Study On Anti-inflammatory Effects And Molecular Mechanisms Of Coccomyxa Gloeobotrydiformis Polysaccharide

Objective:Inflammation is not only the most basic defensive response of the body,but also plays a key role in the occurrence and development of aging and related noncommunicable diseases（NCDs）,such as cardiovascular diseases,cancer,diabetes and so on.Our previous studies indicated that coccomyxa gloeobotrydiformis（CGD）polysaccharide not only showed a therapeutic effect on metabolic syndrome rats and associated cardiovascular complications,but also improved the learning and memory in intrinsic aging rats.The mechanisms might be related to its anti-inflammatory effect.Therefore,in this study,we aim to further investigate the anti-inflammatory mechanism of CGD polysaccharide in order to clarify its therapeutic effect and molecular mechanism on NCDs by observing the alterations of several classic inflammation-related signaling pathways including NF-κB,MAPK,PI3K/AKT,JAK/STAT and Nrf2/HO-1.Methods:1.The cell growth curve was drawn by cell counting method.2.The levels of nitric oxide（NO）in the supernatant of cell culture media was measured with Griess method.3.The proliferation of RAW264.7 cells was evaluated by using MTS assay.4.The LDH release rate of RAW264.7 cells was determined by lactate dehydrogenase（LDH）release assay.5.Morphological changes of the RAW264.7 cells were observed with an inverted microscope.6.Western blotting was used to detect the expressions of inflammatory mediators（PGE₂,iNOS and COX-2）and inflammatory cytokines（TNF-α,IL-6,IL-1βand IL-10）.7.Western blotting was used to detect the expressions of inflammatory pathways including NF-κB,MAPK,PI3K/AKT,JAK/STAT and Nrf2/HO-1 pathway.8.Immunofluorescent assay was used to observe the nuclear translocation of NF-κB p65.Results:1.The growth curve of RAW264.7 cells resembled"S"shape under our experimental conditions.After a 2-day latent adaptive phase,the cells entered the logarithm grow period and reached the peak on day 8,then the number of cells decreased slightly on day 9.2.CGD polysaccharide could inhibit LPS-induced NO production in RAW264.7 cells in the concentration range of 1⁸ mg/mL,and the inhibitory effect was the best when the concentration was 2 mg/mL and 4 mg/mL.3.CGD polysaccharide（2mg/mL,4 mg/mL）inhibited the proliferation of RAW264.7 cells but had no significant effect on cytotoxicity.4.CGD polysaccharide（4 mg/mL）could inhibit the LPS-induced morphological changes in RAW264.7 cells.5.CGD polysaccharide（2mg/mL,4 mg/mL）significantly inhibited the expression of inflammatory mediators including PGE₂,iNOS and COX-2 in LPS-induced RAW264.7 cells.It also suppressed the expression of pro-inflammatory cytokines including TNF-α,IL-6 and IL-1β,and up-regulated the expression of the anti-inflammatory cytokine IL-10 in LPS-stimulated RAW 264.7 cells.6.CGD polysaccharide（2 mg/mL,4 mg/mL）could inhibit the inflammatory pathways including NF-κB,MAPK,PI3K/AKT,JAK/STAT and enhance the anti-inflammatory pathway Nrf2/HO-1 in LPS-induced RAW264.7 cells.Conclusion:Taken together,the results above suggest that CGD polysaccharide significantly inhibits LPS-induced inflammation in RAW264.7 cells.The anti-inflammatory mechanisms of CGD polysaccharide lie in its regulatory effects on the inflammatory signal pathways including NF-κB,MAPK,PI3K/AKT,JAK/STAT and Nrf2/HO-1 pathway,which down-regulate the expressions of inflammatory cytokines（IL-1β,IL-6 and TNF-α）and inflammatory mediators（iNOS,NO,COX-2 and PGE₂）and up-regulate the expression of anti-inflammatory cytokine IL-10,thereby controlling inflammatory response.In addition,as a natural ingredient,CGD polysaccharide has no cytotoxic effect.Therefore,CGD polysaccharide has the potential to be used as a relatively safe and effective anti-inflammatory drug as part of treatment for NCDs.