The Role Of Cholic Acids Injurying The Cardio-vascular Tissues In The Development Of Fetal Complication In Intrahepatic Choliestasis Of Pregnancy

Intrahepatic Choliestasis of Pregnancy (ICP) is the specific complication in the metaphase and late pregnancy, clinic character are itch of skin and jaundice. The disease is very dangerous for fetal, and increase perinatal morbidity and mortality. The mechanisms of its episode have been unclosed, it may be related with the sex hormone, heredity, immunity and environments and so on.Toxic effect of Bile acids is the recent investigational focus, which could cause premature rupture of membranes, fetal distress in uterus, spontaneous premature birth, and meconium contamination. In addition, fetal growth restriction, unexpectedly fetal death, intracranial hemorrhages of newborn, sequela of neonatal nervous system and so on. Because it cause medical dispute, the unexpectedly fetal death has been becoming the important complication, which lead to huge economic lost to society and family.The levels of cholic acids (CA) are the main alteration in bile acids of the maternal serum. So the cholic acids may be the ringleader in fetal mechanisms in ICP. Our researches use the primary culture neonatal rat cardiac myocytes (CMs), study the survival rate of them on the effect of cholic acids by MTT. Furthermore, we study the cytosolic free calcium by laser scanning Confocal microscope (LSCM) and ca²⁺-sensitive fluorescence indicator fluo-3/AM, to find the effect of cholic acid in the fetal sudden cardiac death of ICP. We also study the human umbilical vein endothelial cells (HUVEC), whose proliferation rate, integrity and endocrinein function on the effect of cholic acids, to find how its injury on HUVEC to cause the maternal and fetal complication of ICP. In clinic, we also study the relations between the bile acid and alterations of endothelial cells cytokine and myocardium zymogram in the serum of umbilical vein, to make sure the injury of bile acids in ICP, which could evaluate the fetal condition and give the early intervation. These may be could improve the neonatal prognosis and decrease the perinatal morbidity and mortality.Part I The Studies on the mechanisms of Cellular Damage of Cholic Acids to the Cultured Neonatal Rat CardiacMyocytesObjective To investigate the mechanism of cellular damage of cholic acids on cultured neonatal rat cardiac myocytes (CMs).Methods 1. Using MTT method to detect the effect of cholic acids on the activities of neonatal rat cardiac myocytes.2. CMs cultured in special cell in vitro were incubated with 10 μmol/L ca²⁺-sensitive fluorescence indicator fluo-3/AM without light at 37℃ with 5% co₂ for 40min. Changes of the fluorescence signal of free calcium caused by cholic acids were measured under laser scanning Confocal microscope (LSCM).Results 1. Cholic acids could depress the activities of neonatal rat cardiac myocytes (CMs) in a time -dose-dependent manner.2. Cholic acids caused an increase in the concentration of Cytosolic free calcium in a dose-dependent manner.Conclusions Our research suggests that the cholic acids impair the activities of cardiac myocytes (CMs) by altering Cytosolic free calciuminconcentration.Part II The Studies On the Mechanism of Cellular Damage of Cholic Acids on HUVECObjective To investigates the mechanism of cellular damage of cholic acids onHUVEC.Methods 1. Using MTT method to detect the effect of cholic acids on the growth rate of HUVEC2. Using ELISA method to examine the excretion of VEGF and vWF of the HUVEC.Results 1. The growth rate in the 24h group are not obviously depressed; but the depression are dramatic both in 48h and 72h groups, which are negative correlation with concentration of cholic acids (48h: r=-0.848, P=0.033; 72h: r=-0.979, P=0.001); and the correlation between the consentration and the time in the groups, 1mmol/L groups are negative, respectively(2mmol/Lgroup:r=-0.944,P=0.005; 1mmol/Lgroup: r=-0.817, P=0.047). 2. The excretion of VEGF is decreased with the increasing concentrationof cholic acids; but the concentration of vWF is on the opposite. Conclusions Our research suggests that the cholic acids depress the growth rate of HUVEC with time-concentration dependence. Perhaps the inhibitions are related with direct injuring on HUVEC and indirect decreasing theexcretion of VEGF of them. Part III The study of the correlation between the total bileacid and VEGF vWF and the alteration of myocardiumzymogram in serum of human umbilical veinObjective To investigate the relation between the total bile acid and VEGF vWF and the alteration of myocardium zymogram in serum of human umbilical vein. To study the role of bile acid injuryimg the endothelium cell and the myocardial cell in the pathogenesis of fetal complication.Methods 1.Using blood serum biochemistry to detect the bile acid in serum of humanumbilical vein2.Using ELISA to detect the concentration VEGF and vWF in serum of human umbilical vein3. Using myocardium zymogram detect the alteration of CK-MB LDHResults 1 .Comparing with the normal contral team, the bile acid in ICP is increaseing, P<0.05. 2. Comparing with the normal contral team, the concentration of VEGF andvWF in ICP are increasing, P all<0.05.3.Comparing with the normal contral team, the myocardium zymogram of CK-MB and LDH in ICP are increasing, P all <0.05.4. The correlation between the concentration of bile acid and VEGF are negative correlation, vWF are positive correlation.5. The correlation between the concentration of bile acid and CK-MB LDH are positive correlation.Conclusions Our research suggests that the bile acid could injury the fetal endothelium cell and the myocardial cell. Maybe the bile acid paticipated the pathogenesis of fetal complication in ICP by effecting the struction and function of endothelium cell and the myocardial cell. SUMMARYTaken together, our results indicate that:1. Cholic acids could depress the activities of neonatal rat cardiac myocytes (CMs) in a time -dose-dependent manner;2. Cholic acids caused an increase in the concentration of Cytosolic free calcium in a dose-dependent manner;3. Cholic acids impair the activities of cardiac myocytes (CMs) not only by direct impavt but also by altering Cytosolic free calciumin concentration, which called "calcium overload";4. Cholic acids depress the growth rate of HUVEC with time-concentration dependence;5. The excretion of VEGF inHUVEC is negative correlation with the concentration of cholic acids; but the excretion of vWF is on the opposite;6. The inhibitions of cholic acids are related with direct injuring on HUVEC and indirect decreasing the excretion of VEGF of them;7. The correlation between the concentration of bile acid and VEGF, vWF are positive correlation;8. The correlation between the concentration of bile acid and CK-MB LDH are positive correlation.