Effect of dual blockade of renin-angiotensin system on renal nephrin and vascular endothelial growth factor – A expression in diabetic-hypertensive rats

Objectives: The effects of early renin-angiotensin system (RAS) blockade using angiotensin-converting enzyme (ACE) inhibitor lisinopril and/or angiotensin receptor blocker valsartan on renal nephrin and vascular endothelial growth factor (VEGF)-A gene expression were investigated in diabetic-hypertensive rats. Materials and Methods: Diabetes and hypertension were induced in adult Wistar rats using streptozotocin (45 mg/kg, i.p.) and N?-nitro-L-arginine methyl ester (60 mg/kg/12 h) for 4 consecutive days. Experimental animals were allocated into six groups (n = 6): normal control, diabetic control, diabetic-hypertensive control and lisinopril-, valsartan- and combination-treated diabetic-hypertensive groups (5 mg/kg/drug/day, p.o., for 21 days). Blood glucose, blood pressure, body weight, kidney weight to body weight ratio, serum albumin, creatinine, total protein and urea were measured and recorded every week. Nephrin and VEGF-A gene expression were measured using real-time polymerase chain reaction. Renal nephrin protein was measured using ELISA as well as nephrin immunostaining. Results: Blood pressure was significantly decreased by all treatments (P ? 0.05). All treatments normalised serum albumin and urea. Serum creatinine significantly decreased, while total protein significantly increased (P ? 0.05). Nephrin gene expression had a non-significant decrease in diabetic-hypertensive rats, yet it was statistically increased with individual treatments (P ? 0.05) and normalised with combined treatment. Renal nephrin protein significantly decreased in diabetic-hypertensive rats, normalised by lisinopril and significantly increased by valsartan and combined treatments (P ? 0.05). VEGF-A expression significantly increased in diabetic-hypertensive rats and significantly decreased with lisinopril and valsartan monotherapy and normalised with combined treatment (P ? 0.05). Immunostaining of nephrin also showed an obvious increase in the case of combined treatment. Conclusion: Early dual blockade of RAS in diabetic-hypertensive rats protected against renal damage and improved renal nephrin and VEGF-A gene expression as well as renal nephrin protein expression.

Effect of cassia seed aqueous extract on blood pressure level in n-nitro-l-arginine-methyl ester induced hypertensive rats / 中国组织工程研究

BACKGROUND: The concept of modern diagnosis and treatment of hypertension is to protect target organs, improve clinical symptoms and minimize clinical events through antihypertensive treatment. Cassia seed has an antihypertensive effect that has been confirmed in animal experiment and clinical practice, but whether it can improve the vascular dysfunction, oxidative stress and end organ damage of hypertension remains to be further studied. OBJECTIVE: To evaluate the antihypertensive effect of cassia seed water extract on hypertensive rats induced by N-nitro-L-arginine methyl ester. METHODS: A rat model of hypertension was established by intragastric administration of N-nitro-L-arginine methyl ester to observe the changes of blood pressure and heart rate after the intervention of cassia seed aqueous extract (500 mg/kg per day), once a day, for 4 continuous weeks. Blood pressure and heart rate were measured once a week. At 24 hours after the final administration, serum levels of aspartate aminotransferase, alanine aminotransferase, urea and serum creatinine, and lipid mass spectrometry were detected by an automatic biochemical analyzer. Malondialdehyde, nitric oxide, reduced glutathione levels and angiotensin converting enzyme activity in rat liver and kidney were determined by corresponding assay kits. The expression levels of endothelial nitric oxide synthase and inducible nitric oxide synthase protein and gene in rat kidney tissues were examined by immunohistochemistry as well as real-time quantitative PCR. RESULTS AND CONCLUSION: Compared with the model group, cassia seed aqueous extract treatment significantly reduced mean arterial pressure, diastolic pressure and systolic pressure (P < 0.05) in hypertensive rats, and improved liver and renal markers, lipid distribution and oxidative status. In addition, cassia seed aqueous extract significantly reduced the activity of angiotensin-converting enzyme in the liver and kidney of hypertensive rats (P < 0.05). The protein and mRNA expression levels of endothelial nitric oxide synthase in the kidney of rats treated with cassia seed aqueous extract were significantly higher than those of the model group. In conclusion, cassia seed water extract has shown considerable potential for antihypertension, and its anti-hypertension mechanism includes up-regulation of endothelial nitric oxide synthase expression, antioxidants and inhibition of angiotensin-converting enzymes.

Effect of ASPP2 on regulating GRP78 in apoptosis of placental trophoblast cells induced by L-NAME / 中国药理学通报

Aim: To explore the role of apoptosis stimulating protein 2 of p53 (ASPP2) on L-NAME induced apoptosis of placental trophoblast cells by regulating glucose-regulated protein78(GRP78), and provide a theoretical basis for the study of clinical pregnancy-induced hypertension. Methods: The HTR-8/SVneo human placental trophoblast cells were cultured in vitro, and in the absence (control group) or presence of 100 μmol · L-1 L-NAME (L-NAME group) for 48 h. The effects of L-NAME on placental trophoblast cell apoptosis were tested using flow cytometry and AO/EB assay. The expressions of caspase-12, GRP78 and ASPP2 were detected by Western blot. The ASPP2 interference with adenovirus was used to transfect the cells, and the mRNA expression level of ASPP2 and the protein expression level of GRP78 were detected by qRT-PCR and Western blot, respectively. After treated with 100 (xrnol · L-1 L-NAME for 48 h, the protein expression of caspase-12 and GRP78 was detected by Western blot and immunofluorescence. Results: Compared with control group, the placental trophoblast cell apoptosis significantly increased in L-NAME group (P < 0. 05). AO/EB staining showed that compared with control group, the majority of cells in L-NAME group showed bright orange and the number of late apoptotic cells increased significantly. At the same time, caspase-12, GRP78 and ASPP2 protein expression increased (P < 0. 05, P < 0. 01). After interfering with ASPP2, caspase-12 and GRP78 protein expressions decreased (P < 0. 05). Conclusions: Down-regulation of ASPP2 could decrease GRP78 expression and inhibit L-NAME-induced apoptosis in placental trophoblast cells.

Effects of aqueous leaf extract of Tridax procumbens on contractile activity of corpus cavernosum in N-nitro-l-arginine methyl ester-induced hypertensive male rats / 中西医结合学报

<p><b>OBJECTIVE</b>This study investigated the effects of aqueous leaf extract of Tridax procumbens (ALETP) on contractile activity of corpus cavernosum in N-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive male rats.</p><p><b>METHODS</b>Twenty normal, adult male rats (130-150 g) were divided into four groups of five rats each. Group I (control) was given normal saline (0.6 mL/kg) and group II was given l-NAME (40 mg/kg) for 6 weeks. Groups III and IV also received l-NAME (40 mg/kg) for 6 weeks but were further co-treated with 100 and 200 mg/kg of ALETP, respectively, from week 4 to week 6. All treatments were given orally. Strips of corpus cavernosum from each of the four groups were exposed to increasing concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP) (10-10mol/L) after contraction with phenylephrine (10 mol/L) to test for a dose-response effect. Response to potassium and calcium was also measured after cumulatively adding potassium and calcium (10-50 mmol/L) to potassium- and calcium-free organ chamber. Isometric contractions were recorded through an Ugo Basile data capsule acquisition system.</p><p><b>RESULTS</b>Mean arterial blood pressure was significantly reduced in the ALETP co-treated group compared to the control and l-NAME-only groups (P < 0.05). Cavernosa strips from ALETP co-treated rats exhibited significant inhibition of contraction in response to phenylephrine, potassium chloride, and calcium chloride (P < 0.05). Relaxation in response to Ach and SNP was also significantly impaired in cavernosa strips from the l-NAME-only treated group (P < 0.05), while ALETP co-treated groups showed enhanced percentage relaxation.</p><p><b>CONCLUSION</b>ALETP treatment of l-NAME-induced hypertensive rats promotes a relaxant effect on isolated cavernosa strips. ALETP shows potential in correcting erectile dysfunction in hypertension.</p>

The levels of NO decrease induced apoptosis in human placental trophoblast cells through oxidative stress / 中国药理学通报

Aim To investigate the possible mecha-nisms of the levels of NO decrease induced apoptosis in human placental trophoblast cells. Methods Human placental trophoblast cells ( HTR-8 ) were cultured in 5 ml DMEM-F12 culture medium with 37℃ 5% CO2 . Then, the old culture medium was discarded and re-placed with 10,100,500,1 000 μmol·L-1 L-NAME, and the group without L-NAME was set as the control group, cultured for 48h. The effects of L-NAME on the survival of cells were detected by methylthiazolyldiphe-nyl tetrazolium bromide ( MTT); the content of NO in cells was tested by nitrate reductive enzymatic;trans-mission electron microscopy, flow cytometry analysis and Annexin-V FITC dyeing were used to test the effects of L-NAME on apoptosis in HTR-8 cells;restore Fe3+ colorimetric assay was applied for detection of to-tal antioxidant capacity ( T-AOC ) , xanthine oxidase for detection of superoxide dismutase ( SOD) activity, and thiobarbituric acid colorimetry for determination of content of MDA. Results Compared with the control group, the survival rate of HTR-8 cells and the levels of NO in 100,500,1 000 μmol·L-1 L-NAME group were significantly reduced(P<0.05,P<0.01). Flow analysis and Annexin-V FITC staining showed that L-NAME could induce cell apoptosis in a dose-dependent manner. The number of cell apoptosis was negatively correlated with the content of NO ( r = -0.5210 ) in HTR-8 cells. Transmission electron microscopy results showed that compared with the control group, the ex-perimental group's cell nucleus shape was irregular, nuclear pyknosis in irregular shape, the chromatin ag-glutination or side the collection, mitochondrial swell-ing or enrichment, crest fracture or dissolved, even vanished, forming the vacuole, especially in 100 μmol ·L-1 L-NAME group, the apoptotic bodies obviously appeared. At the same time, T-AOC, SOD levels in HTR-8 cells decreased ( P <0.05 ) , and the MDA content increased ( P<0.05 ) . The number of cell ap-optosis was negatively correlated with the level of T-AOC ( r= -0.3212 ) , SOD ( r= -0.2779 ) in HTR-8 cells , while positively correlated with the content of MDA(r=0.2807). Conclusion Oxidative stress may play an important role in the levels of NO decrease in-duced apoptosis in human placental trophoblast cells.

The Antinociceptive Effect of Intraperitoneally Administered Nonselective Nitric Oxide Synthase Inhibitor on the Rat Formalin Test / 대한통증학회지

BACKGROUND: Nitric oxide (NO) is involved in the transmission and modulation of nociceptive information at the peripheral, spinal cord and supraspinal levels. We conducted this experiment to assess the antinociceptive effects of a nonselective nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), on the modulation of pain in rats subjected to the formalin test. METHODS: Formalin 5% was injected in the right hind paw after intraperitoneal (IP) injection of various doses of L-NAME (0.5 mg/kg, 1.5 mg/kg with and without L-arginine 100 mg/kg, 5.0 mg/kg). The number of flinches was measured. RESULTS: Formalin injected into the rat hind paw induced a biphasic nociceptive behavior. IP injected L-NAME diminished the nociceptive behaviors in a dose-dependent manner during phases 1 and 2. The concomitant injection of L-arginine reversed the antinocipetive effect of L-NAME. CONCLUSIONS: The data demonstrates that a nonselective NOS inhibitor, L-NAME, possesses antinociceptive properties in rats subjected to the formalin test, and the antinociceptive effect of L-NAME is reversed by the concomitant administration of L-arginine.

Neuroprotective Effect of N-nitro-L-arginine Methylester Pretreatment on the Early Stage of Kainic Acid Induced Neuronal Degeneration in the Rat Brain

OBJECTIVE: Kainic acid(KA) enhances the expression of nitric oxide synthase, increases nitric oxide(NO), and thus evokes epileptic convulsion, which results in neuronal damage in the rat brain. NO may stimulate cyclooxygenase type-2 (COX-2) activity, thus producing seizure and neuronal injury, but it has also been reported that KA-induced seizure and neurodegeneration are aggravated on decreasing the COX-2 level. This study was undertaken to investigate whether the suppression of NO using the NOS inhibitor, N-nitro-L-arginine methyl ester(L-NAME), suppresses or enhances the activity of COX-2. METHODS: Silver impregnation and COX-2 immunohistochemical staining were used to localize related pathophysiological processes in the rat forebrain following KA-induced epileptic convulsion and L-NAME pretreatment. Post-injection survival of the rat was 1, 2, 3days and 2months, respectively. RESULTS: After the systemic administration of KA in rats, neurodegeneration increased with time in the cornu ammonis (CA) 3, CA 1 and amygdala, as confirmed by silver impregnation. On pretreating L-NAME, KA-induced neuronal degeneration decreased. COX-2 enzyme activities increased after KA injection in the dentate gyrus, CA 3, CA 1, amygdala and pyriform cortex, as determined by COX-2 staining. L-NAME pretreatment prior to KA-injection, caused COX-2 activities to increase compared with KA- injection only group by 1day and 2days survival time point. CONCLUSION: These results suggest that L-NAME has a neuroprotective effect on KA-induced neuronal damage, especially during the early stage of neurodegeneration.

Nitric Oxide-Mediated Neurotoxicity after Excimer Laser Photorefractive Keratectomy

The aim of this study was to evaluate neurotoxicity of Nitric oxide(NO) on cornea after excimer laser photorefractive keratectomy(PRK). PRK was performed on rabbit eyes. According to the time table, tear samples were collected with microcapillary tubes and corneal sensitivity was measured with a Cochet-Bonnet esthesiometer. No generation in the tear fluid was analyzed. To demonstrate NO Synthase(NOS), immunohistochemical localization was performed on frozen sections from rat eyeball tissue. Western blot analysis was used for detection of peroxynitrite, powerful oxidant of NO. NO generation was increased and reached to a maximum value(0.69+/-0.22micrometer/microgram) after 96 hours of PRK, as compared with in normal subjects(Mean: 0.30+/-0.08micrometer/microgram) and was not increased in the treated group with topical application of Ng-nitro-L-arginine methyl ester(L-NAME), a competitive inhibitor of constitutive NOS(cNOS) and inducible NOS(iNOS). Corneal sensitivity decreased below pretreatment levers after three postoperative days, but it was not observed in the L-NAME applied group. We have confirmed that a very strong iNOS and BNOS immunoreactivity was present in corneal keratocytes. Western blot analysis identifed the bands of nitrotyro-sine-proteins suggesting in vivo peroxynitrite toxicity. Our results suggested that NO generated from the enzyme after PRK decreased corneal sensitivity by damaging corneal sensory nerve through the NO and iths oxidant peroxitrite. Therefore topical application of a NOS inhibitor may be effective in maintaining corneal sensitivity.

Role of nitric oxide in proliferation and secretion effect of vascular endothelial cells induced by vascular endothelial growth factor / 中国病理生理杂志

AIM: To investigate the role of nitric oxide in proliferation and secretion of vascular endothelial cells induced by vascular endothelial growth factorr (VEGF). METHODS: The in vitro cultured vascular endothelial cells of rabbit aorta were divided into control group, VEGF-treated group and VEGF+L-NAME treated group, the absorbance (A) value of vascular endothelial cells, endothelin-1(ET-1) and von Willebrand factor (vWF) in the supernatant were examined by WST-1 assay, radioimmunoassay and ELISA. RESULTS: The A value in VEGF and VEGF+L-NAME treated group were higher than that in control group (P