基于蛋白质组学的透明质酸寡聚糖抗炎活性研究及验证

Research and validation of anti-inflammatory effects of hyaluronic acid oligosaccharide based on proteomics analysis

透明质酸(hyaluronic acid,HA)广泛地分布在人体组织中,是一种具有重要生理功能的多糖,且已被国家卫生健康委员会批准作为“新食品原料”,可用于普通食品添加剂中。对于HA寡聚糖(oligo-HA)抗炎功效的相关研究目前尚存在争议,为明确oligo-HA的抗炎作用机理,同时探索其潜在应用价值,该文对oligo-HA预处理与未处理的经脂多糖刺激的RAW264.7细胞进行蛋白质组学检测,经过数据非依赖采集技术获得数据并筛选,通过GO和KEGG数据库富集分析,对蛋白数据进行处理,并采用斑马鱼刺激模型验证oligo-HA的抗炎功效。结果表明,共鉴定出124个显著差异表达的蛋白,其中显著上调76个,显著下调48个,涉及的生物学过程包括T细胞受体信号通路,癌症相关信号通路,以及多种活性分子的生物合成等。0.5%与1%质量分数的oligo-HA对斑马鱼的处理显著性降低了中性粒细胞的数量,缓解了炎症作用。该文基于对蛋白质组学的分析,可以为oligo-HA在食品原料中的应用提供方向。

Hyaluronic acid(HA)is a kind of polysaccharide broadly distributed in human bodies and tissues.As a major component of the extracellular matrix,it plays an important role in several physiological processes such as immune response and wound recovery.The National Health Commission of China has already approved HA as a‘new food resource’and to be used as additives in common food.Therefore,it is vital to conduct further studies on the functions and mechanisms of HA.Though HA has been proved to take effects in the regulation of inflammatory response by experiments in vitro and in vivo,there are still conflicts about the anti-inflammatory effects of HA,especially with HA oligosaccharides(oligo-HA).This research aims to study the anti-inflammatory effects of oligo-HA and its mechanisms,as well as to discover its potential applications.The lipopolysaccharides-stimulated RAW 264.7 cells pretreated with/without oligo-HA were examined by proteomics analysis to study the change of proteins expression during the cell cycle.Data independent acquisition technique was used to collect the data and differentially expressed proteins were selected according to one-way ANOVA analysis.Differently expressed proteins were further analyzed through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment.After testing the maximum tolerated dose of oligo-HA sample on zebrafish,a sodium lauryl sulfonate(SLS)stimulated zebrafish model was used to support the anti-inflammatory effects of oligo-HA.Our results showed that 124 significantly differently expressed proteins were examined,among which 76 were up-regulated while another 48 were down-regulated.The enrichment result showed that oligo-HA mainly took effects in several biological processes including cell cycle,oxoacid metabolic,amino acid biosynthetic and metabolic,and cell differentiation processes.Also,it regulates osteoclast differentiation,glycosaminoglycan,pantothenate,aminoacyl-tRNA,and CoA biosynthesis.Meanwhile,oligo-HA is involved in thyroid hormone,ErbB,and T cell receptors signaling pathways as well as several cancer processes.Particularly,several differentially expressed proteins related to the regulation of inflammatory responses were picked out.Namely,AKT1,PDCD4,TNFL9,PAK4,RXRA,and KAD1 were down-regulated,and FCGR2,PLCG1,NAL10,ZCCHV,JAG1,while histocompatibility 2 Q region locus 4 were up-regulated.With the regulation of these proteins,oligo-HA sample took effects in regulating inflammatory response.To further prove the anti-inflammatory effects of oligo-HA samples,an SLS-induced zebrafish inflammation model was established.By considering the death rate and fish status,the maximum tolerated dose of oligo-HA sample on zebrafish was set as 1.0%.After treated with 1.0%and 0.5%oligo-HA,the neutrophils detected in zebrafish significantly decreased compared with the model group,indicating that the inflammation in zebrafish is alleviated by oligo-HA treatment.In conclusion,our research detected the proteins that expressed differently in LPS-induced RAW 264.7 cells after treated with oligo-HA through proteomics technique and statistical analysis.Several proteins regulating inflammation and immune response are expressed differently after oligo-HA treatment.Based on the analysis result,we further proved the anti-inflammatory effects of oligo-HA in vivo through an SLS-induced zebrafish inflammation model.The statistically reduced number of recruited neutrophils after oligo-HA treatment suggested the inflammation is suppressed.Further research can be conducted focusing on the effects and mechanisms of oligo-HA according to the proteomics analysis result.