| ObjectiveThe purpose of this study is to identify the Canadian sea cucumber(Cucumaria frondosa)using characteristics and microscopic character.The sulfated polysaccharides from Cucumaria frondosa(CFP)were prepared by extraction,separation,and purification processes according to modern technology.The physical and chemical properties of CFP were analyzed,as well as the structure of CFP,which was the basis for quality control of CFP.According to study the effects of CFP on renal interstitial fibrosis,the effect of CFP on renal interstitial fibrosis was further explored.And the mechanism of CFP on alleviating renal interstitial fibrosis was further explored.The research was to improve the sample information of Cucumaria frondosa,provide scientific basis for the development of resources,and be the foundation for the development of CFP products.Methods1.Identification of Canadian sea cucumberCharacter,microscopy and molecular identification were combined to identify the species of Canadian sea cucumber.And the characteristics and microscopic character were described in detail.2.Study on extraction,separation and purificationThe ultrasonic-assisted extraction method was used to extract the polysaccharides from Cucumaria frondosa,and the process conditions were optimized.In order to obtain the CFP with uniform ion electricity and molecular weight,DEAE-cellulose-52 anion exchange column combined with Sephadex G100 gel column was used to purify CFP.3.Physical and chemical properties and structures identification of CFPPhenol-sulfuric acid method,coomassie bright blue method,barium chloride-gelatin method,sulfuric acid-carbazole method,sugar nitrile acetate derivative-GC spectroscopy,molecular exclusion high performance liquid chromatography(HPLC),UV spectroscopy,IR,methylation reaction,NMR spectrum,and Congo Red reagent method were comprehensive used to systematically analyze the physical and chemical properties and structure of CFP.4.The effect of CFP on renal interstitial fibrosisA common model of renal interstitial fibrosis(unilateral urethral obstruction in rodents,UUO)was used to investigate the intervention effect of CFP on renal interstitial fibrosis.Renal function was evaluated by measuring the content of urinary protein in urine,urea nitrogen and creatinine in serum.Hematoxylin-eosin(H&E)and Masson staining were used to observe the pathological changes and inflammation infiltration of the kidney.Contents of IL-6,IL-1? and TNF-? were used to evaluate changes in inflammatory factors.Immunohistochemical staining was used to determine the content of transforming growth factor ?1(TGF-?1),fibroblast growth factor 2(FGF2),smooth muscle aorta ?(?-SMA),Fibronectin,E-cadherin,Collagen ? and Collagen ? in kidney tissue.5.The mechanism of CFP on renal interstitial fibrosisPharmacological experiment in vivo and in vitro were combined to demonstrate the mechanism of CFP on renal interstitial fibrosis.Western Blot techniques were used to determine the mRNA and protein expression of PI3K-Akt-NF-?B pathway in kidney tissues and HK-2 cell line.Results1.Identification of Cucumaria frondosaThe characteristics,microscopic character and molecular identification results showed that the Canadian sea cucumber was Cucumaria frondosa,which is the dried body from the sea cucumber class.The abdomen and five internal tendons are unique orange or golden.Microscopic identification results indicated that the ossicles of the body wall were perforated plates with different sizes.2.Preparation of CFPThe optimal conditions of ultrasonic-assistant extraction were as follows:ratio of liquid to material(mL:g)of 22:1,extraction time of 30 min,extraction temperature of 62?,ultrasonic power of 120 W.The extraction rate reached 11.72±0.09%.CFP was purified by the combined application of DEAE-cellulose-52 column chromatography and Sephadex G100 gel column chromatography,and two components with relatively uniform ionic conductivity and molecular weights were obtained,named P-1 and P-2.Because of higher content on sulfate and uronic acid,P-2 was selected for subsequent experiments and named as CFP.3.Physical and chemical properties and structures identification of CFPThe content of total sugar,protein,sulfate and uronic acid from CFP was 58.57±0.51%,1.20±0.08%,21.57± 1.09%,5.58±0.16%,respectively.The relative viscosity of CFP was 1.16.CFP was a homogeneous polysaccharide composed of fucose,and the relative molecular mass was 136 kDa.The main sugar residues of CFP were SO3--O-4-?-L-Fucf-(1???3)-?-L-Fucf-(1???2 SO3-,3)-?-L-Fucf-(1???3,4 SO3-)-?-L-Fucf-(1?and?2 SO3-,3,4 SO3-)-?-L-Fucf-(1?.4.The effect of CFP on renal interstitial fibrosisCompared with the Sham group,mice in the UUO group had abnormal renal function parameters.The blood creatinine,blood urea nitrogen,and urinary protein were all significantly increased.And a large number of inflammatory cell accumulation could be observed by HE staining.The levels of inflammatory factors(TNF-?,IL-1?,IL-6)were all increased.The expression of TGF-?1,FGF2,?-SMA,Fibronectin,Collgen I and Collgen IV were all enhanced.After preventive administration of CFP,the kidney function of mice was improved,and the expression of inflammatory factors was reduced.The expression of TGF-?1 and FGF2 was reduced,and meanwhile the synthesis of ECM was reduced.The inflammation and fibrosis of renal were all improved.At the same time,the anti-inflammatory and anti-fibrotic effects of CFP were dose-dependent.5.The mechanism of CFP on renal interstitial fibrosisIn the UUO model,CFP could inhibit the signaling pathway-related proteins of PI3K/AKT/NF-?B and the expression of downstream inflammatory and profibrotic factors.In the model of TGF-?1-induced HK-2 cell fibrosis,CFP could significantly inhibit the high expression of PI3K/AKT/NF-?B pathway-related proteins and phosphorylated proteins caused by TGF-?1,using PI3K inhibitor 3-MA There were no significant differences in the inhibitory effects of PI3K/AKT/NF-?B alone and in combination with CFP.ConclusionIn this paper,the Cucumaria frondosa was used as the object of research,and was identified by the character,microscopy and molecular identification,which laid the foundation for the controllability of the sample.The single-factor and response surface optimization methods were used to optimize the ultrasonic-assistant extraction method to obtain the polysaccharide from Cucumaria frondosa,which was better than traditional water extraction.DEAE-cellulose-52 column chromatography and Sephadex G100 gel column chromatography were combined to purify,and two purified composition with relatively uniform ionic conductivity and molecular weight were obtained.The physical and chemical properties of CFP were studied,and the primary structure was analyzed.The establishment of the UUO model was to study the effect of CFP on renal interstitial fibrosis by inhibiting the PI3K-Akt-NF-?B signaling pathway.In summary,the research provided the basis for quality control of Cucumaria frondosa and CFP,promoted the development and application of Cucumaria frondosa resources,and provided a reference for the prevention of renal interstitial fibrosis. |
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