Trial Research Of Danshensu's Effects On Improved Hemostatic Function And Pregnancy Outcome In Preeclampsia Mice

Preeclampsia(PE) is a serious pregnancy-specific syndrome of unknown etiology associated with vasospasms and pathologic vascular lesion in multiple organs as well as activation of the coagulation system,being one of the leading causes of maternal death and a major contributor of maternal and prenatal morbidity.There is a physiological hypercoagulable state during normal pregnancy,with the activity of "fibrinolysis system" increasing correspondingly and but maintaining dynamic balance of hemostatic functional. Preeclampsia occurs followed by vessel endothelium lesion,coagulation factor and platelet activation,and further tendency to disequilibrium of coagulation and anticoagulation.So far, none of established preeclampsia-like animal models can show overall feature of preeclampsia.Recently,Omatsu established a new preeclampsia-like model in mice.They injected phosphatidylserine/phosphatidylcholine(PS/PC) microvesicles to Institute of Cancer Research(ICR) mice,and induced preeclampsia-like changes,such as hypertension, proteinuria,FGR,diffuse fibrin depositions and microthrombosis in the labyrinth layer of placentas.But no obvious sign of fibrin depositions in the lung,kidney and liver,which indicate that preeclampsia-like changes can be induced by hypercoagulable state in the placenta.The model supports the theoretic evidences of hemostatic functional disorder as pathogenesis and treatment.It has been reported that treatment with the heparin, low-molecular-weight heparin(LMWH),or aspirin has made some desired benefits for partial patients,especially during the early stage of the disease or in severe an recurrent cases.But their clinical applications are difficult because of the controversy of antithrombotic therapy,the insufficient safety evaluation and no evidences to application opportunity and dose.Danshen Root,the dried root from Salvia miltiorrhiza Bunge of Labiate,is one herb clearly studied on the chemical composition.Danshen Injection,Danshensu as the major component,is safely applied to Obstetrics disorders.Danshensu's antithrombotic effects in vitro and in vivo have been confirmed.Recently,Danshensu's multiple pharmacologic actions have been studied and testified,such as protecting from myocardial ischemia, lowering hyperlipemia,inhibiting atherosclerosis,anti-inflammatory,strengthening immunity,protecting liver and anticancer.Traditional medicine considers preeclampsia as disease of asthenia in origin and asthenia in superficiality,and presumes blood stagnation to be pivotal pathogenesis.They provide the probabilities of using Danshensu as prevention and treatment for preeclampsia.In the first part of the article,we successfully duplicated the preeclampsia-like animal model with the features of diffuse fibrin depositions and microthrombogenesis priorly established by Omatsu.The ICR mice were mated at 1:1 female/male ratio in the evenings and presence of a coital plug was taken as an indication of probable pregnancy(day 0.5). PS/PC microvesicles suspension,which can provide catalytic surfaces to coagulation factor and accelerate the coagulation process,were injected into tail veins every day from days 5.5 to 16.5 of pregnancy.Blood pressure(by Non-invasive Blood Pressure controller instrument),urine protein(by 12-hours urine protein quality),platelet counts,plasma antithrombin activity(by immunoturbidimetry),D-D concentration(by immunoprecipitate sandwich method),thrombin time were tested,body weight,nose-breech length,ponderal index(PI) of fetal mice and placental weight were measured,placental fibrin depositions(by PTAH staining) and thrombomodulin(by IHC staining),pathologies of placentas,livers, kidneys and tails were detected on day 17.5.Mice in model group show features,similarly shown by human PE syndrome,as hypertension,proteinuria,FGR,fetal death,decreasing platelet counts and plasma ATⅢactivity,increasing plasma D-D levels,and diffuse fibrin depositions and microthrombogenesis in placentas,but no obvious changes in other organs such as liver and kidney.Reduced plasma ATⅢactivity,platelet counts and thrombin time suggested activation of platelet and coagulation system,while increasing plasma D-D levels indicated thrombogenesis in vessel and activation of secondary fibrolysis.All of these changes look like those occurred in human serious preeclampsia.Placental diffuse fibrin depositions and microthrombogenesis,accompanied by powerful positive expression on thrombomodulin indicate regional activation of coagulation and anticoagulation,but no obvious changes in other organs,which suggest augmented hypercoagulable state in placental circulation in early trimester of pregnancy may be one of pathogenesis and the important process of pathophysiology of preeclampsia and FGR.In the second part of the article,we investigated the availability of anticoagulant therapy by heparin and aspirin,and further compared the that of Danshensu on the condition of animal model established in first part of the article and excluding multiple factors impacts in corpore.Sixty-six pregnant mice were randomly divided into control group(15 animals),preeclampsia group(12 animals),heparin group(9 animals),aspirin group(10 animals),low-dose Danshenau group(10 animals) and high-dose Danshensu group(10 animals).PS/PC microvesicles suspension and saline as control were injected into tail veins every day from days 5.5 to 16.5 of pregnancy.In addition to the injections,1U of common heparin,20μg/g of Aspisol,10μg/g of Danshensu,or 30μg/g of Danshensu was respectively injected into mice simultaneously every day from days 6.5 to 16.5 of pregnancy.The results suggest heparin presents significant effects on maternal syndrome of preeclampsia such as hypertension and proteinuria,and different dose Danshensu also presents the certain effects.High-dose Danshensu and aspirin all process better effects than low-dose Danshensu on decreasing blood pressure to normal level,while high-dose Danshensu process better effects than aspirin and low-dose Danshensu on decreasing proteinuria to normal level.In conlusion,the effects of high-dose Danhsneuon improving maternal syndrome is superior to low-dose Danshensu,but inferior to heparin in all.As to Danshensu's effects on hemostatic function,high- and low-dose Danshensu's marked effects on increasing the plasma ATⅢactivity are same to aspirin and inferior to heparin. High-dose Danshensu's better effect on elevating the platelet counts is superior to low-dose Danshensu and aspirin.Low-dose Danshensu's obvious effect on decreasing D-D levels is close to heparin and superior to high-dose Danshensu and aspirin.High- and low-dose Danshensu's significant effects on reduced thrombin time to normal level are same to heparin.Different anticoagulant all have the improvement roles on placental fibrin depositions,but heparin and high-dose Danshensu's roles on lowering thrombomodulin expression in placentas are superior to low-dose Danshensu and aspirin.But anticoagulant function of high-dose Danshensu is still inferior to heparin.We found long-term use of heparin and aspirin,in spite of low dose administration,can raise the risk of bleeding such as placental abruption and intestinal hemorrhage,but no side effects ware observed in our study.In the second part of the article,we continued to investigate the effects of Danshensu on growth of fetal mice(fetal syndrome).Mice in every group mentioned above were measured on the indices as maternal body weight increasing,body weight,nose-breech length and PI of fetal mice,placental weight,as well as weight of fetal brains and their pathological staining of HE and Nissle's.The results showed reduced maternal body weight, lower pregnancy rate,higher rate of fetal death and absorbed fetal,decreasing placental weight,as well as body weight and nose-breech length of fetal mice,but invariably fetal PI in PE group.Lost fetal brain weight,with reduced cerebral nerves and Nissle's body,was also observed.Features mentioned above showed off symmetric FGR and cerebral dysgenesis.The research in this part further observed and compared the curative effects between heparin,aspirin and different dose Danshensu on FGR,and found following changes:increasing fetal body weight and length in every group,obvious overall improvement in heparin group,greater amelioration equaling to that in heparin group on maternal body weight,fetal nose-breech length and fetal brain weight in high-dose Danshensu group,better changes on survival fetal number in low-dose group than in other groups,and more corrected brain development in high-dose Danshensu group than in aspirin group.We presume the animal model can completely mimic the syndrome of preeclampsia, especially serious preeclampsia,manifesting as either maternal syndrome or fetal syndrome. The model not only corresponds to the hypothesis on hemostatic functional disorder as the pathogenesis of preeclampsia,but also provides the brand-new way to studying clinical diagnosis and treatment.High- and low-dose Danshensu can significantly improve maternal syndrome(hypertension,proteinuria and hemostatic function disorder) in PE mice model, but inferior to heparin.The roles of high-dose Danshensu are superior to low-dose Danshensu and aspirin.Simultaneously,high- and low-dose Danshensu can also obviously improve maternal syndrome(FGR,fetal death and absorbed fetal),and the roles of high-dose Danshensu are equal to those of heparin and but superior to those of low-dose Danshensu,with no similar roles in aspirin.Danshensu process the predominance on better curative effect of cerebral dysgenesis,as well as safety and effectivity in clinical use. Although the trial results is limited because of the short-term administration and small sample size,the experimental exploration provide the theoretic foundation for Danshensu applying to treatment of preeclampsia.