| Preeclampsia is a syndrome characterized by elevatedmaternal blood pressure and proteinuria, and is the principal cause of maternal morbidity and mortality in developing countries. Pathophysiology of preeclampsia is still unclear and numerous theories have been proposed. Among these theories, increasingly importance has been attached to placenta vessel endothelium injury which was considered as the crucial step of the preeclampsia pathophysioloy. Several studies have reported that vasoactive substance NO and lipid metaholism were closely associateed with vessel endothelium injury.Apolipoprotein E (ApoE) is the most important gene related to abnormal metabolism, and iNOS is related to the secretion of NO.So ApoE knockout mice and iNOS knockout mice and ApoE/iNOS double knockout mice were chosen as research object to study whether the iNOS and apoE have the relationship with preeclampsia Firstly, we try to establish animal models with ApoE knockout mice,. iNOS knockout mice and ApoE/iNOS double knockout mice. Pathological changes were observed by HE staining. Ultrastructural of placenta of these mice were observed by electron microscopic. And we studied the expression levels of inflammatory related proteins (IL-6, TNF-α, NF-κB) and apoptosis related proteins(Bax, bcl-2, Caspase-3) in placenta with Western Blot.Try to find out the relationships between ApoE,iNOS and preeclampsia and the pathophysiology of the disease.SECTION1:The Relationship between ApoE and PreeclampsiaPart1:The Preeclampsia-like Syndrome in ApoE Knockout MiceObjective:To observe whether there were the syndromes of increasing of BP, urine protein and changes of the weight of placentas and fetuses in ApoE knockout mice. Method:Animals:ApoE-/-and WT mice were provided by Model Animal Research Center MARC of Nanjing Uniersity.ApoE+/-mice were created by backcrossing ApoE-/-mice with WT mice. The pregnanct mice were divided into three groups according to the genotype of pregnant mice:the ApoE-/-mice, ApoE+/-mice, and WT mice. Genotype were identificated by PCR Technology before the experiments. The blood samples and placentas were taken at19th day of pregnancy. The serum lipidsTG, TC, HDL, LDL were measured. After the mating of the female and male mice of the same genetype, the systolic blood pressures (begin from the Oth day of pregnancy) and urine protein (begin from the4th day of pregnancy) of the pregnant mice were measured every4days. The weight of placenta and fetuses were measured during the19th day of pregnancy.Results:①The serum levels of TC, TG, LDL of apoE-/-mice increased in various degrees compared to WT (P<0.05or0.01), but level of HDL was lower than that of WT mice (P＜0.05). BP (12th day129.3±15.7mmHg,16th day121.7±5.8mmHG) and the level of urine protein (55.3±107.2mg/L) of the ApoE-/-mice were obvious higher than those of WT (P<0.05). The weight of the placentas (110±7.2mg) and fetuses (1004±130mg) of ApoE-/-mice were lower than WT mice.②The serum level of TG in ApoE+/-mice increased than that of WT (P<0.05). But the levels of LDL, HDL and TC were the same with WT mice.There were no differences in BP, urine protein, weight of the placentas and fetuses between ApoE+/-and WT mice (P>0.05).Conclusions:Preeclampsia-like symptoms were seen in ApoE-/-mice. It showed that ApoE may play an important role in pathogenesis of preeclampsiaPart2:The Changes of Expression of Inflammatory Reaction and Apoptosis in placentas of ApoE Knockout MiceObjective:To observed the inflammatory reaction and apoptosis in the placentas and to seek the possible pathogenesis of preeclampsia and the relationship between ApoE and preeclampsiaMethod:Pathological changes were observed by HE staining. Ultrastructural of placenta of these mice were observed by electron microscopic. The expression of TNF-α, IL-6, NF-κB, Bax, Bcl-2and Caspase-3proteins in placentas of ApoE-/-mice, ApoE+/-mice and WT mice were assessed by Western Blot. Results:①Placental villus mediate cells edema was found in ApoE-/-mice by HE staining. Electron microscopic ultrastructural findings showed that there were mitochondria swelling and RER expanded in the placentas of ApoE-/-mice and that of WT mice. The results of Western Blot showed that the expression level of inflammatory related proteins (IL-6:0.917±0.153, TNF-α:0.453±0.020, NF-κB:1.602±0.234) and apoptosis related proteins (Bax:0.789±0.149, bcl-2:0.977±0.349, Caspase-3:2.132±0.173) and the ratio of Bax/bcl-2(0.897±0.301) in placentas of ApoE-/-mice were much higher than those of WT mice(P<0.05).②HE staining and electron microscopic ultrastructural findings showed that there was no morphological change in Apoe+/-placentas. Similar results were obtained in the expression of Bax, caspase-3, IL-6, Bcl-2and NF-κB in apoE+/-and WT (P>0.05), but TNF-α increased apparently (P＜0.05).ConclusionsrPathogenesis of preeclampsia may be caused by the inflammatory reaction and apoptosis in the placentaSECTION2:The Relationship between iNOS and PreeclampsiaObjective:To seek the possible pathogenesis of preeclampsia and the relationship between iNOS and preeclampsia.Method:Animals:iNOS-/-and WT mice were provided by Model Animal Research Center MARC of Nanjing Uniersity. iNOS+/-mice were created by backcrossing iNOS-/-mice with WT mice. The pregnant mice were divided into three groups according to the genotype:the iNOS-/-mice, iNOS+/-mice and WT mice. Genotype was identified by PCR Technology. After the mating of female and male mice of the same genetype, the systolic blood pressures (begin from the Oth day of pregnancy) and urine protein (begin from the4th day of pregnancy) of the pregnant mice were measured every4days. The levels of NO in serum and placenta were measured at the last day of pregnancy. Pathological changes were observed by HE staining. Ultrastructural of placenta of these mice were observed by electron microscopic. The expression of TNF-α, IL-6, NF-κB, Bax, Bcl-2and Caspase-3in placenta of iNOS-/-mice, iNOS+/-mice and WT mice were assessed by Western Blot. iNOS-/-mice, iNOS+/-mice and WT mice were assessed by Western Blot. Results:①There were no difference in BP, urine protein, weight of placentas and fetuses between iNOS-/-and WT mice (P>0.05). No differences were detected between iNOS-/-and WT mice in the levels of NO both in serum and placenta (P>0.05). HE staining and electron microscopic ultrastructural findings showed proliferation of vessel in placenta of iNOS-/-. Compared to WT, the expression of NF-κBand Bax increased (P<0.05) but no s’gnificant changes in Bcl-2, caspase-3, IL-6, TNF-α expression were observed in iNOS-/-.(P＜0.05).②No differences in BP, urineprotein, weight of placentas and fetuses were detected between i NOS+/-and WT mice (P>0.05). Nor were levels of NO in serum or placenta (P>0.05). HE staining and electron microscopic ultrastructural findings showed that there were no morphological changes in Apoe+/-placentas. The expression of Bcl-2in the iNOS+/-mice were higher than WT mice (P＜0.05), but the levels of TNF-α, IL-6, NF-κB, Bax and caspase-3remaned unchanged (P<0.05).Conclusions:Pre-eclampsia-like symptoms were not seen in iNOS-/-or iNOS+/-mice. There seemed no direct relationship between i NOS gene and preeclampsa.SECTION3:Interaction between iNOS and ApoE and the Relationship with PreeclampsiaObjective:To explore the interaction between iNOS and ApoE and the joint effect on preeclampsia Try to find the possible pathogenesis of preeclampsia.Method:Animals:iNOS-/-mice were backcrossed with ApoE-/-mice to get iNOS and ApoE double knockout mice. The pregnant mice were divided into three groups according to the genotype:the ApoE-/-mice, iNOS-/-mice, ApoE-/-iNOS-/-and WT mice. Genotype by PCR technology was identificated before the experiment. Blood samples and placentas were taken at19th day of pregnancy, and levels of TG, TC, LDL and HDL were also measured.After mating of female and male mice of the same genotype, the systolic blood pressures (start from the Oth day of pregnancy) and urine protein (start from the4th day of pregnancy) of the pregnant mice were measured every4days. The weight of placenta, fetuses, NO in serum and NO level of placenta were measured at end of pregnant day. Pathological changes were observed by HE staining. Ultrastructural of placenta of these mice were observed by electron microscopic. The expression of TNF-α, IL-6, NF-κB, Bax, Bcl-2and Caspase-3i n placenta of ApoE-/-iNOS-/-mice were assessed by Western Blot.Results:①The double knockout mice ApoE-/-iNOS-/-were successfully established. Symptoms of preeclampsia were observed in ApoE-/-NOS-/-mice. The serum lipid levels of ApoE-/-iNOS-/-were higher than apoE-/-mice and WT mice(V.S WT P<0.001;VS ApoE-/-,P<0.05).BP (increasing from4th day:117.9±13.4mmHg) and levels of urine protein (12th day432.7±63.56mg/L,16th day06.3±73.18mg/L) in ApoE-/-iNOS-/-mice were obviously higher than those of WT (P<0.05or P<0.01). Besides,BP and levels of urine protein in APOE-/-iNOS-/-were higher than those in apoE-/-(P<0.05). The weight of placentas and fetuses of ApoE-/-iNOS-/-decreased sharply (V.S WT P<0.001;VS ApoE-/-,P<0.05). So was the level of NO in serum and placenta (V.S WT P<0.001;VSApoE-/-,P<0.05).②The apoptosis of placenta cells and injury of vessel wall in the placentas of ApoE-/-iNOS-/-observed by HE staining under optical microscopy. Electron microscopic ultrastructural findings showed that apoptosi s of placental trophoblasticeells and vascul ar endothelial cells damagesin the placentas of ApoE-/-iNOS-/-and that of WT mice. The results of western blot showed that the expression level of inflammatory related proteins (IL-6:0.744±0.101, TNF-α:0.683±0.102, NF-κB:2.376±0.259) and apoptosis related proteins (Bax:1.692±0.214, bcl-2:0.405±0.099, Caspase-3:2.438±0.520) and the ratio of Bax/bcl-2(4.409±1.580) in placentas of ApoE-/-iNOS-/-were much higher than those of WT mice (P<0.05orP<0.01). In addition, the expression levels of TNF-α,NF-κB, Bax and ratio of Bax/bcl-2was higher than that of ApoE-/-(P<0.05).Conclusions:Aggravated Preeclampsia-like symptoms were seen in the ApoE-/-iNOS-/-mice. iNOS may have protective effect on the progression of preeclampsia. |
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