Study On Conjugation Of Superoxide Dismutase To O-quaternary Chitosan Derivatives And Its Protective Effect Against Experimental Colitis In Mice

Reactive oxygen species(ROS)is a collective term of oxygen-derived species and a byproduct of normal oxygen metabolism,mainly including superoxide anion radical(O2·-),hydroxyl radicals(HO-)and hydrogen peroxide(H2O2).ROS exerts a crucial effect on cell proliferation and differentiation by acting as intracellular signaling molecules.However,excessive ROS over the cellular antioxidant defense system can cause deleterious oxidative stress,which has been implicated in diverse severe diseases.Among of the diseases,the inflammatory bowel disease(IBD)is a clinical common intestinal disease,which has the characteristic of recurrence.Although its exact cause is still uncertain,the severity of IBD is related closely to the level of ROS in vivo.It is acceptable that a feasible strategy for alleviation of IBD is to scavenge the accumulation of ROS in body.In the enzymatic defense systems,the most crucial enzyme to overcome the potential toxicity of ROS is superoxide dismutase(SOD).It catalyzes the dismutation of the most dangerous superoxide anion to oxygen and hydrogen peroxide.Thus,SOD enzyme and other ROS scavengers have been found to be potentially effective agents for colitis treatment.In our previous studies,Cu/Zn superoxide dismutase(SOD)was successfully attached to a biodegradable chitosan derivative,O-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride(O-HTCC),to form a novel polymer-enzyme conjugate,O-HTCC-SOD.The present study demonstrated that O-HTCC-SOD had better stability than unmodified SOD under long-term storage at room temperature.O-HTCC-SOD exerted significant anti-inflammatory effects on lipopolysaccharides(LPS)-stimulated mouse peritoneal macrophages by the determination of pro-inflammatory cytokines using ELISA kit and intracellular ROS using fluorescent probe DCFH-DA.In addition,the effect of O-HTCC-SOD on acute colitis was investigted on the experimental mouse model of acute colitis induced by dextran sodium sulfate(DSS).The main results and achievements of the study are as follows:1.Preparation and stability of O-HTCC-SODA water-soluble chitosan derivative was synthesized by grafting 2,3-epoxypropyl trimethyl ammonium chloride(GTMAC)to reactive C6-OH of chitosan through a three-step reaction.Then ultrafiltration was performed to give purified to obtain,O-(2-hydroxyl)-propyl-3-trimethyl ammonium chitosan chloride(O-HTCC)at a yield of 81.2%.The structure was verified by IR and 1H NMR analysis results.The free amino group of O-HTCC is up to 71.5%based on ninhydrin colorimetry assay.O-HTCC was then conjugated with the activated terminal carboxyl of Cu/Zn superoxide dismutase(SOD)via a carbodiimide-mediated reaction.The modification ratio of the enzyme was up to 83.5%.The polymer-enzyme conjugate,O-HTCC-SOD was purified on DEAE Sepharose Fast Flow column to remove unreacted SOD.SDS-PAGE demonstrated that O-HTCC-SOD had higher molecular weight than native SOD.The activity changes of native SOD and O-HTCC-SOD at room temperature were monitored daily for 30 days to estimate their long-term stability for storage.Results showed that the residual activity of SOD was only 44.1%after 30-day storage at room temperature,while that of the conjugate was up to 86.8%.It is obvious that the shelf life of SOD for storage was excellently prolonged via modification of O-HTCC.2.Anti-inflammatory effect of O-HTCC-SOD in vitroPeritoneal macrophages elicited by starch broth were isolated from BALB/c mice.It is no significant difference to the macrophage viability of SOD and O-HTCC-SOD measured by MTT reduction assay at a concentration ranging from 10 U/mL to 1250U/mL.Both SOD and O-HTCC-SOD markedly decreased TNF-α,IL-1β and IL-6 production in LPS-activated macrophages according to ELISA assay(P<0.01).There was no significant dose dependence and difference in inhibitory effects between SOD and O-HTCC-SOD.The effect of O-HTCC-SOD on LPS-induced intracellular ROS production were estimated by using the fluorescent probe DCFH-DA.The results showed that cells pre-treated with O-HTCC-SOD exhibited a remarkable decrease in intracellular ROS levels(P<0.01),while SOD did not show the obvious inhibition(P>0.05).Thus,it was confirmed that O-HTCC-SOD has better scavenging capacity than native SOD as a result of the enhanced ability of entry cell.3.Therapeutic effect of O-HTCC-SOD on DSS-induced colitis in vivoAcute colitis models in BALB/c mice were induced by administration of 5%DSS in drinking water freely for 5 consecutive days.Simultaneously,the mice were treated by intravenous administration of drugs at indicated concentrations into tail vein each day and the treatment effect on of O-HTCC-SOD and native SOD was compared.BALB/c mice fed 5%DSS in drinking water were detected typical clinical symptoms,including body weight loss,increase in DAI score,shortening of the colorectum and increased levels of MPO.Moreover,DSS-induced colonic inflammation and mucosal damage were assessed by detecting the morphological alterations of the mouse colon after H&E staining.The results showed that oral administration of DSS exerted intestinal damage as indicated by loss of epithelial and goblet cells,crypt lesions and massive area with prominent inflammatory cell infiltration in the mucosa and submucosa.Modification of SOD with O-HTCC significantly improved its capability to ameliorate the severity of DSS-induced colitis in mice as observed by decreased DAI score(P<0.01),lowered colon length(P<0.01)and reduced neutrophil infiltration into the colon according to the activity level of MPO(P<0.01),and diminished colonic histopathological damage.Treatment effect of the unmodified SOD statistically lowered DAI scores and the level of MPO than DSS(P<0.05),but did not show obvious therapeutic effect on other symptoms.Therefore,the efficacy of O-HTCC-SOD was much better than the native SOD.The preliminary dose-response profile of O-HTCC-SOD demonstrated that O-HTCC-SOD produced beneficial effect on DAI at doses of 1.5 kU/kg to 6.0 kU/kg,but showed no obvious dose-dependence.There was best inhibitory effect on shortening of colon at 3.0 kU/kg and the conjugate at 3.0～4.5 kU/kg showed better inhibibion on MPO activity.In short,O-HTCC-SOD at a dose of 3.0 kU/kg showed the best treatment effect on acute colitis.4.Immunogenicity of O-HTCC-SODIn our study,immunogenicity of SOD and O-HTCC-SOD was estimated in BALB/c mice.The mice were immunized once every two weeks and there was twice altogether.The serum was collected from the mouse eyeballs of each group and detected by ELISA to determined immunogenicity indirectly.It was showed that the immunogenicity of O-HTCC-SOD was significantly lower than that of unmodified SOD.In summary,modification by the chitosan derivative,O-HTCC,conferred excellent long-term stability for storage,significant reduction of pro-inflammatory cytokines and intracellular ROS levels in LPS-stimulated mouse peritoneal macrophages.In addition,O-HTCC-SOD conjugates showed remarkable enhanced protective effect against colonic damage induced by DSS in vivo.This research has important implications for O-HTCC-SOD as a promising therapeutic option in the management of inflammatory bowel disease or other ROS-related diseases.