| Objective: to observe the activation of nuclear factor-кappa B (NF-кB ) in alveolar macrophages (AMs) induced by lipopolysaccharide(LPS), to reveal the function and mechanism of NF-кB activation in apoptosis of AMs, to investigate the role of protein kinase C(PKC) in NF-кB activation.Method: AMs that collected, pured and cultured with contine method were stimulated by LPS of different concentration(0μg/ml,0.01μg/ml,0.1μg/ml,1μg/ml,10μg/ml) for 60min or by 1μg/ml LPS for different time stage (0min,5min,15min,30min,60min,120min) to observe the dynamic change of NF-кB intranuclear level and NO production, from which the best concentration and time point of LPS stimulation were selected. In the study, all AMs were divided into 4 groups: control group, group stimulated with LPS, group interrupted by Cal C and group inhibited by PDTC. The following parameters were measured: NF-кB level in nuclear protein extraction of AMs detected with sandwich ELISA, Inter-nuclear transposition of NF-кB observed with immunocytochemistry staining, NO content in cell culture medium quantitied with nitric acid reductase assay, Morphologic change of AMs in apoptosis observed with acridine orange (A.O) staining and fragmentation at genome DNA of AMs detected with apoptotic electrophoresis assay.Result: 1. NF-кB intranuclear level and NO production of AMs increased along with the raise of LPS concentration and elongation of treatedtime. Both change were LPS time-dependent and dose-dependent. The peak of NF-кB level was seen when AMs has been stimulated with 10μg/ml LPS for 60min, while NO production showed a peak after treated 48h for 10μg/ml LPS. 2. The results of different group were showed as following: ①NF-кB intranuclear level and NO production of LPS group were higher than that of control group (p<0.01). Pretreated cell with Cal C and PDTC could inhibit both LPS-induced NF-кB activation and NO production. Values of both groups were less than that of LPS group obviously. When cell has been pretreated for 30min with Cal C or PDTC, the LPS-induced NF-кB level was inhibited by 45.2% or 63.8%, while LPS-induced NO production was inhibited 73.8% or 80.6% by Cal C or PDTC. ②Immunocytochemistry assay showed that NF-кB in cells of control group mainly laid in plasma and less in nuclear. Exposure of AMs to LPS caused NF-кB transposition from plasma to nuclear. After pretreated cells with Cal C or PDTC for 30min, NF-кB could be find in plasma or in nuclear. In PDTC pretreated group, there were more cells with NF-кB in plasma than in Cal C group. But the ratios of such cells in both groups were less than in control group. ③The changes in morphology of apoptotic cells: When has been treated with LPS for 24h, AMs became round, plasma dominate or disappeared, chromatin moved to edge of nuclear as a half-moon, nucleolus broke, apoptotic body appeared increasely along with the elongation of stimulation time, cell died and lysised at last. Less apoptotic bodies were seen in Cal C group than in PDTC group; ④The result of apoptotic electrophoresis assay showed a characteristic ladder-shaped line with about 200bp interval (called as ladder pattern) in the electrophoresis path of genome DNA extracted from LPS-treated AMs. Ladder pattern in Cal C group was weakening than that of LPS group, while such line wasn't appeared in control group and PDTC group.Conclusion: 1. LPS can induce the NF-кB activation of AMs, which make it possible for the transcription and expression genes(such as NOS) that would lead to apoptosis of AM at last. 2. It was showed in Cal C inhibitory assay that PKC was involved in the signal transduction pathway of LPS-induced NF-кB activation as up-stream messenger.
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