Studies Of Cu²⁺ Printed Glutaraldehyde Crosslinked Carboxymethyl Chitooligosaccharide’s Protective Effects And Its Mechanism On Uranium Toxicity

Object: Studies of Cu²⁺printed glutaraldehyde crosslinked carboxymethylchitooligosaccharide’s protective effects on depleted uranium toxicity in MC3T3-E1cellsand its mechanism.Methods: The saturated capacity of Cu-Glu-CMC’s chelating DU was detected byICP-MS. EPR, ABTS and FRAP were used to detect Cu-Glu-CMC’s antioxidant activity invitro. Cu-Glu-CMC（50,100,200mg·L-1） were added in MC3T3-E1cells30minitesbefore DU’s administration, Cell viability were assessed with MTT assay. DUaccumulations were measured by ICP-MS. Cell ultrastructure of MC3T3-E1cells wasobserved by electron microscopy. Flow cytometry, DCF staining, MDA and SOD wereused to evaluate cellular redox levels. IL-6secretion and expression of TNF-α wereanalyzed by ELISA and immunohistochemical staining method, respectively. EmployPNPP and ELISA to detect ALP and RANKL, respectively. ARS staining was applied toassess the degree of calcification. Expression of differentiation-related genes was evaluatedwith RT-PCR.Results: The chelating saturation capacity of Cu-Glu-CMC’s was58.6μg·mg^-1238Uand it’s antioxidant activity was significantly higher than CTS. After DU acute toxicity,Cell viability and DU accumulation were concentration-and time-dependent in varieddifferentiation status of MC3T3-E1cells. Cellular ROS and MDA content raisedsignificantly when SOD’s activity declined. IL-6’s secretion decreased while TNF-α’sexpression was increased. Compared with DU group, Cu-Glu-CMC could increase cellviability, decrease DU accumulation in MC3T3-E1cells, significantly, fall cellular ROSand MDA down while enhanced SOD’s activity, elevate IL-6’s secretion and reduce TNF-αstatus. After DU chronic toxicity, the viability of MC3T3-E1cells didn’t change whilethere was DU accumulation, ALP activity decreased, RANKL content increased, theformation of calcified nodules lowered and the expression of runx2、col-1α、bsp、bgp weakened in DU contamination MC3T3-E1cells. Compared with DU group,Cu-Glu-CMC strengthened ALP activity, inhibited RANKL’s secretion, increased thenumber of calcified nodules and enhanced the expression of runx2、col-1α、bsp、bgp in DUcontamination MC3T3-E1cell, significantly.Conclusion: Cu-Glu-CMC could protect MC3T3-E1cells from DU’s toxicity, themechanism are probably related to the factors as follow,①Cu-Glu-CMC decreased DU’scellular accumulation and its toxicity,②Cu-Glu-CMC could reduce ROS level and freeradical damage, improve radical scavenging capacity in DU contamination MC3T3-E1cell,③Cu-Glu-CMC mitigated inflammatory injury in DU contamination MC3T3-E1cell,④Cu-Glu-CMC improved DU’s inhibition on MC3T3-E1’s differentiation and the expressionof differentiation-related genes.