Effect of Huang‐Lian‐Jie‐Du‐Decoction on pharmacokinetics of verapamil in rats

Objectives The aim was to investigate the effect of Huang‐Lian‐Jie‐Du‐Decoction (HLJDD) on the pharmacokinetic behaviour of verapamil in rats. Methods Rats orally received 3.33 g/kg of HLJDD extract for 14 days, and pharmacokinetics of verapamil was investigated after oral and intravenous verapamil. Norverapamil formation for assessing cytochrome P450 3A activity in hepatic and intestinal microsomes of the HLJDD‐treated rats was investigated. The inhibitory effect of berberine on the formation of norverapamil in intestinal and hepatic microsomes was also evaluated. Key findings HLJDD treatment increased the plasma concentration of verapamil and decreased the plasma concentration of norverapamil, resulting in a 24% increase in the AUC_0–480 of verapamil and a 25% reduction in the AUC_0–480 of norverapamil after oral administration. However, HLJDD did not alter the pharmacokinetic behaviour of verapamil after intravenous administration. Norverapamil formation showed biphasic kinetics in both intestinal and hepatic microsomes. HLJDD treatment significantly decreased the intrinsic clearance of verapamil in intestinal microsomes, but had no effect on the hepatic metabolism of verapamil. Berberine also inhibited norverapamil formation in both intestinal and hepatic microsomes; the extent of inhibition was larger in intestinal microsomes. Conclusions HLJDD displayed a route‐dependent effect on the pharmacokinetics of verapamil in rats. HLJDD treatment increased the bioavailability of verapamil partly via inhibiting first‐pass verapamil metabolism in the intestine.     

香菇多糖对巨噬细胞一氧化氮和一氧化氮合酶活性的影响

目的研究香菇多糖(LTN)诱导巨噬细胞的一氧化氮(NO)生成和一氧化氮合酶(iNOS)的活性,探讨LTN的免疫调节作用机理.方法采用Griess反应和荧光法测定不同剂量的LTN作用小鼠腹腔巨噬细胞后NO的生成量和iNOS活性.观察mRNA转录抑制剂、蛋白质合成抑制剂和iNOS抑制剂对巨噬细胞NO的生成和iNOS活性的影响.结果LTN能使小鼠腹腔巨噬细胞NO生成增加,iNOS活性增高,并呈作用剂量依赖关系.3种抑制剂均能抑制LTN诱导的小鼠腹腔巨噬细胞N0的生成和iNOS活性.结论LTN能刺激小鼠腹腔巨噬细胞提高iNOS活性和NO的生成.提示LTN的免疫调节作用机制可能与LTN刺激巨噬细胞NO生成有关.     

罗红霉素通过诱导型一氧化氮合酶/一氧化氮途径抑制哮喘大鼠气道炎症

目的探讨罗红霉素对哮喘大鼠支气管诱导型一氧化氮合酶(iNOS)及一氧化氮(NO)的影响。方法24只成年哮喘大鼠随机分成对照组、哮喘组以及罗红霉素组。对支气管肺泡灌洗液(BALF)细胞总数及嗜酸性粒细胞计数,免疫组织化学检测大鼠支气管上皮细胞iNOS蛋白表达,RT-PCR检测肺组织iNOS mRNA表达,分光光度计检测肺组织iNOS活性及NO含量。双抗体夹心法检测肺组织白细胞介素-4(IL-4)及干扰素-γ(IFN-γ)。结果哮喘组大鼠BALF细胞总数及嗜酸性粒细胞分类分别为(7.28±1.65)×108.L-1、(7.73±1.54)%,均高于对照组(3.76±0.97)×108.L-1、(1.27±0.60)%;罗红霉素组BALF细胞总数及嗜酸性粒细胞分类分别为(5.68±0.95)×108.L-1、(5.54±1.53)%,明显低于哮喘组,差异有统计学意义。哮喘组肺组织IL-4浓度、iNOS活性及NO含量高于对照组,罗红霉素组肺组织IL-4浓度、iNOS活性及NO含量低于哮喘组。哮喘组肺组织IFN-γ浓度低于对照组,罗红霉素组肺组织IFN-γ浓度高于哮喘组。哮喘大鼠支气管上皮细胞iNOS蛋白及肺组织iN-OSmRNA表达分布吸光度值分别为(0.25±0.06)、(0.52±0.14),较对照组[(0.14±0.05),(0.33±0.05)]明显增强;但罗红霉素组iNOS蛋白及mRNA表达为(0.15±0.03)、(0.35±0.07),均明显较哮喘组减弱。结论罗红霉素通过干预哮喘大鼠气道IL-4、IFN-γ以及iNOS/NO体系,抑制哮喘气道炎症反应。     

Role of nitric oxide produced by constitutive and inducible nitric oxide synthases in the mouse gastric fundus

In the present study, the role of nitric oxide (NO) produced by constitutive and inducible nitric oxide synthases (cNOS and iNOS, resepctively) on the contraction and relaxation of fundus in normal and lipopolysaccharide (LPS)-treated mice was examined. A whole fundic ring isolated from mice pretreated with reserpine was mounted in an organ bath containing Krebs' solution with 0.001 mmol/L atropine. Rings were contracted initially by 5-hydroxytryptamine (5-HT; 0.03 mmol/L) before relaxation was induced using ATP (0.03 mmol/L), ADP (0.03 mmol/L), pentoxifylline (0.002 mmol/L), electrical field stimulation (EFS; 50 V, 1 msec, 50 Hz, 3 min) and L-arginine (0.05 mmol/L). All drugs and EFS induced significant relaxation of isolated rings. The relaxations induced were significantly inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; 1.0 mmol/L). However, the iNOS inhibitors L-N(6)-(1-iminoethyl) lysine hydrochloride (L-NIL; 1.0 mmol/L) and amino guanidine (AMG; 1.0 mmol/L) had no significant effect on tissue relaxation. Then, the relaxant effects of 0.03 mmol/L ATP were tested on precontracted isolated fundic rings taken from 10 mg/kg LPS-treated animals. The non-selective NOS inhibitor L-NAME (10 mg/kg), the iNOS inhibitors L-NIL (3 mg/kg) and AMG (20 mg/kg) and betamethasone (0.1 mg/kg) were used to examine the role of NO produced by iNOS in the relaxation responses. It was found that the level of contraction induced by 0.03 mmol/L 5-HT in rings isolated from LPS-treated animals was significantly (P < 0.5) less than that in rings from untreated mice. However, precontracted tissues from LPS-treated mice were significantly relaxed by ATP and the relaxation response to ATP was significantly inhibited by L-NIL, ANG and betamethasone, but not by L-NAME. We suggest that, in LPS-treated mice, the production of NO from iNOS produces a reduction in the contractile response, as well as a decrease in NO formation by cNOS, resulting in changes to smooth muscle cell function.     

Inhibitory effect of fucoidan on nitric oxide production in lipopolysaccharide‐activated primary microglia

1. Microglial activation plays an important role in the pathogenesis of neurodegenerative diseases by producing various pro‐inflammatory cytokines. Microglia‐derived nitric oxide (NO) is critical for the lipopolysaccharide (LPS)‐induced selective loss of dopaminergic neurons. 2. Fucoidan is a sulphated polysaccharide extracted from brown seaweeds. It has a variety of biological actions, including anticoagulant, antiviral and anti‐inflammatory effects. The aim of the present study was to investigate the effects of fucoidan on LPS‐induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. 3. To investigate the effects of fucoidan on LPS‐induced cellular activation in microglia, primary microglial cells were preincubated with fucoidan (31.25, 62.5 and 125 μg/mL) for 10 min, followed by stimulation with LPS (0.01 μg/mL). Then, cell shape and NO production were determined 24 h after LPS stimulation, whereas inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined at 6 and 18 h after LPS stimulation, respectively. To evaluate the inhibitory mechanisms involved, mitogen‐activated protein kinase (MAPK) activation was also evaluated. 4. Lipopolysaccharide transformed cells into an amoeboid shape, whereas 62.5 μg/mL fucoidan inhibited this activation. Moreover, 125 μg/mL fucoidan significantly inhibited microglial NO production to 75% of that in LPS‐treated group and also significantly diminished the expression of iNOS mRNA and protein by nearly 50%. Fucoidan (125 μg/mL) also suppressed phosphorylation of p38 and extracellular signal‐regulated kinase (ERK) by approximately 50%, but not that of c‐Jun N‐terminal kinase. 5. The results provide the first evidence that fucoidan has a potent inhibitory effect against LPS‐induced NO production by microglia. The results also suggest that this inhibitory action of fucoidan involves suppression of p38 and ERK phosphorylation.     

Evidence that inhibition of spinal nitric oxide production contributes to the antinociceptive effects of emulsified isoflurane on formalin-induced pain in rats

The aim of the present study was to investigate the contribution of spinal nitric oxide (NO) to the antinociceptive effects of emulsified isofluane in rats. The formalin test was used to assess nociceptive responses. Immunocytochemistry and histochemistry were performed to determine the effects of emulsified isoflurane on formalin-induced changes in Fos-like immunoreactive (Fos-LI)- and nicotinamide adenine dinucleotide phosphatediaphorase (NADPH-d)-positive neurons, respectively. The results showed that emulsified isofluane, administered intraperitoneally, significantly decreased the formalin-induced paw licking time and that this was attenuated by pretreatment with intrathecal injection of the NO precursor L-arginine. Furthermore, Fos-LI- and NADPH-d-positive neurons were mainly found in the ipsilateral dorsal horn after injection of formalin, some of which were Fos-LI/NADPH-d double-labelled neurons. Administration of emulsified isofluane significantly decreased Fos-LI- and NADPH-d-positive, as well as Fos-LI/NADPH-d double-labelled, neurons. Finally, emulsified isofluane produced a significant reduction of NOS activity and a decrease of NO production in the spinal cord of formalin-treated rats. In conclusion, the results suggest that inhibition of spinal NO production contributes to the antinociceptive effects of emulsified isofluane on formalin-induced pain in rats.     

诱导型一氧化氮合酶在哮喘大鼠肺组织及中性粒细胞中的表达及地塞米松对其的影响

目的：观察诱导型一氧化氮合酶（iNOS）在哮喘大鼠肺组织及血中性粒细胞（PMN）中的表达,探讨PMN参与哮喘炎症的可能作用机制。方法：采用大鼠哮喘模型,随机分成哮喘组、对照组、地塞米松干预组,分离纯化血PMN,免疫组织化学法检测iNOS蛋白的表达水平,测定支气管肺泡灌洗液（BALF）中NO浓度。结果：哮喘组PMNiNOS蛋白光密度（OD）值（0.122±0.017）的表达水平显著高于对照组OD值（0.076±0.014）（P〈0.01）,地塞米松干预组OD值（0.089±0.013）PMNiNOS蛋白的表达水平显著低于哮喘组OD值（P〈0.01）,但与对照组相比差异无统计学意义。哮喘组支气管壁iNOS蛋白OD值（0.243±0.039）的表达水平显著高于对照组（0.119±0.016）（P〈0.01）,地塞米松干预组OD值（0.164±0.016）支气管壁iNOS蛋白的表达水平显著低于哮喘组且高于对照组（P均〈0.01）。哮喘组BALFNO浓度（8.59±1.07）ng/mL显著高于对照组（3.69±1.00）ng/mL（P〈0.01）,地塞米松干预组BALFNO浓度（4.28±0.89）ng/mL显著低于哮喘组（P〈0.01）,但与对照组相比差异无统计学意义。PMNiNOS蛋白的表达水平与BALFNO浓度呈显著正相关（n=29,r=0.770,P〈0.01）。支气管壁iNOS蛋白的表达水平与BALFNO浓度呈显著正相关（n=29,r=0.802,P〈0.01）。结论：哮喘大鼠PMN合成iNOS蛋白的功能增加,PMN可能通过iNOS参与哮喘的发病机制,这种功能可以部分被地塞米松所抑制。     

Involvement of nitric oxide in the inhibition of angiogenesis by interleukin-2

Interleukin-2 (IL-2), an immunoregulatory cytokine possessing antitumour activity, is an inducer of nitric oxide (NO) synthesis in mice and man. In this study, the possibility that IL-2 possesses antiangiogenic properties that account for its antitumour effects in vivo was examined. IL-2 caused a dose-dependent inhibition of angiogenesis in the chick embryo chorioallantoic membrane (CAM). This inhibition was completely reversed by the NO synthase inhibitor N^G-nitro-L-arginine methylester (L-NAME). Furthermore, IL-2 was capable of stimulating NO synthase activity in the CAM in vitro and this effect was suppressed by L-NAME. Addition of IL-2 to human umbilical vein endothelial cells (HUVECs) in culture, had no effect on their growth characteristics. These results suggest that IL-2 may be an important antiangiogenic molecule causing its effect via nitric oxide synthesis. The antiangiogenic activity of IL-2 may be, at least in part, responsible for its antitumour properties.     

Anti‐atherogenic effects of high‐density lipoprotein on nitric oxide synthesis in the endothelium

1. The endothelium is critical in the control of vascular haemodynamics and haemostasis. Endothelial dysfunction, typically characterized by decreased nitric oxide bioavailability and response to endothelium‐dependent agonists, is well accepted as a defining characteristic of early atherosclerosis. 2. Numerous epidemiological studies have reported that increased levels of circulating HDL are vasculoprotective and reduce the incidence of adverse cardiovascular events. Traditionally, these effects have been attributed to the ability of HDL to remove cholesterol from cells via reverse cholesterol transport. However, there is increasing evidence that the beneficial effects on the endothelium by HDL encompass its anti‐inflammatory, antithrombotic and anti‐oxidative properties, which include the release of nitric oxide (NO). 3. This review highlights recent findings on the importance of HDL in reducing atherosclerotic risk. We focus on the beneficial effects of HDL‐induced NO release and how this relates to endothelial dysfunction and on the effect of HDL on vascular repair via endothelial progenitor cells.     

大鼠对氟西泮抗痫耐受和依赖时海马中一氧化氮合酶的变化

目的通过测定大鼠对氟西泮抗痫耐受性和依赖性时海马中一氧化氮合酶(NOS)蛋白表达及活性的变化,探讨一氧化氮(NO)在此过程中可能的作用。方法建立大鼠对氟西泮抗痫耐受性和依赖性的模型。运用免疫印迹及免疫组化法检测海马中NOS蛋白表达,以及比色法检测NOS活性的变化。结果大鼠对氟西泮抗痫耐受组的海马中NOS蛋白表达明显高于对照组;而对氟西泮抗痫依赖组则与对照组差别无统计学意义。两组NOS活性均显著高于各自的对照组。结论NO可能是介导氟西泮抗痫耐受性和依赖性的因素之一。     

Spantide抑制甲醛炎性痛大鼠脊髓一氧化氮合酶表达和一氧化氮含量的增加(英文)

目的　观察鞘内注射P物质 (SP)拮抗剂spantide { [D Arg1,D Trp7,9,Leu11] substanceP}对炎性痛大鼠L5节段脊髓后角一氧化氮合酶 (NOS)表达和腰膨大一氧化氮 (NO)含量的影响 ,以探讨痛及痛过敏时脊髓NOS表达和NO生成增多的机制。方法　大鼠右后掌足底皮下注射 5 %甲醛 0 .2mL诱发炎性痛及痛过敏 ,NADPH d组化法观察脊髓后角NOS表达的变化 ,硝酸还原酶法测定NO含量的变化。结果　皮下注射甲醛 2 4h后 ,双侧L5节段脊髓后角NOS表达及腰膨大部位NO生成明显增加 ;注射甲醛前 5min鞘内注射spantide (5 μg ,10 μL) ,则明显抑制甲醛所致的NOS表达及NO生成增加。结论　初级传入末梢释放的SP在甲醛炎性痛及痛过敏时脊髓NOS表达及NO生成增多中发挥作用     

Adaptation of glycocalyx,nitric oxide synthase expression and vascular cell apoptosis in conduit arteries of tail‐suspended rats

The importance of vascular cell glycocalyx in mechanotransduction has been demonstrated by many studies. The simulated microgravity induced a region‐dependent adaptation of arterial glycocalyx including its thickness, coverage, and gene expression in conduit arteries of tail‐suspended rats has been reported in our previous studies. Herein, we extended this line of research by quantifying the mRNA levels of three nitric oxide synthase (NOSI, NOSII, and NOSIII) and evaluating the apoptotic rates of endothelial cells (ECs) and smooth muscle cells (SMCs) in the common carotid artery, abdominal aorta, and femoral artery of 3 week tail‐suspended rats. Results indicated that the tail suspension of rats induced about 0.36, 0.22, and 0.33 fold down‐regulation of NOSI, NOSII, and NOSIII in the abdominal aorta, while 3.21, and 3.48 fold up‐regulation of NOSII and NOSIII in the carotid artery and no significant effects on three NOS isoforms in the femoral artery. Moreover, the apoptosis of ECs and SMCs were significantly inhibited in both carotid artery and abdominal aorta, while enhanced in the femoral artery of the tail‐suspended rats. A linear positive correlation exists between the normalized coverage of the glycocalyx and the normalized NOSI and NOSIII mRNA levels. These results indicated that the redistribution of haemodynamics in the conduit arteries of 3 week tail‐suspended rats regulated the glycocalyx, NOS expression, and vascular cell apoptosis in a region‐dependent manner, contributing to the final vascular remodelling under simulated microgravity condition.     

Tacrolimus (FK506), an immunosuppressive agent, prevents indomethacin-induced small intestinal ulceration in the rat: inhibition of inducible nitric oxide synthase expression

We examined the effect of tacrolimus (FK506), an immunosuppressive drug, on indomethacin-induced small intestinal ulceration in rats. Animals were given indomethacin (10 mg/kg, s.c.), killed 24 h later, and myeloperoxidase (MPO) activity and thiobarbituric acid reactants (TBARS) were evaluated in intestinal lesions. Tacrolimus (0.3 - 3 mg/kg) was administered p.o. twice 0.5 h before and 6 h after indomethacin injection. The expression of inducible nitric oxide synthase (iNOS) mRNA was determined by a TaqMan real-time RT-PCR, while the activity of nuclear factor (NF)-kappaB DNA-binding was analyzed by electrophoresis mobility shift assays (EMSA) 6 h after indomethacin treatment. Indomethacin provoked severe hemorrhagic lesions in the small intestine, mainly in the jejunum and ileum, accompanied with increases in MPO activity and TBARS. Oral administration of tacrolimus reduced the severity of indomethacin-induced intestinal lesions in a dose-dependent manner. The increases in MPO activity and TBARS were also significantly attenuated by tacrolimus. The expression of iNOS mRNA was markedly enhanced when examined 6 h after indomethacin administration, and this response was counteracted by tacrolimus. Indomethacin also activated NF-kappaB in a tacrolimus-preventable manner. These results suggest that tacrolimus prevents indomethacin-induced small intestinal ulceration in the rat. This effect may be due to inhibition of iNOS induction through suppression of NF-kappaB activation.     

四逆汤对失血性休克家兔一氧化氮（NO）和一氧化氮合酶（NOS）的影响

目的：探讨中药四逆汤对失血性休克家兔一氧化氮的影响。方法：采用动物分组对照实验，测量不同状态下动物血浆一氧化氮和一氧化氮合酶(NOS)的变化。结果：与休克前比较，休克组的家兔血浆一氧化氮水平各时段均明显提高(P＜0．01)，而治疗组仅在治疗后30min家兔一氧化氮及NOS水平明显提高(P＜0．01)，其他时段与休克前比较，未见显著差异(P＜0．01)，且休克后lh、4h、8h休克组一氧化氮水平显著高于治疗组(P＜0．01)。经药物治疗的失血性休克家兔血浆一氧化氮水平明显降低。     

Poly(ADP‐ribose) polymerase 1 deficiency increases nitric oxide production and attenuates aortic atherogenesis through downregulation of arginase II

Poly (ADP ‐ribose) polymerase (PARP ) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular‐related diseases. However, whether PARP regulates nitric oxide (NO ), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP ‐1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E‐deficient (apoE^?/?) mice fed a high‐cholesterol diet for 12 weeks, PARP ‐1 inhibition via treatment with 3,4‐dihydro‐54‐(1‐piperindinyl) butoxy‐1(2H)‐isoquinoline (DPQ ) or PARP ‐1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II ) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells (MAEC s), PARP ‐1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase (eNOS ) was preserved in the aortas and MAEC s when PARP ‐1 was inhibited. Reduced NO production in vitro due to PARP ‐1 deficiency could be restored by treating the MAEC s with oxidized low‐density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP ‐1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity.     

The inhibition of inducible nitric oxide synthase and oxidative stress by agmatine attenuates vascular dysfunction in rat acute endotoxemic model

Vascular dysfunction leading to hypotension is a major complication in patients with septic shock. Inducible nitric oxide synthase (iNOS) together with oxidative stress play an important role in development of vascular dysfunction in sepsis. Searching for an endogenous, safe and yet effective remedy was the chief goal for this study. The current study investigated the effect of agmatine (AGM), an endogenous metabolite of l-arginine, on sepsis-induced vascular dysfunction induced by lipopolysaccharides (LPS) in rats. AGM pretreatment (10 mg/kg, i.v.) 1 h before LPS (5 mg/kg, i.v.) prevented the LPS-induced mortality and elevations in serum creatine kinase-MB isoenzyme (CK-MB) activity, lactate dehydrogenase (LDH) activity, C-reactive protein (CRP) level and total nitrite/nitrate (NOx) level after 24 h from LPS injection. The elevation in aortic lipid peroxidation illustrated by increased malondialdehyde (MDA) content and the decrease in aortic glutathione (GSH) and superoxide dismutase (SOD) were also ameliorated by AGM. Additionally, AGM prevented LPS-induced elevation in mRNA expression of iNOS, while endothelial NOS (eNOS) mRNA was not affected. Furthermore AGM prevented the impaired aortic contraction to KCl and phenylephrine (PE) and endothelium-dependent relaxation to acetylcholine (ACh) without affecting endothelium-independent relaxation to sodium nitroprusside (SNP). In conclusion: AGM may represent a potential endogenous therapeutic candidate for sepsis-induced vascular dysfunction through its inhibiting effect on iNOS expression and oxidative stress.     

Differential effects of selective and non-selective inhibition of nitric oxide synthase on the expression and activity of cyclooxygenase-2 during gastric ulcer healing

Nitric oxide synthases (NOS) and cyclooxygenase-2 (COX-2) are important enzymes involved in ulcer healing but interactions between them have not been clearly defined. The aim of this study was to investigate the effects of selective or non-selective inhibition of NOS on the expression and activity of COX-2 during healing of acetic acid-induced gastric ulcers in rats. N-[3-(aminomethyl)benzyl] acetamidine (1400 W), a potent selective inhibitor of inducible nitric oxide synthase (iNOS), at a dose of 0.1 mg/kg/day, was found to reduce the ulcer sizes at day 3 and 7 post-ulcer induction. On the other hand, 15 mg/kg/day of NG-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor that suppresses both iNOS and endothelial nitric oxide synthase (eNOS), enlarged the ulcer sizes over the same time periods. The expression of COX-2 and COX activity, together with NF-kappaB activation in the ulcer tissues were down-regulated by L-NAME but not 1400 W. It is concluded that iNOS may contribute to ulcer formation while COX-2 and eNOS promote ulcer healing. eNOS enhances COX-2 expression possibly through the activation of NF-kappaB.