| Preeclampsia is one of the leading causes of maternal and fetal mortality and morbidity. The etiology of this disease is still not clear, although the mechanisms of inadequate trophoblast invasion leading to incomplete remodeling of the uterine spiral arteries is wildly accepted and is considered to be a primary cause of preeclampsia. There is evidence that suggested immune disturbance and apoptosis are involved in the etiology of preeclampsia. Low molecular weight heparin is wildly used as a safe and effective anticoagulant in clinical treatment. Recently, accumulated data show that LMWHs have anti-inflammatory and anti-apoptotic effects. LMWH is now used extensively in the treatment of obstetric diseases caused by thrombophilla, such as recurrent abortion. In China, clinical workers tried to use LMWH on the treatment of preeclampsia and found that LMWH could obviously improve the prognosis of this disease. The mechanism is unclear. Nevertheless, rescent studies have suggested that LMWH is efficacious in patients suffering from obstetric disorders with or without thrombophilla. These findings suggested that LMWH may exert other effects independently of its anticoagulant activity. The purpose of this study is trying to test the effect of LMWH on the inflammation and cell apoptosis in a preeclampsia-like rat modle. PART 1 ESTALISHMENT OF PREECLAMPSIA-LIKE RAT MODEL AND EVALUATION OF THE THERAPEUTIC EFFECTS OF LMWH OBJECTIVETo establish a preeclampsia-like rat model by using NOS inhibitor—nitro-L-Arg-methyl ester(L-NAME) and investigate the effects of LMWH on various fetal and physiological parameters, including blood pressure, proteinuria, number of pups, weight of pups and placental.METHODS1. Establishment of preeclampsia-like rat model:L-NAME(200mg/kg, subcutaneous injection) was used to induce preeclampsia-like symptoms from day 13 to 16 of gestation. The blood pressure was measured on day 15. Those rats whose blood pressure was 30mmHg higher on day 15 than that of before pregnant were considered as successful preeclampsia-like models.2. Experimental protocol:Forty Wistar rats were assigned to four groups randomly(n=10 for each group). Normal non pregnant group:physiological saline was given subcutaneously from day 13 to 20 of the experiment; Normal pregnant group:physiological saline was injected subcutaneously from day 13 to 20 of gestation; PIH group:L-NAME(200mg/kg.d, subcutaneous injection) was used from day 13 to 16 of gestation and physiological saline was given from day 17 to 20; LMWH group:L-NAME(200mg/kg.d) was injected subcutaneously from day 13 to 16 of gestation and LMWH was given from day 17 to 20.3. Physiological parameters:Blood pressure was measured before pregnant and on the 15th and 21th day non-invasively. Urine was collected on day 1 and 20 for analysis of protein. Blood was taken on day 21 for blood urea nitrogen(BUN), creatinine(Cr) and ALT test.4. Evaluate of the fetal outcome:Pups and placental were taken out on day 21. The number of normal pups was conducted and the weights of fetus and placental were measured.RESULTS1. Blood pressure measurements:There were no significant differences(P>0.05) between the groups before pregnant. On day 15 blood pressure of PIH and LMWH group was markedly higher than that of control groups(P<0.05). The blood pressure of LMWH was lower significantly than that of PIH group on day 21, but still higher than that of controls(P<0.05). There were also significant differences in different day within each group, i.e. blood pressure was much higher on day 15 and 21 than that of before pregnant(P<0.05) but no difference between day 15 and 21(P>0.05) in PIH group; in LMWH group blood pressure was increased markedly on day 15 compared with that of before pregnant(P<0.05), but decreased significantly on day 21 compared with day 15(P<0.05).2. Urinary protein:There was no statistical significance among all groups on day 1. The PIH and LMWH group both showed an prominent increase in urinary protein from day 1 to day 20(P<0.05). Urinary protein of PIH group was significantly higher than control groups, and that of LMWH was lower markedly than PIH group on day 20(P<0.05).3. Function of kidney and liver:The levels of BUN and Cr of PIH and LMWH group were significant higher than that of tow control groups(P<0.05), and there was significant difference between LMWH and PIH group(P<0.05). There was no difference on levels of ALT between PIH vs. normal control group, PIH vs. LMWH groups(P>0.05).4. Fetal outcomes:The number and weight of normal pups in PIH group were significantly lower than those of normal pregnant control group(P<0.05), and those of LMWH were increased markedly compared with PIH group(P<0.05) but still lower than normal pregnant control group (P<0.05). The weight of placental was significantly lower in PIH and LMWH groups than normal pregnant control group(P<0.05). There was no difference between PIH and LMWH group on placental weight(P>0.05).CONCLUSIONA successful preeclampsia-like pregnant rat model could be established by using L-NAME 200mg/kg.d injected subcutaneously from day 13 to 16 of gestation. LMWH could improve the preeclampsia-like symptoms and fetal outcomes effectively.PART 2 THE ANTI-INFLAMMATORY EFFECTS OF LMWH IN PREECLAMPSIA-LIKE PREGNANT RAT MODEL OBJECTIVEIn this part of study we aimed to test the effect of LMWH on the inflammation in the preeclampsia-like pregnant model in rat through detecting the concentration of cytokines in blood and tissues.METHODS1. Experimental protocol:Forty Wistar rats were assigned to four groups randomly(n=10 for each group). Normal no pregnant control group:physiological saline was given subcutaneously from day 13 to 20 of the experiment; Normal pregnant control group:physiological saline was injected subcutaneously from day 13 to 20 of gestation; PIH group:L-NAME(200mg/kg.d, subcutaneous injection) was used from day 13 to 16 of gestation and physiological saline was given from day 17 to 20; LMWH group:L-NAME(200mg/kg.d) was injected subcutaneously from day 13 to 16 of gestation and LMWH was given from day 17 to 20.2. Sample collection:On day 21, each animal was anaesthetized and blood samples were obtained by femoral artery puncture. The placental, kidney and spleen were rapidly removed, sectioned and either snaped frozen in liquid nitrogen or suspended in formalin for further investigations.3. Test of cytokines:Concentration of IL-2, IL-4, IL-10 and INF-γin blood were measured by Elisa, and the expression of these cytokines in tissues were tested through immunohistochemistry, RT-PCR and Western blot.RESULTS1.Cytokines in blood:The concentration of IL-2 and INF-γin blood in normal pregnant control group were decreased significantly compared with normal non pregnant control group, but IL-4 and IL-10 were increased markedly(P<0.05). In PIH group, the concentration of IL-2 and INF-γwere much higher than normal pregnant control group and LMWH group(P<0.05), however, that of IL-10 and IL-4 were lower than normal pregnant control group (P<0.05). There was significant difference between PIH and LMWH group on the concentration of IL-10 (P<0.05), but no statistically significant on that of IL-4 (P>0.05).2.Cytokines in tissues:The results of immunohistochemistry, RT-PCR and Western blot test of tissues showed various differences among four groups. IL-2 and INF-γin kidney and spleen were lower in normal pregnant control group than normal non pregnant control group(P<0.05), and IL-4 and IL-10 were much higher(P<0.05). In PIH group, IL-2 and INF-γin placenta kidney and spleen were increased significantly compared with normal pregnant control group, but IL-4 and IL-10 were decreased markedly(P<0.05). Compared with PIH group, IL-2 and INF-γin these three kinds of tissues in LMWH group were decreased significantly and IL-10 was increased(P<0.05), but there was no difference in IL-4 expression(P>0.05).CONCLUSIONIt showed increases in proinflammatory cytokines and decreases in anti-inflammatory cytokines in preeclampsia-like pregnant rat model, which was similar to human beings. LMWH may effectively affect the preeclampsia-like animal model by suppressing proinflammatory cytokines and stimulating anti-inflammatory cytokines.PART 3 THE ANTI-APOPTOTIC EFFECT OF LMWH IN PREECLAMPSIA-LIKE PREGNANT RAT MODELOBJECTIVEAberrant and excessive cell apoptosis ia a common pathological process associated with preeclampsia. There were numerous experiments showed that LMWH has the effect of anti-apoptosis. So we aimed to investigate the anti-apoptotic effect of LMWH in preeclampsia-like model in this part.METHODS1. Experimental protocol:Forty Wistar rats were assigned to four groups randomly(n=10 for each group):normal non pregnant control group, normal pregnant control group, PIH group and LMWH group. The methods were same as part 2. 2. Sample collection:On day 21, each animal was anaesthetized. The placenta and kidney were rapidly removed, sectioned and either snap frozen in liquid nitrogen or suspended in formalin for further investigations.3. Investigation of cell apoptosis:The apoptotic index was tested by TUNEL. Expression of cleaved Caspase-3, Bcl-2 and Bax in placenta and kidney were examined by Western blot.RESULTS1. The apoptosis index was increased significantly in PIH group compared with control groups(P<0.05), and decreased markedly in LMWH compared with PIH groups(P<0.05). There was no significant difference between LMWH and normal pregnant control group(P>0.05).2. There were high levels of cleaved Caspase-3 and Bax and low levels of Bcl-2 in PIH group compared with control groups(P<0.05). In LMWH group, the expression of cleaved Caspase-3 and Bax were decreased significantly compared with PIH group, and Bcl-2 increased(P<0.05).CONCLUSIONIt showed an excessive cell apoptosis in placenta and kidney of preeclampsia-like rat model and LMWH could decrease the apoptosis markedly. Enhanced expression of Bcl-2 and suppressed of Bax may be involved in the anti-apoptotic effect of LMWH. |
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