葛根汤与桂枝汤对兔颈椎间盘组织IL-1β、iNOS、TNFα、TGFβ mRNA表达的调节作用

目的 检测兔动物模型风寒湿颈椎病组、低头位颈椎病组、风寒湿加低头位颈椎病组的颈椎间盘组织中白细胞介素1β(interleukin 1β ,IL 1β)、诱导型氮氧化物合酶 (induciblenitric oxidesynthase,iNOS)、肿瘤坏死因子α(tumornecrosisfactor al pha ,TNFα)、转化生长因子 β(transforminggrowthfactor beta ,TGFβ)的mRNA的表达 ,并检测葛根汤、桂枝汤对兔风寒湿颈椎病组颈椎间盘组织中上述细胞因子mRNA的调节作用。方法 36只大白兔随机分为正常对照组、风寒湿颈椎病组、低头位颈椎病组、风寒湿加低头位颈椎病组、葛根汤治疗组、桂枝汤治疗组。葛根汤、桂枝汤治疗组均以风寒湿颈椎病模型为基础。采用逆转录聚合酶链反应法检测颈椎间盘组织中上述细胞因子mRNA的表达。结果 各模型组与正常对照组比较IL 1βmRNA、iNOSmRNA表达上调 (P <0 .0 1) ;葛根汤、桂枝汤治疗组与风寒湿颈椎病组比较 ,IL 1βmRNA、iNOSmRNA表达均下调 (P <0 .0 5或 <0 .0 1) ;低头位加风寒湿颈椎病组TNFαmRNA表达上调 ,与风寒湿及低头位颈椎病组比较 (P <0 .0 1) ;葛根汤下调TNFαmRNA表达 ,与风寒湿颈椎病组比较 (P <0 .0 1) ;各模型组同正常对照组比较 ,TGFβmRNA表达均下调(P <0 .0 1) ;与风寒湿颈椎病组比较 。

eNOS基因重组腺病毒载体在大鼠骨髓来源内皮祖细胞中的表达分析

目的探讨eNOS基因重组腺病毒载体在大鼠骨髓来源内皮祖细胞(EPCs)中的表达与作用。方法将eNOS基因插入载体PSUCMV中构建重组质粒PSUCMVeNOS,经酶切、插入、转染后包装扩增成腺病毒颗粒;大鼠胫骨获取骨髓,制备EPCs,鉴定正确后传代纯化,将三代细胞随机分为PBS对照组(C组)、Lac基因转染组(L组)和eNOS基因转染组(N组)。分别于干预后第1天、第3天、第7天观察各组细胞形态的变化,检测培养液中NO含量和各组细胞eNOS表达的变化。结果 AdCMVeNOS病毒DNA成功包装成完整的病毒颗粒,测其滴度为1.58×1010PFU/mL;将其转染鉴定正确的EPCs,结果于体外转染后1 d内,即可检测到eNOS转基因产物的表达增加;N组同一时点eNOS含量明显高于L组、C组,组间比较差异显著;组内比较,N7、N3与N1组比较差异显著。随时间延长NO生成量递增,第3天与第7天结果差异有显著性。结论 eNOS基因可在大鼠EPCs中高效表达,并可促使培养液中NO含量明显增高。

Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in association with neovascularization in human primary astrocytoma

Objective: To investigate the relationship between the expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and angiogenesis in primary astrocytoma. Methods: Thirty-seven primary astrocytomas and 4 astrocytic hyperplasia samples were collected and divided into three groups according to histological grade. The expression of eNOS, VEGF and factor Ⅷ related antigen (FVIIIRAg) were assayed by immunohistochemistry. Microvascular density was assessed by FVIIIRAg immunoreactivity. The intensity of immunoreactivity was graded according to the percentage of positive tumor cells. Results: No eNOS and VEGF were expressed in the astrocytes and vascular endothelium in astrocytic hyperplasia.The expression of eNOS or VEGF was light in low-grade astrocytoma and strong in glioblastoma. eNOS expression in astrocytoma was very positively correlated with VEGF. eNOS and VEGF expression in anaplastic astrocytoma was median in contrast to the low grade astrocytoma and glioblastoma. Lower microvascular density was found in low grade astrocytoma than that in higher grade malignant ones. The expressions of eNOS and VEGF were correlated with microvascular density and tumor malignancy.Conclusion: This finding suggests that eNOS and VEGF may have cooperative effect in tumor angiogenesis and play an important role in the pathogenesis of primary astrocytoma.

Increased oxidative DNA damage, inducible nitric oxide synthase, nuclear factor кB expression and enhanced antiapoptosis-related proteins in Helicobacter pylori-infected non-cardiac gastric adenocarcinoma

AIM: Several epidemiological studies have demonstrated a dose association between Helicobacter pylori (H Pylon) infection and non-cardiac carcinoma of the stomach. H pylori infection induces active inflammation with neutrophilic infiltrations as well as production of oxygen free radicals that can cause DNA damage. The DNA damage induced by oxygen free radicals could have very harmful consequences,leading to gene modifications that are potentially mutagenic and/or carcinogenic. The aims of the present study were to assess the effect of H pyloriinfection on the expression of inducible nitric oxidative synthase (iNOS) and the production of 8-hydroxy-deoxyguanosine (8-OHdG), a sensitive marker of oxidative DNA injury in human gastric mucosa with and without tumor lesions, and to assess the possible factors affecting cell death signaling due tooxidative DNA damage.METHODS: In this study, 40 gastric carcinoma specimens and adjacent specimens were obtained from surgical resection. We determined the level of 8-OHdG formation by HPLC-ECD, and the expression of iNOS and mechanism of cell death signaling [including nuclear factor-κB(NFκB),MEKK-1, Caspase 3, B Cell lymphomal leukemia-2 (Bcl-2),inhibitor of apoptosis protein (IAP) and myeloid cellleukemia-1 (Mcl-1)] by Western-blot assay.RFSULTS: The concentrations of 8-OHdG, iNOS, NFx3, Mcl-1 and IAP were significantly higher in cancer tissues than in adjacent non-cancer tissues. In addition, significantly higher concentrations of 8-OHdG, iNOS, NFxB, Mcl-1 and lAP were detected in patients infected with H pyloricompared with patients who were not infected with HpylorL Furthermore,8-OHdG, iNOS, NFκB, Mcl-1 and IAP concentrations were significantly higher in stage 3 and 4 patients than in stage1 and 2 patients.CONCLUSION: Chronic Hpylori infection induces iNOS expression and subsequent DNA damage as well as enhances anti-apoptosis signal transduction. This sequence of events supports the rihypothesis that oxygen-free radical-mediated damage due to Hpyloriplays a pivotal role in the development of gastric carcinoma in patients with chronic gastritis.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM: Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins. Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide.Both have constitutive and inducible isoforms. The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis, tumorigenesis and inflammatory processes. This study was to clarify their role in pancreatic adenocarcinomas.METHODS: We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocarcinomas of different grade and stage. The results were compared with microvessel density and clinicopathological data.RESULTS: Twenty-one (52.5%) of the cases showed iNOS expression, 15 (37.5%) of the cases were positive for COX-2.The immunoreaction was heterogeneously distributed within the tumors. Staining intensity was different between the tumors. No correlation between iNOS and COX-2 expression was seen. There was no relationship with microvessel density.However, iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression. There was no correlation with other clinicopathological data.CONCLUSION: Approximately half of the cases expressed iNOS and COX-2. These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas. Due to a low prevalence of COX-2 expression, chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

Increased hepatic expression of nitric oxide synthase type Ⅱ in cirrhotic rats

AIM: To determine the role and effect of nitric oxide synthase type Ⅱ (NOSII) in cirrhotic rats.METHODS: Expression of NOSⅡ mRNA was detected by real time RT-PCR. The activity of nitric oxide synthase and serum levels of NO, systemic and portal hemodynamics and degrees of cirrhosis were measured with high sensitive methods. Chinese traditional medicine tetrandrine was used to treat cirrhotic rats and to evaluate the function of NO.Double-blind method was applied during the experiment.RESULTS: The concentration of NO and the activity of NOS were increased markedly at all stages of cirrhosis, and iNOSmRNA was greatly expressed. Meanwhile the portalvenous-pressure (PVP), and portal-venous-flow (PVF) were significantly increased. NO, NOS and iNOSmRNA were positively correlated to the quantity of hepatic fibrosis.Tetrandrine significantly inhibited NO production and the expression of iNOSmRNA.CONCLUSION: Increased hepatic expression of NOSII is one of the important causes of hepatic cirrhosis and portal hypertension.

Cyclooxygenase 2,pS2,inducible nitric oxide synthase and transforming growth factor alpha in gastric adaptation to stress

AIM:To determine the role of mucosal gene expression of cyclooxygenase 2 (COX-2), pS2 (belongs to trefoil peptides),inducible nitric oxide synthase (iNOS) and transforminggro wth factor alpha (TGFα in gastric adaptation to water immersion and restraint stress (WRS) in rats.METHODS: Wistar rats were exposed to single or repeated WRS for 4 h every other day for up to 6 d. Gastric mucosal blood flow (GMBF-) was measured by laser Doppler flowmeter3. The extent of gastric mucosal lesions were evaluated grossly and histologically and expressions of COX-2, pS2,iNOS and TGFα were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot.RESULTS: The damage to the surface of gastric epithelium with focal areas of deep haemorrhagic necrosis was induced by repeated WRS.The adaptative cytoprotection against stress was developed with activation of cell proliferation in the neck regions of gastric glands. The ulcer index (UI) in groups Ⅱ, Ⅲ and IV was markedly reduced as compared with group Ⅰ (Ⅰ: 47.23±1.20; Ⅳ: 10.39±1.18,P<0.01). GMBF significantly decreased after first exposure to WRS with an adaptive increasement of GMBF in experimental groups after repetitive challenges with WRS. After the 4th WRS,the value of GMBF almost restored to normal level (Ⅰ: 321.87±8.85; Ⅳ: 455.95±11.81,P<0.01). First WRS significantly decreased the expression of pS2 and significantly increased the expressions of COX-2, iNOS and TGFα After repeated WRS, pS2 and TGFα expressions gradually increased (pS2: Ⅰ: 0.37±0.02; Ⅳ: 0.77±0.01; TGFα Ⅰ: 0.86±0.01, Ⅳ: 0.93±0.03, P<0.05) with a decrease in the expressions of COX-2 and iNOS (COX-2: Ⅰ: 0.45±0.02; Ⅳ:0.22±0.01; iNOS: Ⅰ: 0.93±0.01; Ⅳ: 0.56±0.01, P<0.01).Expressions of pS2, COX-2, iNOS and TGFα showed regular changes with a good relationship among them.CONCLUSION: Gastric adaptation to WRS injury involves enhanced cell proliferation,increased expression of pS2 and TGFα, and reduced expression of COX-2 and iNOS.These changes play an important role in adaptation of gastric mucosa after repeated WRS.

唐古特红景天降低大鼠低氧性肺动脉高压的作用及对ET-1,eNOS mRNA和蛋白表达的影响

目的:研究唐古特红景天95%乙醇提取物对大鼠慢性低氧性肺动脉高压(HPH)的作用及其分子机制。方法:雄性Wistar大鼠随机分为对照组、低氧组、低氧+生理盐水组、低氧+低剂量药物组(1.25 g.kg-1.d-1)、低氧+高剂量药物组(2.5 g.kg-1.d-1)5组,每组15只。除对照组外,其余各组动物均置于低压氧舱内(模拟海拔4 500 m的高原环境),每24 h开舱0.5 h进行灌药等处理,连续30 d。检测各组大鼠的肺血流动力学变化、右心室肥厚指数;光镜观察直径50～100μm的肺动脉管壁厚度占外径的百分比(WT%);采用实时荧光定量PCR方法,检测各组动物肺组织内皮素-1(ET-1)和内皮细胞一氧化氮合酶(eNOS)mRNA的表达;采用ELISA法检测各组动物血清ET-1和eNOS蛋白的含量。结果:与低氧对照组和低氧生理盐水组相比,高剂量唐古特红景天干预,具有以下药理作用:①降低大鼠的平均肺动脉压(mPAP),右心室肥厚指数(RV/LV+S),血红蛋白含量(Hb),红细胞压积(Hct)(P<0.01);②减轻大鼠肺动脉的肌化程度(P<0.05);③降低大鼠肺组织ET-1 mRNA表达,上调大鼠肺组织内皮细胞(eNOS)mRNA表达(P<0.05);④降低大鼠血清ET-1蛋白的含量,上调大鼠血清内皮细胞eNOS蛋白的含量(P<0.05)。结论:唐古特红景天对于HPH的形成具有一定的防治作用,其药理作用机制与上调eNOS mRNA和蛋白表达、抑制ET-1 mRNA和蛋白表达有关。