The Study On The Structures Of Polysaccharides From Angelica Sinensis (Oliv.) Diels And Their Inhibiting Mechanism On LPS-induced Inflammatory Response

Objective:Sepsis and septic shock contribute to the most common causes of death in intensive care unit and bacterial infection.The lipopolysaccharide（LPS）,a compound of the outer cell membrane of gram-negative bacteria,is specifically recognized by TLR4,and triggers innate immune responses through TLR4 activation.However,over-activation of TLR4 with LPS can lead to acute systemic disease mainly include sepsis,endotoxic shock,multi-organ injury,and death.Therefore,TLR4 has been considered as the most promising therapeutic target for sepsis and sepsis complications.Many TLR4 small-molecule inhibitors have been developed.However,many of them failed in further clinical trials because of their limited therapy and adverse reactions.TLR4 has been confirmed to be the receptor of polysaccharides,one of the well-sourced bio-macromolecules.Recent data indicated that TLR4 is closely involved in the immunomodulatory effects of several polysaccharides.Polysaccharides from the root of Angelica sinensis（Oliv.）Diels has been reported with significant immunomodulatory effect.However,the structures analysis of Angelica polysaccharides has not been perfected,whether Angelica polysaccharide could inhibit the LPS-induced macrophage inflammatory activation and the specific mechanism have not been thoroughly explored.Therefore,we intend to isolate series of homogeneous Angelica polysaccharides（APSs）,analyze their primary structural characters,investigate the mechanism of the active APSs on LPS-induced macrophage inflammation,and further evaluate the in vivo treatment effect of the bioactive APSs on sepsis model mice.Methods:（1）The crude Angelica polysaccharides（APS）was extracted using the traditional water extraction and alcohol precipitation method.The pigments and protein impurities in the crude APS were removed by H₂O₂ and sevag reagents.Then the total APS was further isolated and purified by DEAE Sephadex A-25 column and Sephadex G-100column.（2）The homogeneity and molecular weights of APSs were determined by high-performance size-exclusion chromatography（HPSEC）.The total carbohydrate contents of seven APSs were measured by sulfuric acid-phenol method.The uronic acid contents in seven APSs were determined by sulfuric acid-carbazole method.The protein contents of APSs were measured via coomassie brilliant blue method,and the FT-IR spectra were performed for the identification of characteristic groups in APSs.（3）The monosaccharide compositions of APSs were determined by high-performance liquid chromatography（HPLC）.The glycosidic linkages of APSs were determined by methylation and GC-MS.The 1D and 2D NMR spectra were employed to analyze sugar residues composition and linkage sequence of APSs.（4）Cytotoxicity of APSs on RAW264.7 cells was measured using CCK-8 kit.MST was used to determine the binding affinity of APSs for TLR4.The secretions of cellular inflammatory factors and NO under APSs and LPS treatment were revealed by ELISA.Western blot analysis was applied to determine the expressions of NF‐κB and IRF3 in the cytosolic and nuclear fractions,and the expressions of TLR4,MD-2,TIRAP,My D88,TRAM,and TRIF in LPS‐induced RAW264.7 cells treatment with/without APS-2I.The association of TLR4 with TIRAP,TRAM and MD-2were determined through co-IP experiment.（5）A septic mice model was established by the cecal ligation and puncture（CLP）method.Effect of APS-2I on survival of CLP sepsis model mice were determined.The heart,lung and kidney injuries in CLP mice were accessed by HE staining.The expression of F4/80 and Ly6G were determined to evaluate the inhibiting effect of APS-2I on the infiltration of inflammatory cells in lungs of CLP model mice.Results:（1）Seven APSs named APS-1I,APS-1II,APS-2I,APS-2II,APS-3I,APS-4I and APS-4II with molecular weight of 1.7×10⁴,2.3×10³,7.2×10⁵,1.0×10⁴,5.9×10⁵,8.1×10³ and 1.1×10³ Da respectively were isolated from Angelica sinensis（Oliv.）Diels.The total sugar contents of seven APSs are 97.6%,97.4%,96.3%,94.6%,98.3%,95.7%and 96.1%successively;the uronic acid contents of them are 1.2%,1.3%,2.7%,2.1%,2.4%,1.8%and 2.1%,respectively,and the protein content of each is 1.4%,0.8%,1.1%,1.8%,1.7%,1.5%and 1.1%.In addition,FT-IR spectra of seven APSs all displayed the featured signals of the polysaccharide.（2）HPLC,GC-MS and NMR analysis showed that APS-1I,APS-1II,APS-2I,and APS-4II are branched polysaccharides:（1）The main chain of APS-1I is→2-α-Glcp-1→4-α-Galp-1→[6-α-Glcp-1]_n→3,6-α-Glcp-1→3-Rhap-1→4-α-Galp-1→[6-α-Glcp-1]_n→3,6-α-Glcp-1→3-Rhap-1→,and the three side chains of APS-1I are→3-Rhap-1→4-α-Galp-1→6-α-Glcp-1→6-α-Glcp-1→,→4-β-Glcp-1→4-α-Galp-1→and→3-β-Galp-1→3,5-α-Araf-1→3-α-Araf-1→;（2）The major chain of APS-1II is→3-α-Araf-1→3,5-α-Araf-1→3-α-Araf-1→3,5-α-Araf-1→6-α-Glcp-1→3-α-Araf-1→,and the two side chains of APS-1II areα-Glcp-1→5-α-Araf-1→4-β-Glcp-1→5-α-Araf-1→andα-Fucp-1→3-α-Fucp-1→3-α-Fucp-1→3-α-Fucp-1→;（3）The main chain of APS-2I is→4)-α-D-Galp A-（1→3）-α-L-Rhap-（1→2,6）-α-D-Manp-（1→4,6-OMe）-α-D-Galp A-（1→3）-α-L-Rhap-（1→2,6）-α-D-Manp-（1→α-D-T-Galp,and the two branches of APS-2I are→5）-α-L-Ara?-（1→3）-α-L-Ara?-（1→3,5）-α-L-Ara?-（1→3）-α-L-Ara?-（1→4）-β-Galp-（1→and→6）-β-Galp-（1→6）-β-Galp-（1→;（4）The backbone of APS-1I is→6）-α-Glcp-（1→6）-α-Glcp-（1→5）-α-Araf-（1→5）-α-Araf-（1→3,5）-α-Araf-（1→3）-β-Galp-（1→3）-β-Galp-（1→4）-α-Galp-（1→3）-α-Araf-（1→3,5）-α-Araf-(1→,and traces ofα-Araf-1→were found at their branch point.APS-2II,APS-3I and APS-4I areα-glucans,all of them had a backbone composed of consecutiveα-1,6-Glcp with traces ofα-1,2-Glcp,α-1,3-Glcp andα-T-Glcp in their terminal sequences.（3）APS-2I and APS-3I displayed notable affinity to TLR4 with Kd values of 7.18±2.2μM and 46.36±6.1μM respectively among the seven APSs.Furthermore,APS-2I showed higher affinity to TLR4 than that of LPS to TLR4（27.68±6.7μM）.APS-2I dose dependently inhibited LPS‐induced inflammatory cytokine（TNF-α,IFN-β）and NO increase in RAW264.7 cells,and inhibited NF-?B and IRF3 nuclear translocation in LPS induced RAW264.7 cells.Also,APS-2I decreased TIRAP,My D88,TRAM and TRIF expressions and inhibited the formation of TLR4-MD-2 complex in LPS stimulated RAW24.7 cells.（4）APS-2I treatment markedly prolonged the survival time of CLP mice from 20.3 h to 65.5 h.APS-2I reduced TNF-α,IL-6,IFN-βand NO levels in serum of CLP model mice and protected them from heart,lung and kidney injury,and significantly inhibited inflammatory cells infiltration in lung after CLP challenge.Conclusions:（1）Seven APSs including APS-1I,APS-1II,APS-2I,APS-2II,APS-3I,APS-4I and APS-4II with molecular weights of 1.7×10⁴,2.3×10³,7.2×10⁵,1.0×10⁴,5.9×10⁵,8.1×10³ and 1.1×10³ Da separately were isolated from the root of Angelica sinensis（Oliv.）Diels.（2）APS-1I,APS-1II,APS-2I,and APS-4II were found to be novel structural polysaccharides,and APS-2II,APS-3I and APS-4I were found to beα-glucan.（3）APS-2I is the bioactive APSs among seven APSs,which targeted TLR4 and blocked its downstream signaling pathways to inhibit LPS-induced inflammatory response in RAW264.7 cells.Mechanistically,APS-2I bind to TLR4 and inhibited the formation of TLR4-MD-2 complex,reduced expressions of TIR proteins to block the downstream signaling pathway of TLR4,and play a treatment role in LPS stimulated sepsis model cells.（4）APS-2I had a significant sepsis treatment effect in vivo.Taken together,this study systematically elucidated the primary structure of seven APSs,and explored the anti-sepsis mechanism of the bioactive polysaccharide,APS-2I.This research provided a good basis for fully explore the medicinal value of Angelica sinensis and promoted the modernization of traditional Chinese medicine.