| There is a rich resource of macro algae.Several active substances extracted from macro algae,have been proved to be of intense biological activities,ranging from anti-viral,anti-tumor,anti-coagulation and immunity-enhancing biological activities.In this paper,we chose six common macro algae as the raw material,namely Grateloupia filicina,Eucheuma spinosum,Enteromorpha prolifera,Ulva pertusa,Laminaria japonica and Sargassum Pallidum,belong to Rhodophyta,Chlorophyta and Phaeophyta respectively.We conducted sulfated polysaccharides extraction,followed by measurement and comparison of total sugar content,monosaccharide composition,the content of sulfate,molecule structures and biological activity(in vitro and in vivo experiments).Polysaccharide with best immunomodulatory activity,Sargassum Pallidum,was isolated,purified and derivatized.After determination of monosaccharide composition,sulfate content,structure along with its in vitro inhibiting activity in tumor cell growth,we explored the inhibition mechanism by RNA-sequencing technology.The main findings are as follows:1.We extracted six seaweed sulfated polysaccharides for chemical property analysis.We found that physicochemical property varies among different algae sulfated polysaccharides.For the total sugar content,the highest is Enteromorpha prolifera polysaccharide,up to 63.11%.In these seaweed tested,total sugar content of brown algae polysaccharides was slightly lower than red and green algae polysaccharides.The Enteromorpha prolifera polysaccharide has the highest sulfate content,which is 21.35%.Red algae(Grateloupia filicina and Eucheuma spinosum)polysaccharides have relatively higher sulfate content than brown algae(Sargassum Pallidum and Laminaria japonica)polysaccharide.The highest content of monosaccharides in Chlorophyta polysaccharide is rhamnose,the highest content monosaccharide of red algae is galactose,and fucose for brown algae.In addition,the monosaccharide composition is different in polysaccharides extracted from algae of the same catogory.FT-IR analysis showed that the six sulfated polysaccharides share several absorption peaks,but there are some differences,which means they have different structures.2.We investigated the immunomodulatory activity and antiviral activity of six kinds of seaweed sulfated polysaccharides in vitro,found that all of the seaweed sulfated polysaccharides can significantly enhance lymphocyte proliferation,and also,reduce the hemagglutination titer of virus.3.From the results above,we chose the sulfated polysaccharides from Grateloupia filicina,Ulva pertusa and Sargassum Pallidum for further research.We took inactivated H9N2 avian influenza virus as an immune stimulator to investigate the immunomodulatory activity of Grateloupia filicina,Ulva pertusa and Sargassum Pallidum sulfated polysaccharides in vivo,and found that the three kinds of polysaccharide could significantly enhance the AIV specific antibodies and the humoral immunity level,of which 50 mg/kg of Grateloupia filicina and 50 mg/kg of Sargassum Pallidum sulfated polysaccharide got better effects;for stimulating the expression of immune-related cytokine experiments,the expression of IFN-? and IL-4 levels were significantly increased in the experimental polysaccharide group,where 10 mg/kg seaweed sulfated polysaccharide groups are more effective to stimulate INF-?;as for IL-4 stimulation,the 50 mg/kg groups were more preferable than the 10 mg/kg groups;and seaweed sulfated polysaccharides have regulatory roles on T lymphocyte subsets,they could significantly increase the CD3+CD4+ levels,but only Sargassum Pallidum sulfated polysaccharide has significant stimulating effect on CD3+CD8+.Experimental results revealed that the Sargassum Pallidum sulfated polysaccharide have the most significant immunomodulatory effect,which might be related to their abundance in fucose content.4.In vitro antiviral activity results showed that all of the sulfated polysaccharides could inhibit the virus to a certain extent.Different antiviral effects were compared,namely blocking effect,inhibition effect,and direct killing effect,in which the effect of directly kill was lower than the other two.This showed that the polysaccharides could directly kill the virus,but the blocking effect,inhibition effect had better effect.In the anti-H9N2 AIV experiment,all three kinds of seaweed sulfated polysaccharides could significantly inhibit the replication of the H9N2 virus,in which,the Grateloupia filicina sulfated polysaccharide had the most significant effect which might due to its highest sulfate content.5.The Sargassum Pallidum sulfated polysaccharides were further separated and purified.Three components 0.5M,1M and 2M,were obtained respectively.We studied the physical and chemical properties and in vitro tumor cell inhibition activity of the three components.2M components was found to be the most effective in inhibition of tumor cells in vitro and can significantly induce apoptosis of Hela cells.To investigate the molecular mechanisms of tumor cell growth inhibition caused by the adding of polysaccharide,we used RNA-sequencing technology to analyze the transcriptome changes of Hela tumor cells after adding of Sargassum Pallidum sulfated polysaccharides and 2M component.It was found that,with the adding of polysaccharides,the expression of apoptosis-related genes,such as NFBKIA,Caspase gene were increased;and the apoptosis inhibition related genes,such as NF-kB,THBS1 were down regulated.GO and KEGG analysis showed that the addition of 2M components could significantly inhibit the proliferation of tumor cells,showing the expression of tumor suppressor genes increased,ribosome binding activity and laminin-related gene significantly down-regulated,and biological pathways associated with the termination of translation and mRNA metabolism down-regulated significantly.The results show that the addition of 2M component can significantly reduce the Hela tumor cell activity,showing reduced levels of growth,increased levels of apoptosis;transcriptome results consistent with the experimental results,a number of apoptosis,cell differentiation and metabolism related genes/ pathway is presumably to be the anti-tumor molecular mechanisms.6.We derivatized Sargassum Pallidum polysaccharide,and obtained sulfated,acetylated,phosphorylated,benzoylated and aminated five kinds of derivatives in total.Derivatization was proved to be successful by the FT-IR,and the activity of tumor cell growth inhibition in vitro were compared.In terms of inhibition of tumor cells.The highest activity was found in vitro in acetylated derivative,which can induce apoptosis in Hela cells.To explore the molecular mechanisms of inhibition activity of the polysaccharide derivative on tumor cell growth,we analyzed the Hela tumor cells' transcriptome changes after adding Sargassum Pallidum acetylated derivative via RNA-seq.It was found that the adding of Sargassum Pallidum acetylated derivative could significantly reduce the Hela tumor cell activity,showing reduced levels of growth,increased level of apoptosis;transcriptome results revealed that acetylated derivatives can significantly improve the expression of tumor suppressor,apoptosis,immune and skeletal control related genes/ pathways,as well as reduced the ribosomal activity related,metabolic related and oxidative phosphorylation related genes/ pathways,real-time quantitative PCR was used to verify the results obtained by transcriptome,and prove to be accurate and credible.The results of transcriptome and in vitro experments matches,further expound the molecular mechanisms of Sargassum Pallidum acetylated derivative inhibits tumor cell differentiation. |
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