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Preliminary Study On The Inhibition Of NLRP3 Inflammasome By Water-soluble Laminaria Polysaccharide And Its Mechanism Of Relieving Ulcerative Colitis

Posted on:2023-11-23Degree:MasterType:Thesis
Country:ChinaCandidate:Y P FanFull Text:PDF
GTID:2530307046494284Subject:Biological engineering
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Background and Objective:Laminaria Polysaccharide is the most abundant substance in kelp.The extraction method of laminarin not only affects the extraction efficiency,but also has a great influence on the molecular mass,structure,pharmacological activity and biological function properties of laminarin.Biomacromolecules follow the general principle of structure and function adaptation.Based on existing research,it has been found that active polysaccharides with triple helical structure can exhibit better relative biological activity.The extraction method is expected to fill the gap of industrial production.Among the many bioactive functions of laminarin,although some scholars have suggested that laminarin may have anti-inflammatory activity,the current research is not enough to confirm this result,and more research results are needed to support it.Inflammasome plays an important regulatory role in the occurrence and development of inflammation,and the imbalance of inflammasome regulation has become a close factor that induces a variety of inflammatory diseases.Therefore,the search for natural compounds as inflammasome inhibitors has attracted much attention in recent years.Our research group has explored the activity ofβ-glucan with triple helical structure in the early stage.Based on this,this subject evaluated the structure and anti-inflammatory activity of water-soluble laminarin(W-LP)extracted by the improved preparation method.And the association between it and the inflammasome NLRP3 was preliminarily explored.Methods:1.Optimization of the boiling alcohol precipitation reaction conditions for the extraction of polysaccharides solution from laminarin.The dried lamina was first ground to powder,extracted hot water around 90°C for 2 h,Ca Cl2and ethanol were added for precipitation of impurities,and then concentrated by rotary evaporation.Then using the phenol sulfuric acid method、NMR、Ion chromatograph、Circular dichroism and evaluation of aqueous solubility to characterization of polysaccharide content,composition,monosaccharide composition,higher order structure,and aqueous solubility of W-LP2.A cell inflammation model was constructed by stimulating RAW264.7 cells with 100ng/ml lipopolysaccharide(LPS).Probing the cytotoxicity of W-LP with CCK8 and The anti-inflammatory activity and preliminary anti-inflammatory mechanism of W-LP were investigated by RT-q PCR,Griess reagent method,fluorescence microscopy,flow cytometry,WB and laser confocal.3.Lipopolysaccharide(LPS)and nigericin co stimulation of mouse bone marrow-derived macrophages(BMDMs)and murine macrophage cell line J774A.1 were employed to construct a cellular model of NLRP3 inflammasome activation.The effects of W-LP on NLRP3 activation were explored by WB,ELISA and IF experiments.4.Dextran sulfate sodium(DSS)was adopted to construct acute inflammatory colitis in mice.Low dose(200 mg/kg),medium dose(400 mg/kg)and high dose(800 mg/kg)of water-soluble laminarin polysaccharide were administered one week before the start of modeling.Treatment with polysaccharide administration was continuously performed during the modeling period.To evaluate the relevant indicators of the colitis disease model to explore the effect of water-soluble laminarin in relieving ulcerative colitis.Results:1.The polysaccharide content of the phenol-sulfuric acid extract is 72%~73%.The carbon NMR spectrum showed characteristic peaks of polysaccharides,and the monosaccharide composition of the extract was consistent with the monosaccharide composition of brown algae polysaccharides.W-LP has a distinct triple helix conformation and good water solubility.2.The anti-inflammatory activity of W-LP was obvious,and it was completed by inhibiting NF-κB signal transduction by inhibiting the entry of NF-κB into the nucleus.3.By inhibiting the NF-κB signaling pathway,W-LP affects the activation stage of the first messenger of the NLRP3 inflammasome,inhibits ASC oligomerization,affects the activation of mature Caspase-1,and finally inhibits the activation and release of IL-1β.4.W-LP inhibits the activation of NLRP3 by inhibiting NF-κB signaling,thereby achieving the effect of relieving ulcerative colitis.Experimental conclusion and significance:In this study,the method of polysaccharide extraction was optimized to obtain a polysaccharide with high purity,high content,stable triple helix structure,small component and better water solubility.Subsequent animal experiments showed that the water-soluble laminarin polysaccharide we extracted had a better ability to alleviate DSS induced inflammatory lesions in mouse colon,and the mechanism may be related to the inhibition of NF-κB signaling pathway related.It was verified at the cellular level that the water-soluble laminarin active polysaccharide has anti-inflammatory effects and is able to inhibit NF-κB signaling pathways.To provide a theoretical basis for the more full use of the active polysaccharide constituents of lamina criba and provide new ideas for the exploitation and utilization of marine resources in China.
Keywords/Search Tags:Laminaria Polysaccharide, triple helix, water soluble, NLRP3 inflammatory, NF-κB signaling pathway
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