Preparation And Characterization Of Sulfated Polysaccharides From Cipangopaludina Chinensis And Its Action To Stabilize Atherosclerotic Plaques

As the common pathological basis of all cardiovascular and cerebrovascular diseases,atherosclerotic vulnerable plaques are the main causes of coronary heart disease,cerebral infarction and peripheral vascular disease,and the coronary atherosclerotic heart disease caused by its further involvement in the heart ranks the first in the world in terms of causes of death.Therefore,the effective therapeutic drugs and methods should be developed to promote the stable change of plaques according to the formation and rupture mechanism of vulnerable plaques.It is not only an important method to prevent atherosclerosis and secondary diseases,but also a key subject for the cardiovascular science at home and abroad.It is one of the main principles for the treatment of atherosclerotic plaques in traditional Chinese medicine by the "lipid-lowering and resolving phlegm,supporting healthy energy and removing stasis".Sulfated polysaccharides play a significant role in immune regulation and anticoagulation.Their effects meet the theoretical and therapeutic requirements of the "lipid-lowering and resolving phlegm,supporting healthy energy and removing stasis" for atherosclerotic plaques.It has shown great potential in the prevention and treatment of atherosclerotic plaque.Aquatic animals often contain unique structure basis of polysaccharides with high sulfuric acid group content by the special survival environment,which is one of the main sources of natural sulfated polysaccharides.Cipangopaludina chinesis is a kind of mollusc belonging to the cipangopaludina.The meat from Cipangopaludina chinensis has been a favorite delicacy of urban and rural residents in China since ancient times.Meanwhile,it was also a medicine frequently used by many Chinese ancient doctors.As a kind of common edible and pharmaceutical aquatic animal,its growing environment meets the natural synthesis conditions of sulfated polysaccharides in its body.Thus,it is a very theoretical value and practical significance to prepare and develop sulfated polysaccharides from Cipangopaludina chinensis and further study its role and mechanism on atherosclerotic plaque.ObjectiveThe purpose of this study is to establish the systematic preparation method of sulfated polysaccharides from Cipangopaludina chinensis(CCPSs)by optimizing manufacture technology of CCPSs on extraction,protein removal,classification and purification.The physical and chemical properties and primary structure of CCPSs were identified and analyzed systematically in order to provide reference for its quality control,pharmacodynamics and structure-activity study.Effect of CCPSs on anti-atherosclerosis and plaque stability has been comprehensively evaluated by studying its effects on arteriosclerosis index(AI)and vulnerable index(VI).The effects of CCPSs on angiogenesis in atherosclerotic plaques and its regulation of PI3K/Akt/mTOR signaling pathway were thoroughly studied to clarify its mechanism in stabilizing atherosclerotic plaques.Finally,the four-dimensional research results of CCPSs on the"preparation-structure-efficiency-mechanism" would be formed to provide theoretical support for its comprehensive development and utilization.Methods1.Process study of CCPSs on extraction,deproteinization,classification and purification.The crude CCPSs was extracted by ultrasound coupled enzymatic hydrolysis assisted extraction method(UCEH),and its process conditions were optimized by single factor experiment and response surface experiment.A novel method based on UCEH was constructed by comparing with the traditional hot water extraction method.On this basis,FTT was used to remove the proteins from the crude CCPSs.And,single-factor experiment was employed to optimize and screen the extraction process parameters.A green deproteinization method from FTT was established by comparing the traditional Sevag method.The crude CCPSs was purified by Q Sepharose Fast Flow and Sephacryl s-400 gel column chromatography.The sulfuric acid group content and molecular weight were used as the screening indexes.The separation components were followed up and optimized to obtain CCPSs,a target purified polysaccharide component.2.Identification of physical and chemical properties and structures for CCPSs.The appearance was evaluated by visual observation method.The solubility was determined by static equilibrium solubility test.The contents of total sugar,protein,sulfuric acid group and uronic acid were measured by phenol-sulfuric acid method,coomase bright blue method,ion chromatography and sulfuric acid-m-hydroxybiphenyl method,respectively.UV-vis,IR,sugar nitrile acetate derivatives from hydrolyzed polysaccharide-GC,molecular exclusion HPLC,periodate oxidation degradation and Smith,methylation reaction-GC,1 D and 2 D NMR were used to identify proteins and nucleic acids,functional groups and sugar ring form,the monosaccharide composition and their proportion,molecular weight and homogeneity,hydroxyl replace situation and glycosidic bond connection site,the anomeric carbon configurations,hydroxyl replace situation and sugar-based link order,respectively.On this basis,a comprehensive spectrum analysis was carried out to identify the primary structure of CCPSs.3.The stability effect of CCPSs on atherosclerotic plaques.ApoE-/-mice were fed a high-fat diet to replicate atherosclerosis.TG,TC,HDL and LDL were measured by the special kit.AI was calculated based on blood lipid index.Hematoxylin-eosin(H&E)staining was used to observe plaque morphology and measure the area of lumen and plaque tissue.And,the ratio of lumen and plaque tissue area was calculated.The contents of Collagen and lipid were determined by skywolf scarlet and oil red O staining method,respectively.Antibody of ?-SMA and CD68 were used for immunofluorescence staining of smooth muscle cells and macrophages and determine their contents.VI was calculated via plaque composition.The effects of CCPSs on anti-atherosclerosis and plaque stability were evaluated by comparing ratio of plaque to lumen area,AI and VI of each group.4.The stability mechanism of CCPSs on atherosclerotic plaques.This experiment was performed in vivo and in vitro.The densities of angiogenesis in the plaques were determined by immunofluorescence staining with CD34 antibody in ApoE-/-atherosclerosis model mice fed with high-fat diet to demonstrate the effect of CCPSs on angiogenesis in plaques.In order to demonstrate the influence of CCPSs on the PI3K/Akt/mTOR pathway,which regulate angiogenesis in arterial plaques,Western Blot and qRT-PCR were used to detect the protein and mRNA expression from signal transduction molecules of PI3K/Akt/mTOR pathway and its downstream key angiogenic factors in arterial plaques.On this basis,HUVECs was taken as the research object.And the relationship between the inhibition of angiogenesis by CCPSs and the regulation of PI3K/Akt/mTOR pathway was further researched from both positive and negative aspects through the combination of pathway activators and blockers.Results1.Process study of CCPSs on extraction,deproteinization,classification and purification.(1)The optimum conditions of UCEH extraction for crude CCPSs were as follows:liquid ratio(mL:g)of 22.5:1,enzyme dosage of 238 U/g,pH value of 5.0,extraction temperature of 58? and ultrasonic power of 400 W.Under optimized conditions,the extraction rate of CCPSs reached 13.57 ± 0.48%.Compared with the traditional hot water extraction method,the extraction of CCPSs can not only save time,reduce solvent and energy consumption,but also obtain higher yield,polysaccharide content and extraction rate of CCPSs.(2)The optimum conditions of FTT deproteinization for crude CCPSs solution were as follows:freezing temperature of-40?,freezing time of 96 h,thawing temperature of 10?,the freeze-thaw cycle processing times of 11 times.Under the optimal conditions,Dr%,Rr%and Kc of crude CCPSs by FTT method were 87.10 ± 1.91%,84.91±2.45%and 5.77±0.43,respectively.Compared with the traditional Sevag method,FTT method not only has the advantages of high protein removal rate and stable polysaccharide structure,but also has the characteristics of high selectivity and no pollution.(3)The crude CCPSs was purified by Q Sepharose Fast Flow and Sephacryl s-400 gel column chromatography.The target polysaccharide CCPSs with relatively uniform ionic conductivity and molecular weight was obtained through component screening and optimization.Its average yield reached 1.42%.2.Identification of physical and chemical properties and structures for CCPSs.CCPSs was white powders easily soluble in water with an average total sugar content of 91.88%.There is no protein and no uronic acid in CCPSs.The main chains of CCPSs were formed by(1?3)linked ?-D-Glc.Its branched chain was consisted of a(1?3)linked?-D-Glc and terminal ?-D-Glc-4-O-SO3-.The branch chain was connected to C-4 of?-D-Glc from the main chain.The branching degree of CCPSs was 16.73%.The C-1 of the branched chain of ?-D-Glc-4-O-SO3-was connected to O-3 of ?-D-Glc by(1?3)linked.CCPSs was a sulfated polysaccharide with an average sulfonic content of 9.12%.3.The stability effect of CCPSs on atherosclerotic plaques.CCPSs had a significant stability effect on atherosclerotic plaques.Its stability effect on plaques is mainly manifested as follows:CCPSs can significantly decrease TC,TG,LDL content in serum of model mice(P<0.01 or P<0.05),markedly increase HDL content in serum of model mice(P<0.01),significantly reduce plaque area(P<0.01),obviously inhibit lipid content and macrophage content(P<0.01 or P<0.05)and remarkably increase the content of smooth muscle cells and collagen in plaque tissue(P<0.01 or P<0.05).AI index was significantly reduced by improving the disorder of lipid metabolism in atherosclerosis model mice(P<0.01).CCPSs can significantly decrease VI value via inhibiting plaque vulnerable components and promoting plaque stability factors(P<0.01).4.The stability mechanism of CCPSs on atherosclerotic plaques.CCPSs significantly inhibited angiogenesis in aortic plaques of ApoE-/-atherosclerotic mouse models(P<0.01 or P<0.05).CCPSs can significantly suppress the expression of mRNA and phosphorylated protein from PI3K/Akt/mTOR pathway signaling molecules,and VEGF and MMP-9 of its downstream angiogenesis regulators(P<0.01 or P<0.05).In HUVECs cell model,CCPSs significantly inhibited enhance expression of phosphorylated protein from PI3K/Akt/mTOR signaling molecules and high tube formation rate of HUVECs in Matrigel gel induced by VEGF pathway activator(P<0.05).Moreover,the inhibitory effect of CCPSs can be blocked by LY294002 of PI3K pathway blocker.There was no significant difference between LY294002 in combination with CCPSs and LY294002 alone on the expression of phosphorylated protein of PI3K/Akt/mTOR signaling molecules and tube formation rate of HUVECs in Matrigel gel(P>0.05).ConclusionIn this paper,the UCEH,which was characterized by rapid cell breaking and efficient hydrolysis of glycoprotein glycopeptide bonds,was used to extract crude CCPSs from meat of Cipangopaludina chinensis.A novel extraction method of crude CCPSs based on UCEH was established through the design of extraction scheme and parameter optimization.It was proved that UCEH was a desirable extraction method for crude CCPSs.Based on solubility difference between proteins and polysaccharides in the process of FTT,a novel green deproteinization method of crude CCPSs solution was constructed via program design and parameter optimization.As an environmentally friendly and green method,FTT could replace the traditional Sevag method for deproteinization of crude CCPSs.The Q Sepharose Fast Flow column chromatography and Sephacryl S-400 gel column chromatography were employed to purify the crude CCPSs according to the characteristics of strong electronegativity of CCPSs induced by high sulfuric acid group content,and the chemical nature of polysaccharide as a kind of macromolecular polymer.The target CCPSs with relatively uniform ionic conductivity and molecular weight were obtained using this method.Chemical analysis,UV-vis,IR spectrum and NMR spectrum were applied to identify and analyze the physical and chemical properties and primary structural characteristics of CCPSs.It has been demonstrated that CCPSs has a significant stabilizing effect on atherosclerotic plaques through in vivo and in vitro experiments.And,it has also been clarified that the mechanism of CCPSs to stabilize atherosclerotic plaques by intervening angiogenesis based on PI3K/Akt/mTOR pathway.The theoretical hypothesis,CCPSs has the function of stabilizing atherosclerotic plaques and its stable mechanism is intraplate angiogenesis mediated by PI3K/Akt/mTOR pathway,was proposed and verified theoretically through above these innovative studies.In terms of the experimental method,a series of novel technologies,including FTT,UCEH and Q Sepharose Fast Flow chromatography combined with Sephacryl S-400 column chromatography,was introduced for the preparation of CCPSs.At the research level,the four-dimensional research results of CCPSs on the "preparation-structure-efficiency-mechanism" had been formed.In terms of research contents,the preparation method of CCPSs was established for the first time.Its primary structure was also analyzed.The role of CCPSs in stability atherosclerotic plaques was confirmed and its related mechanism was also clarified.These studies have important theoretical reference and practical significance for promoting the in-depth development of Cipangopaludina chinensis,providing novel intervention measures for the prevention and treatment of atherosclerotic diseases with integrated traditional Chinese and western medicine,and supplying new ideas for the development of aquatic animal polysaccharides.