Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.

Association between endothelial nitric oxide synthase(ENOS) G894T polymorphism and high altitude(HA) adaptation： a meta-analysis

Objective: Highland natives adapt well to the hypoxic environment at high altitude(HA). Several genes have been reported to be linked to HA adaptation. Previous studies showed that the endothelial nitric oxide synthase(ENOS) G894 T polymorphism contributed to the physiology and pathophysiology of humans at HA by regulating the production of NO. In this meta-analysis, we evaluate the association between the ENOS G894 T polymorphism and HA adaptation through analyzing the published data. Methods: We searched all relevant literature about the ENOS G894 T polymorphism and HA adaptation in Pub Med, Medline, and Embase before Step 2015. A random-effects model was applied(Revman 5.0), and study quality was assessed in duplicate. Six studies with 634 HA native cases and 621 low-altitude controls were included in this meta-analysis. Results: From the results, we observed that the wild-type allele G was significantly overrepresented in the HA groups(OR=1.85; 95% CI, 1.47–2.33; P<0.0001). In addition, the GG genotype was significantly associated with HA adaptation(OR=1.99; 95% CI, 1.54–2.57; P<0.0001). Conclusion: Our results showed that in 894 G allele carriers, the GG genotype might be a beneficial factor for HA adaptation through enhancing the level of NO. However, more studies were needed to confirm our findings due to the limited sample size.

The emerging role of nitric oxide in the synaptic dysfunction of vascular dementia

With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic rate.However,few therapeutic options exist that can markedly improve the cognitive impairment and prognosis of vascular dementia patients.Similarly in Alzheimer’s disease and other neurological disorders,synaptic dysfunction is recognized as the main reason for cognitive decline.Nitric oxide is one of the ubiquitous gaseous cellular messengers involved in multiple physiological and pathological processes of the central nervous system.Recently,nitric oxide has been implicated in regulating synaptic plasticity and plays an important role in the pathogenesis of vascular dementia.This review introduces in detail the emerging role of nitric oxide in physiological and pathological states of vascular dementia and summarizes the diverse effects of nitric oxide on different aspects of synaptic dysfunction,neuroinflammation,oxidative stress,and blood-brain barrier dysfunction that underlie the progress of vascular dementia.Additionally,we propose that targeting the nitric oxide-sGC-cGMP pathway using certain specific approaches may provide a novel therapeutic strategy for vascular dementia.

Simvastatin Increases the Activity of Endothelial Nitric Oxide Synthase via Enhancing Phosphorylation

3-hydroxy-3-methylgulutaryl-coenzyme A （HMG-CoA） reductase inhibitors or statins are a kind of lipid-lowering agents and have been used for the prevention and treatment of Cardiovascular diseases. Recent studies suggested that statins, besides lowering cholesterol, may protect vessels by enhancing the activity of endothelial nitric oxide synthase （eNOS）. In the present study, we investigated if simvastatin increases eNOS activity through its phosphorylation in 293 cells （293-eNOS） with stable expression of eNOS. The results showed that incubation of 293-eNOS cells with simvastatin （10 μm/L） for 2 h significantly increased in the activity of eNOS as shown by the conversion of L-arginine to L-citrulline （2889.70±201.51 versus 5630.18＋218.75 pmol/min . mg proteins） （P〈0.01）. Western blotting revealed that simvastatin increased phosphorylation of eNOS at 1177 （ser） and also 495 （thr） but did not affect the overall expression of eNOS or inducible NOS. Further study found that simvastatin raised phosphorylation levels of Akt and AMPK, and such effect could be antagonized by Akt inhibitor or AMPK inhibitor. These results suggest that simvastatin could stimulate,the activity of eNOS via its phosphorylation by Akt and AMPK, which provides a new mechanism, other than lipid-lowering effect, for the cardiovascular protection of statins.

Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanolinduced liver injury

AIM： To study the expression and activity of inducible nitric oxide synthase （iNOS） and endothelial nitric oxide synthase （eNOS） in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-KB （NF-κB） and tumor necrosis factor-α （TNF-α） expression in the liver. METHODS： Female Sprague-Dawley rats were given fish oil （0.5 mL） along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase （ALT） activity and pathological analysis. Liver malondialdehyde （MDA）, nitric oxide contents, iNOS and eNOS activity were determined. NF-κB p65, iNOS, eNOS and TNF-α protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction （RT-PCR）. RESULTS： Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration （6 wk） enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-κB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-KB, and TNF-α mRNA expression. CONCLUSION： iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-KB and TNF-α expression, eNOS activity is reduced, but its mRNA expression is not affected.

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 withendothelial 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 tissuemalondialdehyde (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 vs 45.3±10.1μmol/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±0.18 μg/Land 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). TheNOS 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 eNOSderived NO.

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 factorⅧ 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.

C1q/TNF-related protein 1 promotes vasodilatory dysfunctions by increasing arginase 1 activity and uncoupling of endothelial nitric oxide synthase

Objective C1q/TNF-related protein(CTRP)1 was initiallyidentified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins.Previously,we showed that CTRP1promotes the development of atherosclerosis by increasing endothelial adhesiveness.Here,we sought to investigate whether CTRP1 also influences vascular dilatory functions.

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells

AIM： To study the expression of endothelial and inducible nitric oxide synthases （eNOS and iNOS） and their role in inflammatory bowel disease （IBD）. METHODS： We examined the effect of sera obtained from patients with active Crohn＇s disease （CD） and ulcerative colitis （UC） on the function and viability of human umbilical vein endothelial cells （HUVEC）. HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10^-4 mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS： In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control （18%±16% vs 23%± 15% P〈0.01）. UC or CD serum caused a significant increase in iNOS compared to control （UC： 300%±21%; CD： 275% ± 27% vs 108% ± 14%, P〈0.01）. Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION： IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinflammatory protection of endothelial cells.

NITRIC OXIDE SYNTHASE AND VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION IN HEPATOCELLULAR CARCINOMA AND THE CORRELATION WITH ANGIOGENESIS

Objective: To analyze the expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) and its relation to angiogenesis. Methods: Tissue sections from 71 HCC patients were examined immunohistochemically for protein expression of iNOS, eNOS, and VEGF. Microvessal density (MVD) was counted by endothelial cells immunostained by anti-CD34 antibody. Results: Positive immunostaining for iNOS, eNOS was detected in 83.1% and 85.9% of HCC respectively. INOS and eNOS were not detected in normal hepatic tissue. MVD was 34.3±1.5/HP and 38.6±1.6/HP in HCC with positive staining for iNOS and VEGF while it was 31.2±2.8/HP, and 22.4±2.0/HP in HCC with negative staining for iNOS and VEGF (P<0.01). A correlation between NOS expression and VEGF in HCC was not observed. Conclusion: iNOS and eNOS may play a role in malignant transformation f post-hepatic cirrhosis. The expression of iNOS and VEGF favors angiogenesis of HCC.

Expression of Endothelial Nitric Oxide Synthase Traffic Inducer in the Placenta of Pregnancy Induced Hypertension

The expression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） in the placenta of the patients with pregnancy induced hypertension （PIH） was detected and its role in the pathogenesis of PIH was studied. The pathological changes in placental vessels were observed by HE staining. NO2-/NO3- , the stable metabolic end products of NO, was measured with nitrate reductase. The eNOS activity in placental tissues was assayed by spectrophotometry. Western blot analysis was applied to detect NOSTRIN expression. The incidence of thickening and fibronoid necrosis of placental vessels was significantly higher in women with PIH than in the normal group （P〈 0.01）. The levels of placental NO2-/NO3- in PIH patients （27. 53±7.48 μmol/mg） were significantly lower than in normal group （54. 27±9.53 μmol/mg, P〈0.01）. The activity of eNOS was significantly decreased in PIH group （12. 826±3.61 U/mg） as compared with that in normal group （21. 72±3.83 U/mg, P〈0.01）. Western blot analysis revealed that both groups expressed 58 kD NOSTRIN, but the protein level was significantly higher in women with PIH than in the normal group （P〈0.01）. A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in placental tissue of women with PIH （r=-0.57, P〈0.01）. It was concluded that the level of NOSTRIN expression in placenta of women with PIH was increased, which may play an important role in the pathogenesis of PIH.

Changes in Human Umbilical Vein Endothelial Cells Induced by Endothelial Nitric Oxide Synthase Traffic Inducer

This study investigated the changes in human umbilical vein endothelial cells （HUVECs） induced by overexpression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS （eNOS） activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid （P〈0.01）. The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells （P〈0.01 and P〈0.01, respectively）. Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase

Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase （eNOS）. Methods ＆ Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin （BH4）. Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes： GTP cyclobydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin （BH2） as well as BH4：BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4： eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality （i.e., coupled or uncoupled）, the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Endothelial nitric oxide synthase deficiency influences normal cell cycle progression and apoptosis in trabecular meshwork cells

AIM： To clarify how the endothelial nitric oxide synthase （eNOS, NOS3） make effect on outflow facility through the trabecular meshwork （TM）. METHODS： Inhibition of NOS3 gene expression in human TM cells were conducted by three siRNAs. Then the mRNA and protein levels of NOS3 in siRNA-treated and negative control （NC） cells were determined, still were the collagen, type IV, alpha 1 （COL4A1） and fibronectin 1 by real-time PCR and Western blot analysis. In addition, NOS3 concentrations in culture supernatant fluids of TM cells were measured. Cell cycle and cell apoptosis analysis were performed using flow cytometry. RESULTS： The mRNA level of NOS3 was decreased by three different siRNA interference, similar results were obtained not only of the relative levels of NOS3 protein, but also the expression levels of COL4A1 and fibronectin 1. The number of cells in S phase was decreased, while contrary result was obtained in G2 phase. The number of apoptotic cells in siRNA-treated groups were significant increased compared to the NC samples. CONCLUSION： Abnormal NOS3 expression can make effects on the proteins levels of extracellular matrix component （e.g. fibronectin 1 and COL4A1）. Reduced NOS3 restrains the TM cell cycle progression at the G2/ M-phase transition and induced cell apoptosis.

Shear stress effect on endothelial nitric oxide synthase in cultured human umbilical vein endothelial cells

Background: Low shear stress caused by disturbed or turbulent flow at arterial branch points is known to associate with atherosclerosis. However, shear stress at the venous valve location and its association with deep vein thrombosis are less understood due to the complex and poorly understood bi-directional flow in the valve pocket region. We investigated how venous endothelial cells respond to flow shear stress around the venous valve region using a novel in vitro system that mimics venous flow. Results: Human umbilical vein EAhy. 926 cells were cultured on a flexible silastic membrane that mimicked venous tissue. Confluent cells were exposed to sinusoidal uni-and bi-directional pulsatile shear stress (0.1 to 1 dyne/cm2) for up to 6 h. Western-blot analyses indicated that endothelial nitric oxide (eNOS) expression levels decreased regardless of all tested flow patterns, stress magnitude, and shearing time. In contrast, the expression levels of inhibitor of κB (kappa B) and α (alpha)-tubulin were unaffected by the shear stress. Conclusions: Our results indicate that shear stress causes a decrease specifically in eNOS expression, suggesting that it may play a significant role in regulating inflammation related protein expression in endothelial cells.

基于血清iNOS和eNOS水平建立慢性阻塞性肺疾病急性加重患者机械通气撤机预测模型

目的 基于血清诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)水平建立慢性阻塞性肺疾病急性加重(AECOPD)患者机械通气撤机的预测模型。方法 选择2020年1月至2023年6月在河北北方学院附属第一医院接受机械通气治疗的166例AECOPD患者,按照撤机结局分为撤机成功组(n=112)和撤机失败组(n=54)。比较两组患者的临床资料及入院时、自主呼吸试验(SBT)前的血清iNOS、eNOS水平。采用Logistic回归分析撤机失败的影响因素,采用受试者工作特征(ROC)曲线分析iNOS、eNOS预测撤机失败的价值。结果 与撤机成功组比较,撤机失败组SBT前24 h内的急性生理学与慢性健康状况评价Ⅱ(APACHEⅡ)评分、肌酐(Cr)、iNOS、eNOS水平较高,白蛋白(Alb)水平较低(P<0.05);多因素Logistic回归分析显示,APACHEⅡ、Alb、iNOS、eNOS是撤机失败的影响因素(P<0.05);ROC曲线分析显示,iNOS、eNOS预测撤机失败的ROC曲线下面积为0.648(95%CI 0.563~0.733,P=0.002)、0.755(95%CI 0.683~0.827,P<0.001),以4.418 ng/mL、3.821 ng/mL为最佳截断值,预测敏感度分别为68.52%、83.33%,特异度分别为51.82%、66.36%;iNOS、eNOS与Alb、APACHEⅡ联合预测撤机失败的ROC曲线下面积为0.961(95%CI 0.928~0.993),优于单一指标(Z=7.412、6.682、4.323、4.951,P<0.05),敏感度和特异度分别为94.44%和90.91%。结论 AECOPD患者SBT前血清iNOS、eNOS水平增加与撤机失败相关,SBT前检测iNOS、eNOS联合Alb、APACHEⅡ能够较好地预测撤机结局。

秋水仙碱通过激动PI3K/AKT/eNOS信号通路对急性心肌梗死大鼠心功能的影响

目的:探究秋水仙碱通过调节磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/内皮型一氧化氮合酶(eNOS)信号通路对急性心肌梗死(AMI)大鼠心功能的影响。方法:78只大鼠中随机选取12只作为假手术组,剩余大鼠构建AMI模型,造模成功大鼠分为模型组、秋水仙碱组[4 mg/(kg·d)]、秋水仙碱[4 mg/(kg·d)]+LY294002(20 mg/mL)组、秋水仙碱[4 mg/(kg·d)]+MK-2206(60μg/mL)组、秋水仙碱[4 mg/(kg·d)]+L-NAME(1.6 mg/mL)组,每组12只。超声心动图检测大鼠左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)、射血分数(EF)及短轴缩短率(FS)。处死大鼠,苏木素-伊红(HE)染色检测大鼠心肌组织石蜡切片病理学变化;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测心肌细胞凋亡率;酶联免疫吸附实验(ELISA)测定大鼠血清肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、肌酸激酶同工酶(CK-MB)以及心肌组织匀浆中超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)水平;免疫印迹法(Western Blot)测定大鼠心肌组织PI3K/AKT/eNOS通路蛋白表达。结果:与假手术组相比,模型组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率升高,EF、FS水平,心肌匀浆SOD、CAT,心肌组织磷酸化PI3K(p-PI3K)/PI3K、磷酸化AKT(p-AKT)/AKT、磷酸化eNOS(p-eNOS)/eNOS降低(P<0.05);与模型组相比,秋水仙碱组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率降低,EF、FS水平,心肌匀浆SOD、CAT,心肌组织p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS升高(P<0.05);与秋水仙碱组相比,秋水仙碱+LY294002组、秋水仙碱+MK-2206组、秋水仙碱+L-NAME组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率升高,EF、FS水平,心肌匀浆SOD、CAT,心肌组织p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS降低(P<0.05)。结论:秋水仙碱可能通过激活PI3K/AKT/eNOS信号通路对AMI大鼠发挥心功能保护作用。

Association of Glu298Asp Polymorphism of the En-dothelial Nitric Oxide Synthase Gene with Essential Hypertension in Elderly People

Objective: To investigate the association of Glu298Asp polymorphism of theeNOS gene with essential hypertension in elderly people. Methods: Ninety-five cases of essentialhypertension were randomly chosen from outpatients and inpatients as the study group, and an equalnumber of sexes, age-matched healthy people as the control group. Their height, weight and bloodpressure were recorded and their fasting plasma lipid concentrations were measured. Glu298Asppolymorphism of the eNOS gene was measured using the methods of PCR and RFLP. Results: Theconstituent ratio of Genotype Glu/Asp in the study group (26.3%) was higher than that in the controlgroup (12.6%, x^2 = 5. 67, P<0.05), the allelic frequency of 298Asp in the study group (13.2%) wassignificantly higher than that in the control group (6.3%, x^2 = 5.06, P<0.05). Conclusion: Glu298Asp variant of the eNOS gene may be an independent predictor in essential hypertension.

海南地区eNOS基因单核苷酸多态性与系统性红斑狼疮的相关性分析

目的探讨海南地区内皮型一氧化氮合酶(eNOS)基因单核苷酸多态性(SNP)与系统性红斑狼疮(SLE)遗传易感性的关系。方法收集2020年1月-2022年12月在海南医学院第一附属医院及海南省人民医院的海南籍SLE患者(SLE组,n=214)及健康对照者(对照组,n=214)的血液样本,通过SNaPshot测序技术检测两组eNOS基因rs3918188、rs1799983及rs1007311位点的碱基情况,并采用logistic回归分析eNOS基因上述3个目的位点的基因型、等位基因及基因模型(显性模型、隐性模型、超显性模型)与SLE遗传易感性之间的相关性。采用HaploView4.2软件分析各位点的单倍体与SLE遗传易感性的关系。结果Logistic回归分析结果显示,rs3918188位点中,CC基因型及C等位基因是SLE遗传易感性的危险因素(CCvs.AA:OR=2.449,P<0.05;Cvs.A:OR=2.133,P<0.001)。在rs3918188位点的隐性模型中,与AA+AC基因型携带者比较,CC基因型携带者的SLE发生风险增高(OR=2.774,P<0.001);而在该位点的超显性模型中,与AA+CC基因型携带者比较,AC基因型携带者的SLE发生风险降低(OR=0.385,P<0.001)。此外,在基因型、等位基因型以及3种基因模型中,rs1799983和rs1007311的多态性均与SLE的易感性无明显相关(P>0.05)。单倍型分析显示,eNOS基因的rs1007311和rs1799983位点之间存在强连锁不平衡,但单倍型与SLE的遗传易感性无明显相关(P>0.05)。结论海南地区eNOS基因rs3918188位点的CC基因型和C等位基因可能是SLE的危险因素,而在超显性模型下该位点AC基因型携带者的SLE发生风险降低。

ET-1/eNOS表达差异在牦牛隐睾发生中的作用分析

旨在比较内皮素-1(endothelin-1,ET-1)及内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)在牦牛正常睾丸与隐睾组织中的分布,分析其表达差异在牦牛隐睾发生中的作用。本研究采集健康和病理性成年(4岁)雄性牦牛睾丸共20对,分为3组:正常组(10对)、单侧下降组(6对)及隐睾组(4对),应用H.E染色、Masson’s三色染色、Gomori’s染色结合形态计量学统计软件比较正常睾丸与隐睾的组织化学特点;通过免疫组织化学方法、免疫组织荧光技术及实时荧光定量PCR(quantitative real-time PCR,qPCR)检测ET-1和eNOS在正常睾丸与隐睾中的表达量并比较组间差异。结果表明:与正常组相比较,牦牛单侧下降组间质面积/管腔面积之比无显著性差异(P>0.05);隐睾组管腔面积明显减小,胶原纤维和网状纤维含量增多。免疫组化和免疫荧光结果显示,ET-1表达于间质细胞、各级生精细胞,隐睾组上皮未见明显表达,主要表达于间质细胞,正常组ET-1的平均光密度与单侧下降组差异显著(P<0.05),与隐睾组差异极显著(P<0.01);eNOS表达于生精小管、间质细胞,正常组eNOS的平均光密度与单侧下降组差异极显著(P<0.001),与隐睾组差异显著(P<0.05)。qPCR结果显示,ET-1在正常组中相对表达量显著高于单侧下降组(P<0.01)和隐睾组(P<0.01),eNOS在单侧下降组中的相对表达量相比于正常组升高(P<0.05)。高原低氧环境下,牦牛隐睾睾丸生精小管皱缩,间质细胞减少,局部分泌调节受到抑制,最终导致精子发生阻滞。ET-1和eNOS共同作用于间质细胞,在单侧下降组和隐睾组间质细胞中表达失衡,应是间质细胞及血管分布异常引起。