The Research Of EN01 Polypeptide And Four Phospholipids On Cell Growth And Metabolism

Because of its unique growing environment,Marine organisms contain new,complex and diverse natural active substances with unique functions that are beneficial to human health.The results show that Marine active substances have remarkable pharmacological stability and strong potency,and have broad research prospects in nutrition,health care,biological pharmacy and even clinical.In addition,the preparation process of active substances is constantly optimized,which provides a technical means for its research and development.The marine bioactive substances mainly include peptide,lipid,glycoside,protein and alkaloid.As our research group Dai et al^[1]shown that the n-hexane layer of starfish can promote cell proliferation,cell migration and protease-autophagy pathway,while the methanol layer can inhibit cell proliferation and activate autophagy and apoptosis pathways in varying degrees.The results suggest that the methanol water layer mixture may be the protein and peptides,and the n-hexane layer mixture may be the fatty acids and other lipids.However,the types of polypeptides and lipids and their mechanisms of action have not been thoroughly studied.On the basis of previous studies,this project induces the release of active substances by cutting the wrist of starfish,and then further extract and separat the active proteins to screen out,and discuss its effect on cell growth and metabolism,providing a basis for the study of functional polypeptides.In order to further study how the starfish heterologous fat-soluble extract influence on cell growth and metabolism,we choose Eriocheir Sinensis with limb regeneration ability.Because of its hepatopancreas,the center of lipid metabolism,has a high level of lipid up to 32.79%.We extract the hepatopancreas of lipid to probe into its active lipid,laying the foundation for the research of nutrition and biology pharmacy.The main experimental contents are as follows:（1）For the extraction method of active substances,we adopt the repeated cold extraction method.First,the lipid soluble and water-soluble components in the body of Eriocheir Sinensis and sea stars are extracted with 95%ethanol.Then,the methanol with low polar and methanol with highly polar are further extracted by ultrasonic cleaning.On the one hand,ultrasonic extraction can destroy the cell membrane and accelerate the dissolution of cell contents;on the other hand,the combination of three organic solvents can extract more compounds of different polarity,which is a relatively efficient extraction method.（2）The starfish extracts are extracted by method of extraction and separation of natural compounds,and the water-soluble layer with highly polarretained.The water-soluble layer is further separated by reversed-phase silica gel column,and then the purified protein is purified by semi-preparative HPLC and mass spectrometry,and the active protein EN01 with strong functions in the water-soluble layer mixture is screened out.Four peptides,methylated,acetylated,phosphorylated and unmodified,are synthesized by chemical modification.Firstly,Cell counting Kit-8（CCK-8）assay is used to analyze the effect of polypeptide on the proliferation and cell activity of zebrafish embryonic fibroblasts（PAC2）cells.Secondly,intracellular Lactate Dehydrogenase（LDH）assay is used to evaluate the effect of polypeptides on glycolysis in glucose metabolic pathway.The results show that the four peptides reduce the metabolic of glycolysis,the acetylated EG-2 peptides enhance the tricarboxylic acid cycle,the methylated EG-1 peptides inhibite the metabolic pathway of the tricarboxylic acid cycle,and the other two peptides show no significant changes in the metabolic pathway of the cell tricarboxylic acid cycle.ENO1 is an enzyme that catalyzes the conversion of 2-phosphoglycerate into phosphoenolpyruvate during glycolysis.In order to verify whether the treatment of four polypeptides will directly affect glucose metabolism,we test the change of 2-phosphoglycerol level.The results show that the production of pyruvate is decreased after four kinds of peptides are modified.We use PCR ARRAY to screen out differentially expressed genes to further analyze the effects of four polypeptides on glucose metabolism pathways.Genes with over 2 Fold Change are screened,and the differentially express genes in the PCR ARRAY of glucose metabolism pathway is enriched by DAVID.The results of CCK-8 and LDH show that EG-1 polypeptide treatment inhibite the metabolic pathway of glycolysis and the tricarboxylic acid circulation pathway,and 2-PG test show that pyruvate is decreased.We speculate that PAC2 cells treated with EG-1polypeptide are more prone to the metabolic pathway of pentose phosphate or gluconeogenesis.The cells treated with EG-2 polypeptide inhibit the metabolic pathway of glycolysis and promot the metabolism of tricarboxylic acid in mitochondria.Cells treated with the polypeptide EG-3inhibit glycolysis and reduce pyruvate production,possibly through pentose phosphate metabolic pathway or glycogen metabolic regulation pathway to provide energy.Finally,the effects of four peptides on the subcellular structure are detected by specific mitochondrial and lysosomal fluorescence staining.Mitochondria are aggregated by methylated and acetylated peptides,and dissolved by phosphorylated and unmodified peptides.（3）The Eriocheir Sinensis extracts are extracted by the same method,and the liposoluble layer with lowpolar retained.The lipid soluble layer is further separated into 9 parts（HE1-9）with different polarity by column chromatography,the fatty acid species in the hepatopancreatic of the amputated Eriocheir Sinensis are determined by GC-MS,including 22 fatty acids,9 saturated fatty acids,7 monounsaturated fatty acids and 6polyunsaturated fatty acids.Moreover,the solubility and polarity of lipids decrease as the increase of the hydrocarbon chain length of fatty acids.Liquid phase time of flight mass spectrometry is used to further identify the lipid compounds.The major types are glycerophospholipids,containing a few types of sphingolipids and cholesterol.Through literature review,four lipid compounds that may play a role were screened out:C-8Ceramide-1-phosphat,N-dodecanoyl-L-Homoserine lactone,Oleyloxyethyl Phosphorylcholine,Palmitoyl-L-carnitine.The effects of four lipids on cell growth,glycolysis and aerobic oxidation of tricarboxylic acid cycle are evaluated by CCK-8,LDH release and total LDH content in cells.At the same time,the changes of the structure of mitochondria and lysosomal subcells are detected by the fluorescence staining of the specific markers of mitochondria and lysosomal,which further verified the stability of cell membrane and the changes of energy metabolism of the tricarboxylic acid cycle in mitochondria.To sum up,our experiment shows that the artificially induced peptides in regenerated starfish and lipids in Eriocheir Sinensis both contain active substances that affect cell growth and metabolism,suggesting the possibility of the application of natural bioactive substances in clinical treatment and promoting the development of studies on lipid and polypeptide active substances.Meanwhile,our experiments verify the effect on cell growth and metabolism from the cellular and subcellular levels.There is a certain relationship between the cell proliferation and cytotoxicity of active substances and the integrity of cell mitochondria and lysosome.A detection system is established for effective screening of active ingredients in vitro,which is to provide theoretical basis and technical support for the study of functional active ingredients.