Effects Of Agaricus Blazei Murill Polysaccharide On TLR4 Receptor Signal Transduction Pathways

[Objective]To study the effects of Agaricus Blazei murill polysaccharide(ABPS)on TLR4 receptor signal transduction pathways,and discuss the immune regulation mechanisms of ABPS.[Methods](1)Using ABPS whose concentration respectively was 2000 ?g/mL,1000?g/mL,500?g/mL,250 ?g/mL,125 ?g/mL,62.5 ?g/mL,31.2?g/mL and 15.6 ?g/mL acting on macrophage RAW264.7 12 h,24 h and 36 h,and then we detected the macrophage proliferation by MTT.(2)Macrophage RAW264.7 was treated with the same concentration of lipopolysaccharide(LPS)and ABPS for 3 h,6 h,12 h,and 36 h,at the same time we used different concentrations of LPS and ABPS acting on macrophage RAW264.7 for 24 h.We treated with macrophage RAW264.7 by TLR4 antibody for an hour,and then cultured the cells for 24 h using the new cell culture medium which contained the ABPS and LPS.Collecting the cell culture and the cells respectively,then we detected the production of NO in cell culture supernatant;collected the macrophages' whole proteins and tested the content of iNOS;detected the content of cytokines IL-1?,TNF-?,IFN-? by ELISA;extracted the total RNA and determined the TNF-?,IL-1?,IL-6,TLR4,MyD88,TRAM,TRAF-6 mRNA expression of macrophages by fluorescence quantitative RT-PCR.[Results](1)When macrophage RAW264.7 was treated with ABPS for 12 h,24 h and 36 h,ABPS could promote the proliferation of macrophage.When the concentration of ABPS was 1000 ?g/mL,500 ?g/mL,250 ?g/mL,125?g/mL and 62.5?g/mL,there were the extremely significant differences in the proliferation of macrophage(P<0.01).Among them,the ABPS whose concentration was 1000?g/mL had the strongest effect on cell proliferation.(2)The NO generated from macrophage treated by ABPS and positive control(LPS),was higher than the negative control,which had the significant differences(P<0.05 or P<0.01).In each period,the NO produced from LPS group was higher than that in the ABPS group and the difference was significant(P<0.01).(3)After macrophage RWA264.7 was treated with different concentrations of ABPS and LPS for 24 h,the production of NO was higher than that in the negative control group,which had the extremely significant differences(P<0.01).The NO production of LPS group was higher than that in the ABPS group.There were extremely significant differences in the NO production of the ABPS group whose content respectively was 2000 ?g/mL,500 ?g/mL and 250 ?g/mL(P<0.01).There was significant difference of the NO production when the content of ABPS was 1000 ?g/mL(P<0.05).In the certain concentration range of ABPS,the production of NO increased with the increasing of ABPS concentration.The production of NO was the largest when the polysaccharide concentration was 1000 ?g/mL.After macrophage RAW264.7 was treated by TLR4 antibody,the production of NO is lower than that of untreated group.And the differences were significant(P<0.05)or extremely significant(P<0.01).But the productions of NO in treated groups were still higher than that in the negative control group which had the extremely significant differences(P<0.01).(4)After macrophage RWA264.7 was treated with ABPS and positive control(LPS)for 24 h,the iNOS content of macrophage was higher than that in the negative control group and there were extremely significant difference among them(P<0.01).The iNOS content of positive control group was significantly higher than ABPS(P<0.05 or P<0.01).The iNOS content increased with the concentration of ABPS increasing in the certain concentration range.The iNOS content was the largest when the polysaccharide concentration was 1000?g/mL.(5)After macrophage RWA264.7 was treated with ABPS and positive control(LPS)for 24 h,the IL-1?,TNF-?,IFN-? contents in the cell culture supernatant were significantly higher than those in the negative control group(P<0.01).The IL-1?,TNF-?,IFN-? contents in the positive control group were significantly higher than those in the ABPS group.In the certain concentration range of ABPS,the IL-1?,TNF-?,IFN-? contents in the cell culture supernatant increased with the concentration of ABPS increasing.The IL-1?,TNF-?,IFN-?contents were the maximum when the concentration of ABPS was 1000?g/mL.After macrophage RAW264.7 was treated with TLR4 antibody,the IL-1?,TNF-?,IFN-? contents were lower than those in the untreated groups which had extremely significant differences(P<0.01).(6)Compared with the control group,the mRNA expression levels of TNF-??IL-1??IL-6?TLR4?MyD88?TRAM?TRAF-6 increased in polysaccharide group with the concentration of ABPS increasing.After macrophage RAW264.7 was treated with TLR4 antibody,the mRNA expression levels of TNF-??IL-1??IL-6?TLR4?MyD88?TRAM?TRAF-6 were lower than those in the untreated group,and the differences were significant(P<0.05 or P<0.01).[Conclusion]ABPS could promote the proliferation of macrophage RAW264.7,and it coud adjust the immune function of macrophage RAW264.7 by means of the TLR4 receptor signal transduction pathways.TLR4 is one of the receptors of ABPS.