Study On The Underlying Mechanisms Of Hemolysis Induced By Puerarin Injection

Puerarin {7-hydroxy,3-(4-hydroxyphenyl)-8β-D-glueopyranosyl},is the most abundant and active ingredient in Radix Puerariae Lobatae which is a very common used herb in China at folk and at Chinese clinic.Puerarin Injection(PI) is widely used for treating coronary heart diseases,angina,high blood pressure,miocardial infarction,retina vas infarction,sudden deafness and so on.Although puerarin is well-known on low toxicity and high therapeutic effect,it has caused a great anxiety because of the serious side effects(ADR) induced by PI. Primary symptoms of ADR induced by PI are fever,anthesma,renal and hepatic injury acute hemolysis,et al.The acute hemolysis usually may be produded quickly and then the risk is dangerous.Some reports said that the occurrence of ADR is associated with age,manufacturers, dosage,course of and combination of administer.Some researchers considered the hemolysis was the second type of allergic reaction.However,the mechanism for the hemolysis reaction is still unclear.So the aims of this study were to reveal the mechanisms as well as try to bring out measures to decrease the hemolysis incidence induced by PI.We found that increasing concentration of puerain and extending reaction time,the incidence of hemolysis was obviously higher.The optimal reaction time should be more than 6 hours.Thus hemolysis was tested after 24 hours' incubation in vitro.For the solvent in Puerarin Injection is propylene glycol(PP),hemolysis induced by PP and the acitity of preparation was studied simultaneously.But no hemolysis occurred.Thus it was concluded that hemolysis was mainly caused by peurarin.Experiments were further carried on to investigate the feature of hemolysis induced by PI at the clinic.There are mainly three mechanisms underlying the drug induced hemolysis, oxidative hemolysis,immune hemolysis as well as non-immune hemolysis.Guinea pigs were administrated intravenously for several days at the dose of 50mg/kg per day which is equal to the dose of about 400 mg/d given at clinic.The malondialdehyde(MDA) and the superoxide dismutase(SOD) in the RBC were tested.The level of MDA obviously decreased compared to the control group so did the SOD too.The lower MDA indicated the fewer free radicals in the RBC.The reduction of SOD could be explained by the formation of complex produced by peurarin and metal ions such as Mg²⁺ or Mn²⁺ from SOD.No allergic reaction and no immune hemolysis occurred.No antibody of globulin was found too.However,the shape of RBC changed after injecting PI.Echinocytes were examed by the microscope.Combining the results in vitro that peurarin could induce RBC of human,dogs or guinea pigs at a dose-dependent manner,the type of PI-caused hemolysis was defined as the non-immune one.Because of hemolysis induced directly by the PI in vitro and morphological change of RBC in vivo,the influence caused by PI on the RBC fragility,energy metabolism as well as calcium influx was analyzed.Puerarin had no effect on the RBC fragility in the hyposmosis saline solution or hot iso-osmia saline solution at the temperature of 50℃.Neither calcium ionic channel blocker Nifedipine nor Varapamil could inhibit the hemolysis induced by puerarin.Flow cytometer and fluorescent probe Fluo 3/AM were employed to detect the level of calcium ion(Ca²⁺) in cytoplasma.No elevation of Ca²⁺ in the puerarin group indicated that puerarin did not cause the influx of Ca²⁺,which was a very important contributor for the death of cells.ATP also plays an important role in keeping normal shape and function of RBC. ATP-depleted can cause the erythrocytes become crenated,which are called echinocytes. Puerarin had no effect on the Na⁺-K⁺-ATP activity and the hemolysis could not be attenuated by the addition of both of glucose and adenosine.Therefore the hypothesis that puerarin inducing hemolysis by accelerating APT-depletion was excluded.To our surprise,puerarin dissolved in the non-buffered saline solution(pH 7.0) could induce hemolysis and the hemolysis could be inhibited by Lidocaine,while puerarin in phosphate buffered saline(pH 7.4) had no destroy on RBC.For any hemolytic reaction is inevitably to involve the impairment of erythrocyte membrane.The change of membrane fluidity induced by puerarin was tested too.In non-buffered physiological saline solution(pH 7.0),puerarin dramatically improved the membrane fluidity compared to the control group.When the concentration was more than 1mmol/L,fluidity no longer increased along with the elevation of puerarin concentration. This result indicated that puerarin nonspecifically inserted into the lipidic part of the membrane and changed membrane fluidity.The higher fluidity might be due to the increasing membrane surface electric density and the repulsion of phospholipid molecules and then abrupted the on membrane organization.In PBS iso-osmia solution(pH 7.4),puerarin at the concentration ranging from 0.05～1mmol/L also increased membrane fluidity obviously. However,when the concentration became higher,the fluidity decreased quickly.This could result from the formation of hydrogen bonding between puerarin and phospholipid molecules, which was then packing the membrane bilayer and restraining its movement,and that may be the mechanism why puerarin can not induce hemolysis in PBS.What's more,the addition of Lidocaine could inhibit the increment of membrane fluidity induced by puerarin in the non-buffered saline solution(pH 7.0) and prevented hemolysis.For Lidocine is a cationic compound which interacts with erythrocyte membranes inducing internalization or stomatocytosis by their incorporation in the inner monolayer and performs the opposite effect to the one induced by neutral or ionic drugs such as puerarin,the effect on the membrane fluidity should be taken into consideration as a contributor to hemolysis induced by puerarin.To further investigate whether membrane protein was damaged by puerain,experiments were carried out In vitro,human erythrocytes were suspended in puerarin injection containing 2mmol/L puerarin and 0.8%PP at 37℃for 2 hours and then membrane protein was extracted by kits and separated by SDS-PAGE.Compared to the normal control groups,the amount of protein expression of band 2(spectrin),band 2.1(ankyrin),band 3,band 4.1,band 5(actin) decreased along with some other unknown bands increased in intensity after staining. Similar result in the PP control group(containing 0.8%PP) but change was much more moderate.Theses results confirmed the effect of pruerin at high concentration on the organization of RBC membrane protein especially the skeleton protein and the PP also had gentle effect too.In vivo,puerarin injection was given at the dose of 50 mg/kg intravenously for ten days.The PP control group was administrated with the same amount of PP.Both in PI or PP group,the change of band 2(spectrin),band 2.1(ankyrin),band 3,band 4.1 was very slight vs.the normal control group which was injected with the same volume of saline.The difference between the results of in vitro to the ones of In vivo might be explained by the quick elimination of puerarin in animals and shorter interaction with erythrocyte membrane. Thus it could be concluded that generally puerarin injected intravenously had slight effect on RBC in vivo.The causes for acute hemolysis induced by PI at the clinic were probably associated with the dysfunction of metabolism of liver and/or kidney in some old patients who were given high dose of puerarin for several days,which resulted into the accumulation of puerarin in the serum and longer disturbance on the RBC and occurrence of hemolysis.Taurine is a common amine acid in animals or human.It can stabilize the cell membrane. In the study,taurine inhibited the hemolysis induced by purarin and the change in conformation of RBC after the injection of puerarin.This inferred the disturbance on stability of RBC membrane induced by puerarin.In summary,this study revealed some mechanisms underlying the hemolysis caused by Puerarin Injection through experiments carried out in vitro and in vivo.The feature of the hemolysis seemed to belong to the non-immune one,which showed dose-dependent as well as temporal-dependent manners and had little relationship with oxidation reaction or allergic reaction.A large amount of puerarin attached to membrane and then induced the increment of membrane surface electric density and the repulsion of phospholipid molecules and disturbed membrane organization.Consequently,morphological change of RBC occurred and hemolysis was induced.Reduction on concentration/dose or addition of taurine could prevent against hemolysis.The present results may contribute to supply some interventions to avoiding hemolysis induced by Puerarin Injection.