Heptadecane is a volatile component of Spirulina platensis, and blocks the synthesis of fatty acids and ameliorates several oxidative stress-related diseases. genes) and reactive species (RS) production in aged kidney tissue. In the second part of the study, YPEN-1 cells (an endothelial cell line) were used to explore the molecular mechanism underlying the anti-inflammatory effect of heptadecane by examining its modulation of NF-kB and NF-kB signal pathway. Results showed that heptadecane exhibited a potent anti-oxidative effect by protecting YPEN-1 cells from tert-butylhydroperoxide induced oxidative stress. Further molecular investigations revealed that heptadecane attenuated RS-induced NF-kB via the NIK/IKK and MAPKs pathways in YPEN-1 cells and aged kidney tissues. Based on these results, we conclude that heptadecane suppresses age-related increases in pro-inflammatory gene expressions by reducing NF-kB activity by upregulating the NIK/IKK and MAPKs pathways induced by RS. These findings provide molecular insight of the mechanisms by which heptadecane exerts its antiinflammatory effect in aged kidney tissues. We conclude that heptadecane suppresses age-related increases in pro-inflammatory gene expressions then travel upstream set by step by reducing NF-kB activity by downregulating the NIK/IKK and MAPKs pathways induced by RS. Introduction Heptadecane is major component of Spirulina platensis , which contains high levels of proteins, amino acids, vitamins, beta-carotene, and other pigments , and it has been shown that the strong antioxidative effects of have therapeutic benefit in a rat renal disease model , and that it inhibits the proliferation of human liver cancer cells . Moreover, heptadecane was found to almost completely block the synthesis of fatty acids studies on an endothelial cell line. Here, we report the suppressive effect of heptadecane on NF-kB and on the ability of heptadecane to reduce age-related oxidative stress and to modulate the NIK/IKK and MAPKs cascades. Results Modulation of Age-related NF-kB Activation by Heptadecane To assess overall age-related oxidative status and its modulation by heptadecane, total RS was measured in kidney homogenates using a DCFDA probe. The results showed that RS level increased with age and that this increase was significantly suppressed by treatment with high-dose heptadecane (Fig. 1A). To determine whether or not NF-kB activation is increased during aging, we examined nuclear protein levels by Western blotting using p65- and p50-specific polyclonal antibodies. The results shown in Fig. 1B clearly reveal that the nuclear translocation of NF-kB was significantly greater in aged rats, but that aged heptadecane-fed rats showed dose-dependently lower levels of NF-kB. Furthermore, as shown in Fig. 1B, aged heptadecane-fed rats showed higher levels of IkB and IkB proteins in cytoplasmic extracts than aged rats, while Thus, these findings indicate that NF-kB translocation during aging is probably elicited by age-related increases in the degradations of IkB and IkB. Figure 1 Heptadecane suppressed age-related increases in NF-kB activity. To verify NF-kB DNA-binding, EMSA was carried out using nuclear proteins isolated from young, aged, and aged rats fed heptadecane. The results shown in Fig. 1C indicate that the binding activity of NF-kB RAD001 was upregulated during aging, and that heptadecane suppressed this upregulation. Additionally, we performed immunohistochemistry analysis on aged rat renal tissue for the immunoreactivity using anti-NF-kB (p65) antibody. As shown in Fig. 1D, intensive NF-kB increased compared with that of tubular cells of young controls, indicating the suppression RAD001 of the over expression of NF-kB by heptadecane. Suppression of NIK/IKK and of MAPK Activation by Heptadecane during Aging We investigated whether heptadecane can regulate the phosphorylations of the NIK/IKK and MAPKs pathways which lead to NF-kB activation during aging. The results obtained showed that the phosphorylations of NIK/IKK and MAPKs were significantly increased in aged rats; however, aged, heptadecane-fed rats showed dose dependent lower levels than their aged, non-heptadecane-fed counterparts (Fig. 2). Figure 2 Heptadecane suppressed the age-related activations of NIK/IKK and MAPKs. These results show that heptadecane supplementation significantly inhibited the oxidative stress-induced phosphorylations of NIK/IKK and MAPKs during aging, and that heptadecane blocks the phosphorylation and degradation of IkB, which elicit the nuclear translocation of NF-kB. Inhibition of NF-kB-responsive Gene Expression by Heptadecane Above all, our findings data indicate that NF-kB activation is involved in age-related oxidative stress (Fig. IL6 RAD001 3), which leads to age-related inflammation and that this age-related NF-kB activation is strongly inhibited by heptadecane. In addition, we examined the gene expressions of iNOS and COX-2 (NF-kB-dependent genes), which both have a kB-site located in their promoter regions. Our results showed that the protein levels of these two genes were positively related to NF-kB activity and that heptadecane down-regulated their expressions. Figure 3 Heptadecane suppressed expressions of NF-kB-dependent genes in aged rats. Effect of Heptadecane on Oxidative Stress The oxidant, t-BHP, is a known to induce oxidative stress . To determine the defensive effect of heptadecane against oxidative stress, t-BHP was used.