Extraction,Purification,Structural Elucidation And Anti-colitis Mechanisms Of Smilax China L.Polysaccharide

Smilax china L.is a medicinal plant belonging to the Liliaceae family.The rhizome of Smilax china L.is commonly known as‘Baqia’in China and has been widely used in traditional Chinese medicine for treatment of inflammatory diseases,such as pelvic inflammation and rheumatic arthritis.In this work,two polysaccharides（SCLP1 and SCLP3-2）were extracted,isolated and purified from the rhizomes of Smilax china L.Then,the structures and anti-inflammatory activity in vitro of SCLP1 and SCLP3-2 were investigated.SCLP3-2 showed better anti-inflammatory activity than SCLP1,so the preventive effect and underlying mechanisms of SCLP3-2 on ulcerative colitis（UC）were further studied.Two highly purified polysaccharides SCLP1 and SCLP3-2 were obtained from the rhizomes of Smilax china L.by extraction-alcohol precipitation,freeze-thaw,dialysis,DEAE-52 cellulose column purification and Sephadex G-50 column purification.SCLP1was a neutral polysaccharide with a weight-average molecular weight of 42.1 k Da.SCLP3-2 was an acidic polysaccharide with a weight-average molecular weight of 16.8k Da.The content of total sugar of SCLP1 and SCLP3-2 were 98.9%and 92.6%,respectively.The purity identification results indicated that both SCLP1 and SCLP3-2were highly purified and homogeneous polysaccharides.The structures of SCLP1 and SCLP3-2 were investigated by a combination of chemical and spectral analyses.SCLP1 was composed of glucose and mannose（54.5:1.0）.The backbone of SCLP1 was 1,4-linkedα-Glcp interspersed with 1,2-linkedα-Glcp and Manp.The branches were supposed to be 1,6-linkedα-Glcp and terminated withα-Glcp residue.SCLP3-2 was composed of galacturonic acid,arabinose,galactose and rhamnose（23.3:2.1:1.7:1.0）.SCLP3-2 was a type of pectic polysaccharide,and its backbone was composed of 1,4-linkedα-Galp A,which was partially methyl-esterified at C-6 carboxyl and slightly acetylated at the position of O-2 and（or）O-3.The side chains consisted of 1,2-linkedα-Rhap,1,3-linkedα-Rhap,1,4-linkedβ-Galp,1,5-linkedα-Araf and T-α-Araf.LPS-stimulated RAW264.7 cells were used as a model of inflammation to investigate anti-inflammatory effects of SCLP1 and SCLP3-2.Treatment with SCLP1 or SCLP3-2 significantly and dose-dependently inhibited the LPS-induced NO,IL-6 and TNF-αproduction,which suggested that SCLP1 and SCLP3-2 might possess a potential anti-inflammatory activity.SCLP3-2 showed better anti-inflammatory activity than SCLP1.Therefore,the effects of SCLP3-2 on inflammatory disease were further investigated.UC in BALB/c mice was induced by free drinking 3%dextran sulfate sodium（DSS,w/v）for 9 days.To explore whether SCLP3-2 could alleviate UC,the mice received SCLP3-2（250 and 500 mg/kg,intragastric administration）one week before and during DSS treatment.Administration of SCLP3-2 significantly ameliorated symptoms,decreased disease activity index（DAI）score and alleviated colonic shortening of UC mice.Hematoxylin and eosin staining results showed that SCLP3-2 obviously mitigated colonic damage and inflammatory cell infiltration in colons of UC mice.All the above results indicated that SCLP3-2 could significantly ameliorate DSS-induced UC in mice.In addition,SCLP3-2 effectively decreased myeloperoxidase activity and proinflammatory cytokines（IL-6 and TNF-α）,and increased anti-inflammatory cytokine IL-10 in colon tissues of UC mice.The regulatory effects of SCLP3-2 on inflammation in UC mice suggested that the effect of SCLP3-2 on alleviating UC may be related to its anti-inflammatory activity.SCLP3-2 is a pectic polysaccharide.Pectic polysaccharides show a good binding ability with galectin-3（Gal-3）and inhibit its activities.Therefore,Gal-3/NLRP3inflammasome/IL-1βpathway may be associated with the protective effect of SCLP3-2on UC.In vivo and in vitro experiments were used to explore and verify the underlying mechanisms of SCLP3-2 ameliorating UC.Phorbol 12-myristate 13-acetate（PMA）-differentiated THP-1 cells were exposed to LPS and DSS to construct an in vitro cell model,and SCLP3-2 was simultaneously added.Localized surface plasmon resonance（LSPR）analysis result showed that the equilibrium dissociation constant（KD）of SCLP3-2 and Gal-3 was 5.37×10^-8 M,indicating a good binding affinity between SCLP3-2 and Gal-3.The western blot,immunohistochemistry,immunofluorescence,enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction analyses of colon tissues and（or）cells proved that SCLP3-2 significantly inhibited Gal-3 and its downstream NLRP3inflammasome pathway.Moreover,coimmunoprecipitation assay confirmed that SCLP3-2 could inhibit the interaction of Gal-3 and NLRP3.Gal-3 was overexpressed by infecting PMA-differentiated THP-1 cells with Gal-3-lentivirus.SCLP3-2 treatment significantly suppressed the elevated Gal-3 and NLRP3protein levels and IL-1βsecretion caused by Gal-3 overexpression.These results suggested that Gal-3 was the target molecule of SCLP3-2 and that the inhibitory effect of SCLP3-2 on the Gal-3/NLRP3 inflammasome/IL-1βpathway was at least partly via direct regulation of Gal-3.On the basis of the results obtained above,it was concluded that SCLP3-2 could alleviate UC by inhibiting the Gal-3/NLRP3 inflammasome/IL-1βpathway from two aspects:（1）Suppressing the expression of Gal-3;（2）Directly binding to Gal-3,thereby suppressing the interaction of Gal-3 and NLRP3.