Study On The Preparation Of Hep-SOD And TMC-SOD Conjugates And Their Effect On ROS Induced Injury And The Mechanisms

Reactive oxygen species(ROS),such as superoxide anion(O₂^-) and H₂O₂, cause oxidative stress in the body,a condition implicated in the pathogenesis of many diseases such as pulmonary diseases and liver diseases.Antioxidant enzyme, superoxide dismutases(SOD),converting O₂^- into H₂O₂,is a candidate agent for augmentation of antioxidant defenses.However,anti-inflammatory and anti-fibrotic effects of the major oxygen free radical scavenging enzyme SOD are not satisfactory in vivo studies.The discouraging results of animal and clinical studies can be attributed,at least in part,to unfavorable pharmacokinetic profiles of SOD(t_1/2 is approximately 6 min).To improve its pharmacokinetic properties and effects on suppressing ROS-mediated injury,long half-life and tissue or cell-specific targeting SOD derivertives,cationized SOD(TMC-SOD) and anionized SOD(Hep-SOD), were designed and prepared by chemical modification with N,N,N-trimethyl chitosan chloride(TMC) and heparin in this research.In this study,the effects and the mechanisms of Hep-SOD and TMC-SOD conjugates on ROS induced injury were also studied.All the results of our research were summarized as follows.1 Synthesis of Hep-SOD and TMC-SOD derivativesSOD was modified with NaIO₄ activated heparin,in pH9.5,0.3 mol/L carbonate buffer,the product was determined by electrophoresis.After pretreatment,terminal solution of modification was added to DEAE-Sepharose Fast Flow column,and Hep-SOD conjugate was collected.SOD was modified with TMC using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) as coupling agent.In order to increase its solubility in water and its cation property,chitosan was degraded to low molecular product with H₂O₂,and then TMC-SOD was synthesized.Through pretreatment,the terminal solution of modification was added to DEAE-Sepharose Fast Flow column,and TMC-SOD conjugate was collected.The enzyme activity of native SOD,TMC-SOD and Hep-SOD was 3250,2830 and 2770 U/mg,respectively.The methods of was performed,It was proved with MTT assay that the conjugated SOD was safe to cells.2 Secondary structure analysis by Circular dichroism(CD) measurementsThe secondary structure of Hep-SOD and TMC-SOD was analyzed by CD technique.The secondary structures of Hep-SOD and TMC-SOD were slightely changed compared with those of native SOD.Compared with native SOD,it was found that theα-helix of Hep-SOD was increased(23.50%vs 24.50%),both theβ-sheet (49.8%vs 48.3%) and random coil(31.90%vs 30.80%) were decreased.The secondary structure of SOD was changed partly after modified with TMC also.The results showed thatα-helix decreased a little by modification with TMC(23.50%vs 20.70%); random coil portion increased a little(31.90%vs 37.90%);β-conformation's portion of TMC-SOD,includingβ-sheet andβ-turn,were higher than that of native SOD(53.7%vs 49.8%).But generally speaking,the change of secondary structure after chemical modification was weak.3 Targeting of SOD derivatives to activated macrophages.3.1 Intracellular SOD,T-AOC activity and inhibitory effect on superoxide anion generation examination following incubation with SOD and SOD derivativesElicited macrophages were collected from the peritoneal cavity of Kunming mice 3 days after intraperitoneal injection of 1 mL of 6.0%starch medium.Macrophages were incubated with SOD and SOD derivatives,and intracellular SOD,total antioxygen capacity(T-AOC) activity and inhibitory effect on superoxide anion generation were analyzed with test kits.The results showed that the level of endogenous SOD activity was 48.34±5.39U/mg protein in untreated macrophages.When the cells were incubated with TMC,heparin,native SOD, TMC-SOD or Hep-SOD,the intracellular SOD activity was 50.40±5.71, 72.13±6.99,65.92±6.00,408.4±15.91 and 162.2±7.25 U/mg protein, respectively.A similar effect as the intracellular SOD activity was observed for T-AOC.T-AOC was markedly increased after incubated with TMC-SOD or Hep-SOD,consistent with the notion that these changes reflected increased antioxidant capacity of macrophages incubated with SOD derivatives.The T-AOC of macrophages showed significant changes after incubated with SOD or SOD conjugates.The T-AOC levels in the TMC-SOD and Hep-SOD groups were notably higher than that in other groups after incubated for 2 h.Our research proved that both TMC-SOD and Hep-SOD derivatives inhibited superoxide anion release from macrophages.The inhibitory effect of TMC-SOD was markedly higher than other groups.In this study,we found that SOD modified with TMC or heparin had a higher sensitivity to colchicine,which was contradicted to the early report that cationized SOD with hexamethylenediamine had a lowered sensitivity to colchicine.The different sensitivities of polysaccharide and hexamethylenediamine used as modifier to colchicine on effecting the uptake abilities of macrophages led us postulate that there might be receptors with polysaccharide specificity in macrophages.It had been reported that the mannose receptor could also bind chitosan.In this study,we speculated that SOD after modified with TMC could bind to mannose receptors,while hexamethylenediamine could not.Further study is necessary to test these speculations.3.2 Flow cytometry analysis of the binding ability of SOD conjugates to macrophagesThe binding ability of SOD conjugates to cells was determined by flow cytometry.The results indicated that fluorescence intensity increased after incubation with FITC-labeled SOD derivatives,and that of TMC-SOD-FITC was the highest.The fluorescence intensity of macrophages incubated with FITC-SOD for 1 h was only 22%;while incubated with Hep-SOD-FITC and TMC-SOD-FITC for 1 h,the fluorescence intensities were 29.21%and 95.36%respectively.Extending the incubation time with SOD and Hep-SOD to 2 h,the fluorescence intensities were 37.64%,51.81%respectively.3.3 Study on intracellular distribution of FITC-labeled SOD and SOD derivatives in HUVEC by confocal laser scanning microscopyMacrophages were incubated with 200μg/ml FITC-SOD and SOD derivatives at 37℃for 1 h or 2 h.The images were obtained by confocal laser scanning microscopy. One hour after incubation,the fluorescent signals of FITC-labeled native SOD were minimal,and that of FITC-labeled Hep-SOD were slightly higher than that of native SOD.In contrast,FITC labeled TMC-SOD were observed to have stronger fluorescent signals than native SOD and Hep-SOD.Two hour after incubation,all the fluorescent signals were enhanced compared with that of 1 h after incubation.4 The preventive effects of Hep-SOD on carbon tetrachloride(CCl₄)-induced acute liver failure and hepatic fibrosis in mice4.1 The effects of Hep-SOD on CCl₄-induced acute liver failureCCl₄ dissolved in sesame oil(2%v/v) was administered to the peritoneal cavity of mice to induce acute liver failure.To examine the hepatoprotective effect of medication,Hep-SOD,heparin+SOD,SOD or heparin were injected intravenously via the tail veins of mice respectively,immediately after CCl₄ administration.Twenty four hours after CCl₄ administration,blood was collected and serum was separated. Serum LDH and GOT/GPT activities were assayed using commercial kits.The liver was isolated,washed with saline,weighed,and homogenized.The total MDA contents of the supernatants were determined.Determination data demonstrated that GOT/GPT content in serum of all medication groups except heparin group and SOD group was lower than that of control group,but higher than that of normal group.The test also showed that administration of CCl₄ to mice resulted not only in a significant increase in serum GOT/GPT and LDH activities,but also in a significant reduction in the total GSH content.Furthermore,treatment with Hep-SOD also reduced the serum LDH and increased total GSH significantly.Determination data also demonstrated that MDA content of heparin group was lower than control group, but the effect of Hep-SOD was considered extremely high.Compared with native SOD,it was showed that Hep-SOD had a significant protection in terms of serum LDH and total GSH content(P＜0.05).Collectively,these findings indicated that Hep-SOD had preventive effects on necro-inflammatory responses following acute CCl₄ administration,while heparin had no effect.4.2 The effects of Hep-SOD on CCl₄-induced hepatic fibrosisKunming mice received intraperitoneal injection of CCl₄ twice a week for 12 weeks.At the same time,the Hep-SOD was administered by intraperitoneal injection. Twenty four hours after the final injection of CCl₄,blood was collected from the vena cava.Liver samples were collected and frozen in liquid nitrogen.(1) Hepatic hydroxyproline content determinationLiver collagen concentration was determined by measuring hydroxyproline content in liver samples.Results were expressed as micrograms per gram of wet tissue. The results showed that there was a significant increase in hepatic hydroxyproline levels in CCl₄-treated mice compared to normal controls(548±39 vs.168±14μg/g wet liver,P＜0.05,respectively).Compared with control group,the liver tissue hydroxyproline content of each treated group was decreased.Administration of Hep-SOD,heparin+SOD,SOD or heparin could prevent the increase in hepatic hydroxyproline content in CCl₄-treated mice(206±16;248±13;320±57;250±17μg/g wet liver,respectively).Compared Hep-SOD group with control group,the liver tissue hydroxyproline content of Hep-SOD group was significantly lower(P＜0.01).(2) Hepatic histopathology of mice hepatic tissue after Hep-SOD co-administered with CCl₄ for 12 weeks.Repeated CCl₄ administration for 12 weeks caused overt bridging fibrosis in livers as expected.In sharp contrast,fibrotic changes caused by CCl₄ were prevented dramatically when Hep-SOD or heparin+SOD was given.In contrast to normal mouse liver morphology,CCl₄-induced fibrosis was evidenced by disruption of tissue architecture,extension of fibers,large fibrous septa formation and collagen accumulation.In the control group,the damage was found throughout the liver and the fibrotic infiltrations appeared in both pericentral and periportal regions,and the bridging fibrosis was completely formed.These alterations were partly reduced in the liver sections of the mice that received administration-treatment.The formation of fibrosis,marked fatty degeneration,necrosis,inflammation of hepatocytes,and infiltration of inflammatory cells including macrophages and lymphocytes were seen in heparin-administered mouse livers.On the other hand,mice treated with Hep-SOD showed marked suppression of the CCl₄-induced inflammatory cell infiltration and the formation of fibrosis.Compared to the control group,the hepatic necrosis,particularly in the pericentral region,was diminished in the heparin+SOD group.Compared with the control group,Hep-SOD significantly reduced the scores of liver fibrosis,which verified that the scoring of the Hep-SOD-treated group had been obviously improved.(3) Effects of Hep-SOD on related mRNA transcription in mouse liver tissueTGF-β1,MMP-2,fibronectinand collage-I mRNA expressions in liver tissue were determined by Real-time PCR,and the results showed that TGF-β1 mRNA in the liver increased about 4-fold after repeated injections of CCl₄ for 12 weeks. However,Hep-SOD prevented this increase almost completely.The result also showed that MMP-2,fibronectin and collage-I mRNA in the liver was increased by chronic CCl₄ administration,and the mRNA levels were decreased sharply when Hep-SOD was given simultaneously.(4) Effects of Hep-SOD on TGF-β1 expression in mouse liver tissueWestern blotting analysis confirmed that chronic CCl₄ treatment increased TGF-β1 in the liver significantly,whereas Hep-SOD,heparin+SOD,SOD and heparin prevented this increase partially.CCl₄ induction of TGF-β1 in the liver was prevented noticeably in the Hep-SOD treatment group.5 The preventive effects of TMC-SOD on CCl₄-induced acute liver failure and hepatic fibrosis in miceThe effects of TMC-SOD on CCl₄-induced acute liver failure and hepatic fibrosis were evaluated.To investigate the effects of TMC-SOD on acute liver failure, TMC-SOD was administered to CCl₄-treated mice by intravenous injection. Biochemical indicators,such as GOT/GPT,GSH,LDH and MDA were determined 24 h after CCl₄ treatment.The development of CCl₄-induced acute liver failure altered the redox state with a decreased GSH and increased formation of lipid peroxidative products,which were partially normalized by treatment with TMC-SOD or TMC+SOD.Compared with other groups,the acute liver injury of TMC-SOD group was significantly lessened(reduced activities of GOT/GPT,MDA and increased activities of GSH).To investigate the effects of TMC-SOD on hepatic fibrosis, TMC-SOD and CCl₄ were co-administered by intraperitoneal injection twice a week for 12 weeks.Histological and hepatic hydroxyproline examination revealed that TMC-SOD could significantly prevent the progression of hepatic fibrosis.Moreover, real-time PCR was used to determine TGF-β1,MMP-2,fibronectin and collagen-I expression.Significantly greater fibrosis and TGF-β1,MMP-2,fibronectin,collagen-I expression were found in the liver of CCl₄-induced mice at the end of 12th week. TMC-SOD could markedly attenuate the mRNA expression of TGF-β1,MMP-2 and collagen-I.Western blots of tissue homogenates revealed that the protein expression of TGF-β1 was substantially reduced also by TMC-SOD treatment.These results demonstrate that administration of TMC-SOD may be useful in the treatment and prevention of acute liver failure and hepatic fibrosis.6 Pharmacokinetics and Biodistribution StudiesTo determine the pharmacokinetic clearance of SOD after chemically modified with heparin and TMC,plasma concentrations of ¹²⁵I-Hep-SOD and ¹²⁵I-TMC-SOD were investigated and compared with that of ¹²⁵I-SOD in Kunming mice.The radioactivity in plasma or tissues was determined following trichloroacetic(TCA) precipitation after a single intravenous injection.The concentration-time curves of ¹²⁵I-Hep-SOD and ¹²⁵I-TMC-SOD were proved to comply with two-compartment model.The study proved that the half-life of TMC-SOD or Hep-SOD was longer than that of native SOD.At the same dosage,the distribution half life t_1/2α,the elimination half life t_1/2β and the AUC for plasma were increased after a single i.v. with¹²⁵I-Hep-SOD and ¹²⁵I-TMC-SOD compared with¹²⁵I-SOD.¹²⁵I-Hep-SOD was aggregated in most tissues especially in liver while ¹²⁵I-TMC-SOD in lung.High lung-retention was observed for the conjugate of TMC-SOD at 5min(24.98±1.87%ID/g) and 1 h(16.13±1.19%ID/g),and high liver-retention was observed for the conjugate of Hep-SOD at 5min (21.38±3.35%ID/g) and 1 h(14.38±2.67%ID/g).7 The main results and conclusions in this paper are summarized as follows:(1) TMC-SOD and Hep-SOD were prepared and separated;the secondary structures of Hep-SOD and TMC-SOD were analyzed by CD technique.(2) Cationic polysaccharide TMC or anionic polysaccharide heparin,might be useful protein modification materials for macrophage targeting delivery of enzymes,especially TMC.(3) Hep-SOD prevents severe hepatic damage and fibrogenesis induced by chronic CCl₄ administration through up-regulation of GSH,down-regulation of TGF-β1, possibly through its antioxidant and anti-inflammatory action.(4) TMC-SOD may be useful in the treatment and prevention of acute liver failure and hepatic fibrosis.The administration of TMC-SOD may be an optional therapeutic and preventive measure against oxidative stress-induced liver injury and hepatic fibrosis.(5) Half-life of SOD was increased,compared with native SOD,after modified with heparin or TMC.Biodistribution studies showed that TMC-SOD targeted to lung,Hep-SOD targeted to liver.(6) Chemically modified SOD with TMC or heparin could be effective agents for the treatment of inflammation mediated by ROS.Both TMC-SOD and Hep-SOD are promising agents for the treatment of ROS induced injuries.