Effects Of Polysaccharide From Inonotus Obliquus On Signaling Pathway In RAW264.7 Cells Infected With Toxoplasma Gondii

Inonotus Obliquus Polysaccharide(IOP)is a polysaccharide component extracted and isolated from the traditional Chinese medicine Inonotus Obliquus,and has a wide range of biological activities.Previous studies in this laboratory have shown that IOP has significant anti-toxoplasmosis and immune enhancement functions in vivo.However,the research on the anti-toxoplasma mechanism of IOP in vitro is still unclear.Therefore,this study will study the regulation mechanism of NF-?B and MAPK signaling pathways in RAW264.7 macrophages infected with Otoliths in vitro.In this study,macrophage RAW264.7 was used as a test object to study the effect of IOP on the expression of inflammatory factors and gene transcription levels in RAW264.7 macrophages infected with Toxoplasma gondii.By detecting the expression of key proteins in NF-?B and MAPK signaling pathways,the immune regulation mechanism of IOP against Toxoplasma gondii was studied from the level of molecular immunity,which provided a scientific basis for the development and application of IOP.First,the MTT method was used to determine the safe test dose of the IOP.The results showed that IOP with 0?180 ?g/mL had no obvious cytotoxicity(P>0.05).Therefore,the concentrations of IOP in the high,medium,and low groups of IOP were 100 ?g/mL,50 ?g/mL,and 25 ?ig/mL,respectively.The expression levels of related inflammatory factors IL-1?,IL-4,IL-6,IFN-y and TNF-a in the supernatant of RAW264.7 cell culture supernatant were detected by ELISA.The inflammatory factors and chemokine mRNAs of RAW264.7 cells were detected by RT-PCR.The effect of IOP on the activity of nuclear transcription factor AP-1(c-jun)and N-NF-?B p65 in NF-?B signal transduction pathway was detected by laser confocal method.The key proteins of NF-?B and MAPK signal transduction pathway,TLR2,TLR4,p-IKK?/?,p-I?B?,C-NF-?B p65 and N-NF-kB p65 and p-p38,p-JNK and p-ERK were determined by Western-blo method.ELISA results showed that IOP inhibited the overexpression of related inflammatory factors to some extent.Compared with the model group,the cytokines IL-1?,IL-4,IL-6,IFN-y and TNF-a in the cell culture group showed a downward trend.RT-PCR results showed that IOP inhibited the overexpression of IL-1?,IL-6,TNF-a and chemokines MIP-la and MCP-1 mRNA in RAW264.7 cells infected with Toxoplasma gondii in a dose-dependent manner.IOP could regulated the expression of cytokines and chemokine mRNA in RAW264.7 cells infected with Toxoplasma gondii at the transcriptional level of cells,and then regulated the immune response of the body.The results of laser confocal microscopy showed that IOP could inhibit the nuclear transcription factor AP-1(c-jun)and N-NF-?B p65 from cytoplasmic dissociation to nucleus in RAW264.7 cells infected with Toxoplasma gondii in a dose-dependent manner.Western-Blot results showed that IOP up-regulated the expression of C-NF-?B p65 in RAW264.7 cells infected with Toxoplasma gondii,and down-regulated TLR2,TLR4,p-IKK?/?,p-I?B?,N-NF-?B p65 and overexpression of p-p38,p-JNK and p-ERK proteins,key proteins of MAPK signaling pathway(P<0.05 or P<0.01).The results showed that IOP could regulated the over-expression of TLR2,TLR4 and inhibited the phosphorylation level of IKK?/?,I?B?,p38,JNK and ERK in NF-?B signaling pathway and regulated the shift of NF-?B p65 from cytoplasm to nucleus.In summary,the results showed that IOP showed anti-toxoplasma effect in Toxoplasma gondii RAW 264.7 macrophages.IOP regulates the expression of cytokines and the expression of key proteins in mRNA expression and pathway by regulating the cascade of signal factors in NF-?B and MAPK signal transduction pathways,thereby exerting its regulatory role against Toxoplasma gondii in vitro.