Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by butein in RAW 264.7 cells

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.     

Inhibitory effects of Irigenin from the rhizomes of Belamcanda chinensis on nitric oxide and prostaglandin E(2) production in murine macrophage RAW 264.7 cells

In the present study, we investigated antiinflammatory effects of six flavonoids isolated from the rhizomes of Belamcanda chinensis (Iridaceae) in RAW 264.7 macrophages. The results indicated that irigenin concentration dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin (PG) E(2) production. Furthermore, this compound inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins and mRNAs without an appreciable cytotoxic effect. Treatment of the transfectant RAW 264.7 cells with irigenin reduced the level of nuclear factor-kappaB (NF-kappaB) activity, also effectively lowered NF-kappaB binding measured by electrophoretic mobility shift assay (EMSA), which was associated with decreased p65 protein levels in the nucleus. On the basis of the above data, we suggest that the effect of irigenin in decreasing LPS-induced NO and PGE(2) synthesis is due to diminish the mRNA and protein expression of iNOS and COX-2, respectively, also may be due to under the suppression of NF-kappaB activation. Therefore, irigenin isolated from the rhizomes of Belamcanda chinensis could be offered as a leading compound for anti-inflammation.     

核因子κB在精氨酸血管升压素诱导大鼠心肌成纤维细胞一氧化氮合成中的作用

本文探讨了精氨酸血管升压素(AVP)刺激下体外培养的大鼠心肌成纤维细胞(CFs)内一氧化氮(NO)含量、一氧化氮合酶(NOS)活性、诱导型一氧化氮合酶基因表达的变化及其与核因子κB(NF-κB)的关系。用胰酶消化法分离培养Sprague Dawley仔鼠的CFs,分别采用硝酸还原酶法、分光光度法、逆转录-聚合酶链式反应(RT-PCR)、免疫荧光-共聚焦显微镜和蛋白质印迹检测AVP干预下CFs的NO含量、NOS活性、iNOS mRNA表达和NF-κB的活化。结果显示,AVP浓度依赖性(0．001—0．1μmol／L)地增加CFs的NO含量,提高NOS活性,增加iNOS mRNA表达;AVP能够活化NF—κB,使其由细胞浆转位于细胞核;NF-κB特异性抑制剂吡咯啉烷二甲基硫脲(PDTC)能够抑制AVP诱导的CFs NO含量增加、NOS活性提高和iNOS mRNA表达增加。上述结果提示,AVP干预下CFs iNOS mRNA表达增加、NOS活性增高、NO合成增多可能通过NF-κB激活途径,NF-κB激活参与心肌纤维化的发生和发展。     

8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and C/EBPbeta in Raw 264.7 cells

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPbeta DNA-binding activity and NF-kappaB activation.     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

Age-related decline of inducible nitric oxide synthase gene expression in primary cultured rat hepatocytes

Hepatocytes exhibit a non-specific immune response by expressing the enzyme inducible nitric oxide (NO) synthase (iNOS, NOS2) through the stimulation of a mixture of cytokines, or a single cytokine such as interleukin-1beta. We examined the age-dependent inducibility of the iNOS gene expression and the capacity of NO production in response to lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) in primary cultured rat hepatocytes that were isolated from the livers of rats, 3 (young) and 24 (aging) months of age. NO production (NO2-), indicating iNOS activity, was much higher in the young rat hepatocytes following stimulation with LPS or IL-1beta. Likewise, in the young hepatocytes, Western blot analyses showed much higher protein levels in the iNOS expression; it was also a little higher in mRNA levels that were analyzed by RT-PCR. Furthermore, after stimulation with IL-1beta, the levels of transactivation of nuclear factor-KB (NF-kappaB) that were involved in the induction of the iNOS gene were reduced without a significant difference in the aged cells. Therefore, the decrease of NO formation in the aged hepatocytes was due to the belated and incomplete inducibility of the iNOS protein expression, together with a minor contribution of the reduced-transactivation of NF-kappaB. These results suggest that the age-related decline of the iNOS gene expression in primary rat hepatocytes may be associated with the increased incidence of many infective diseases with aging.     

Hepatic ischemia/reperfusion in rats induces iNOS gene transcription by activation of NF-kappaB.

It has been known that many immediately early genes are expressed during ischemia/reperfusion (I/R) injury. Here, employing a model of hepatic I/R, we show that inducible nitric oxide synthase (iNOS) is induced via the activation of nuclear factor kappaB (NF-kappaB) after I/R in rat liver. When liver was subjected to ischemia followed by reperfusion, but not ischemia alone, an NF-kappaB complex composed of p50/p65 heterodimer and p50 homodimer was rapidly activated within 1 h and remained elevated for up to 3 h, and then tended to decline after 5 h of reperfusion. Also, the expression of iNOS mRNA was initiated after 1 h and continued to increase after 5 h of reperfusion during the time course studied. This upregulated iNOS mRNA expression coincides with increased iNOS enzyme activity and NF-kappaB binding activity after hepatic I/R. Administration of N-acetylcysteine (NAC, 20 mg/kg i.v. 10 min before reperfusion), an antioxidant, not only significantly inhibited the expression of iNOS mRNA but also blocked upregulated NF-kappaB binding activity after reperfused liver. These results suggest that NF-kappaB is activated by oxidative stress during hepatic I/R and may play a significant role in the induction of the iNOS gene.