?.The Mechanism Of Hypothermia-induced Oxidative Damage In Platelets ?.Toxic Effects Of DMSO

Platelets(PLTs),as one of the main components in hematological system,play an irreplaceable role in the human coagulation function and maintenance of vascular integrity.PLTs transfusion is an effective measure for thrombocytopenia,so that providing adequate and safe PLTs products is of great significance for clinical and war-wounded therapy.However,there lies imbalance between supply and demand in clinical application of PLTs.On one hand,FDA provides a shelf life of PLTs preservation only for 5 days(22? ± 2?);on the other hand,the demand for PLTs is on the increase in the case of emergency and wartime.Therefore,long-term preservation of PLTs has been a research hotspot in transfusion medicine.At present,there are three methods for prolonging shelf life of PLTs,including 4?cold preservation,freeze-drying preservation and-80 ? cryopreservation,but low-temperature damage was unavoidable and unclear.Mazur reported that there are two main damage hypothesis for-80? cryopreservation: "ice crystal" and "salt effect".The formation of ice crystal in PLTs results in irreversible mechanical damage during the cryopreservation process.Elevated salt concentration by dehydration also caused damage to PLTs.Scientists have developed a variety of protective agents,including DMSO and the second messenger effector(Thrombo Sol),but the damages are still existed.In recent years,researchers have put forward a new hypothesis "hypothermia stress",considering that sudden changes in temperature may induce oxidative stress in cells,whose damaging and apoptosis effects on cells may be associated with the overproduction of oxygen free radicals and ROS accumulation.Whether the apoptosis of PLTs induced by hypothermia is associated with the "hypothermia stress" remains to be further explored.FDA has not approved-80?-cryopreserved PLTs for clinical use.On one hand,PLTs function was significantly decreased after cryopreserved,on the other hand,cyroprotective agent DMSO has not yet be approved for clinical infusion.Toxicological data show that DMSO was found to be a toxic chemical agent.A large number of toxicity experiments focused on oral,skin and inhalation.However,few data on toxic effects of DMSO following intravenous infusion of cryopreserved PLTs are available.The aim of this study was to explore the mechanism of hypothermia-induced damage to PLTs,and also to explore dose-related effects of DMSO on RBCs,PLTs,vascular endothelial cells in vitro and metabolic organs in vivo.Our studies provide a theoretical basis for the research of PLTs cryoprotectant and the clinical application of cryopreserved PLTs.Part I.The Mechanism of Hypothermia-induced Oxidative Damage in PLTs1.Effects of hypothermia and ROS on PLTs apoptosis.In this study,we chose 22? as the control group,the hypothermia group was 4?,0?,-20?,-30? and-80?.Compared to control group,apoptosis levels of PLTs were significant increased after treatment for 2h in hypothermia group.ROS(H2O2 and TBHP)also increased PLTs apoptosis after incubated for 30 min,showing dose-effect relationship.Hypothermia and ROS both can lead to increased apoptosis of PLTs.Whether there is a necessary link between the two factors remains to be further studied.2.The production and main source of ROS in hypothermia-incubated PLTs.The results showed that hypothermia induced intracellular ROS production in a time-dependent manner in PLTs,compared to control group.Four ROS inhibitors,DPI,L-NAME,NAC and Mito-TEMPO,were chose to comfirm the main source of ROS.Compared to the first three inhibitors,the mitochondria-targeted ROS scavenger Mito-TEMPO can be more effective in inhibiting intracellular ROS and mitochondrial ROS generation.Hypothermia decreased PLT Mn SOD protein levels and enzyme activity,resulting in reduced degradation of ROS.3.The damages of ROS to PLTs.Hypothermia-induced malonyldialdehyde(MDA)production and cardiolipin peroxidation in platelets were inhibited by Mito-TEMPO,indicating that hypothermia-induced peroxidation damages were mediated by mitochondrial ROS.At the same time,Mito-TEMPO significantly inhibited PS externalization,decrease of mitochondrial membrane potential(MMP)and shedding of membrane glycoprotein Ib?,indicating that Mito-TEMPO ameliorates hypothermia-impaired platelet apoptosis and function.4.The mechanism of hypothermia-induced PLTs apoptosis.In our experiments,Bax translocated to the mitochondria and cyt C released from the mitochondria are key events that regulates hypothermia-induced PLTs downstream apoptotic events,especially when the temperature was below 0 ?.These results showed that mitochondria death signal pathway has been identitied to control the apoptosis of hypothermia-induced PLTs.The activated caspase-3 accelerated PLTs apoptosis.Mito-TEMPO significantly inhibited hypothermia-induced caspase-3 activation.These observations showed that mitochondrial ROS play important roles in hypothermia-induced PLTs apoptosis.Part II.Toxic effects of DMSO1.The effect of DMSO on human RBCs.DMSO(0.2%,0.4% and 0.6%)increased the supernatant plasma free hemoglobin(FHb)level of RBCs in a dose-dependent manner.The longer the incubation with DMSO,the greater the content of FHb.Apoptosis levels of RBCs did not have any significant differences between control group and DMSO incubation groups.The results suggested that DMSO-induced hemolysis belongs to the dose-related non-immune hemolysis.DMSO influenced the stability of RBCs by changing RBCs internal and external balance,causing acute adverse reactions.2.The effect of DMSO on human PLTs.The results showed that DMSO reduced ADP,THR and TRAP-induced PLTs aggregation starting at concentrations of 0.5%,2% and 0.25%,respectively.DMSO increased PLTs aggregation inhibition rates in a dose-dependent manner.When the concentration of DMSO was 6%,the inhibition rates of ADP,THR and TRAP-induced PLTs aggregation reached a 90%-plus percent.In the meanwhile,CMFDA fluorescence intensity endogenous thrombin levels associated with PLTs aggregation function and were reduced by DMSO,which were consistent with the results of aggregation test.3.The effect of DMSO on vascular endothelial cells.With the addition of DMSO(0.2%,0.4% and 0.6%)to the culture medium for 5 consecutive days,the proliferation of the vascular endothelial cell line EAhy926 cells was inhibited by blocking the G1 phase to the S phase.The apoptosis of EAhy926 cells cultivated in cell culture media with DMSO stimulation(0.2%,0.4% and 0.6%)were increased,compared to the control group.All results showed time-dose-effect relationship.4.Animal experiments: The male Bal B/c mice were injected 6% DMSO(150 ?l,300 ?l)continuously for 5 days.Compared with the control group(without any treatment),the appetite of Bal B/c mice was affected and weight gain was reduced.Howerver,the functions and pathological observation of liver and kidney did not have any significant differences between control group and DMSO-incubated groups.The small dose and short time may be lead to the occurrence of these results.It will be worth to further study when changing injection dose,manner and time.In summary,determining the role of mitochondrial ROS as contributory factors in hypothermia-induced PLTs apoptosis is critical in providing a rational design of PLTs cyroprotective agent.To the toxic effects of DMSO,our group have developed a cryopreservation PLTs dialysis machine(Patent No.: 201320735032.7),aiming at most maintaining the function of PLTs while removing DMSO of cryopreserved PLTs,the ultimate goal is to provide safe and effective PLTs products to clinical and war-wounded therapy.