Effects Of Pine Pollen Polysaccharides Pre-sulfation And Post-sulfation And DSS On LPS-induced Oxidative Stress And Inflammation In RAW264.7 Cells

Polysaccharides and sulfated polysaccharides from Pinus massoniana pollen could improve immune function.Previous studies showed that pine pollen polysaccharides and sulfated polysaccharides could alleviate DSS（Dextran sulfate sodium）-induced ulcerative colitis,which was related to the regulation of Th17 and Treg cells imbalance and the RIPK3-mediated necroptosis pathway.However,DSS also belonged to sulfated polysaccharides basically,and the reason why DSS could induce colitis but pine pollen sulfated polysaccharides could alleviate colitis was not clear.Previous studies in the laboratory confirmed that the pine pollen sulfated polysaccharides could improve the immune activity by increasing[Ca²⁺]i.Therefore,it was firstly speculated that DSS could induce colitis probably related to the reduction of[Ca²⁺]i.However,the pre-experimental results showed that DSS increased[Ca²⁺]i just like the pine pollen sulfated polysaccharides,so the hypothesis was not valid.Inflammation is closely relevanted to oxidative stress,and the effects of pine pollen polysaccharides,sulfated polysaccharides and DSS on LPS-induced oxidative damage and inflammation of RAW264.7cells have not been studied.Therefore,the research of this thesis was based on oxidative stress and inflammation to explored the reason.Whether pine pollen polysaccharides and sulfated polysaccharides could alleviate LPS-induced oxidative stress and inflammation but DSS could aggravate LPS-induced oxidative stress and inflammation was unknown.In this thesis,we focused on this issue.The research included the following seven parts:1.Polysaccharides were extracted from pine pollen,purified by column separation and sulfated.Polysaccharides were extracted by the previous laboratory method and named as PPM60.PPM60 was purified by Sephacryl ^TMS-400 column to gain the third component and named as PPM60-Ⅲ.PPM60-Ⅲwas sulfated by chlorosulfonic acid-pyridine way to gain sulfated polysaccharides and named as SPPM60-Ⅲ.2.RAW264.7 cells were cultured,cell activity was determined by CCK-8,and apoptosis was determined by TUNEL method.For the activity of RAW264.7 cells,PPM60-Ⅲand SPPM60-Ⅲincreased first and then decreased,and DSS showed inhibition that depend on concentration.For LPS-induced apoptosis,PPM60-Ⅲ,SPPM60-Ⅲand DSS all played a role in alleviating it.3.The level of ROS（Reactive oxygen species）,the ability of resisting·O₂^-（Superoxide anion Free radicals）and·OH（Hydroxide free radicals）and the level of MDA（Malondialdehyde）in cells were measured by the kit.Both PPM60-Ⅲand SPPM60-Ⅲsignificantly inhibited the level of ROS and MDA in RAW264.7 cells,but DSS did not show this phenomenon.PPM60-Ⅲand SPPM60-Ⅲboth improved the ability of cells to resist·O₂^-and·OH,so played an antioxidant role.However,DSS did not significantly improve the ability of cells to resist·O₂^-and·OH.4.The contents of IL-1β,IL-6,TNF-α,IL-10 and NO in cells were tested by the kit.Both PPM60-Ⅲand SPPM60-Ⅲdiminished the standards of factors IL-1β,IL-6,TNF-αand NO that promote inflammation in RAW264.7 cells,added the content of the factor IL-10 that resist inflammation,so restrained cell inflammation.However,DSS further intensified the generation of NO induced by LPS,further reduced the content of IL-10 that resist inflammation,so intensified the inflammatory damage of cells to some extent.5.SOD2（Superoxide dismutase 2）,CAT（Catalase）,GR（Glutathione reductase）,GPx4（Glutathione peroxidase 4）,NOX（NADPH oxidase）and i NOS（Inducible nitric oxide synthase）oxidoreductase were analyzed by western blot.For antioxidant enzymes SOD2,CAT,GR,GPx4,PPM60-Ⅲand SPPM60-Ⅲall enhanced their expression levels;On the contrary,DSS weakened their expression levels.For oxidases NOX and i NOS,both PPM60-Ⅲand SPPM60-Ⅲweakened their expression levels;On the contrary,DSS strengthened their expression levels.It was further verified that PPM60-Ⅲand SPPM60-Ⅲcould resist LPS-induced oxidative stress,but DSS aggravated LPS-induced oxidative stress.6.The levels of Keap1,Nrf2,Nrf2（nucleus）,Nrf2（cytoplasm）,HO-1,NQO-1,GCLM and GCLC in Keap1-Nrf2 signal pathway were analyzed by western blot.Keap1-Nrf2 signaling pathway is an important mechanism for the body to defend oxidative stress and inflammatory injury,and also played an important role in cell metabolism.Both PPM60-Ⅲand SPPM60-Ⅲenhanced the expression standards of peroxiredoxins Nrf2,NQO-1,GCLM,GCLC and HO-1,while DSS further weakened the levels of peroxiredoxins Nrf2,NQO-1,GCLM,GCLC and HO-1 decreased by LPS.PPM60-Ⅲand SPPM60-Ⅲboth reduced the expression level of Keap1induced by LPS,while DSS further enhanced the expression level of Keap1.In addition,both PPM60-Ⅲand SPPM60-Ⅲcould promote the nuclear translocation of Nrf2,but DSS could restrain the nuclear translocation of Nrf2.It showed that PPM60-Ⅲand SPPM60-Ⅲcould resist oxidative stress and inflammation,while DSS could aggravate oxidative stress and inflammation,which was relevanted to Keap1-Nrf2 pathway.7.Metabolomics was used to find differential metabolites.Oxidative stress and inflammationis were also closely relevanted to metabolic changes.Multivariate statistical analysis displayed that the differential metabolites between each group could be well divided,and the metabolic pathways primary contained glycometabolism and amino acid metabolism.PPM60-Ⅲand SPPM60-Ⅲcould balance the disorder of glycometabolism and amino acid metabolism induced by LPS,thus alleviated oxidative stress and inflammation to some extent,while DSS could further disorder glycometabolism and amino acid metabolism,thus aggravated oxidative stress and inflammation to some extent.To sum up,PPM60-Ⅲand SPPM60-Ⅲcould resist oxidative stress and inflammation by activating Keap1-Nrf2 signaling pathway,thus enhanced cell activity.DSS could aggravate oxidative stress and inflammation by inhibiting Keap1-Nrf2 signaling pathway,thus inhibited cell activity,which was closely relevanted to glycometabolism and amino acid metabolism.