Protective effect of trans-nerolidol on vascular endothelial cell injury induced by lipopolysaccharides / Efectos protectores del trans-nerolidol en lesiones inducidas por lipopolisacáridos en células endoteliales vasculares

This article aimed to discuss the protection of trans - nerolidol on vascular endothelial cells (ECs) injured by lipopolysac charides. ECs were divided into four groups: normal, model, low and high dose trans - nerolidol treatment groups. The cell survival rate and the contents of NO in the cell culture supernatant were determined. The protein expression and transcript level of pe roxisome proliferator - activated receptor - γ (PPARγ), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were determined by western blotting and RT - PCR respectively. Compared with the normal group, cell livability, protein e xpression and mRNA transcript level of PPARγ and eNOS decreased, NO contents, protein expression and mRNA transcript tlevel of iNOS increased in model group significantly. Compared with model group, all the changes recovered in different degree in treatmen t groups. Hence, it was concluded that trans - nerolidol can alleviate the ECs injuryby the regulation of iNOS/eNOS through activating PPARγ in a dose - dependent manner

Este artículo tiene como objetivo discutir la protección del trans - nerolidol en las células endoteliales vasculares (CE) dañadas por lipopolisacáridos. Las CE se di vidieron en cuatro grupos: normal, modelo, grupos de tratamiento con trans - nerolidol de baja y alta dosis. Se determinó la tasa de supervivencia de las células y los contenidos de óxido nítrico (NO) en el sobrenadante del cultivo celular. La expresión de p roteínas y el nivel de transcripción del receptor activado por proliferadores de peroxisomas - γ (PPARγ), el óxido nítrico sint et asa endotelial (eNOS) y el óxido nítrico sint et asa inducible (iNOS) se determinaron mediante western blot y RT - PCR, respectivamen te. En comparación con el grupo normal, la viabilidad celular, la expresión de proteínas y el nivel de transcripción de PPARγ y eNOS disminuyeron, los contenidos de NO, la expresión de proteínas y el nivel de transcripción de iNOS aumentaron significativam ente en el grupo modelo. En comparación con el grupo modelo, todos los cambios se recuperaron en diferentes grados en los grupos de tratamiento. Por lo tanto, se concluyó que el trans - nerolidol puede aliviar el daño en las CE regulando iNOS/eNOS a través d e la activación de PPARγ de manera dependiente de la dosis.

Placental expression of striatin & endothelial nitric oxide synthase in women with & without pre-eclampsia

Background & objectives: Striatin is a multi-domain scaffolding protein essential for activating endothelial nitric oxide synthase (eNOS). However, its role in pre-eclampsia remains use explored. Hence, this study aimed to investigate the association between striatin and eNOS in regulating nitric oxide (NO) production in the placenta of women with and without pre-eclampsia. Methods: Forty pregnant women each without (controls) and with pre-eclampsia (cases) were enrolled in the study. Blood striatin and NO concentrations were detected by the ELISA. Protein expression of striatin, phosphorylated eNOS (peNOS), inducible NOS (iNOS) and phosphorylated NF-?B were measured in the placental tissues by Western blot. Twenty four hour urinary protein and serum urea, uric acid and creatinine were analyzed as an autoanalyzer. Placental histology was analyzed by haematoxylin and eosin staining. Results: Compared to normotensive pregnant women, the levels of serum NO and striatin were decreased in pre-eclamptic women. The protein expression of striatin and peNOS was significantly reduced (P<0.05) while p65NF-?B and iNOS were upregulated considerably (P<0.05) in the placenta of cases compared to controls. Interpretation & conclusions: Our results show for the first time that decreased striatin expression was associated with decreased peNOS protein expression in the placental tissue of pre-eclamptic women. Interestingly, no significant difference was found in blood striatin or NO levels between controls and cases. Thus, therapies that improve placental striatin expression are attractive possibilities, both for prevention as well as treatment of endothelial dysfunction in pre-eclampsia.

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes / 中南大学学报(医学版)

OBJECTIVES@#Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy.@*METHODS@#The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels.@*RESULTS@#Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05).@*CONCLUSIONS@#p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

Effect of Xileisan temperature-sensitive gels on endothelial nitric oxide synthase, vascular endothelial growth factor A and tumor necrosis factor-α expression in rats with bleeding internal hemorrhoids / 预防医学

Objective@#To investigate the effect of Xileisan temperature-sensitive gels on endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor A (VEGF-A) and tumor necrosis factor-α (TNF-α) expression in rats with bleeding internal hemorrhoids, so as to provide insights into the illustration of the pathogenesis of internal hemorrhoid hemorrhage. @*Methods@#Thirty six-week-old SPF-graded rats of the SD strain were randomly divided into the normal group, model group and Xileisan temperature-sensitive gel group, of 10 rats in each group (half male and half female). Cotton balls were soaked with 0.16 mL of croton oil mixture and then inserted into the anus of rats in the model group and Xileisan temperature-sensitive gel group for 10 s. After 6 h when the rectal mucosa tissues presented remarkable swelling, the perianal mucosa was rubbed repeatedly with a rough glass rod until the glass rod was bloody. Following successful modeling, rats in the Xileisan temperature-sensitive gel group was given rectal administration of Xileisan temperature-sensitive gel at a dose of 0.5 mL/d, while animals in the normal group and model group were given rectal administration of the blank gel at the same dose. Following administration for 7 successive days, rats were sacrificed, and the hemorrhoids tissues were collected for pathological examinations. The eNOS, VEGF-A and TNF-α expression was determined using immunohistochemistry and compared among groups.@*Results@#Compared with the normal group, the rat hemorrhoids mucosa showed inflammatory changes in the model group, with submucosal congestion and edema, blood vessel congestion and dilation, and visible new blood vessels, and remarkable improvements were seen in the hemorrhoid mucosal inflammation in the Xileisan temperature-sensitive gel group. There were significant differences in the integrated option density (IOD) of eNOS and VEGF-A expression in rat hemorrhoids tissues among the three groups (P<0.05), and no gender-specific differences were seen (P>0.05). The IOD values of eNOS (45.84±13.66) and VEGF-A expression (45.89±9.06) were higher in rat hemorrhoids tissues in the model group than in the normal group (23.11±5.64 and 27.91±11.65) and the Xileisan temperature-sensitive gel group (27.41±8.89 and 33.44±6.20) (P<0.05), while no significant differences were detected in the IOD of TNF-α expression in rat hemorrhoids tissues among the three groups (P>0.05).@*Conclusion@#Xileisan temperature-sensitive gel may alleviate inflammation and internal hemorrhoids hemorrhage through inhibiting eNOS and VEGF-A expression in rat hemorrhoids tissues.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Fasudil alleviates lung injury via targeting Rhoa/ROCK signal pathway in septic mice / 中华急诊医学杂志

Objective:To determine the protective effect of fasudil on acute lung injury in septic mice.Methods:Forty-five 4-6-week-old male C57BL mice were randomly(random number) assigned to three groups ( n=15 each group): control group, lipopolysaccharide (LPS) group and Fasudil intervention group (FAS+LPS). Acute lung injury model of septic mice was established with an intraperitoneal injection and intratracheal infusion of LPS. The mice in the FAS+LPS group were injected with fasudil hydrochloride intraperitoneally 30 min before intraperitoneal LPS injection and 1 h after intratracheal LPS infusion, respectively. All mice were sacrificed at 4 h after modeling, and lung tissues were collected. Hematoxylin-eosin staining was preformed to observe the morphological changes in the lung tissue. The wet /dry weight (W/D) ratio, malondialdehyde (MDA) content and the activity of myeloperoxidase (MPO) in the lung tissues were detected. Caspase-3 expression was examined by immunohistochemical (IHC) staining. Western blot was employed to detect the expression of RhoA, ROCK1, endothelial nitric oxide synthase (eNOS), and p-eNOS. Results:Inflammatory cell infiltration and erythrocyte exudation were significantly reduced, and the degree of interstitial oedema and derangement of alveolar structure appeared in a decreasing degree after FAS intervention. Compared with the LPS group, the W/D ratio, MDA content, MPO activity and the expression of Caspase-3 in the FAS+LPS group were significantly reduced (all P<0.01). Meanwhile, the expression of RhoA and ROCK1 of the LPS group were obviously higher than those in the control group ( P<0.05), and p-eNOS was obviously lower than that in the control group ( P<0.05). Furthermore, the expression of RhoA and ROCK1 of the FAS+LPS group were obviously lower than those in the LPS group, and p-eNOS was obviously higher than that in the LPS group. There was no significant difference on the expression of eNOS among the three groups. Conclusions:Fasudil can alleviate the degree of inflammatory cell infiltration, reduce apoptosis in lung tissue, inhibit the RhoA/ROCK1 signaling activity, and promote the phosphorylation expression of eNOS in septic mice.

Involvement of ET-1/eNOS in the ameliorating effect of electroacupuncture on cardiac dysfunction in rats with spontaneously hypertensive / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on cardiac function of ventriculus sinister in rats with spontaneously hypertensive (SHR), and to explore the mediation effect of endothelin-1 (ET-1)/endothelial nitric oxide synthase (eNOS).@*METHODS@#Six 12-week-old male Wistar Kyoto (WKY) rats were taken as the normal group. Eighteen 12-week-old SHR were randomly divided into a model group, an EA group and a sham EA group, 6 rats in each group. The rats in the EA group were treated with EA (disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity) at "Neiguan" (PC 6), 30 min each time, once a day for 8 weeks. The rats in the sham EA group were treated with superficial needling at "Neiguan" (PC 6) with no electrical stimulation applied. After treatment, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were tested by echocardiographic analysis. The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), the maximum rate of increase/decrease of left ventricular pressure (±dp/dtmax) were detected. The serum content of ET-1 was detected by ELISA. Western blot was used to evaluate the expression of ETAR, eNOS in myocardial tissue of left ventricular.@*RESULTS@#Compared with the normal group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were decreased (P<0.01, P<0.05), while LVSP, LVEDP, +dp/dtmax and -dp/dtmax were increased (P<0.01) in the model group. Compared with the model group, LVEF, LVFS, +dp/dtmax/LVSP and -dp/dtmax/LVSP were increased (P<0.01, P<0.05), and LVSP and LVEDP were decreased (P<0.01) in the EA group. Compared with the normal group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were increased (P<0.01), whereas expression of eNOS was decreased (P<0.01) in the model group. Compared with the model group, the serum content of ET-1 and the expression of ETAR in myocardial tissue were decreased (P<0.05), whereas expression of eNOS was increased (P<0.05) in the EA group.@*CONCLUSION@#EA intervention may alleviate hypertensive cardiac function damage by up-regulating the expression of eNOS protein in myocardial tissue, down-regulating the serum content of ET-1 and the expression of ETAR protein in myocardial tissue.

Human umbilical cord Wharton's jelly-derived mesenchymal stem cell transplantation could improve diabetic intracavernosal pressure / 亚洲男科学杂志(英文版)

Mesenchymal stem cells (MSCs) secrete various cytokines with angiogenic and neuroprotective effects. This study aimed to assess the effects of human umbilical cord Wharton's jelly-derived MSCs (hWJ-MSCs) on diabetes-related intracavernosal pressure (ICP) impairment in rats. hWJ-MSCs were isolated from human umbilical cord Wharton's jelly and transplanted into the corpus cavernosum of streptozotocin (STZ)-induced diabetic rats by unilateral injection. The erectile function was evaluated at 4 weeks, as well as the expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS), and insulin-like growth factor 1 (IGF1). STZ-induced diabetic rats showed impaired ICP, which was significantly improved by hWJ-MSC treatment. VEGF, eNOS, IGF1, and bFGF expression levels were higher in hWJ-MSC injection sites than those in control ones in STZ-induced diabetic rats. These results suggest that hWJ-MSC transplantation might improve diabetic erectile dysfunction through increased production of paracrine growth factors, highlighting a novel potential therapeutic option for erectile dysfunction.

Sesamin and vitamin E ameliorate aortic endothelial dysfunction rats induced by D-galactose and aluminum trichloride via regula?tion of endothelial nitric oxide synthase and NAD(P)H oxidase 4 / 中国药理学与毒理学杂志

OBJECTIVE To observe the protective effect of sesamin (Ses) and vitamin E (Vit E) against aortic endothelial dysfunction in rats induced by D-galactose (D-gal) and aluminum trichloride (AlCl3), and explore its conceivable mechanisms. METHODS A model of aortic endothelial dysfunction rats was established by D-gal (180 mg · kg-1, ip) combined with AlCl3 (15 mg · kg-1, ig) for 84 d. Model rats were randomly divided into model, model+Vit E 10 mg·kg-1, model+Ses 160 mg·kg-1, and model+Ses 160 mg · kg-1+Vit E 10 mg · kg-1 groups. After 70 d of treatment with Ses and Vit E, systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP) were measured by tail cuff. The rats were anesthetized by sodium pentobarbital (30 mg·kg-1, ip). Thoracic aortas from the rats were removed and divided into two parts (3 mm in length). The relaxation of the aortic ring induced by acetylcholine (ACh) and sodium nitroprusside was measured. The primary pathologic changes in the aorta were observed by HE staining. Total antioxidant capacity (T-AOC), hydrogen peroxide (H2O2) and nitric oxide (NO) in serum were measured by colorimetric analysis. The expression of endothelial nitric oxide synthase (eNOS) positive cells in the aorta were measured by immunohistochemistry. The expres?sions of eNOS and NAD(P)H oxidase 4 (NOX4) protein in the aortal were detected by Western blotting. RESULTS Compared with the model group, the relaxation response with increase in ACh concentra?tion (1×10-7-1×10-4 mol·L-1) was enhanced (P<0.01) in model+Ses+Vit E, SBP, DBP and MBP decreased (P<0.01), the serum T-AOC and NO level were increased (P<0.01), the serum H2O2 levels were reduced (P<0.01), the eNOS expression was increased (P<0.01) and NOX4 expression was reduced (P<0.01) in each treatment group. Compared with model+Ses, the SBP, DBP and MBP were lower (P<0.01 or P<0.05), the serum H2O2 level was lower (P<0.01), the serum NO level was increased (P<0.05), the eNOS expression level was higher (P<0.01) and the NOX4 expression level was reduced (P<0.05) in model+Ses+Vit E. Compared with the model+Vit E, the serum T-AOC and NO levels were increased (P<0.05), the serum H2O2 level was lower (P<0.01), eNOS expression was increased (P<0.01) and NOX4 expression was reduced (P<0.05) in model+Ses+Vit E group. CONCLUSION Ses and Vit E can ameliorate aortic endothelial dysfunction of rats induced by D-gal and AlCl3 via the regulation of eNOS and NOX4.

Effect of Danggui Buxuetang on Functional Activity of Endothelial Progenitor Cells Exposed to Shear Stress / 中国实验方剂学杂志

Objective:To investigate the effect of Danggui Buxuetang（DGBX）on the functional activity of rat endothelial progenitor cells（EPCs）exposed to different luminar shear stress （SS）. Method:EPCs isolated from rat bone marrow were incubated on a parallel plate flow chamber at a steady SS of 0， 0.12， 1.2， 2.4 Pa for 6 h，then the cells exposed to different SS were randomly divided into 8 groups： control group （perfused with serum free medium），simvastatin group（0.1 μmol·L^-1simvastatin），3 DGBX groups（low，medium，high-dose DGBX）and 3 inhibitor groups（3 DGBX groups with LY294002）. After 12 h，the samples were collected for the detection of cell proliferation ，migration，tubule formation ，the secretion of nitric oxide （NO） ，and the expressions of endothelial nitric oxide synthase（eNOS） mRNA and protein kinase B（Akt），respectively. Result:Compared with the control group，simvastatin and DGBX（high-dose）could both promote the functional activities and NO secretion，and up-regulate the expressions of eNOS mRNA and Akt protein in EPCs exposed to different SS（<italic>P</italic><0.05），while DGBX（mid-dose）could do these only at 0 Pa. However，LY294002 could inhibit all effects of DGBX on EPCs. Conclusion:SS seems to play an important role in the effect of DGBX on EPCs，and DGBX could promote the functional activity of EPCs exposed to SS by up-regulating the expressions of NO/eNOS/Akt.

Effect of Sanhuang Xiexintang in Inhibiting 7-Ketocholesterol-induced Endothelial Disfunctional by NIRP3 Inflammasome Pathway / 中国实验方剂学杂志

<b>Objective::To study whether Sanhuang Xiexintang (SHXXT) can restore endothelial function by inhibiting the activation of NOD-like receptor protein 3 (NIRP3) induced by 7-ketocholesterol (7-keto) in vascular endothelial cells. <b>Method::The aortic rings of mice were cultured in normal group, model (7-keto) group, SHXXT groups (1%, 2% and 5% drug-containing serum). Vasodilation function of mice was observed. Microvascular endothelial cells were cultured according to the above experimental groups, and NIRP3 inhibitor isoglycyrrhizin (ISO) group, was also set. Western blot was used to detect the expressions of endothelial nitric oxide synthase (eNOS), NIRP3, cysteinyl aspartate specific proteinase-1 (Caspase-1), interleukin-1<italic>β</italic> (IL-1<italic>β</italic>) protein. In addition, nitric oxide (NO) quantitative kit was used to detect the concentration of NO. <b>Result::Compared with the normal group, the endothelium-dependent vasodilation function of vascular rings was significantly reduced in model group (<italic>P</italic><0.01), and the drug group significantly restored the endothelium-dependent vasodilation function in a concentration-dependent manner (<italic>P</italic><0.05, <italic>P</italic><0.01). Meanwhile, microvascular endothelial cells were also studied. Compared with the normal group, the content of eNOS protein in the model group decreased (<italic>P</italic><0.05), while the concentration of NO decreased significantly (<italic>P</italic><0.01). After treatment with SHXXT serum, eNOS and NO could be restored, with significant differences in the concentration of NO with 5% (<italic>P</italic><0.05) and 10% (<italic>P</italic><0.01) SHXXT serum. At the same time, the expressions of NIRP3 (<italic>P</italic><0.05), cle-Caspase-1 activation (<italic>P</italic><0.01) and IL-1<italic>β</italic> production (<italic>P</italic><0.01) in endothelium were significantly increased under 7-keto stimulation, and the SHXXT serum could significantly inhibit the expression and activation of relevant proteins. Subsequently, endothelial cells were treated with NIRP3 inhibitor ISO. Compared with the model group, eNOS expression increased, and NO concentration increased significantly (<italic>P</italic><0.01) after treatment with ISO, but ISO had no synergistic effect on SHXXT serum. <b>Conclusion::SHXXT can improve endothelium-dependent vascular dysfunction induced by 7-keto, which is achieved by NO signaling pathway mediated by inhibiting the activation of endothelial NIRP3-related proteins.

Protective effect of Bifidobacterium on endothelial nitric oxide synthase in neonatal necrotizing enterocolitis model / 解放军医学杂志

Objective: To investigate the effect of Bifidobacterium on endothelial nitric oxide synthase and its potential protective mechanism in neonatal necrotizing enterocolitis using an induced mouse model. Methods: Sixty 8-day newborn C57BL/6 mice were randomized into three groups (n=20): control group, necrotizing enterocolitis group (NEC) and Bifidobacterium intervention group (NEC+BIF). Samples were collected 3 days after NEC modeling. HE staining pathological score was used to evaluate the modeling effect; the level of endothelial nitric oxide synthase (eNOS) in the intestine was detected with Western blotting; the eNOS activity, nitric oxide (NO) and superoxide (O2•) content were measured by Colorimetry. Real-time quantitative PCR was used to detect the mRNA level of interleukin-6 (IL-6) and interleukin-1β (IL-1β); the protein expression of IL-6 and IL-1β was assessed by ELISA. Results: No significant difference was observed in the protein levels of eNOS between the three groups. Compared with control group, in NEC group, the eNOS activity and NO content were significantly decreased; the O2 content was substantially increased; both IL-6 and IL-1β were dramatically increased (P0.05). Conclusion: Bifidobacterium can alleviate the severity of intestinal damage in NEC mouse model, the mechanism may be related to the enhancement of eNOS activity.

Avaliação de mecanismos de modificação pós-traducional da óxido nítrico sintase endotelial (eNOS) associados a biodisponibilidade do óxido nítrico em artérias de ratas espontaneamente hipertensas (SHR) ao final da prenhez / Evaluation of post-translational modification mechanisms of endothelial nitric oxide synthase (eNOS) associated with the nitric oxide bioavailability in arteries from spontaneously hypertensive rats (SHR) in the end of pregnancy

A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas endoteliais da aorta e da artéria mesentérica na presença ou ausência da metil-ß-ciclodextrina (dextrina, 10 mmol/L) por microscopia eletrônica. Em estudos funcionais, avaliamos a participação da enzima OGA, pela inibição com PugNAc (100 µmol/L) e das cavéolas, utilizando um desorganizador de cavéolas, a dextrina (10 ou 20 mmol/L), na menor reatividade vascular à PE observada em aortas de ratas P. Observamos que o conteúdo de proteínas O-GlcNAciladas estava diminuído em aorta e em leito mesentérico de ratas Wistar P e SHR P. Apesar da expressão da OGT e da OGA não estar alterada, a atividade da OGA foi aumentada em aorta e leito mesentérico de ratas Wistar P, mas, encontra-se diminuída em aorta e aumentada em leito mesentérico de SHP P. A incubação com PugNAc reverteu a reduzida reatividade à PE em aorta e artéria mesentérica de ratas Wistar P mas este efeito não foi observado em vasos SHR P, demonstrando que a OGA parece ter um papel importante na redução da O-GlcNAcilação de proteínas vasculares em Wistar P. Em vasos incubados com PugNAc, a remoção do endotélio ou a incubação com L-NAME, não alterou significativamente a reatividade à PE. Juntos estes resultados sugerem que a maior atividade da eNOS observada em vasos de Wistar P, fica prejudicada na presença do PugNAc, e depende da atividade da OGA. Como não houve alteração da resposta contrátil à PE em vasos de SHR P incubados com PugNAc, possivelmente um mecanismo diferente, envolvendo a menor atividade da OGT, ocorre nestas artérias para a redução da O-GlcNAcilação da eNOS. A desorganização das cavéolas por meio da dextrina causou aumento de contração à PE e redução de potência da ACh em aortas de Wistar NP e SHR NP, porém não houve alteração em aortas de ratas Wistar P e SHR P. A dextrina não alterou o número de cavéolas em artérias de Wistar P e SHR P quando comparado com ratas NP. SHR NP apresentam um reduzido número de cavéolas das aortas em relação a Wistar NP bem como expressão reduzida de Cav-1, p-Cav-1 e CaM. A prenhez não foi capaz de alterar a expressão da Cav-1, CaM e Hsp90 em aorta e leito mesentérico de ratas normotensas e hipertensas. Estes resultados sugerem que a prenhez não altera a expressão das proteínas Cav-1, CaM e Hsp90 e possivelmente a interação com a eNOS em aorta e artérias mesentéricas de ratas normotensas e hipertensas. Em conclusão, entre os mecanismos estudados de modificação pós-traducional da eNOS, a redução da O-GlcNAcilação da eNOS, por mecanismos que envolvem a atividade da OGA e possivelmente da OGT, favoreceria a fosforilação da eNOS e consequente maior biodisponibilidade de NO, contribuindo desta forma para modulação da resposta contrátil da PE nas artérias de ratas P(AU)

Reduction of vascular reactivity to phenylephrine (PE) in aorta of spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higher production and/or higer bioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation, by PI3K/Akt. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOS on serine site leads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this study was to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteries of pregnant rats. The O-GlcNAc-protein content and also the enzymes expression that participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2nd or 3rd branch) of non-pregnant (NP) and pregnant (P), normotensive rats (Wistar) and SHR. Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolae in the aorta and the mesenteric artery in the presence or absence of methyl-ß-cyclodextrin (dextrin, 10 mmol/L) by electronic microscopy. In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 µmol/L) and of the caveolae, using a caveolae disassembler, dextrin (10 or 20 mmol/L), in the reduced vascular reactivity observed in aortas or mesenteric arteries of P rats. We observed that the content of O-GlcNAcylated proteins was decreased in the aorta and in the mesenteric bed of Wistar P and SHR P rats. Although OGT and OGA expression is not altered, OGA activity was increased in the aorta and mesenteric bed of Wistar P rats but was decreased in the aorta and increased in the mesenteric bed of SHP P. Incubation with PugNAc reversed the reduced reactivity to PE in the aorta and mesenteric artery of Wistar P but this effect was not observed in SHR P arteries, demonstrating that OGA appears to play an important role in reducing O-GlcNAcylation of vascular proteins in Wistar P. In arteries incubated with PugNAc, endothelial removal or incubation with L-NAME did not significantly alter reactivity to PE. Together, these results suggest that the greater eNOS activity observed in Wistar P vessels was impaired in the presence of PugNAc, and it depends on OGA activity. As there was no change in the contractile response to PE in SHR P arteries incubated with PugNAc, possibly a different mechanism, involving the lower activity of OGT, occurs in these vessels for the reduction of O-GlcNAcylation of eNOS. Dextrin caused increased contraction of PE and decreased ACh potency in Wistar NP and SHR NP aortas, but there was no change in aortas of Wistar P and SHR P. Dextrin did not alter the number of cavelae in Wistar P and SHR P arteries compared to NP rats. SHR NP showed a lower number of caveolae than to NP Wistar as well reduced expression of Cav-1 and CaM. Pregnancy was not able to alter the expression of Cav-1, CaM and Hsp90 in the aorta and mesenteric bed of normotensive and hypertensive rats. These results suggest that pregnancy does not alter the expression of Cav-1, CaM and Hsp90 proteins and possibly interaction with eNOS in the aorta and mesenteric arteries of normotensive and hypertensive rats. In conclusion, among the studied mechanisms of post-translational modification of eNOS, the reduction of O-GlcNAcylation of eNOS, by mechanisms that involve OGA activity and possibly OGT, would favor eNOS phosphorylation and consequent greater NO bioavailability, contributing in this way for modulation of the contractile response to PE in the arteries of P rats(AU)

Remifentanil alleviates renal ischemia/reperfusion injury by activating PI3K/Akt/eNOS pathway / 第二军医大学学报

Objective: To explore the effect and mechanism of intraoperative remifentanil (RF) on renal ischemia/reperfusion (I/R) injury. Methods: Fifty male C57/BL mice were randomly divided into 5 groups: sham group, I/R group, I/R+LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor) group, I/RRF group and I/R + RF+LY294002 group, with 10 mice in each group. Venous blood or renal tissue samples were collected from the mice of each group 6 h after I/R operation. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected using automatic biochemical analyzer. The expression levels of PI3K/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway-related proteins in renal tissues of mice were detected using Western blotting. The aggregation of inflammatory cells was observed by H-E staining. The expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and IL-10 in renal tissues of mice were detected by enzyme-linked immunosorbent assay. The mRNA expression levels of anti-apoptotic factor Bcl2 and apoptotic factor Caspase-3 in renal tissues were determined by real-time quantitative PCR. Results: Compared with the sham group, the BUN and SCr levels in venous blood were increased in the I/R group, the PI3K expression, phosphorylated-Akt (p-Akt)/Akt ratio and phosphorylated-eNOS (p-eNOS)/eNOS ratio in renal tissues were decreased, the release levels of inflammatory factors (TNF-α, IL-1β, IL-6 and IL-10) were increased, Bcl2 mRNA expression was decreased, and Caspase-3 mRNA expression was increased; and the differences were significant (all P < 0.05). The mice of the I/R group had increased inflammatory cell recruitment in renal tissues. After RF treatment, the mice of the I/R + RF group had decreased levels of BUN and SCr in venous blood, increased PI3K expression, p-Akt/Akt ratio and p-eNOS/eNOS ratio in renal tissues, decreased release levels of inflammatory factors (TNF-α, IL-1β, IL-6 and IL-10), increased Bcl2 mRNA expression, and decreased Caspase-3 mRNA expression; and the differences were significant compared with the mice of the I/R group (all P < 0.05). The inflammatory cell recruitment was decreased in the I/R RF group. Moreover, compared with the mice of the I/RRF group, the mice of the I/RRF LY294002 group had increased levels of BUN and SCr in venous blood, decreased p-eNOS/eNOS ratio in renal tissues, increased IL-1β and IL-6 release, and increased Caspase-3 mRNA expression; and the differences were significant (all P<0.05). The inflammatory cell recruitment was increased in the I/R + RF + LY294002 group. Conclusion: RF exerts protective effect on kidney with I/R injury by alleviating renal inflammation and cell apoptosis through activating PI3K/Akt/eNOS pathway.

Effect of Buyang Huanwu Tang on Cerebral Vasospasm After Subarachnoid Hemorrhage in Rats Based on PI3K/Akt/eNOS Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the molecular mechanism of Buyang Huanwu Tang in improving cerebral vasospasm after subarachnoid hemorrhage. Method:Eighty male Sprague-Dawley rats were randomly divided into sham operation group, model group, Buyang Huanwu Tang low and high dose (13, 26 g·kg-1·d-1) group. According to 10 mL·kg-1, the drug was administered twice a day for 7 days. The subarachnoid hemorrhage model was made by double occipital pool injection method. The neurological function scores of rats in each group were evaluated at 1, 3, 5 and 7 days. The diameter of basilar artery was measured by hematoxylin-eosin (HE)staining. The expressions of phosphp-phosphoinositide 3-kinases(p-PI3K), phosphp-protein kinase B(p-Akt),endothelial nitric oxide synthase(eNOS) and neuronal nitric oxide synthase(nNOS) protein in basilar artery brain tissue were detected by Western blot. The expression of nitric oxide(NO) and endothelin-1(ET-1) in rat cerebrospinal fluid was detected by enzyme-linked immunosorbent assay (ELISA). Result:Compared with sham operation group, the neurological function scores of the model group were significantly decreased (PPPPP-1·d-1) increased the neurological function scores 3 to 5 days after treatment, and the basilar artery diameter was significant increased (PPPPPPPPPConclusion:The protective effect of Buyang Huanwu Tang on cerebral vasospasm after subarachnoid hemorrhage may be related to up-regulation of p-PI3K, p-Akt and eNOS expression in PI3K/Akt/eNOS signaling pathway, thereby increasing NO production.

Telmisartan Inhibits Nitric Oxide Production and Vessel Relaxation via Protein Phosphatase 2A-mediated Endothelial NO Synthase-Ser¹¹⁷⁹ Dephosphorylation

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser¹¹⁷⁹ (p-eNOS-Ser¹¹⁷⁹). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser¹¹⁷⁹ levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser¹¹⁷⁹ levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser¹¹⁷⁹ levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser¹¹⁷⁹ dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.

Effect of astragaloside on cardiac function,inflammatory-related cytokines and endothelial nitric oxide synthase signaling pathway in rats with acute myocardial infarction / 新乡医学院学报

Objective To evaluate the cardio protective effect of astragaloside in rats with acute myocardial infarction and investigate the potential molecular mechanisms.Methods Forty-five Wistar rats were randomly divided into sham operation group,model group and astragaloside group,with 15 rats in each group.The acute myocardial infarction model was prepared by permanently occluding the left anterior descending artery in the model group and the astragaloside group,while the rats in the sham operation group were threaded only.Twenty-four hours after operation,the rats in the astragaloside group received astragaloside 20 mg · kg-1 · d-1 by intraperitoneal injection,once a day for two weeks.The rats in sham operation group and model group received isodose carboxymethylcellulose sodium by intraperitoneal injection,once a day for two weeks.Electrocardiogram was recorded to observe the changes of ST segment before ligation,after ligation immediately,1,3,7,14 d after intervened by astragaloside.Echocardiography was used to detect the left ventricular internal diameter at end-diastole (LVDD),left ventricular internal diameter at end-systole (LVDS),left ventricular end diastohc volume (LVEDV),left ventricular end systolic volume (LVESV),left ventricular fractional shortening (LVFS),left ventricular ejection fraction (LVEF).The levels of tumor necrosis factor alpha (TNF-α),interleukin (IL)-1 β,IL-6,nuclear factor-κB (NF-κB) in serum were measured by enzyme linked immunosorbent assay and the level of nitric oxide(NO) in plasma was measured by reagent kid.The expressions of toll-like receptor(TLR) 2,TLR4,nucleotide binding oligomerization do main(NOD) 1,NOD2,endothelial nitric oxide synthase (eNOS) mRNA were detected by quantitative real-time polymerase chain reaction.Results During the operation,there was 0,4,2 dead rats in the sham operation group,model group and astragaloside group.The ST segment in the model group and astragaloside group was higher than that in the sham operation group after operation immediately,1,3,7,14 d after intervented by astragaloside(P ＜ 0.01).The ST segment in the astragaloside group was lower than that in the model operation group at the time of 1,3,7,14 d after intervented by astragaloside(P ＜ 0.05).Compared with the sham operation group,the LVDD,LVDS,LVEDV,LVESV increased (P ＜ 0.01),LVFS,LVEF decreased (P ＜ 0.01),the levels of TNF-α,IL-1β,IL-6 and the expression of TLR2,TLR4,NOD1,NOD2 mRNA in cardiac muscle tissue were higher in the model group and astragaloside group (P ＜ 0.01),the level of NO was lower (P ＜ 0.05).The level of serum NF-κB in the model group was higher than that in the sham operation group(P ＜0.01),the expression of the eNOS mRNA in cardiac muscle tissue was lower than that in the sham operation group(P ＜ 0.05).There was no significant difference in the level of serum NF-κB and the expression of the eNOS mRNA in cardiac muscle tissue between the astragaloside group and sham operation group (P ＜ 0.05).Compared with the model group,in the astragaloside group the LVDD,LVDS,LVEDV,LVESV decreased (P ＜ 0.05),but LVFS,LVEF increased (P ＜ 0.05),the levels of TNF-o,IL-1 β,IL-6,NF-κB and the expression of TLR2,TLR4,NOD 1,NOD2 mRNA in cardiac muscle tissue were lower(P ＜ 0.05),the level of NO and the expression of the eNOS mRNA in cardiac muscle tissue were higher(P ＜ 0.05).Conclusion Astragaloside has protective effect against to acute myocardial infarction induced injury by decreasing the expression of TLR signal path and activating eNOS signaling.

Effect of Valproic Acid on Nitric Oxide and Nitric Oxide Synthase in Trabecular Meshwork Cell

PURPOSE: To investigate the effects of valproic acid on the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0.25, 0.5, and 1.0 mM valproic acid for 6, 12, and 24 hours. Expression of eNOS mRNA was assessed with Reverse transcription-polymerase chain reaction, and production of NO was assessed with Griess assay. Cellular survival was assessed with the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Valproic acid at concentrations of 0.25, 0.5, 1.0 mM did not affect the cellular survival of HTMC significantly after exposure for 24 hours. Valproic acid increased NO production in a dose- and time-dependent manner. Also, valproic acid increased the degree of eNOS mRNA expression in a dose-dependent manner in HTMC. CONCLUSIONS: Valproic acid increases production of NO and expression of eNOS mRNA in HTMC. Thus, valproic acid might increase aqueous outflow through the trabecular meshwork.

The effects of co-culture of L-iminoethyl ornithine and excessive fluorine on the expression of nitric oxide/endothelial nitric oxide synthase in human neuroblastoma SH-SYSY cells / 中华地方病学杂志

Objective To investigate the effects of endothelial nitric oxide synthase (eNOS) specific inhibitor L-iminoethyl ornithine hydrochloride (L-NIO) for all the fluorine in vitro cultivation of SH-SY5Y cells apoptosis,eNOS mRNA and protein expression,and effect of nitric oxide (NO) content and nitric oxide synthase (NOS) activity change.Methods The SH-SY5Y cells cultured in vitro were divided into control group,low fluoride group,high fluoride group,L-NIO group,low fluoride with L-NIO group,high fluoride with L-NIO group,n =3.The control group added equal volume culture liquid with the experimental group,the concentration of sodium fluoride (NaF) in the low fluoride group and the high fluoride group were 0.2 and 2.0 mmol/L,respectively.The L-NIO group added 3 μmol/L L-NIO,the low fluoride with L-NIO group and the high fluoride with L-NIO group were added to 0.2 mmol/L NaF and 3 μmol/L L-NIO,2.0 mmol/L NaF and 3 μmol/L L-NIO,respectively.The incubation time was 48 h.The expression level of eNOS protein in cells was detected by Western blotting.The expression level of eNOS mRNA in cells was detected by Real-time fluorescence quantitative PCR method.The apoptosis of cells was detected by flow cytometry,NO content and NOS activity in cell culture liquid were detected by nitrate reductase and colorimetric assay.Results Compared with the control group (1.000 ± 0.026),the expression of eNOS protein in the low and high fluoride groups (1.108 ± 0.071,1.349 ± 0.057) increased (P ＜ 0.05),and the L-NIO group (0.755 ± 0.148) decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group (0.802 ± 0.115) decreased (P ＜ 0.05);compared with the high fluoride group,high fluoride with L-NIO group (0.988 ± 0.135) decreased (P ＜ 0.05).Compared with the control group (1.000 ±0.018),the expression of eNOS mRNA in the low and high fluoride groups (1.809 ± 0.099,2.416 ± 0.295) increased (P ＜ 0.05),the L-NIO group (0.609-± 0.077) decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated(P ＜ 0.05),the low fluoride with L-NIO group (1.040 ± 0.034) decreased (P ＜ 0.05);compared with the high fluoride group,high fluoride with L-NIO group (1.233 ± 0.152) decreased (P ＜ 0.05).Compared with the control group [(1.66 ± 0.07)％],the cell apoptosis rate in the low and high fluoride groups [(8.81 ± 0.27)％,(17.60 ± 0.20)％] increased,L-NIO group [(1.03 ± 0.04)％] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group [(6.03 ± 0.10)％] decreased (P ＜ 0.05);compared with the high fluoride group,the high fluoride with L-NIO [(12.12 ± 0.08)％] decreased (P ＜ 0.05).Compared with the control group [(2.773 ± 0.145)μmol/L],the content of NO in the cell culture medium in the low and high fluoride groups [(8.251 ± 1.047),(14.287 ± 1.062) μmol/L] increased (P＜ 0.05),and the L-NIO group [(1.648 ± 0.155) μmol/L] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated (P ＜ 0.05),the low fluoride with L-NIO group [(4.622 ± 0.252) μmol/L] decreased (P ＜ 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(7.899 ± 0.385) μmol/L] decreased (P ＜ 0.05).Compared with the control group [(0.507 ± 0.041) U/ml],the activity of NOS in the cell culture medium in the low and high fluoride groups [(0.772 ± 0.032),(2.258 ± 0.062) U/ml] increased (P ＜ 0.05),and the L-NIO group [(0.346 ±0.015) U/ml] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated (P ＜0.05),the low fluoride with L-NIO group [(0.637 ± 0.026) U/ml] decreased (P ＜ 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(1.161 ± 0.071) U/ml] decreased (P ＜ 0.05).Conclusions Excessive fluoride can lead to overexpression of eNOS protein and mRNA in SH-SY5Y cells,increase of apoptosis rate,increase the content of NO in cell culture and enhance the activity of NOS.After co-culture of L-NIO and fluorine,it can antagonize the damage of fluorine to SH-SY5Y cells and play a certain neuroprotective effect.

AngⅡ/AT1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation / 中国病理生理杂志

AIM:To investigate the mechanism of angiotensinⅡ(AngⅡ)/angiotensinⅡ type 1 receptor (AT1R)pathway activating protein phosphatase 2A(PP2A)which leads to down-regulation endothelial nitric oxide syn-thase(eNOS)phosphorylation level in mesenteric arteries of rats.METHODS: METHODS: The mesenteric arteries of adult male SD rats(weighing 160~180 g;n=90)were isolated under aseptic conditions.Firstly,to determine the effect of angiotensinⅡdown-regulated eNOS(Ser1177)phosphorylation level,the mesenteric arteries were randomly divided into normal control(control)group and AngⅡgroup.The mesenteric arteries in AngⅡgroup were incubated with AngⅡat 1×10 -7mol/L,1×10 -6mol/L and 1×10 -5mol/L for 6 h,12 h and 24 h,respectively.Secondly,to investigate the mo-lecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS(Ser1177)phosphorylation level,the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan(CAN; a specific AT1R blocker)+AngⅡgroup.The mesenteric arteries were pretreated with 1×10 -5mol /L CAN for 1 h,then incubated with 1×10 -7mol/L AngⅡfor 12 h in CAN+AngⅡgroup.The protein levels of eNOS,p-eNOS(Ser1177),PP2Ac,p-PP2Ac(Tyr307)and protein phosphatase 2A inhibitor 2(IPP2A2 )in the arteries were determined by Western blot.The ac-tivity of PP2A in the arteries was detected by PP2A activity kit.RESULTS:Compared with the control group,the protein level of p-eNOS(Ser1177)in the mesenteric arteries was decreased after incubated with AngⅡfor 6 h,12 h and 24 h(P<0.05).The decreasing tendency of p-eNOS(Ser1177)showed concentration-dependently,especially in 12 h and 24 h groups.The expression of eNOS protein showed no significant difference in each group.Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10-7mol/L for 12 h in vitro, the protein levels of p-eNOS(Ser1177)were down-regulated(P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS(Ser1177)(P<0.05);the protein levels of eNOS showed no significant difference in each group.Compared with the control group,the protein levels of p-PP2Ac(Tyr307)and IPP2A2 were decreased after the mesenteric arteries were trea-ted with AngⅡat 1×10 -7mol/L for 12 h(P<0.05).Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307)and IPP2A2 (P<0.05),however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group,the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡat 1× 10-7mol/L for 12 h(P<0.05).Candesarten pretreatment inhibited the activity of PP 2A significantly(P<0.05).CON-CLUSION:AngⅡincreases PP2A activity via AT1R pathway,thus leading to down-regulation eNOS(Ser1177)phospho-rylation level in mesenteric arteries.The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac(Tyr307)and IPP2A2.