| Oligochitosan, having an average molecular weight of 1,000 Da and a degree of N-acetylation below 15%, can be obtained by either chemical or enzymic hydrolysis of chitosan. Oligochitosan is not only water-soluble, non-toxic, biocompatible but also possesses versatile functional properties of chitin and its derivatives, such as polyelectrolite properties, the presence of reactive functional groups, gel-forming ability, high adsorption capacity, bacteriostatic, fungistatic and antitumour influence, and thus it has attracted more and more attention in the field of biology. Previous investigations have shown that chitin and its derivatives have immunoenhancement activities. They can induce the immunocytes to secret numbers of immune-functional molecules, which mediate the stimulation of the whole immune system. There is difference that whether chitin and its derivatives alone can induce macrophages to produce nitric oxide (NO) and tumor necrosis factor (TNF). The data gained from Giuseppe Balsamo et al. showed that chitosan alone can induce the production of NO in murine peritoneal exudates macrophages, however, Hyung-Min Kim et al. and Hun-Taeg Chung et al. reported that chitosan or oligochitosan can synergistically induces NO production by RAW264.7 cells when the cells are treated with recombinant r-IFN. Chitosan or oligochitosan alone has no effect on the production of NO.The information above led us to determine whether oligochitosan can enhance NO release in macrophages and to further determine whether nuclear factor-kappa B (NF-kB)is involved in the course of the activation. Macrophage-like line RAW264.7 cells were cultured. The cells were then stimulated with various concentrations and incubation times of oligochitosan to study dose-dependent and time-dependent manners. The effect of oligochitosan on the production of NO released by RAW264.7 cells was evaluated by Griess method, Which was also used to evaluate the effect of NF-kB inhibitor on NO production induced by oligochitosan. Western blot was performed to detect the protein content of NF- k B in the nuclear extract. The activity of inducible nitric oxide synthase (iNOS) was determined using iNOS assay kit.The data we obtained show that in resting macrophages, the basal levels of iNOS activity and NO production are relatively low; nevertheless, iNOS activity and NO production can be significantly induced in response to oligochitosan stimulation. Oligochitosan (80 (ug/ml) significantly induced the release of NO 12 h after incubation, and the amount of NO increased with time. RAW264.7 cells were treated with various doses of oligochitosan (5, 50, 100, 500 ug/ml) for 18 h, we found that oligochitosan caused marked increase in iNOS activity and NO production in a dose-dependent manner. In this paper, the western blot results demonstrated that NF-kB is involved in the course of the activation induced by oligochitosan. The protein level of NF-kB in nucleus peaks at 6 h after stimulation and persists for at least 6 h. PDTC (pyrrolidinedithiocarbamate), an inhibitor of NF-kB, the effective concentration of it for NF-kB inhibition without any toxicity on cells is up to 100 umol/L, can significantly block NO production induced by oligochitosan.Although the study reported herein reveals that oligochitosan induces NO production via NF-kB in macrophage, which is an important signaling mechanism of oligochitosan inducing immuno-enhancement of macrophage in vitro. There are still many questions remain unanswered, including the mechanisms that specify oligochitosan recognition, the participation of additional molecules in transmembrane signaling, and the repertoire of downstream pathways activated by oligochitosan. It was also observed that PDTC did not totally block NO production, which gave us another evidence that there must be some other signal transduction pathways (perhaps MAPK, PKC, Ca2+ dependent signaling) involved in the activation, so further investigation is necessary to clarify molecular mechanism of oligochitosan inducing immunological en... |
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