Alleviation Of Cyclophosphamide-mediated Blood Hypercoagulation By Sepia Esculenta Ink Polysaccharide And Its Derivatives

Sepia ink polysaccharide(SIP)is a type of glycosaminoglycans derived from sepia ink.Studies have demonstrated that SIP has chemoprotective,chemosensitizing,antitumour,anticoagulant,antioxidant and immunomodulatory activities,and that its antioxidant and anticoagulant activities can be enhanced by chemical modifications.Cancer,the second leading cause of death,puts human life at great risk.As one of the important means of cancer treatment,chemotherapy drugs,while killing tumour cells,can also induce hypercoagulation of the body’s blood and consequent thrombogenesis,resulting in increase of death risk in cancer patients.In order to investigate the anticoagulant activity of SIP and its derivatives with a view to developing drugs for clinical treatment or adjuvant therapy of cancer,this study employed chloroacetic acid method,phosphorylation method and chlorosulfate-pyridine method for the molecular modification of SIP-IV,the main component of Sepia esculenta ink polysaccharide,by carboxymethylation,phosphorylation and sulfation,respectively.First,the effects of the above chemical modifications on SIP-IV activity were evaluated based on antioxidant,anticoagulant and thrombolytic activities.Secondly,an animal model of chemotherapy drug-mediated blood hypercoagulation was established using cyclophosphamide(CTX)to assess the alleviating effect of SIP-IV and its derivatives on chemotherapy drug-mediated blood hypercoagulation and to provide a preliminary elucidation of its anticoagulant mechanism.Infrared spectroscopic analysis showed that the carboxymethylated,phosphorylated and sulfated modifications,with characteristic absorption peaks,respectively,indicated the successful preparation of the derivatives carboxymethyl SIP-IV(C-SIP-IV),SIP-IV phosphate(P-SIP-IV)and SIP-IV sulfate(S-SIP-IV)with degrees of substitution(DS)of1.02,0.09 and 1.60,respectively.In vitro antioxidant and anticoagulant studies showed that in vitro antioxidant activity of P-SIP-IV and S-SIP-IV was enhanced,while that of C-SIP-IV was reduced.The anticoagulant activity of SIP-IV,C-SIP-IV,P-SIP-IV and S-SIP-IV was enhanced sequentially and in a dose-dependent manner,indicating that SIP-IV has anticoagulant activity and that carboxymethylation,phosphorylation and sulphation modifications all enhanced this activity,with the sulphation modification having the strongest effect.In vivo studies revealed that SIP-IV significantly increased SOD activity in the liver of normal mice(P<0.01)and that its derivatives significantly upregulated CAT and SOD activity and GSH content(P<0.05).In addition,CTX-induced hepatic oxidative damage was significantly improved by SIP-IV and its derivatives(P<0.05),and the three derivatives were more effective than the natural polysaccharides in protecting the liver.SIP-IV and its derivatives prolong APTT,PT,TT and bleeding time in vivo,exhibiting in vivo anticoagulant effects.The results of the plasma coagulation factor assay revealed that SIP-IV and its derivatives could elevate the level of antithrombin III,thereby prolonging blood clotting time.Chemotherapy mice protected by SIP-IV and its derivatives showed significant improvement in coagulation factors II and X and antithrombin III levels(p<0.05).These results suggest that SIP-IV and its derivatives are effective in alleviating the hypercoagulable state of blood mediated by chemotherapeutic agents.In addition,in vitro thrombolytic assays showed that both SIP-IV and its derivatives had thrombolytic activity,with sulfated modification resulting in significantly enhanced thrombolytic activity compared to SIP-IV,carboxymethylated modification resulting in reduced thrombolytic activity,and phosphorylated modification having no effect on thrombolytic activity.In conclusion,SIP-IV and its derivatives can effectively alleviate chemotherapeutic drug-induced blood hypercoagulation and exhibit anticoagulant and thrombolytic activities,which proves the feasibility of SIP-IV and its derivatives as adjuvant drugs in chemotherapy and provides a preliminary theoretical basis for the development of adjuvant drugs in chemotherapy.