Radix asari extract protects pancreatic beta cells against cytokine-induced toxicity: implication of the NF-kappaB-iNOS signaling cascade

In this study, we assessed the preventive effects of Radix asari extract (RAE) against cytokine-induced beta-cell destruction. Cytokines secreted by immune cells that have infiltrated pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. Treatment of RINm5F (RIN) cells with interleukin (IL)-1beta and interferon (IFN)-gamma resulted in a reduction of cell viability and proliferation. However, treatment of RIN cells with RAE protected the IL-1beta and IFN-gamma- mediated viability and proliferation reduction in a concentration-dependent manner. Incubation with RAE also resulted in significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, and this reduction was correlated with reduced levels of mRNA and protein associated with the inducible form of NO synthase (iNOS). The molecular mechanism by which RAE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation as a result of RAE's suppression of IL-1beta and IFN-gamma-induced IkappaBalpha degradation. The protective effects of RAE were verified via the observation of reduced NO generation and iNOS expression, as well as the observation of normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated rat islets. These results suggest that RAE protects beta cells from cytokine toxicity by suppression of NF-kappaB activation.     

Inhibitory effect of Artemisia capillaris extract on cytokine-induced nitric oxide formation and cytotoxicity of RINm5F cells

Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. Cytokines stimulate an inducible form of nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, leading to insulin insufficiency. In the present study, the effects of Artemisia capillaris extract (ACE) on cytokine-induced beta-cell damage were examined. Treatment of RINm5F (RIN) rat insulinoma cells with interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) induced cell damage. ACE completely protected IL-1beta and IFN-gamma-mediated cytotoxicity in a concentration-dependent manner. Incubation with ACE resulted in a significant reduction in IL-1beta and IFN-gamma-induced NO production, a finding that correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which ACE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in the nucleus, and IkappaBalpha degradation in cytosol compared to unstimulated cells. Furthermore, ACE restored the cytokine-induced inhibition of insulin release from isolated islets. These results suggest that ACE protects beta-cells by suppressing NF-kappaB activation.     

Protective effect of retinoic acid on interleukin-1 beta-induced cytotoxicity of pancreatic beta-cells

Cytokines produced by immune cells in pancreatic islets infiltrating are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the effects of retinoic acid (RA) on cytokine-induced beta-cell dysfunction were examined. RA significantly protected interleukin-1 beta (IL-1) and interferon-gamma (IFN-gamma)-mediated cytotoxicity of rat insulinoma cell (RINm5F), and also reduced in IL-1 and IFN-gamma-induced nitric oxide (NO) production, which correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism, by which RA inhibited iNOS gene expression, appeared to involve the inhibition of NF-kappa B activation. Our results suggest possible therapeutic value of RA for the prevention of diabetes mellitus progression.     

No evidence for linkage in the promoter region of the inducible nitric oxide synthase gene (NOS2) in a Danish type 1 diabetes population

Exposure of human pancreatic islets to a mixture of cytokines induces expression of inducible nitric oxide synthase (iNOS), impairs beta-cell function and induces apoptosis. Exposing human islets to high amounts of NO from chemical NO-donors causes DNA strand breaks and mitochondrial damage, suggesting that NO is deleterious to human beta-cells. Hence, we consider the gene encoding iNOS in beta-cells, NOS2, a candidate gene for type 1 diabetes in humans. In the present study we have tested three identified polymorphisms within the promoter sequence of the human NOS2 gene in a type 1 diabetic family material comprising 154 affected sib-pair families and 103 affected simplex families (1143 individuals in total). PCR-based amplification of the polymorphic loci were established. Linkage analysis was performed using the extended transmission disequilibrium testing (ETDT). A Bsal RFLP was found not to be polymorphic in 20 type 1 diabetic patients and 14 healthy control subjects and was not analysed further. In affected cases a nine allele CCTTT repeat and a bi-allelic TAAA repeat revealed allelewise Petdt of 0.52 and 0.60, respectively. ETDT applied to (TAAA)n; (CCTTT)n haplotypes demonstrated random transmission from heterozygous parents to affected offspring. In conclusion, the tested polymorphisms within the NOS2 gene promoter did not show evidence for linkage to type 1 diabetes in a Danish family material.     

Fructus Benincasae Recens extract prevents cytokine-induced nitric oxide formation and cytotoxicity of RINm5F cells

Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of β-cell destruction in insulin-dependent diabetes mellitus. In this study, the effects of Fructus Benincasae Recens (FBR) extract on cytokine-induced β-cell dysfunction were examined. Fructus Benincasae Recens extract completely protected interleukin-1β (IL-1β) and interferon-γ (IFN-γ)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with FBR extract resulted in a significant reduction of IL-1β and IFN-γ-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which FBR extract inhibited iNOS gene expression appeared to involve the inhibition of NF-κB activation. Our results revealed the possible therapeutic value of FBR extract for the prevention of diabetes mellitus progression.     

Herpes Simplex Virus 1 Suppresses the Function of Lung Dendritic Cells via Caveolin-1

Caveolin-1 (Cav-1), the principal structural protein of caveolae, has been implicated as a regulator of virus-host interactions. Several viruses exploit caveolae to facilitate viral infections. However, the roles of Cav-1 in herpes simplex virus 1 (HSV-1) infection have not fully been elucidated. Here, we report that Cav-1 downregulates the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) in dendritic cells (DCs) during HSV-1 infection. As a result, Cav-1 deficiency led to an accelerated elimination of virus and less lung pathological change following HSV-1 infection. This protection was dependent on iNOS and NO production in DCs. Adoptive transfer of DCs with Cav-1 knockdown was sufficient to confer the protection to wild-type (WT) mice. In addition, Cav-1 knockout (KO) (Cav-1^−/−) mice treated with an iNOS inhibitor exhibited significantly reduced survival compared to that of the nontreated controls. We found that Cav-1 colocalized with iNOS and HSV-1 in caveolae in HSV-1-infected DCs, suggesting their interaction. Taken together, our results identified Cav-1 as a novel regulator utilized by HSV-1 to evade the host antiviral response mediated by NO production. Therefore, Cav-1 might be a valuable target for therapeutic approaches against herpesvirus infections.     

Is mortalin a candidate gene for T1DM ?

Mortalin has been found to be up-regulated by 2D-protein gel analysis in isolated rodent islets exposed to cytokines. In islets from two rat strains with different sensitivity to the toxic effects of cytokines we observed a significant difference in IL-1beta mediated mortalin expression. Constitutive over-expression of rat mortalin in NIH3T3 cells reduced cellular survival in accordance with mortalin being associated to cellular senescence. Hence we consider the gene encoding for mortalin at chromosome 5q31.1 a putative candidate gene in cytokine induced beta-cell destruction. We scanned the human mortalin gene for polymorphisms and identified three novel polymorphisms. Neither the SNPs individually nor as constructed haplotypes showed disease association tested by (E)TDT in a Danish type 1 diabetes (T1DM) population. Furthermore, we tested the D5S500 microsatelite located close to 5q31.1 without finding linkage to (T1DM). In conclusion, the functional data identifying a difference in mortalin expression in IL-1beta stimulated islets between two rat strains and over-expression of mortalin in NIH3T3 cells associated with decreased viability suggests a functional role for mortalin in cytokine mediated beta cell destruction; however, the identified polymorphisms did not reveal any association in the presence of linkage disequilibrium of mortalin to T1DM in the Danish population.     

Interferon-gamma-induced nitric oxide causes intrinsic intestinal denervation in Trypanosoma cruzi-infected mice

In this study, the role of nitric oxide (NO) in neuronal destruction during acute-phase Trypanosoma cruzi infection was evaluated in male C57BL/6 (WT, wild-type) mice and knockout mice [inducible nitric oxide synthase (iNOS)(-/-) and interferon (IFN)(-/-)]. Selected animals were infected by intraperitoneal injection of 100 trypomastigote forms of the Y strain of T. cruzi. Others were injected intraperitoneally with an equal volume of saline solution and served as controls. Our findings support those of previous studies regarding myenteric denervation in acute-phase T. cruzi infection. In addition, we clearly demonstrate that, despite the fact that parasite nests and similar inflammatory infiltrate in the intestinal wall were more pronounced in infected iNOS(-/-) mice than in infected WT mice, the former presented no reduction in myenteric plexus neuron numbers. Neuronal nerve profile expression, as revealed by the general nerve marker PGP 9.5, was preserved in all knockout animals. Infected IFN(-/-) mice suffered no significant neuronal loss and there was no inflammatory infiltrate in the intestinal wall. On days 5 and 10 after infection, iNOS activity was greater in infected WT mice than in controls, whereas iNOS activity in infected knockout mice remained unchanged. These findings clearly demonstrate that neuronal damage does not occur in NO-impaired infected knockout mice, regardless of whether inflammatory infiltrate is present (iNOS(-/-)) or absent (IFN(-/-)). In conclusion, our observations strongly indicate that myenteric denervation in acute-phase T. cruzi infection is because of IFN-gamma-elicited NO production resulting from iNOS activation in the inflammatory foci along the intestinal wall.     

Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways

Nitric oxide (NO*) expression by inducible nitric oxide synthase (iNOS) is an important host defense mechanism against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this investigation was to examine the role of mitogen-activated protein (MAP) kinase (MAPK) and nuclear factor kappaB (NF-kappaB) signaling pathways in the regulation of iNOS and NO* by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM). Specific pharmacologic inhibition of the extracellular-signal-regulated kinase (ERK) or NF-kappaB pathway revealed that both these signaling cascades were required in gamma interferon (IFN-gamma)-ManLAM-induced iNOS protein and NO2- expression in mouse macrophages. Transient cotransfection of dominant-negative protein mutants of the c-Jun NH2-terminal kinase (JNK) pathway revealed that the MAP kinase kinase 7 (MKK7)-JNK cascade also mediated IFN-gamma-ManLAM induction of iNOS promoter activity whereas MKK4 did not. Overexpression of null mutant IkappaBalpha, a potent inhibitor of NF-kappaB activation, confirmed that the IkappaBalpha kinase (IKK)-NF-kappaB signaling pathway enhanced IFN-gamma-ManLAM-induced iNOS promoter activity. By contrast, activated p38mapk inhibited iNOS induction. These results indicate that combined IFN-gamma and ManLAM stimulation induced iNOS and NO. expression and that MEK1-ERK, MKK7-JNK, IKK-NF-kappaB, and p38mapk signaling pathways play important regulatory roles.     

Inhibitors of phosphodiesterase isoforms III or IV suppress islet-cell nitric oxide production

The general phosphodiesterase (PDE) inhibitor pentoxifylline (PTX), and the PDE type IV inhibitor rolipram (ROL), both increase intracellular cAMP levels and suppress inflammatory cytokine production by T cells and macrophages. We have previously shown that PTX and ROL protect from autoimmune diabetes in nonobese diabetic (NOD) mice. These drugs may mediate some of their anti-inflammatory effects by blocking nitric oxide (NO) production by macrophages. In this study, we investigated the effect of PDE inhibitors in blocking NO production by insulin-secreting NIT-1 insulinoma cells and mouse islet cells in vitro and in vivo. Insulinoma cells and islet cells produced NO when stimulated with a combination of inflammatory cytokines and lipopolysaccharide (LPS). We found that both PTX and ROL markedly suppressed this induced NO production. Islet cells express PDEs III and IV and, accordingly, the PDE III inhibitor cilostamide (CIL) also suppressed NO production, and a combination of ROL and CIL had a synergistic effect. This suppression appeared to be mediated, at least in part, by elevating cAMP level and was mimicked by other cAMP-elevating agents, ie, membrane-permeable cAMP analogs (dibutyryl cAMP and 8-bromo cAMP) and an adenylate cyclase stimulator (forskolin). PDE inhibitors suppressed the expression of inducible nitric oxide synthase (iNOS) mRNA. In vivo treatment with PTX or ROL prevented iNOS protein expression in the islets of NOD mice with cyclophosphamide-accelerated disease. Our findings suggest that PDE inhibitors can protect islets against autoimmunity.     

Antidiabetogenic effect of pentoxifylline is associated with systemic and target tissue modulation of cytokines and nitric oxide production

We have shown recently that xanthine derivative pentoxifylline (PTX) downregulates an inflammatory autoimmune process triggered in genetically susceptible Dark Agouti rats by multiple low doses of streptozotocin (MLD-SZ, 20 mg/kg/day ip for 5 days). We studied the cellular and molecular consequences of PTX treatment during MLD-SZ-induced diabetes with special emphasis on local vs. systemic production of inflammatory mediators. Administration of PTX (200 mg/kg/day for 10 days) during induction of the disease reduced clinical signs of diabetes and protected rats from development of destructive intrainsulitis. Pentoxifylline did not affect diabetogenic effect of single high dose of SZ (100 mg/kg SZ). Ex vivo analysis of the islets of Langerhans performed in early disease development revealed that PTX downregulates production of proinflammatory cytokines IFN-gamma and TNF, as well as inducible nitric oxide synthase (iNOS) expression and NO production. In addition, PTX treatment suppressed splenocyte autoreactivity, as well as the frequency of cells expressing IL-2R and MHC class II antigens. There was no evidence of any changes in proportion of ICAM-1 and LFA-1 expressing splenocytes in comparison to control MLD-SZ-treated animals. In contrast to suppressed intraislet production, high peripheral expression of both iNOS mRNA and NO was found in MLD-SZ rats treated with PTX. Taken together, the data indicate that the effect on both systemic and intra-islet production of NO, suppression of autoreactive cell activation and of local type 1 cytokine release may contribute to the therapeutic benefit achieved by PTX in the rat.     

Effects of acrylamide on oxidant/antioxidant parameters and CYP2E1 expression in rat pancreatic endocrine cells

Oxidative stress is one of the principle mechanism of acrylamide-induced toxicity. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide or by direct conjugation with glutathione. Bearing in mind that up to now the effects of acrylamide on oxidative stress status and CYP2E1 level in endocrine pancreas have not been studied we performed qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase (CAT) and CYP2E1 expression in islets of Langerhans of rats subchronically treated with 25 or 50 mg/kg bw of acrylamide. Since the majority of cells (>80%) in rodent islets are beta cells, in parallel studies, we employed the Rin-5F beta cell line to examine effects of acrylamide on redox status and the activity of CAT, SOD and glutathione-S-transferase (GST), their gene expression, and CYP2E1, NF-E2 p45-related factor 2 (Nrf2) and iNOS expression. Immunohistochemically stained pancreatic sections revealed that acrylamide induced increase of iNOS and decrease of CYP2E1 protein expression, while expression of antioxidant enzymes was not significantly affected by acrylamide in islets of Langerhans. Analysis of Mallory-Azan stained pancreatic sections revealed increased diameter of blood vessels lumen in pancreatic islets of acrylamide-treated rats. Increase in the GST activity, lipid peroxidation and nitrite level, and decrease in GSH content, CAT and SOD activities was observed in acrylamide-exposed Rin-5F cells. Level of mRNA was increased for iNOS, SOD1 and SOD2, and decreased for GSTP1, Nrf2 and CYP2E1 in acrylamide-treated Rin-5F cells. This is the first report of the effects of acrylamide on oxidant/antioxidant parameters and CYP2E1 expression in pancreatic endocrine cells.     

Radix clematidis extract protects against cytokine- and streptozotocin-induced beta-cell damage by suppressing the NF-kappaB pathway

Although Radix clematidis has commonly been used in Chinese medicine for the treatment of arthralgia, the anti-diabetic effects of Radix clematidis have not yet been reported. In the present study, we demonstrated that Radix clematidis extract (RCE) could prevent cytokine-induced beta-cell damage and streptozotocin (STZ)-induced diabetes in mice. Treatment of RINm5F insulinoma cells with interleukin-1beta and interferon-gamma reduced cell viability; however, RCE protected the cells from this cytokine-mediated viability reduction in a concentration-dependent manner. Additionally, incubation with RCE resulted in a significant suppression of cytokine-induced nitric oxide (NO) production, which was correlated with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which RCE inhibited iNOS gene expression appeared to involve inhibition of NF-kappaB activation. Furthermore, RCE abolished the cytokine-induced increases in NF-kappaB binding activity and p65 subunit levels in the nucleus, as well as IkappaBalphadegradation in the cytosol when compared to unstimulated cells. The protective effect of RCE was further demonstrated by the observed suppression of NF-kappaB-dependent iNOS expression and normal insulin secreting responses to glucose in cytokines-treated islets. The anti-diabetic effect of RCE was even more striking in vivo, where nearly complete protection against STZ-induced diabetes was observed. Treatment of mice with STZ resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by immunohistochemical staining; however, pretreatment of mice with RCE blocked the destruction of STZ-induced islets and the development of type 1 diabetes.     

SHR和WKY大鼠心肌细胞凋亡、HSP70、iNOS水平的比较研究

目的:检测自发性高血压大鼠(SHR)和正常血压大鼠(WKY)心肌细胞凋亡和诱导型一氧化氮合成酶、热休克蛋白70水平变化,并探讨其机制。方法:透射电镜、TUNEL法检测SHR和WKY大鼠心肌细胞凋亡;免疫组化检测其iNOS、HSP70蛋白表达。结果:透射电镜示SHR组可见凋亡特征的心肌细胞;TUNEL法示SHR组凋亡明显高于WKY组(P<0.01);免疫组化示SHR组iNOS、HSP70蛋白表达明显高于WKY组(P<0.01)。结论:SHR心肌细胞凋亡在自发性高血压病发病过程中起重要作用;SHR的iNOS、HSP70蛋白表达增加与细胞凋亡同时存在,可能也参与自发性高血压心肌细胞凋亡的调控。     

Ischemic damage increases nitric oxide production via inducible nitric oxide synthase in the cochlea

The present study was designed to elucidate the dynamic changes of nitric oxide (NO) production in the perilymph and to investigate the immunostaining for inducible nitric oxide synthase (iNOS) in the cochlea for 7 days after transient cochlear ischemia. Moreover, aminoguanidine, which is a selective iNOS inhibitor, was administrated immediately following ischemia and every 24h thereafter for 7 days to investigate whether the production of NO is dependent on the iNOS pathway. Significant increases in the oxidative NO metabolites, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), were measured on day 1 using an in vivo microdialysis and on-line high performance liquid chromatography (HPLC) system. The immunostaining for iNOS was strongly expressed on days 1 and 4 and returned to normal on day 7 after the ischemia. The administration of aminoguanidine reduced the oxidative NO metabolites on day 1 and suppressed the expression of iNOS. These findings suggest that transient ischemia causes a remarkable increase in NO production in the perilymph, which might be attributable to the iNOS pathway.