The Research Of Signal Mechanism Of Macrophage Stimulated By Whole Peptidoglycan Of Bifidobacterium

Aim: Whole Peptidoglycan (WPG) is the main component of Bifidobacterium which can inhibit the growth of tumours,resist to infection and enhance immunity. It is also a regulator that has no toxin and side effects.WPG can activate macrophages,and facilitate it to secret a lot of cytotoxic molecules.At present, there is no much knowledge about this mechanisms.In this study,we want to explore the signal mechanisms of WPG activating macrophages by investigating the effects of WPG which purified from Bifidobacteria bifidum on the pathway of PKC→NF-κB and PKC → ERK1/2→AP-1 and the transcription of TNF-α mRNA of rat peritoneal macrophages.Methods: SD rat peritoneal macrophages were separated and cultivated.The experiment included control group ,WPG stimulating group and preincubating group.(1)The translocation of NF-kB of macrophages was observed by using laser scanning confocal microscopy(LSCM).(2)NF-κB and AP-1 DNA binding activity of macrophages was evaluated by employing electrophoretic mobility shift assay (EMSA). (3)The protein expression of phosphorylation of ERK1/2 and IκB α of macrophages was detected by using western blot. (4)The expression of TNF-α mRNA was evaluated by using semi-quantity of RT-PCR.Results: NF-kB situated in the cytoplasm of normal rat peritoneal macrophages. NF-kB was significantly activated and translocated from cytoplasm to nuclei after the macrophages were stimulated by WPG. By using EMSA, NF-kB DNA binding activity of macrophages in WPG stimulating group was significantly higher than that in control group(P<0.01). By employing LSCM,The positive rate of NF-kB in the nuclei of macrophages in WPG stimulating group was obviously higher than that in control group(P<0.01).By using western blot,the protein expression of IκB α in the cytoplasm of macrophages in WPG stimulating group was markedly lower when compared with control group(P<0.01). After the macrophages were pretreated with Chelerythrine, its NF-kB translocation,DNA binding activity and degradation of IκB α was significantly lower when compared with WPG stimulating group(P<0.01).(2) The ERK activity was low in normal rat peritoneal macrophages.After the macrophageswere stimulated by WPQits protein expression level of phosphorylation of ERKl/2 was obviously higher when compared with control group(P<0.01). After the macrophages were pretreated with Chelerythrine, its protein expression level of phosphorylation of ERKl/2 was markedly lower when compared with WPG stimulating group(P<0.01). (3) AP-1 situated in the cytoplasm of normal rat peritoneal macrophages.After the macrophages were stimulated by WPG , AP-1 was significantly activated.Its DNA binding activity of macrophages in WPG stimulating group was significantly higher than that in control group by using EMSA(P<0.01). After the macrophages were pretreatment with PD98059,its AP-1 DNA binding activity was significantly lower when compared with WPG stimulating group(P<0.01). (4)There was low expression of TNF-a mRNA in normal rat peritoneal macrophages.The expression of TNF-a mRNA of macrophages in WPG stimulating group was markedly higher than that in control group(P<0.01). After the macrophages were pretreatment with NAC and PD98059 respectively, the expression of TNF-a mRNA was significantly lower when compared with WPG stimulating groupCPO.Ol).Conclusion: (1) WPG of Bifidobacteria could elevate the activity of NF-kB^ ERKl/2 and AP-1 of macrophages.(2) WPG of Bifidobacteria could degrade IkB a and activate NF-kB of macrophages through PKC.(3) WPG of Bifidobacteria could activate ERKl/2 of macrophages through PKC.(4) WPG of Bifidobacteria could activate AP-1 of macrophages through PKC.(5) WPG of Bifidobacteria could regulate the expression of TNF-a mRNA of macrophages by activating NF-kB and ERKl/2.