NF-kappaB inhibitors stabilize the mRNA of high-affinity type-2 cationic amino acid transporter in LPS-stimulated rat liver

BACKGROUND: Induction of inducible nitric oxide synthase (iNOS) results in nitric oxide (NO) overproduction during endotoxemia. Cellular uptake of L-arginine, modulated by the isozymes of type-2 cationic amino acid transporters (CAT), including CAT-2, CAT-2A and CAT-2B, has been reported to be a crucial factor in the regulation of iNOS activity. We sought to elucidate the expression of CAT-2 isozymes and the role of nuclear factor-kappaB (NF-kappaB) in this expression in lipopolysaccharide (LPS)-treated rat liver. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous (i.v.) injections of normal saline (N/S), LPS, LPS preceded by an NF-kappaB inhibitor (PDTC, dexamethasone or salicylate) or an NF-kappaB inhibitor alone. After injection, rats were sacrificed at different times and enzyme expression and liver injury were examined. Hepatic and systemic NO production were also measured. RESULTS: CAT-2, CAT-2A and CAT-2B were constitutively expressed in un-stimulated rat liver. LPS stimulation not only significantly increased iNOS mRNA and NO concentrations but also decreased the mRNA concentrations of CAT-2 and CAT-2B, but not CAT-2A, in a time-dependent manner. LPS-induced hepatic and systemic NO overproduction was associated with hepatocellular injury. Pre-treatment with NF-kappaB inhibitors significantly attenuated LPS-induced iNOS induction as well as CAT-2/CAT-2B mRNA destabilization, which was associated with significant inhibition of NO biosynthesis and less liver injury. CONCLUSION: NF-kappaB inhibitors stabilize CAT-2 and CAT-2B mRNA in LPS-stimulated rat liver. The hepatic CAT-2/CAT-2B pathway may be a constitutive part of cytoprotective mechanisms against sepsis.     

Platonin attenuates LPS-induced CAT-2 and CAT-2B induction in stimulated murine macrophages

BACKGROUND: Platonin, a cyanine photosensitizing dye, is a potent immunomodulator that suppresses acute inflammation. Platonin not only inhibits interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha production but also improves circulatory failure in septic rats. In addition, platonin reduces plasma nitric oxide (NO) formation during sepsis. However, the effects of platonin on inducible NO synthase (iNOS) and cationic amino-acid transporter (including CAT-2, CAT-2 A, and CAT-2B) expressions during sepsis remain uninvestigated. METHODS: Five groups of confluent murine macrophages (RAW264.7 cells) were randomly allocated to receive a 1-h pretreatment of one of five doses of platonin (0.1 microM, 1 microM, 10 microM, 100 microM, or 1000 microM) followed by lipopolysaccharide (LPS; 100 ng ml(-1)). For negative, positive, and platonin control, three other groups of cell cultures were randomly allocated to receive phosphate-buffered saline, LPS, or platonin (1000 microM). The cultures were harvested after exposing them to LPS for 18 h or a comparable duration in those groups without LPS. NO production, L-arginine transport, and expression of the relevant enzymes were then evaluated. RESULTS: Platonin significantly attenuated LPS-induced up-regulation of iNOS expression and NO production in stimulated murine macrophages in a dose-dependent manner. Platonin also significantly inhibited up-regulation of CAT-2 and CAT-2B expression as well as L-arginine transport in LPS-stimulated murine macrophages in a dose-dependent manner. In contrast, CAT-2 A expression in murine macrophages was not affected by LPS and/or platonin. CONCLUSIONS: Platonin attenuates NO production and L-arginine transport in LPS-stimulated murine macrophages possibly through inhibiting iNOS, CAT-2, and CAT-2B expression.     

Microdialysis for measurement of hepatic and systemic nitric oxide biosynthesis in septic rats

BACKGROUND: We sought to compare two techniques, microdialysis and repeated blood withdrawal, for serial assessment of hepatic and systemic nitric oxide (NO) biosynthesis in septic rats. METHODS: Rats were randomly allocated to either microdialysis or blood withdrawal groups. Two microdialysis probes, one in liver and the other in right atrium, were placed in rats in the microdialysis group. Half of the rats from each group were then given lipopolysaccharide (LPS) to induce NO production. The other half of the rats from each group were injected with vehicle (normal saline) to serve as controls. In the microdialysis group, dialysate (30 microl) was collected every 30 min. In the blood withdrawal group, 0.3 ml of blood was drawn every 30 min. Sampling was performed up to 6 h after injection of LPS or vehicle. Hemodynamics, hepatic and systemic NO concentrations, and iNOS expression in harvested liver tissues were assayed. RESULTS: Repeated blood withdrawal by itself caused a significant decrease in blood pressure and induced hepatic iNOS expression. Microdialysis, on the contrary, reliably detected LPS-induced NO production without resulting either in hemodynamic changes or in iNOS induction in liver tissue. CONCLUSIONS: Microdialysis provides serial measure of hepatic and systemic NO concentrations in LPS-treated rats without the need for removal of tissue.     

Ketamine suppresses endotoxin-induced NF-kappaB activation and cytokines production in the intestine

BACKGROUND: Ketamine has been advocated for anesthesia in endotoxemic and other severely ill patients because it is a cardiovascular stimulant. However, ketamine also suppresses serum levels of endotoxin-induced tumor necrosis factor-alpha, and reduces mortality in mice in endotoxin shock. Our study was designed to investigate the protective effect of ketamine on the endotoxin-induced proinflammatory cytokines and nuclear factor kappa B (NF-kappaB) activation in vivo. METHODS: Adult male Wistar rats were randomly divided into six groups: saline controls; rats challenged with endotoxin (5 mg kg(-1)) and treated with saline; challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (0.5 mg kg(-1)); challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (5 mg kg(-1)); challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (50 mg kg(-1)); and saline injected and treated with ketamine (50 mg kg(-1)). TNF-alpha, IL-6 and NF-kappaB were investigated in the tissues of the intestine (jejunum) after 1, 4 and 6 h. RESULTS: Endotoxin caused transient production of TNF-alpha and IL-6 and activation of NF-kappaB in the intestine at peak times of 1, 4 and 1 h, respectively. Ketamine 0.5 mg kg(-1) suppressed endotoxin-induced TNF-alpha elevation and inhibited NF-kappaB activation in the intestine; a dose of 5 mg kg(-1) was required to inhibit IL-6. CONCLUSION: Ketamine suppresses the production of proinflammatory cytokines such as TNF-alpha and IL-6 in the intestine, possibly via inhibition of NF-kappaB.     

Acupuncture stimulation of ST-36 (Zusanli) significantly mitigates acute lung injury in lipopolysaccharide-stimulated rats

BACKGROUND: We sought to investigate the potential therapeutic effects of acupuncture stimulation of ST-36 (Zusanli) on endotoxemia-induced acute lung injury in lipopolysaccharide (LPS)-stimulated rats. METHODS: Sixty rats were randomized into six groups (n = 10): (i) lipopolysaccharide (LPS) control group, (ii) normal saline (N/S) control group, (iii) LPS plus ST-36 group, (iv) N/S plus ST-36 group, (v) LPS plus sham point (Sham) group, and (vi) N/S plus Sham group. Manual acupuncture stimulation of ST-36 (designated as 'ST-36') or a 'non-acupoint' (designated as 'Sham') was performed in lightly immobilized rats for 30 min. Then, LPS injection was employed to induce sepsis. Rats were killed at 6 h after LPS injection and lung injury, nitric oxide (NO) biosynthesis and inducible NO synthase (iNOS) expression were assayed. RESULTS: Significant lung injury, pulmonary iNOS expression and systemic and pulmonary NO biosynthesis were noted in the LPS groups. Rats in the LPS plus Sham group had lung injury, pulmonary iNOS expression, systemic and pulmonary NO biosynthesis similar to those observed in the LPS group. However, the degree of lung injury, pulmonary iNOS expression and pulmonary NO biosynthesis, but not systemic NO biosynthesis, were significantly attenuated in the LPS plus ST-36 group as compared with those in both the LPS group and the LPS plus Sham group. CONCLUSION: Acupuncture stimulation of ST-36 may be effective as a prophylaxis measure against sepsis. However, results from this study do not support the use of acupuncture for the treatment of sepsis.     

Induced nitric oxide impairs relaxation but not contraction in endotoxin-exposed rat pulmonary arteries

BACKGROUND: Many patients with severe acute lung injury do not respond to nitric oxide (NO) inhalational therapy with alleviation of pulmonary arterial hypertension and hypoxemia, so this treatment remains controversial. MATERIALS AND METHODS.: We investigated in endotoxin-exposed Wistar rat pulmonary arteries whether endogenous NO alters contractile and relaxing responses, by electrochemical NO and isometric force measurements. RESULTS: Receptor-independent contraction was similar in control and endotoxin-exposed arteries, while thromboxane analogue (TxA)-dependent contraction was less in the latter. Neither non-selective NO synthase (NOS) inhibition by N(G)-nitro-l-arginine (l-NA) or selective inducible-NOS2 inhibition by aminoguanidine (AG) improved TxA-induced contraction in endotoxin-exposed arteries. Acetylcholine-induced relaxation was impaired in endotoxin-exposed pulmonary arteries, despite a comparable acetylcholine-induced NO release in control arteries. Additionally, NO solution-induced relaxation of endotoxin-exposed arteries was impaired, but could be improved by l-NA or AG. Application of a phosphodiesterase-insensitive cyclic guanosine monophosphate analogue induced similar relaxation in both control and endotoxin-exposed arteries. CONCLUSIONS: Endotoxin-associated NOS2-derived NO is thus associated with impaired NO-mediated relaxation, but does not underlie reduced receptor-mediated pulmonary contractile responses. An increased phosphodiesterase activity may underlie the former, so this route can be explored to replace or improve the effect of inhalational NO therapy in severe sepsis-induced acute lung injury in patients.     

The effects of erdosteine on lung injury induced by the ischemia-reperfusion of the hind-limbs in rats

BACKGROUND: [corrected] The goal of this experimental study was to investigate whether erdosteine has a protective effect against lung injury as a remote organ after hind-limb ischemia-reperfusion (I/R). MATERIALS AND METHODS: The rats were divided into three groups: control, I/R, and I/R + erdosteine. After the experimental procedure, nitric oxide (NO) levels, myeloperoxidase (MPO), adenosine deaminase (ADA), and the activities of xanthine oxidase (XO) were determined on the lung tissue. The levels of NO and activities of MPO were also measured on the bronchial alveolar lavage (BAL). In addition, the lung tissue was examined by histopathology. RESULTS: The lung tissue ADA and XO activities were increased in the I/R group compared with the control group (P < 0.05). In the I/R group, the levels of NO were higher than the control group (P < 0.05), whereas the erdosteine treatment did not alter the NO levels (P < 0.05). The MPO activities increased after I/R in the I/R group compared to both control and I/R + erdosteine group (P < 0.05). The activity of MPO increased in the IR group in comparison with the control group in BAL (P < 0.05). The activity of MPO in the I/R + erdosteine group was significantly lower than the I/R group in BAL (P < 0.05). NO levels increased in all I/R groups compared to control group in BAL (P < 0.05). However, treatment of erdosteine significantly decreased NO levels compared to I/R group (P < 0.05). The animals of the I/R group had total destruction of normal alveolar structure with the intense presence of infiltrating neutrophils and mononuclear phagocytes in histopathological examination. The rat lung exhibited mild degrees of destruction in the erdosteine group. CONCLUSIONS: As a result, erdosteine may be a protective effect for lung injury, decreasing oxidative stress and neutrophil accumulation after hind-limb I/R in rats.     

雌激素对大鼠阴道组织NOS活性的影响

目的　研究雌激素对阴道组织一氧化氮合酶 (NOS)活性的影响 ,探讨雌激素在维持女性性反应中的分子学机制。方法　 40只雌性成年SD大鼠随机分为 3组 :卵巢切除去势组 (10只 )、雌激素替代组 (10只 )及假手术对照组 (2 0只 ) ,通过阴道涂片筛选出待取材的动物。于术后第 14天取出阴道组织、用分光光度计测定其一氧化氮合酶(NOS)活性。结果　卵巢切除去势组NOS活性显著低于对照组 (P <0 .0 1) ,雌激素替代能使NOS活性显著上升 (P <0 .0 1) ,并接近对照组正常水平 (P >0 .0 5 )。结论　雌激素可能通过调节阴道NOS活性而维持女性性反应。     

The effect of high protein diet and exercise on irisin,eNOS, and iNOS expressions in kidney

Long-term effects of high protein diets (HPDs) on kidneys are still not sufficiently studied. Irisin which increases oxygen consumption and thermogenesis in white fat cells was shown in skeletal muscles and many tissues. Nitric oxide synthases (NOS) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. We aimed to investigate the effects of HPD, irisin and NO expression in kidney and relation of them with exercise and among themselves. Animals were grouped as control, exercise, HPD and exercise combined with HPD (exercise-HPD). Rats were kept on a HPD for 5 weeks and an exercise program was given them as 5 exercise and 2 rest days per week exercising on a treadmill with increasing speed and angle. In our study, while HPD group had similar total antioxidant capacity (TAC) levels with control group, exercise and exercise-HPD groups had lower levels (p?p?相似文献   

Modulation of macrophage nitric oxide production by prostaglandin D2

BACKGROUND: Nitric oxide and prostaglandins readily become activated in response to inflammatory events. The overproduction of nitric oxide is detrimental to the host. The present study was conducted to examine whether prostaglandin D(2) (PGD(2)) modulates nitric oxide production in macrophages in response to an inflammatory stimulus. METHODS: Cultures of RAW 264.7 murine macrophages were exposed to Escherichia coli lipopolysaccharide (LPS, 0.01 and 1.0 microg/ml) before and after exposure to PGD(2) (0.01 to 10 nmol). After 24-h incubation, supernatants were collected and nitrite was quantitated by Greiss reaction as a measure of nitric oxide synthesis. Inducible nitric oxide synthase (iNOS) protein was measured by Western blot analysis. RESULTS: Macrophages exposed to 0.01 and 1.0 microg/ml LPS produced 8.3 +/- 0.2 and 15.0 +/- 1.4 nmol/1.1 x 10(6) cells/24 h of nitrite, respectively. The simultaneous addition of PGD(2) with LPS inhibited nitrite production in a dose-dependent fashion and suppressed iNOS protein expression. A strong time effect was also exhibited when macrophages were incubated with PGD(2) 1 hour before as compared to 7 hours after the addition of LPS (0.01 or 1.0 microg/ml), indicating that the earlier the time PGD(2) was added to the culture media, the greater the inhibition. Prostaglandin D(2) had the capacity to block nitrite synthesis even when added as much as 7 hours after an LPS challenge. Blocking endogenous prostaglandins, using indomethacin (10 microM), suppressed nitrite production. CONCLUSION: Exogenous PGD(2) caused dose- and time-dependent decreases in LPS-stimulated nitrite production by RAW 264.7 macrophages by hindering iNOS protein expression. Conversely, the endogenous prostaglandins released by these same cells in response to an LPS challenge stimulated nitrite production, which may consequently dampen the inhibitory actions of exogenous PGD(2).     

Experimental varicocele in the rat: early evaluation of the nitric oxide levels and histological alterations in the testicular tissue

The relationship between varicocele and male infertility remains to be explained. Oxidative damage because of the testicular venous backflow may represent one of the causes of gonad injury and seems to precede the histological alteration. Therefore measuring the values of spermatic or intratesticular nitric oxide (NO) could be useful in evaluating this oxidative distress. The aim of this study is to assess the role of testicular NO in early detection of the damages induced by an experimental varicocele in the Wistar rat. A left varicocele was induced in 10 animals (group A). A control group of 10 rats was performed (group B). Animals were killed 3 months after the operation. Both testicles were harvested, weighed and sectioned in two equal parts: one for the evaluation of the NO level and the other one for histological examination. All the rats in group A showed a conspicuous dilatation of the left spermatic vein. The histopathological analysis was normal in both the groups. Biochemistry showed a meaningful statistical difference (P < 0.001) in the concentrations of NO among the specimens of the left and right gonads in group A but no difference was found in group B. The increase in NO values and the presence of other oxidant agents represent the first sign of testicular distress and it seems to anticipate histopathological changes. As it is well known that a great difference exist between human and animal sperm, NO could therefore in the future be taken into consideration together with others parameters for the evaluation of patient who is affected by varicocele.     

饮酒对大鼠生精细胞iNOS,Bcl-2基因表达的影响

目的 探讨酒精对大鼠睾丸诱导型一氧化氮合酶（iNOS）、Bcl-2基因表达和生精细胞凋亡的影响。方法30只成年健康SD雄性大鼠随机均分为对照组、低剂量组和高剂量组，用不同剂量的酒精灌胃成年大鼠26d（两个生精周期）后，免疫组织化学法（SP法）检测睾丸iNOS、BCl-2基因表达的变化；原位缺口末端标记法（TUNEL法）检测细胞凋亡指数（A1）的变化。结果 与对照组相比，低剂量组大鼠睾丸iNOS、Bcl-2基闪表达强度和细胞凋亡指数（A1）无明显变化（P〉0．05）：而高剂量组与对照组和低剂量组相比，iNOS表达显著增强（P〈0．01），Bcl-2基因表达明显减弱（P〈0．01，P〈0．05），细胞凋亡指数则增加（P〈0.01）。结论 长期大量饮酒可以诱导睾丸生精细胞凋亡增加，iNOS与Bcl-2基因表达的改变是重要原因之一。     

Effects of human cathelicidin antimicrobial peptide LL-37 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro

BACKGROUND: Lipopolysaccharides (LPS), released by Gram-negative bacteria, cause vascular expression of inducible nitric oxide synthase (iNOS) leading to nitric oxide (NO) production and septic shock. Human cathelicidin antimicrobial peptide (LL-37) can bind and neutralize LPS. We wanted to study whether LL-37 affects LPS or interleukin-1beta (IL-1beta)-induced production, release and function of NO in intact rat aorta rings and cultured rat aorta smooth muscle cells. METHODS: Isolated segments of thoracic aorta and cultured cells were incubated in the presence of LPS, LL-37, LPS + IL-37, IL-1beta, IL-1beta + IL-37 or in medium alone. Smooth muscle contraction in response to phenylephrine and accumulation of the sdegradation products of NO, nitrate and nitrite, were measured on aorta segments. Levels of iNOS were assessed by Western blot and cytotoxic effects were detected by measurement of DNA fragmentation in cultured cells. Number of viable cells were determined after Trypan blue treatment. RESULTS: Both LPS and IL-1beta reduced contractility in response to phenylephrine and increased NO production as well as iNOS expression. LL-37 inhibited the LPS depression of vascular contractility induced only by LPS. LL-37 reduced both the LPS- and IL-1beta-induced NO production and iNOS expression. LL-37 at high concentrations induced DNA fragmentation and decreased the number of living cells. CONCLUSION: IL-37 reduces NO production induced by LPS and IL-1beta. The reduction does not seem to result only from neutralization of LPS but also from a cytotoxic effect, possibly via induction of apoptosis.     

In vivo transfer of a neuronal nitric oxide synthase expression vector into the rat bladder by electroporation

OBJECTIVE: To investigate the possibility of in vivo gene transfer by attempting to transfer the neuronal nitric oxide synthase (nNOS) gene into rat bladder using electroporation. MATERIALS AND METHODS: The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of the marker genes green fluorescent protein (GFP) and luciferase, and the nNOS gene, was then injected into the subserosal space of the bladder and electroporation applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder and immunohistochemical staining used to detect nNOS; NO(x) released from isolated bladder strips was also assessed using microdialysis and high-performance liquid chromatography. RESULTS: From the luciferase assay, 45 V, 1 Hz, 50 ms and eight pulses were selected as the optimum conditions for electroporation. Bladder specimens with GFP genes injected by electroporation showed bright and numerous sites of GFP expression in the smooth muscle layer. In rats with the nNOS gene injected by electroporation there was marked nNOS immunoreactivity, and NO(x) released from bladder strips was significantly greater than in the control groups. CONCLUSIONS: These results suggest that electroporation is a useful technique for in vivo gene transfer into rat bladder smooth muscles, and that the nNOS gene transferred by this procedure functionally expresses and contributes to NO production.     

Down-regulation of inducible nitric oxide synthase (iNOS) in rat with congenital hydronephrosis

BACKGROUND: Most of our knowledge concerning obstructive uropathy has been derived mainly from surgically manipulated animal models, and the pathogenesis of congenital obstructive hydronephrosis is not fully elucidated. Nitric oxide (NO) acts as an important biological modulator with diverse physiological functions, which can be either toxic or protective depending on the situation. NO is synthesized from l-arginine by nitric oxide synthase, and in the kidney iNOS is expressed spontaneously. The aim of our study is to investigate the expression of iNOS protein and its relationship with tubulointerstitial fibrosis and tubular cell apoptosis in congenital hydronephrosis. METHODS: We conducted histological studies on 18 kidneys of six-week-old-rats from an inbred colony of congenital hydronephrosis with reference to the histological grading of the affected kidney, tubulointerstitial fibrosis, renal tubular atrophy, and tubular cell apoptosis. Renal transforming growth factor-beta1 (TGF-beta1) level was determined by a sandwich ELISA assay and the expression of iNOS was analyzed by western blotting. RESULTS: Most of the hydronephrotic kidneys were markedly enlarged with dilatation of the collecting system, parenchymal thinning, tubular atrophy, interstitial infiltration and fibrosis. Renal TGF-beta1 level was higher in hydronephrotic kidneys than normal control kidneys (364.81 +/- 52.60 vs. 221.19 +/- 22.53 pg/mg protein, P < 0.05). Tubular apoptotic score in hydronephrotic kidneys was also significantly higher than in the normal control kidneys (1.97 +/- 0.42 vs. 0.14 +/- 0.02/HPF, P < 0.01). The expression of iNOS protein was lower in the affected kidneys compared with the normal control kidneys (8.79 +/- 0.78 vs. 14.00 +/- 0.83 arbitrary unit, P < 0.01). There was a negative correlation between iNOS expression and histological grading in congenital hydronephrosis. The iNOS expression also correlated negatively with renal interstitial fibrosis, TGF-beta1 level and tubular cell apoptosis. CONCLUSION: Our study confirmed the down-regulation of iNOS expression in affected kidneys from rats with congenital hydronephrosis, in which the cytoprotective effect of NO may be lost or weakened.     

Glycyrrhizin treatment is associated with attenuation of lipopolysaccharide-induced acute lung injury by inhibiting cyclooxygenase-2 and inducible nitric oxide synthase expression

Glycyrrhizin (GL), a major active constituent of licorice root, has been attributed numerous pharmacologic effects, including anti-inflammatory, anti-viral, anti-tumor, and hepatoprotective activities. In this study, we investigated the anti-inflammatory effect of GL on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. ALI was induced in Balb/c mice by intratracheal instillation of LPS (1 mg/kg). Before 1 h of LPS administration, the mice received intraperitoneal injection of GL at varied doses (10, 25, and 50 mg/kg). The severity of pulmonary injury was evaluated 12 h after LPS administration. GL pretreatment led to significant attenuation of LPS induced evident lung histopathologic changes, alveolar hemorrhage, and neutrophil infiltration with evidence of reduced myeloperoxidase (MPO) activity. The lung wet/dry weight ratios, as an index of lung edema, were markedly reduced by GL pretreatment. The concentrations of pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α were elevated in bronchoalveolar lavage fluid (BALF) after LPS administration, which were significantly inhibited by GL pretreatment. GL pretreatment also reduced the concentrations of nitric oxide (NO) in lung tissues. Furthermore, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was suppressed by GL pretreatment. In conclusion, GL potently protected against LPS-induced ALI, and the protective effects of GL may attribute partly to the suppression of COX-2 and iNOS expression.     

腺病毒介导的激活大鼠阴茎海绵体平滑肌细胞中NO／cGMP通路的实验研究

目的：探讨靶向大鼠iNOS基因的shRNA重组腺病毒载体对大鼠阴茎海绵体平滑肌细胞iN0s基因的激活作用,为阴茎勃起功能障碍（ED）的基因治疗提供实验依据。方法：将前期构建的重组腺病毒AdS—iN—OSrshRNA-EGFP（AdU6／shiNOS）和对照病毒AdU6／shControl,分别转染大鼠阴茎海绵体平滑肌细胞,分别在不同病毒MOI（25,50,75）值下72小时后采样检测。采用realtimeRT-PCR半定量检测AdU6／shiNOS对细胞iNOS基因mRNA表达影响;Western—blot法检测海绵体平滑肌细胞iNOS蛋白表达变化。然后培养基中加L—Arg（10mmol／L）,用酶联免疫法检测病毒转染72小时后海绵体平滑肌细胞内cGMP的浓度变化,记录AdU6／shiNOS对平滑肌细胞内cGMP的影响。结果：AdU6／shiNOS转染大鼠阴茎海绵体平滑肌细胞72小时后,和空白对照组、阴性对照组相比iN0s基因在mRNA和蛋白表达水平均显著上调（P〈O．05）,呈剂量依赖性,MOI一75时RNAa效果最好。而且转染72小时后,实验组原代平滑肌细胞内cGMP水平显著高于对照组及空白组（Pd0．05）。结论：利用腺病毒介导的RNAa技术,提高海绵体平滑肌细胞iN0s基因表达获得成功,可以增加阴茎海绵体平滑肌细胞cGMP水平,激活了NO／cGMP通路,这为勃起功能障碍的基因治疗研究开辟了新的方向。