Endogenous nitric oxide synthase inhibitor increase skeletal muscle contractility and mitochondria biosynthesis in 4-week running rats

AIM: To observe the effect of endogenous nitric oxide synthase(NOS) inhibitor asymmetric dimethylarginine(ADMA) and its signaling pathways on NO levels and skeletal muscle contractility in 4-week running rats. METHODS: The 4 weeks running rat model was established. The twitch tension, tetanic tension and the fatigue test of soleus muscle induced by electrical stimulation ex vivo were detected. The ATP content, mitochondrial DNA levels and the mRNA expression of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α), nuclear respiratory factor(NRF) were measured to reflect the mitochondrial function and biosynthesis in the skeletal muscle. Serum ADMA concentration was detected by high performance liquid chromatography. The endogenous ADMA enzymes PRMT1 and 2 subtypes of ADMA metabolism enzyme DDAH, 3 subtypes of NOS protein expression in the skeletal muscle were determined by Western blot. NOS activity and nitric oxide(NO) content were analyzed by colorimetric method. RESULTS: Compared with normal control group, the twitch tension, tetanic tension and the anti-fatigue capability of soleus muscle in running group were significantly enhanced, ATP content, mitochondrial DNA content and the mRNA expression of PGC-1α and NRF were significantly increased(P<0.01). In addition, the protein expression of constitute type NOS(cNOS) and NOS activity were significantly increased(P<0.01), but the increase in NO content was relatively smaller in soleus muscle in exercise group(P<0.05). Moreover, serum ADMA concentration in running group was increased, while the DDAH2 expression in skeletal muscle was decreased.CONCLUSION: Short-term endurance exercise enhances the twitch tension, tetanic tension and fatigue resistance of soleus muscle. The mechanism may be that increased cNOS expression feedbacks to increase ADMA concentration, thus maintaining the increase in NO synthesis at a relatively low level, and resulting in promoting skeletal muscle mitochondria biosynthesis and mitochondrial function.     

Effects of carvedilol on metabolic mechanism of endogenous NOS inhibitor in human umbilical vein endothelial cells treated with oxidized free radical

AIM: To observe the effects of oxidized free radical (OFR) on dimethylarginine dimethylaminohydrolase (DDAH) activity and concentration in human umbilical vein endothelial cells (HUVECs), and to investigate the metabolic mechanism of endogenous NOS inhibitor and the role of carvedilol. METHODS: HUVECs of 3-6th passage, cultured with modified Jaffes method, were divided into three groups: (1) cells cultured with equivalent DMEM medium as control; (2) OFR intervention groups, 0.01 mmol/L, or 0.1 mmol/L OFR was added respectively; (3) drug intervention groups: 0.1 mmol/L OFR plus 10 μmol/L carvedilol. ADMA, nitric oxide (NO), endothim (ET), L-citrulline concentrations and the activity of NOS in conditioned medium were measured after 24 h exposure. ADMA concentration in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. RESULTS: Compared with control group, ADMA and ET concentrations were increased, while the level of NO and the activity of NOS decreased and relevant to the concentrations of OFR. We assayed DDAH activity by determining L-citrulline formation from ADMA. The concentration of L-citrulline was decreased, while the DDAH expression had no obvious change. With the role of carvedilol, ADMA, ET concentrations were decreased, while the level of NO, L-citrulline and the activity of NOS increased. CONCLUSION: Endothelial dysfunction induced by OFR is associated with the increase in ADMA concentration and reduction of DDAH activity, but not DDAH expression. Carvedilol promotes the degradation of ADMA through increasing activity of DDAH and improving endothelium function.     

Hyperlipidemia induces endothelial dysfunction in rats by increasing oxidative and nitrative stresses

AIM: To observe the effect of high-fat diet on the endothelial functions in rats. METHODS: Male SD rats (8 week old) were randomly divided into 2 groups to receive a regular or a high-fat diet, respectively. After 14 weeks, the animals were anesthetized and caval blood was collected to determine the lipid profile, fasting blood glucose and insulin levels. The aorta of the animals was isolated to observe the response of vasorelaxation to endothelium-dependent vasodilator acetylcholine (ACh) and endothelium-independent vasodilator SNAP(S-nitroso-N-acetyl penicillamine). In addition, the production of nitric oxide(NO) and superoxide, the expression of gp91^phox, and the activity of NO synthase(NOS) in the aortic tissues were measured. RESULTS: The lipid profile, the levels of fasting blood glucose and insulin were significantly increased in the plasma of rats fed with high-fat diet. A dose-dependent vasorelaxation to ACh was reduced, and the expression of gp91^phox, the production of superoxide and the activity of iNOS were enhanced in the aortic tissues of the rats fed with high-fat diet. CONCLUSION: High-fat diet induces endothelial dysfunction by increasing the oxidative and nitrative stresses.     

Asymmetric dimethylarginine and atherosclerosis

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). It reduces nitric oxide production. ADMA is correlated with the risk factors of atherosclerosis, such as hypercholesterolemia,hypertension and hyperglycaemia. Accumulating evidence suggests that a derangement of the NOS pathway plays a critical role in atherogenesis and ADMA may participate in the process.     

Effect of asymmetric dimethylarginine on blood pressure and renal function in sympathectomized spontaneously hypertensive rats

AIM: To investigate the effect of asymmetric dimethylarginine (ADMA) on blood pressure and renal function in sympathectomized spontaneously hypertensive rats (SHR). METHODS: The neonatal SHR were sympathectomized by guanethidine monosulfate. Systolic and diastolic blood pressure was monitored by tail-cuff method. Urine excretion of norepinephrine (NE) was measured by metabolic cage collection. The levels of ADMA and NE in the kidneys were analyzed by HPLC. Nitric oxide (NO) content in SHR kidney was detected by colorimetry. The protein expression of endothelial nitric oxide synthase (eNOS) was determined by Western blot. Glomerular filtration rate (GFR) was examined to evaluate the renal function. RESULTS: Neonatal chemical sympathectomy produced significant decreases in urinary NE excretion, renal NE and ADMA contents, and systolic and diastolic blood pressure compared with the sympathetically intact SHR (P<0.05). Moreover, the level of NO content and protein expression of eNOS in the kidneys were significantly increased (P<0.05). However, no significant difference was observed in microalbumin, urinary sodium excretion and GFR between the sympathetically intact SHR and the sympathectomized SHR. CONCLUSION: Inhibition of sympathetic nervous system affects blood pressure by reducing the release of ADMA and NE, and increasing NO synthesis and eNOS expression. The regulation of ADMA generation by sympathetic nervous system does not influence renal function.     

Advances in PDE5 inhibitors and diseases related to cGMP signaling pathway

Phosphodiesterase 5 （PDE5） inhibitors， as drugs to dilate the corpora cavernosa of the penis through sexual stimulation， are used to treat erectile dysfunction， and are also approved for treating pulmonary arterial hypertension. With the in-depth studies， PDE5 inhibitors have been approved to treat a variety of diseases， such as cardiovascular diseases， Alzheimer disease， etc， but there is currently no overall review on clinical applications of PDE5 inhibitors. As known， the classic pathway of PDE5 inhibitors promotes cyclic guanosine monophosphate （cGMP） production. However， reports on downstream pathways regulated by cGMP are still unclear. This review summarized the research and clinical application progress of PDE5 inhibitors in different diseases， and the pharmacological bases of PDE5 inhibitors through cGMP signaling pathway are discussed.     

Role of nitric oxide in aortic contractile function of renal hypertensive rats

AIM:To investigate the changes of aortic contractile function in renal hypertension with two-kidney, one-clip (2K1C) rats and its interrelation with nitric oxide (NO). METHODS:Animals were divided into 5 groups, sham operation group, 2K1C group, captopril group, L-arginine group and L-NAME group. At 4th week after operation, isometric tension changes of aortic rings were recorded, aortric cGMP content were also measured. RESULTS:Phenylephrine, acetylcholine, angiotensin Ⅱ and high potassium chloride solution-induced contraction was significantly increased, and aortric cGMP content was lowered in aortic rings from 2K1C rats compared with rings from sham rats. These changes were abolished in 2K1C rats treated with captopril. L-arginine partially reversed the change of aortic contractibility of 2K1C rats, and elevated aortic cGMP content. In 2K1C rats treated with nitric oxide synthase inhibitor L-NAME, aortric cGMP content were decreased further, but phenylephrine-induced contractile response were unaffected. After blockade of NO production with L-NAME, the maximal responses of aortric rings relaxation to sodium nitroprussidum were not significantly different in all five groups. CONCLUSION:These results indicate that the deficiency of nitric oxide production and the increase in the contractive factor in vascular tissue may contribute to changes in aortic contractile function of 2K1C rats.     

Alterations of nitric oxide synthase in cardiac sarcoplasmic reticulum from rats with myocardial calcification

AIM: To study alterations of nitric oxide synthase (NOS) in cardiac sarcoplasmic reticulum from rats with myocardial calcification, and to explore the mechanism of inhibition of SR function in the rats with myocardial calcification. METHODS: Compared with control, myocardial calcium content in the 6 weeks increased by 408%（Ｐ＜0.01）, the NO production, NOS activity and NOS protein expression in the SR with myocardial calcification increased versus control（Ｐ＜0.01）.Myocardial calcium content was not alterations significantly, but the NOS/NO pathway in the SR was up-regulated slightly in the 2 weeks. RESULTS: Compared with control, myocardial calcium content in the 6 weeks increased by 408%(P＜0.01), the NO production, NOS activity and NOS protein expression in the SR with myocardial calcification increased versus control(P＜0.01).Myocardial calcium content was not alterations significantly, but the NOS/NO pathway in the SR was up-regulated slightly in the 2 weeks. CONCLUSION: The up-regulated NOS/NO system in the SR with myocardial calcification is the important mechanism of function inhibition of the SR.     

Effects of protein kinase C inhibitor,chelerythrine chloride,on nitric oxide synthase expression and nitric oxide content in spinal cord of rats with inflammatory pain induced by formalin

AIM: To study the effects of protein kinase C (PKC) inhibitor， chelerythrine chloride (CH)， on nociceptive response, nitric oxide synthase (NOS) expression and nitric oxide (NO) content in spinal cord of rats with inflammatory pain. METHODS: Inflammatory pain was induced by formalin injection into right hind paw. NADPH-d histochemistry was used to investigate the changes of NOS expression. Nitrate/nitrite (NO2-/NO3-) was assayed to represent NO content. RESULTS: Compared with the control group, the number of NADPH-d positive cells increased significantly in the superficial layer (LaminaeⅠ-Ⅱ) of the spinal cord dorsal horn and the grey matter surrounding the central canal (Laminae Ⅹ) in rats with inflammatory pain, the reactive degree of NADPH-d positive soma and fibers and NO content of the lumbar enlargement of spinal cord also increased significantly. Intrathecal injection of CH inhibited the spontaneous pain response in the second phase induced by formalin injection, and prevented the increases in the number and reactive degree of NADPH-d positive cells, as well as NO content of the lumbar enlargement of spinal cord. CONCLUSION: It is suggested that the activation of PKC promotes NOS expression and NO production in the nociceptive neurons of spinal cord during formalin-induced inflammatory pain.     

Behavior changes of learning and memory related to the levels of NO and nNOS in brain of rats with acute alcoholism

AIM: In order to investigate the molecular mechanism of alcoholism acting on learning and memory, the dysfunction of learning and memory function was observed and the content of nitric oxide (NO) and neuronal nitric oxide synthase (nNOS) were determined in rats with acute alcoholism.METHODS: The mature male Sprague-Dawley rats were randomly divided into two groups. The experimental group animals were intraperitoneally administered with ethanol. The control group animals were injected with saline in the same way. The tests of learning and memory were performed at Y-maze after 6 h. Then brains were removed and the content of NO in brain tissue and nNOS expression in hippocampus CA1, corpus striatum were determined, respectively.RESULTS: (1) The training times to reach qualifying standards of Y-maze in experimental group (34.33±13.04) were higher than those in control group (27.50±8.79, P<0.05). (2) The content of NO in experimental group (23.09±9.60) in hippocampus CA1 was significantly increased (P<0.01) compared with that in control group (8.46±5.67). The content of NO in experimental group (19.46±8.25) in corpus striatum was also higher than that in control group (8.22±4.46, P<0.01). (3) The levels of nNOS expression in experimental group (34.33±13.04) in hippocampus CA1 increased significantly (P<0.05) compared with that in control group (27.50±8.79). nNOS positive neurons in experimental group (18.22±7.47) in corpus striatum were also higher than those in control group (10.15±4.24, P<0.05).CONCLUSION: These findings suggest that the mechanism of ethanol neurotoxicity may be partly involved in the signal pathway of NOS and NO in the brain.     

Effect of lentinan on myocardial impairment in diabetic rats

AIM:To study the protective effect of lentinan against myocardial impairment in diabetic rats.METHODS:Morphology of myocardium from streptozocin induced diabetic rats treated with lentinan was observed under light microscopy(LM) and transmission electron microscopy(TEM). Activity of superoxide dismutase(SOD), nitric oxide synthase (NOS) and contents of malondialdehyde (MDA) and nitric oxide (NO) were detected biochemically in myocardial homogenate.RESULTS:Vacuolar degeneration, local lysis of myocardium and interstitial proliferation under LM and expansion of mitochondria, shortening of mitochondrial crest, lysis of myofibril and proliferation of interstitial collogenous fiber under TEM were observed. The activity of SOD decreased and the activity of NOS, the contents of NO, MDA increased, but the morphological change became slight in LNT-treatment group. Activity of SOD increased while activity of NOS and contents of MDA, NO decreased in LNT-treated rats compared with diabetic rats.CONCLUSION:LNT protectes diabetic myocardium, and the anti-lipid peroxidation and decreasing of NO level may be involved in it.     

Asymmetric dimethylarginine protects PC12 cells against glutamate-induced excitotoxic damage

AIM: To investigate the effects of asymmetric dimethylaoyoinine (ADMA) on glutamate-induced PC12 cell damage and its mechanisms. METHODS: PC12 cells were treated with different concentrations of glutamate as an in vitro excitotoxic trauma model. The cell viability was measured by MTT assay. Glutamate cytotoxicity was evaluated by lactate dehydrogenase (LDH) release assay. Intercellular reactive oxygen species (ROS) was detected by dihydrorhodamine123 (DHR) staining and flow cytometric (FCM) analysis. Nitric oxide synthase (NOS) activity and nitric oxide (NO) production were detected by using commercial kits with a spectrophotometer. RESULTS: Glutamate at concentrations of 1 mmol/L to 6 mmol/L dose-dependently decreased PC12 cell viability. Pretreatment 30 min with ADMA prior to administration of glutamate significantly attenuated the inhibition of cell viability, LDH release and ROS accumulation induced by glutamate. Pretreatment with ADMA significantly inhibited the increases in NOS activity and NO production caused by glutamate. CONCLUSION: ADMA obviously protects PC12 cells against glutamate-induced excitotoxicity by inhibiting NOS activity, overproduction of NO and accumulation of intracellular ROS.     

Protective effect of curcumin on myocardium in diabetic rats

AIM: To study the effects of curcumin (Cur) on diabetic cardiomyopathy (DCM) in rats. METHODS: Male Wistar rats (n=75) were divided into control group and diabetes model group, in which the rats were fed with high-fat diet and then intraperitoneally injected with streptozotocin (STZ, 40 mg/kg). Fasting blood glucose was measured 72 h and 1 week after STZ injection. The diabetic rats were diagnosed when sustained fasting blood glucose levels ≥ 11.6 mmol/L. The diabetic rats were randomly divided into DCM group, DCM+Cur 100 mg/kg group and DCM+Cur 200 mg/kg group. After treatment for 16 weeks, glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) level were measured, and the level of cardiac troponin I (cTnI) in the serum was determined by enzyme-linked immunosorbent assay. The protein expression of protein kinase C (PKC) was detected by Western blotting. RESULTS: Curcumin significantly decreased the blood glucose level, increased the body weight, inhibited MDA content and up-regulated the GSH-Px activity in the diabetic rats. Furthermore, curcumin treatment inhibited the diabetes-induced protein expression of PKC. CONCLUSION: Curcumin may have a protective effect on diabetic cardiomyopathy by attenuating oxidative stress.     

Alleviation of aortic vascular dysfunction in STZ/HFD-induced type 2 diabetic rats by nFGF1

AIM: To investigate the protective effect of non-mitogenic fibroblast growth factor 1 (nFGF1) on the aortic vascular function in streptozotocin (STZ)/high-fat diet (HFD)-induced type 2 diabetic rats and its underlying mechanisms. METHODS: Five-week-old male SD rats (n=30) were randomly divided into 3 groups (n=10 in each group), including normal control group, type 2 diabetic group and nFGF1 treatment group (type 2 diabetic rats were intraperitoneally injected with 0.5 mg/kg nFGF1 every other day for 4 weeks). After the rats were sacrificed, blood glucose, cholesterol and triglyceride levels, aorta diastolic function and superoxide dismutase (SOD) level in the aorta of each group were measured. Besides, the protein levels of cyclooxygenase-2 (COX-2), phosphorylated extracellular signal-regulated kinase (p-ERK) and endothelial nitric oxide synthase (eNOS) in the aorta were determined by Western blot. RESULTS: nFGF1 markedly lowered blood glucose, cholesterol and triglyceride levels, enhanced aorta SOD activity and upregulated protein level of eNOS in the type 2 diabetic rats. Furthermore, the increased protein levels of COX-2 and p-ERK in the type 2 diabetic rats were largely abrogated by nFGF1. CONCLUSION: nFGF1 effectively attenuates aortic vascular dysfunction in the type 2 diabetic rats, which may be associated with decreasing blood glucose, cholesterol and triglyceride levels, reducing inflammation and oxidative stress response, and activating eNOS signaling pathway.     

Effects of extract of gingko biloba on the lipid metabolism and the function of macrophages from diabetic rats

AIM: To study effect of extract of ginkgo biloba (EGb) on the lipid metabolism and the function of macrophages from diabetic rats.METHODS: Sprague-Dauley rats were divided into four groups: normal control group, high-fat group, diabetic group and EGb treatment group.At the end of experiment, the rats were sacrificed, the blood glucose, blood insulin and serum lipid were measured.The activity of superoxide dismutase (SOD), content of malondialdehyde (MDA), nitric oxide (NO) in alveolar macrophages (AM) and peritoneal macrophages (PM) were assayed.In addition, peroxisome proliferator activated receptor γ (PPARγ), CD36 mRNA expression in AM was measured by RT-PCR.RESULTS: The concentration of the blood glucose, blood insulin and total cholesterol (TC), total triglycerides (TG), low-density lipoprotein- cholesterol (LDL-C) in blood increased significantly in type 2 diabetic group.The supplement of EGb decreased blood glucose, blood insulin and TC, TG, LDL-C levels.The activity of SOD decreased, while the content of NO, MDA increased in the diabetic macrophages, the activity of SOD became increased, but the content of NO and MDA decreased in EGb-treated group.The mRNA expression level of CD36 and PPARγ in alveolar macrophages from diabetic group increased, while expression level of CD36 and PPARγ mRNA in EGb treated rats continued to rise.CONCLUSIONS: EGb corrected insulin resistance and ameliorated disturbance of lipid metabolism caused by type2 diabetes in rats.Adjustment of PPARγ and CD36 mRNA expression of as well as reduction of lipid peroxidation and NO level may be involved in this process.     

Screening of lentiviral vectors carrying effective siRNA targeting S1P2 gene

AIM:To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2(S1P2) gene expression in the primarily cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).METHODS:SHR and SD rats (n=5 each) were used for primarily culturing corpus cavernosum smooth muscle cells.The cells were randomly divided into 6 groups:SHR siRNA-1,SHR siRNA-2,SHR siRNA-3,SHR GFP,SHR control (SHR non-transfection group),and SD control (SD rat control group).Each group had 5 samples with 1.0×10⁵ cells of each sample.At 72 h after transfection (MOI=60) with lentiviral vectors carrying S1P2 siRNA into the SHR corpus cavernosum smooth muscle cells,the expression of GFP was observed under fluorescence microscope.The protein expression of S1P2,ROCK1,ROCK2 and eNOS in the corpus cavernosum smooth muscle cells,and the mRNA expression of S1P2,ROCK1 and ROCK2 were determined by by Western blot and RT-PCR.RESULTS:The transfection efficiency of the corpus cavernosum smooth muscle cells in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were>80%.Compared with SHR control group,the mRNA levels and the protein expression of S1P2,ROCK1 and ROCK2 in SHR GFP group showed no remarkable changes,while those in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SD control groups were significantly lower than those in SHR control group (P<0.05).The protein expression of eNOS in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were not significantly changed as compared with SHR control group,but that in SD control group was significantly higher than that in SHR control group.CONCLUSION:Three groups of siRNA lentiviral vectors targeting S1P2 inhibit the expression of S1P2 in the corpus cavernosum smooth muscle cells of SHR,and by silencing the S1P2 expression,the expression of ROCK1 and ROCK2 is inhibited.Among them,siRNA-1 has the highest inhibitory efficiency.     

Changes of nitric oxide synthase and nitric oxide in lung of smoking rats

AIM:To observe the changes of iNOS and eNOS in lung tissue and NO in bronchoalveolar lavage fluid (BALF) in smoking rats.METHODS:80 Wistar rats were divided into control, smoking group, L-NIL group and L-NAME group (rats were exposed to smoke and injected (i.p.) with selective iNOS inhibitor L-NIL or NOS inhibitor L-NAME). iNOS and eNOS protein levels in whole lung were detected by immunohistochemical staining, and NOS mRNA was quantified using RT-PCR. In addition, NO₂^-/NO₃^- was determined using Griess assay.RESULTS:The expression of iNOS mRNA and protein in smoking rats increased, the expression of eNOS mRNA and eNOS protein decreased, and the total cell count and the level of NO₂^-/NO₃^-in BALF increased(P＜0.05). In vivo, L-NIL reduced the total cell count and NO₂^-/NO₃^- in BALF (P＜0.05), while L-NAME had no effect on them.CONCLUSION:Cigarette smoke increased expression of iNOS mRNA and protein and decreased expression of eNOS mRNA and protein. The large amount of NO generated by iNOS may amplify inflammation in lung tissue.     

Effect of curcumin derivative B06 on liver from type 2 diabetic rats

AIM:To observe the protective effect of curcumin derivative B06 on the liver from the rats with hyperlipidemia and type 2 diabetes mellitus. METHODS:Male Sprague-Dawley rats (n=35) were divided randomly into 5 groups: normal control group, high-fat group, high-fat+B06-treated group, diabetic group and diabetic +B06-treated group. After fed with a high-fat diet for 4 weeks, the rats in the later 2 groups were injected with streptozotocin intraperitoneally to induce type 2 diabetes mellitus. The rats in B06-treated groups were given B06 by gavage at a dose of 0.2 mg· kg-1·d-1 for 8 weeks. After the treatment, the morphology of the liver was observed under light and transmission electron microscopes. The protein expression of AMP-activated protein kinase α (AMPKα) and phosphorylated AMPK α (p-AMPKα) was detected by Western blotting. RESULTS:Fatty degeneration, hepatocellular necrosis, inflammatory cell infiltration and hyperplasia of fibrous tissue were observed in the liver from the rats in high-fat group and diabetic group,and were relieved after B06 treatment. The protein expression of p-AMPKα was decreased in the liver of the rats in diabetic group and high-fat group, and it was increased in the liver of the high-fat and diabetic rats in B06-treated group. CONCLUSION:Curcumin derivative B06 exerts a protective effect on the liver in type 2 diabetic rats, and the increased expression of p-AMPKα may be involved in the mechanism of protection.     

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.