| Objective:Purple sweet potato is rich in anthocyanins,polysaccharides,carotenoids and other active substances,and has many biological functions such as anti-cancer,anti-oxidation,enhancing immune function,anti-tumor and so on.It also has many biological effects such as anti-cancer,antioxidation,reducing blood fat,blood sugar and enhancing immunity.Most experiments show that polysaccharides can bind to macrophage surface receptors,activate macrophage cells through signal transduction,and release cytokines to play the role of immune regulation.Although purple sweet potato polysaccharides are important active components in Purple Sweet Potato,there are few studies on its biological function,especially the immune regulation function.PSPP-1 was isolated and purified from Purple Sweet Potato by our research group.The content of PSPP-1 was 97.65%.PSPP-1 contained α-glycoside bond without three stranded helix structure and had amorphous morphology.Its molecular weight is 18300 Da,the main chain is→4)-α-D-Glcp(1→,the branched chain is α-DGlcp(1→,and a small amount of→4,6)α-D-Glcp(1→;→3,4)α-D-Glcp(1→;→2,4)α-D-Glcp(1→,The purple sweet potato polysaccharide PSPP-1 not only has no cytotoxic effect on a variety of human normal cells,but also has a better inhibitory effect on tumor cells was proved in vitro experiments proved.Based on the previous research of our group,we further studied the immunomodulatory function of purple sweet potato polysaccharide PSPP-1,investigated the related immunomodulatory activity and mechanism,clarified the biological activity of polysaccharide through receptor cell effect signal transduction pathway,and provided scientific basis for studying the effect of purple sweet potato polysaccharide PSPP-1 on innate immune system and its immunomodulatory mechanism This study provides a theoretical basis for screening more targets of purple sweet potato polysaccharides and futher development and utilization of purple sweet potato polysaccharides.In this study,the mouse macrophage RAW264.7 cells were used as the research object.MTT method was used to detect the effect of purple sweet potato polysaccharide PSPP-1 on the activity of mouse macrophage RAW264.7 cells.Inverted microscope was used to detect the effect of purple sweet potato polysaccharide PSPP-1 on the morphology of mouse macrophages.Neutral red method was used to detect the effect of purple sweet potato polysaccharide PSPP-1 on the phagocytic function of mouse macrophage RAW264.7,the effect of purple sweet potato polysaccharide PSPP-1 on cell cycle of mouse macrophage RAW264.7 detected by Flow Cytometry.The effect of PSPP-1 on NO secretion of RAW264.7 macrophages was detected by Griess method.The effect of PSPP-1 on ROS secretion of RAW264.7 macrophages detected by Flow Cytometry.The levels of cytokines such as IL-1β,IL-6,TNF-α and IFN-β in cell culture supernatant were detected by ELISA.Western blot was used to detect the effect of PSPP-1 on the expression of TLR4、TLR2、TIRAP、MyD88、IRAK4、p-IRAK4、IRAK1、p-IRAK1、TRAF6、TAK1、p-TAKl、IKKα/β、p-IKKα、pIKKβ、IκBα、p-IκBα、NF-κB、p-NF-κB、p-MEK1/2、ERK、p-ERK、p-MKK3/6、P38、pP38、p-MKK7、JNK、p-JNK、TRAM、TRIF、TRAF3、TBK1、p-TBK1、IRF3、p-IRF3 protein in Toll like receptor 2/4(tlr2/4)signaling pathway in RAW264.7 cells.The results are as follows:(1)PSPP-1 has no cytotoxic effect on RAW264.7 cells and can stimulate the proliferation and activation of macrophages,increase the volume of RAW264.7 cells,change from the resting round to the spindle with pseudopodia,and promote the migration of RAW264.7 cells from G1 phase to S phase and G2/M phase.(2)PSPP-1 can enhance the phagocytic function of mouse macrophage RAW264.7 cells and promote the secretion of NO,ROS,IL-1β,TNF-α,IL-6 and IFN-β.(3)PSPP-1 could up regulate TLR4、TLR2、TIRAP、MyD88、IRAK4、p-IRAK4、IRAK1、p-IRAK1、TRAF6、TAK1、p-TAK1、p-IKKα、p-IKKβ、p-IκBα、p-NF-κB protein Level and down regulate IκBα protein level in RAW264.7 cells.(5)PSPP-1 could up regulate the protein levels of p-MEKl/2、p-ERK、p-MKK3/6、p-P38、pMKK7、p-JNK in RAW264.7 cells.Conclusion:PSPP-1 can pass through TLR2/4 receptor MyD88 dependent signaling pathway(TLR4/TLR2/TIRAP/MyD88/IRAK4/p-IRAK4/IRAK1/p-IRAK1/TRAF6/TAK1/p-TAK1),NF-κB pathway(IKκα/β/P-IKKα/β-IKKβ/IκBα/p-IκBa/NF-κB/p-NF-κB),MAPKs pathway(pMEK1/2/ERK/p-ERK、p-MKK3/6/p38/p-P38、p-MKK7/JNK/p-JNK)and TRIF dependent signaling pathway(TLR4/TLR2/TRAM/TRIF/TRAF3/TBK1/p-TBK1/IRF3/p-IRF3)synergistically activated mouse macrophage RAW264.7 cells to produce immune response,secrete NO,ROS,IL-1β,IL-6,TNF-α and IFN-β,enhance the immune function of mouse macrophage RAW264.7.It provide scientific basis for further development and utilization of purple sweet potato polysaccharide. |
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