro-vessel density (MVD) on consecutive sections. The pathological parameters were also analyzed, the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBi), and direct bilirubin (DBi). RESULTS: The number of liver tumors decreased significantly in groups treated with As2O3, especially in medium-dose (1 mg/kg) group (t = 2.80, P><0.01). As203 caused HCC cell death via apoptosis; necrosis was seen and apoptosis was common when the dose was 1 mg/kg. Proliferation index decreased sharply in medium-dose (1 mg/kg) group (7.87±4.11 vs 24.46±6.49, t= 2087, P<0.01), but not in 0.2 mg/kg group. However, S-phase fraction decreased dramatically in both groups, it reached the bottom level only when the dose was 1 mg/kg compared with control (0.40±0.13 vs .01±0.51, t= 2.97, P<0.01), and it was obviously accompanied with accumulation of cells in G0/G1 (G0/G1 restriction). The expressions of VEGF and MVD in medium-dose (1 mg/kg) group were significantly lower than normal saline group (0.63±0.74 vs 2.44±0.88,P<0.05; 15.75±3.99 vs 47.44±13.41, t = 2.80, P<0.01). Compared with normal saline group, mediumand low-dose groups As2O3 and matrine lowered the levels of ALT in serum (61.46±9.46, 63.75±20.40, 61.18±13.00 vs 108.98?9.86, t = 2.14, P<0.05), but had no effect on the level of serum AST, TBi, and DBi. CONCLUSION: As2O3 had inhibitory effect on growth of experimental HCC in rats induced by 2-FAA, but had no obvious effect on normal hepatic cells. The mechanisms may involve decrease of cell division, accumulation of cells in G0/G1 phase, apoptosis of tumor cells, and inhibitory effect on angiogenesis through blocking VEGF.     

吉法酯对三硝基苯磺酸诱导大鼠实验性结肠炎的作用

Objective To investigate the effects of Gefarnate on expression of myeloperoxidase (MPO),cyelooxygenase-1 (COX-1) and COX-2 in trinitrobenzene sulphonic acid (TNBS) induced experimental colitis in rats and its therapeutic effects on ulcerative colitis. Methods Forty female Sprague-Dawley (SD) rats were randomly divided into 4 groups with 10 each. The rats in group A, B and C were infused with TNBS/alcohol by enema. After the production of colitis, the rats in group A or B were treated daily with 1 ml of normal saline or with 1 ml of 5-ASA (100 mg/kg) by enema,and those in group C were treated daily with 1 ml of Gefarnate by gavage. Group D was served as normal control. After the production of colitis,animals were sacrificed at day 7 and 14 with 5 in each group. The macroscopic changes of the colon were evaluated according to disease activity index (DAD scoring and histological change was assessed by HE staining. MPO activity of the mucosa was detected by biochemical methods. Expressions of COX-1 and COX-2 in tissues were detected by immunohistochemistry. Results Compared with group A, macroscopic and histological scores and MPO activity were significantly decreased in group B and C (P<0.05). The expressions of COX-1 at day 7 and 14 were 1.86±0.51 and 1.96±0.41 in group B, 1.73±0.68 and 1.79±0.6 in group C, 1.91±0.34 and 1.99±0.45 in group D, respectively, which were significantly higher than those in group A (0.87±0.18 and 0.93±0.15, P<0.05). Whereas the expressions of COX-2 at day 7 and 14 were 1.53±0.19 and 0.73±0.15 in group B, 1.73±0.94 and 0.86±0.29 in group C, 0.24±0.18 and 0.18±0. 16 in group D, respectivley, which were significantly lower that those in group A (3.50±0.2;3 and 3.06±0.27). There was a significant difference between group D and group B or C (P<0.05). Conclusions Gefarnate provides a therapeutic effect during TNBS-induced colitis in rats, which is similar to that of 5-ASA. The mechanisms are involved in decreasing the concentration of colonic MPO and regulating the expression of COX-1/COX-2.     

Protective effect of L-arginine preconditioning on ischemia and reperfusion injury associated with rat small bowel transplantation

AIM: To investigate the protective effect and possible mechanism of L-arginine preconditioning on ischemia and reperfusion injury associated with small bowel transplantation (SBT). METHODS: Male inbred Wistar rats weighting between 180 and 250 g were used as donors and recipients in the study. Heterotopic rat SBT was performed according to the techniques of Li and Wu. During the experiment, intestinal grafts were preserved in 4℃ Ringer's solution for 8 h before being transplanted. Animals were divided into three groups. In group 1, donors received intravenous L-arginine (50 mg/kg, 1 mL) injection 90 min before graft harvesting. However, donors in control group were given normal saline (NS) instead. In group 3, six rats were used as sham-operated control. Specimens were taken from intestinal grafts 15 min after reperfusion. Histological grading, tissue malondialdehyde (MDA) and myeloperoxidase (MPO) levels were assessed. The graft survival of each group was monitored daily until 14 d after transplantation. RESULTS: Levels of MDA and MPO in intestine of sham-operated rats were 2.0±0.22 mmol/g and 0.66±0.105 U/g. Eight hours of cold preservation followed by 15 min of reperfusion resulted in significant increases in tissue MDA and MPO levels. Pretreatment with L-arginine before graft harvesting resulted in lower enhancement of tissue levels of MDA and MPO and the differences were significant (4.71±1.02 mmol/g vs 8.02±3.49 mmol/g, 1.03±0.095 U/g vs 1.53±0.068 U/g, P<0.05). Besides, animals in L-arginine pretreated group had better histological structures and higher 2-wk graft survival rates comparing with that in NS treated group (3.3±0.52 vs 6±0.1, 0/6 vs 6/6, P<0.05 or 0.01). CONCLUSION: L-arginine preconditioning attenuates ischemia and reperfusion injury in the rat SBT model, which was due to antioxidant activities partially.     

Effects of Radix Puerariae flavones on liver lipid metabolism in ovariectomized rats

AIM:To study the effects of Radix Puerariae flavones (RPF)on liver lipid metabolism in ovariectomized (OVX) rats.METHODS:Forty adult female Wistar rats were randomlydivided into four groups:OVX group;sham-OVX group;OVX estrogen group and OVX RPF group.One week afteroperation rats of the first two groups were treated withphysiological saline,rats of OVX estrogen group withestrogen (1 mg/kg.b.w.) and rats of OVX RPF group withRPF (100 mg/kg.b.w.),respectively for 5 weeks.Alter therats were killed,their body weight,the weight of theabdominal fat and uterus were measured,and the levels oftotal cholesterol (TC) and triglycedde (TG) in liver homogenatewere determined.RESULTS:Compared with the sham-OVX group,the bodymass of the rats in OVX group was found increased significantly;more abdominal fat in store;TC and TG in liver increased anduterine became further atrophy.As a result,the RPF wasfound to have an inhibitive action on those changes of variousdegrees.CONCLUSION:RPF has estrogen-like effect on lipidmetabolism in liver and adipose tissue.     

Effects of Guiyuanfang and autologous transplantation of bone marrow stem cells on rats with liver fibrosis

AIM: To investigate the therapeutic effects of Guiyuanfang and bone marrow stem cells (BMSCs) on rats with liver fibrosis. METHODS: Liver fibrosis model was induced by carbon tetrachloride, ethanol, high lipid and assessed biochemically and histologically. Liver function and hydroxyproline contents of liver tissue were determined. Serum hyaluronic acid (HA) level and procollagen Ⅲ level were performed by radioimmunoassay. The VG staining was used to evaluate the collagen deposit in the liver. Immunohistochemical SABC methods were used to detect transplanted BMSCs and expression of urokinase plasminogen activator (uPA). RESULTS: Serum transaminase level and liver fibrosis in rats were markedly reduced by Guiyuanfang and BMSCs. HA level and procollagen Ⅲlevel were also reduced obviously, compared to model rats (HA: 47.18±10.97 ng/mL, 48.96±14.79 ng/mL; PCⅢ: 22.48±5.46 ng/mL, 26.90±3.35 ng/mL; P<0.05). Hydroxyproline contents of liver tissue in both BMSCs group and Guiyuanfang group were far lower than that of model group (1 227.2±43.1 μg/g liver tissue, 1390.8±156.3 μg/g liver tissue; P<0.01). After treatment fibrosis scores were also reduced. Both Guiyuanfang and BMSCs could increase the expression of uPA. The transplanted BMSCs could engraft, survive, and proliferate in the liver. CONCLUSION: Guiyuanfang protects against liver fibrosis. Transplanted BMSCs may engraft, survive, and proliferate in the fibrosis livers indefinitely. Guiyuanfang may synergize with BMSCs to improve recovery from liver fibrosis.     

CYP2J2基因和EPHX2基因多态性与缺血性脑卒中的遗传易感性

目的 研究我国汉族人群细胞色素P450表氧化酶2J2(CYP2J2),可溶性环氧化物水解酶2(EPHX2)基因多态性与缺血性脑卒中的关系.方法 选择200例缺血性脑卒中患者和350例健康人群,采用PCR-RFLP技术分析两个基因CYP2J2 G-50T,EPHX2 G860A多态性的基因型.结果 仅EPHX2 860A等位基因频率在缺血性脑卒中组与对照组比较有显著性差异.多元Logistic回归分析表明,携带EPHX2 860A等位基因的人群患缺血性脑卒中相对风险率下降50%(OR=0.5).当个体同时携带CYP2J2-50GG和EPHX2 860A (A 示A等位基因)联合基因型时,其患缺血性脑卒中相对风险率下降53.9%(OR=0.461).结论 虽然EPHX2 860A等位基因与缺血性脑卒中有相关性并且为缺血性脑卒中一个独立的保护因子,但联合基因型CYP2J2-50GG/EPHX2 860A 的协同作用对缺血性脑卒中有更强的保护作用.     

双歧杆菌脂磷壁酸延缓细胞衰老的作用机制

目的 探讨双歧杆菌脂磷壁酸(Lipoteichoic acid,LTA)在延缓H2O2诱导细胞衰老中的作用.方法 H2O2诱导WI-38细胞衰老,β-半乳糖苷酶细胞化学染色计算衰老细胞百分率变化,RT-PCR和Western印迹检测衰老细胞p21、细胞周期蛋白E(cyclin E)和周期蛋白依赖性蛋白激酶2(CDK2)表达水平的变化.结果 双歧杆菌LTA处理后,β-半乳糖苷酶细胞化学染色阳性细胞百分率较衰老模型组降低(P＜0.01).与年轻对照组相比,衰老模型组细胞中p21的表达增高,cyclin E和CDK2表达降低,而双歧杆菌LTA能够逆转上述变化(P＜0.01).结论 双歧杆菌能延缓H2O2诱导的细胞衰老,机制可能与改变p21,cyclin E和CDK2表达水平有关.     

不同浓度脂联素对心肌细胞氧化应激损伤后GRP78及Caspase-12表达的影响及意义

目的 通过原代培养SD大鼠的乳鼠心肌细胞建立H_2O_2心肌细胞氧化应激损伤模型,观察脂联素对心肌细胞氧化应激所致内质网应激的保护作用.方法 采用酶消化法原代培养乳鼠心肌细胞,倒置相差显微镜下观察细胞生长状态,通过α-肌动蛋白免疫荧光法对培养的心肌细胞进行鉴定.选用原代培养3～4天的心肌细胞,随机分为对照组、H_2O_2组、H_2O_2+10 mg/L脂联素组、H_2O_2+20 mg/L脂联素组和H_2O_2+30 mg/L脂联素组.实验终止后,在倒置相差显微镜下观察心肌细胞形态的变化,采用化学比色法测定乳酸脱氢酶的释放,通过流式细胞术来检测心肌细胞的凋亡,用RT-PCR与western Blotting方法检测内质网应激指标GRP78和Caspase-12的表达.结果 与对照组相比,给予H_2O_2后,细胞凋亡率显著增加(70.7%±6.4%比1.0%±0.6%,P<0.05),LDH释放增加(1411.5 ±189.7 U/L比353.3 ±50.3 U/L,P<0.05),内质网伴侣蛋白GRP78以及Caspage-12在mRNA(分别为1.25±0.50比0.18 ±0.10和1.32±0.15比0.26±0.06)及蛋白水平(分别为0.92±0.50比0.37±0.10和1.24 ±0.50比0.51±0.01)表达增加(P<0.05),30 mg/L脂联素预处理后给予H_2O_2,可较大程度地逆转上述指标变化,细胞凋亡率显著下降(43.6%±3.8%),LDH释放减少(686.7±61.1 U/L),内质网伴侣蛋白GRP78以及Cagpase-12在mRNA(分别为0.56±0.03和0.83±0.04)及蛋白水平(分别为0.66±0.03和0.64±0.03)表达减少(P<0.05).结论 氧化应激使GRP78和Caspase-12表达增强,启动内质网应激,脂联素可以通过减轻内质网应激逆转H_2O_2所致的心肌细胞损伤及凋亡作用,对心肌细胞有保护作用.     

CYP2J2基因和EPHX2基因多态性与肺癌易感性研究

目的探讨CYP2J2基因启动子区G-50T多态性和EPHX2基因G860A多态性与肺癌发生的关系。方法经病理检查确诊为肺癌患者150例（肺癌组）,300名本院健康体检者作为对照组,检测其CYP2J2基因启动子区G-50T多态性和EPHX2基因G860A多态性,并进行统计学分析。结果肺癌组和对照组比较,CYP2J2启动子区G-50T多态性差异无统计学意义,而肺癌组患者EPHX2 860G等位基因频率显著高于对照组人群（96％比78．3％,P〈0．01）,多元回归分析方法显示,肺癌的发生与EPHX2 G860A多态性显著相关（校正OR值=0．164,95％CI 0．079～0．342,P〈0．001）。结论EPHX2 G860A多态件与肺痛密切相关．可作为肺癌高毹患者的预涮指标．     

外阴阴道念珠菌病阴道乳酸杆菌的分布及作用

目的 通过对外阴阴道念珠菌病（vulvovaginal candidiasis，VVC）患者与正常妇女阴道乳酸杆菌优势菌种和产H2O2能力的比较，初步探讨乳酸杆菌在VVC发病中的作用。方法 以66例VVC患者为实验组，50例正常妇女为对照组。1．将阴道分泌物接种在MRS乳酸杆菌琼脂培养基上，在CO2环境下培养，采用微量生化试验鉴定乳酸杆菌；2．将乳酸杆菌接种在H2O2鉴定培养基上，根据显色鉴定是否产H2O2。结果1．两组在乳酸杆菌的分布上无差别（P〉0．05），均以嗜酸乳酸杆菌、詹氏乳酸杆菌和卷曲乳酸杆菌为优势菌种；2．对照组H202阳性乳酸杆菌的分离率明显高于实验组，分别为23例（46％）和6例（9．09％），有显著性差异（P〈0．01）；3．两组内三种优势乳酸杆菌产酸能力相似（P〉0．05），但两组间相比，VvC组嗜酸乳酸杆菌和卷曲乳酸杆菌产H202能力较对照组下降（P〈0．05）。结论 1．VVC患者与正常妇女阴道内乳酸杆菌菌种分布相似；患VVC后阴道嗜酸乳酸杆菌和卷曲乳酸杆菌产酸能力下降；2．VVC的发生与H2O2阳性乳酸杆菌的数量有关。     

恶性肿瘤外周血基质金属蛋白酶2及其抑制物表达的临床研究

目的：研究基质金属蛋白酶2（MMP-2）及金属蛋白酶抑制剂-2（TIMP-2）表达在胃癌、肠癌转移及预后判断中的作用。方法：采用逆转录荧光实时定量PCR的方法检测58例结肠癌患者，52例胃癌患者外周血中MMP-2和TIMP-2 mRNA的表达水平。结果：胃癌、肠癌患者MMP-2表达均高于正常对照（P〈0．01）；其中，有局部浸润、淋巴结肿大、远处脏器转移者的MMP-2 mRNA和TIMP-2／MMP-2比值与未转移者差异有统计学意义（P〈0．01）；TIMP-2／MMP-2比值对判断胃癌、肠癌瘤转移的灵敏度分别为86．6％、89．2％，特异性为81．8％、83．3％。结论：基质金属蛋白酶2的表达升高与恶性肿瘤的转移密切相关，TIMP-2／MMP-2比值是恶性肿瘤转移和预后判断的良好指标。     

选择性环氧合酶-2抑制剂NS-398对人肝癌细胞株HepG2增殖和凋亡的影响

目的探讨选择性环氧合酶-2抑制剂NS-398对人肝癌HepG2细胞株的生长抑制、诱导凋亡及其对bcl-2表达的影响。方法采用MTT法检测细胞增殖，流式细胞术检测细胞周期、凋亡及凋亡相关蛋白bcl-2的表达。结果NS-398抑制HepG2的增殖活性，经20、40、80和160μmol／L的NS-398处理细胞48h后，其抑制率分别为6．72％、16．21％、20．86％和25．34％，呈剂量依赖效应关系；细胞经160bLmol／L的NS-398处理24h、48h和72h后，G。／G1期细胞由76．07±0．75％分别减少至62．27±0．74％、59．17±1．47％和53．03±1．60％（P〈0．05），S期细胞由11．40±0．79％分别增加至13．23±0．81％、16．20±1．95％和16．60±1．25％（P〈0．05），G2／M期细胞无明显变化；凋亡细胞增多，凋亡率分别为8．47％、16．3％和23．9％；细胞经160μmol／L的NS-398处理48h后bcl-2蛋白与对照组比，表达下调（P〈0．01）。结论NS-398对人肝癌细胞株HepG2有抑制增殖、诱导凋亡作用，细胞凋亡的机制可能与细胞凋亡相关基因bcl-2表达下调有关。     

7-二氟甲氧基金雀异黄素对H_2O_2诱导血管内皮细胞与单核细胞黏附的抑制作用

目的 探讨7-二氟甲氧基金雀异黄素对氧化应激诱导血管内皮细胞与单核细胞黏附的抑制作用及其机制.方法 荧光分光光度计检测单核细胞与血管内皮细胞的黏附,酶联免疫吸附法检测E选择素和细胞间黏附分子1等黏附分子的释放,Western blotting检测P38丝裂原活化蛋白激酶的磷酸化水平.结果 H2O2处理血管内皮细胞24 h后,内皮细胞-单核细胞的黏附率显著增加,内皮细胞E选择素和细胞间黏附分子1的释放增加;加入7-二氟甲氧基金雀异黄素后,血管内皮细胞-单核细胞的黏附率以及内皮细胞释放E选择素和细胞间黏附分子1等黏附分子呈浓度依赖性减少.H2O2处理血管内皮细胞24 h后,可显著激活P38丝裂原活化蛋白激酶,这一作用可被7-二氟甲氧基金雀异黄素所抑制.给予P38丝裂原活化蛋白激酶特异性抑制荆SB203580亦可阻断H2O2诱导的内皮细胞-单核细胞黏附及内皮细胞E选择素和细胞间黏附分子1的释放.结论 7-二氟甲氧基金雀异黄素对氧化应激诱导的血管内皮细胞与单核细胞黏附具有拮抗作用,其机制可能与其抑制P38丝裂原活化蛋白激酶的激活进而阻断内皮细胞释放黏附分子E选择素和细胞间黏附分子1有关.     

氯膦酸二钠脂质体对大鼠腹腔巨噬细胞功能的影响

目的:探讨氯膦酸二钠脂质体(liposomal clodronate,LC)的制备及对大鼠腹腔巨噬细胞(peritoneal macrophages,PM)功能的影响.方法:利用薄膜法制备LC;分离培养大鼠PM,用不同浓度的LC(50,100,150 μmol/L)分别作用于大鼠PM,然后采用MTT比色法,中性红比色法,检测NO,O2-和H2O2的含量来评估巨噬细胞功能.结果:在三种不同浓度LC的干预下,MTT结果显示巨噬细胞的生长受到明显抑制( P<0.01);巨噬细胞吞噬中性红结果表明其吞噬功能受到明显抑制,抑制率分别为17.4%,25.8%和38.0%;巨噬细胞分泌NO、O2-的能力也受到明显抑制,与对照组相比差异具有显著性(48.398±0.667,27.664±0.566,25.626±0.184vs 91.714±1.726;0.289±0.017,0.256±0.013,0.216±0.003 vs 0.342±0.022,均P<0.05);巨噬细胞产生H2O2的能力仅在100 μmol/L与150μmol/L时受到明显抑制(0.261±0.002,0.213±0.015 vs 0.277±0.003,均P<0.05).结论:LC对大鼠PM功能具有抑制作用.