Mechanism Of Hemolysis Reaction In Vitro Induced By Puerarin

Puerarin injection has an excellent therapeutic effect in clinical treatment of cardiovascular diseases in China,but it can lead to severe intravascular hemolysis.This obvious side effect restricts its application.Although there have been many reports about the mechanism（s）of hemolysis induced by puerarin injection,at present the unified understanding about the type and classification of puerarin hemolysis were divergent yet.In this study,the morphologic changes of cells were observed by ordinary optical microscope,ordinary dark-field microscope,scanning and transmission electron microscope,live cell imaging system and inverted fluorescence microscope;the extra-and intercellular osmotic pressure of erythrocytes were measured;antagonistic experiments with a variety of compounds and the protein-protein interactions analysized by SDS-PAGE electrophoresis,non-reducing electrophoresis,protein co-immunoprecipitation and mass spectrometry were employed to to explore the mechanism（s）of hemolysis induced by puerarin.The purpose of this study is to re-recognize target（s）,the participants and courses of the puerarin hemolysis,identify the available compounds that can antagonize puerarin hemolysis and provide some theoretical evidences for the continued application of puerarin.The main results obtained in this study are as follows:1.Establishment of one hemolysis model induced by puerarin in vitro in accordance with the Chinese Pharmacopoeia method and calculation the rate of hemolysis.Using dimethyl sulfoxide（DMSO）as a solvent,the final concentration of puerarin solution in this experiment was determined to be 2,4 and 8 mmol/L for sheep erythrocytes as the experimental material in this section.The results showed that the hemolysis rate induced by DMSO with the corresponding concentration of the puerarin dose was much lower than that of puerarin,indicating that the hemolytic reaction in vitro of puerarin solution was caused entirely by itself and independent solvent.For sheep erythrocytes,2 mmol/L puerarin solution did not cause hemolysis,4 mmol/L puerarin solution had caused slight hemolysis,and 8 mmol/L puerarin solution caused about 10%hemolysis rate.Therefore,8 mmol/L puerarin solution were confirmed for sheep erythrocytes as models in the follow-up experiment of this part.2.To determine whether the puerarin hemolysis was relevant to the puerarin concentration,the volume of hemolytic system,different species of erythrocytes and osmotic pressure.Erythrocytes were treated with puerarin at the final concentrations of 2,4,8,20,25and 50 mmol/L respectively.The results showed that the hemolysis rate induced by puerarin were significantly different from that of the control group from 4 mmol/L.The hemolysis rate increased accompanied with puerarin concentration raised.However,when the concentration exceeded 25 mmol/L,the hemolysis rate was constant.The hemolysis rate of each group showed an"S"curve with the increase of puerarin concentration,suggesting that puerarin hemolysis may be related to osmotic pressure.The incubation volume was set at 0.2,0.4,0.6,0.8,1.0,1.2,and 1.4 m L with 8 mmol/L puerarin.The results showed that hemolysis rate increased firstly and then decreased.It indicated that hemolysis was related to volume change,suggesting that hemolysis is related to osmotic pressure.Erythrocytes of mice,sheep,New Zealand rabbits,chickens and toads were treated by puerarin,resulting in hemolysis（the data of chickens and toads were not shown）.It confirmed that puerarin hemolysis had the same hemolytic mechanism with no species specificity.The extracellular and intracellular osmotic pressure of erythrocytes were measured,respectively.The results showed that the extracellular and intracellular osmotic pressure of erythrocytes treated 1mmol/L puerarin increased by 1.72 times and 3.10 times compared that of the control group,8mmol/L puerarin increased by 1.57 times and 2.59 times.The results showed that puerarin caused osmotic pressure to increase significantly.The extracellular and intracellular concentrations of calcium,phosphorus,sodium,potassium and chloride ions were measured.The results showed that the concentrations of intracellular and extracellular ions expressed little effect on osmotic pressure,suggesting that the increase of intracellular osmotic pressure caused by puerarin was independent of intracellular ionic concentration.The extracellular and intracellular protein contents of erythrocytes were measured,respectively.The results showed that the intracellular protein contents in 2 mmol/L puerarin group was 0.05 times higher than that of the control group;4mmol/L puerarin group was 0.04times higher;8mmol/L puerarin group decreased to 0.93 of the control group.It was indicated that the increase in intracellular osmotic pressure caused by puerarin was mainly due to protein content,suggesting that puerarin could increase intracellular colloid osmotic pressure.This may be related to the release and degradation of cell membrane proteins and cytoplasmic proteins.The erythrocytes were pretreated with hypertonic glucose solution,fructose solution and sodium chloride solution,and then hemolysis was induced by puerarin.The results showed that 5 times osmotic pressure of hypertonic solution could antagonize puerarin hemolysis,also suggesting that puerarin hemolysis was associated with increased intracellular osmotic pressure.The same treatment was to puerarin group.The results showed that hemolysis aggravated and the rate reached 42.63%.The above results suggested that puerarin caused rapidly and dramatically in intracellular colloid osmotic pressure leading to hemolysis,and its mechanism（s）was similar to that of hypertonic hemolysis.3.The sheep erythrocytes treated with 2,4 and 8mmol/L puerarin were observed by ordinary optical microscope,scanning electron microscope and transmission electron microscope,and the sheep erythrocytes treated with 1mmol/L puerarin were observed by ordinary dark-field optical microscope,and the mice erythrocytes treated with 1mmol/L puerarin were observed by living cell image system and fluorescence inversion microscope.It was observed that the morphological changes of red blood cells mainly included edge rolling,cell contraction,volume reduction,uneven distribution of cell membrane and intracellular protein components,and subsequent leakage;cell membrane gap.The main cell types were spiny,spherical,hemolytic erythrocytes and blood ghost cells.In all the phenomena of red blood cells,the cytoskeleton changed significantly.When treated with low concentration puerarin,red blood cells became spiny,spherical,small in volume,and cell shrinkage,suggesting actin polymerization;the uneven distribution of cell membrane and intracellular protein components,as well as the presence of ghost cells,suggested the cytoskeleton depolymerization and were released outside together with hemoglobin.The results of above experiments further suggested that the membrane and intracellular protein components were released into and/or outside the cell,leading to the osmotic pressure sharply increased,actin shrinkage and cytoskeleton depolymerization.Therefore,it was speculated that most of the membrane and intracellular proteins were involved in the above processes,including hemoglobin,enzymes,transporters and skeleton proteins.Puerarin indeed caused the depolymerization and release of cell membrane and intracellular protein components,which finally participated in hemolysis,through dynamic and static morphological observation.4.To determine whether various transport channels were related to puerarin hemolysis.100m M Ca²⁺and its chelating agents could counteract the hemolysis of puerarin,indicating that the hemolysis was independent of the enzyme regulation.The transport substrate of Band3 protein(10 m M Cl^-、K⁺、Na⁺、HCO₃^-、SO₄^2-,respectively),even DIDS as Band 3 protein inhibitor could antagonize the hemolysis of puerarin.All ligands,furosemide as Na⁺-K⁺-2Cl^-co-transporter channel inhibitor,NPPB as K⁺-Cl^-co-transporter channel inhibitor,verapamil as the inhibitor multidrug resistant protein transporter channel,amino acid transporter substrate glutamate sodium and arginine,could antagonize the hemolysis of puerarin.The results showed that all the channels in the experiment were involved in the hemolysis of puerarin,which may be related to channel binding,not pure transport function.It was speculated that the binding of substrate or inhibitor to the channel affected the protein-protein interactions network of erythrocytes treated with puerarin.5.It has been confirmed that the puerarin hemolysis has been proved to be closely related to cytoskeletal protein.Cytochalasin B as the inhibitor of actin polymerization state,aprotinin,dithiothreitol,leucostatin and benzylsulfonyl fluoride as inhibitors of serine protease,cysteine and tyrosine protease were used to pretreat erythrocytes,respectively.The results showed that the above intervention agents could significantly antagonize puerarin hemolysis,suggesting that this hemolysis could be antagonized as long as intersected with the directly or indirectly protein-protein interactions network induced by puerarin.6.It was now necessary to determine whether puerarin had a target in erythrocytes membrane.Three TSPO ligands,including inhibitor PK11195,agonist Ro5-4864 and substrate diazepam,were used to pretreat erythrocytes.The results showed that three ligands could significantly antagonize puerarin hemolysis.TSPO,VDAC（Voltage-dependent anion channel,VDAC）,ANT（Adenine nucleoside transporter,ANT）and cyclophilin D（Cyp D）formed a"TSPO complex".Rotenone as VDAC’s inhibitor,carboxylic atractyloside as ANT’s inhibitor and cyclosporine A as Cyp D’s inhibitor were pretreated erythrocytes and they could also significantly antagonize the puerarin hemolysis.It suggested that the"TSPO complex"on the erythrocyte membrane was involved in the hemolysis process of puerarin and its mechanism was related to ligand affinity,independent of ligand function.As long as the ligands could bind to the"TSPO complex"and affected the structure of TSPO or protein-protein interactions,resulting in antagonizing the hemolysis of puerarin.7.To determine the effect of puerarin on erythrocytes’respiration and energy metabolism was necessary.To reflect the energy metabolism induced by puerarin through measuring the following four indicators,total ATP content,Na⁺-K⁺-ATPase activity,ATPase activity and NADH/NAD⁺ratio,respectively.The results showed that the total ATP content,Na⁺-K⁺-ATPase activity and ATPase activity were significantly decreased and NADH/NAD⁺ratio was increased after 8 mmol/L puerarin treatment.It indicated that puerarin reduced ATP production by attenuating Na⁺-K⁺-ATPase activity and the glycolysis process;and decreased ATP consumption by inhibited the activity of ATPase.It suggested that the respiratory function of erythrocytes was weakened,which provided free electrons for the occurrence of oxidative stress.Antioxidant vitamin C,vitamin E and oxidant hydrogen peroxide,daunorubicin,uric acid（anti-nitrification stress）and sodium nitroprusside（enhanced nitrification level stress）were pretreated to red blood cells.The results showed that they could antagonize puerarin hemolysis,indicating that the hemolysis mechanism was independent of the oxidation state.It was suggested that the anti-hemolytic effects of vitamin C and vitamin E was not from anti-oxidation mechanism.The total nitrosation level and total nitrosylation level of erythrocyte treated puerarin were tested.The results showed that puerarin significantly increased the level of total nitrosation and total nitrosation in erythrocytes.It indicates that the puerarin hemolysis was accompanied by a nitrative stress state.8.These results indicated that a variety of channel proteins and membrane proteins which existed interaction with each other were involved in the erythrocyte hemolysis induced by puerarin.It was suggested that puerarin increased the protein-protein interactions by TSPO as target.Therefore,the following co-immunoprecipitation and mass spectrometry identification was for the interaction of TSPO or Band 3 protein after puerarin treatment.The results showed that classification and quantities of proteins interacting with TSPO and Band 3protein were significantly increased after puerarin treatment.The above targets were identified by mass spectrometry.It has been proved that a variety of intervention agents could significantly antagonize the hemolysis of puerarin,revealing that the hemolytic effect of puerarin was enhibited by increasing protein-protein interactions,which formed a very complex network.In summary,the hemolytic mechanism of puerarin was as follows:puerarin combined with TSPO on erythrocyte membrane,leading to the excessive increase of non-specific protein-protein interactions and the structural change,inactivation of binding protein,cellular respiratory function weakened and energy production impaired.As well as oxidative stress and nitration stress would further damage the protein,resulting in these binding protein release and/or degradation.Then cytoskeleton depolymerization or intracellular colloid osmotic pressure increased,which lead to hemolysis finally.