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Expression of LncRNA-DPT and its correlation with the pathogenesis of intrahepatic cholestasis of pregnancyOBJECTIVE:To investigate the expression of LncRNA-DPT and inducible nitric oxide synthase(iNOS)in patients with intrahepatic cholestasis of pregnancy(ICP).To analysis of LncRNA-DPT induces apoptosis and down-regulates the expression of iNOS and participates in the pathogenesis of ICP.METHOD:1.There were 80 normal pregnant women and 77 cases of intrahepatic cholestasis of pregnancy randomly included in the two groups,to analysis the general clinical data and biochemical indicators of the subjects;2.Reverse transcription real-time PCR(RT-qPCR)was used to detect the expression level of LncRNA-DPT in the maternal-fetal interface of the two groups;3.RT-qPCR was used to detect the expression level of iNOS in the placenta and umbilical tissue cells of the two groups;4.Immunohistochemistry(IH)was used to detect the expression of iNOS in the placenta of the two groups.The expression of iNOS in the maternal and umbilical cord plasma was detected by enzyme-linked immunosorbent assay(ELISA);5.Western blot(WB)was used to detect the expression of iNOS in placenta and umbilical tissue cells;6.The apoptosis of placenta and umbilical tissue cells were detected by terminal deoxynucleotidyl transferase mediated nick end labeling(Tunnel)and mitochondrial reactive oxygen species(ROS)detection.RESULTS:1.There were no significant difference in the gestational age,BMI,gravidity and parity between the two groups(p>0.05).The gestational weeks,plasma concentration of iNOS,neonatal birth weight,placental weight and neonatal APGAR scores in the ICP group were smaller than the control group(p<0.05).There were significant difference in biochemical parameters(TBA,CG,ALT,AST,TB,CB,UCB,TC,TG),cesarean section rate,the rate of fetal distress,premature birth rate,the rate of fetal growth restriction,and NICU rate of neonates,the ICP group were higher than the control group(p<0.05).2.The expression of LncRNA-DPT in placenta of ICP patients was significantly lower than that of normal placenta by RT-q PCR(p<0.05).3.There was a negative correlation between iNOS and TBA expression(r=-0.450,p<0.05);iNOS was negatively correlated with CG expression(r=-0.367,p<0.05);iNOS was negatively correlated with ALT expression(r=-0.359,p<0.05);iNOS was negatively correlated with AST expression(r=-0.329,p<0.05);4.iNOS was mainly expressed in placental syncytiotrophoblasts,and its expression in placenta was significantly decreased in ICP patients(p<0.05).iNOS was mainly expressed in umbilical cord endothelial cells,and its expression was lower in umbilical cord tissue of ICP than normal pregnancy,the difference was statistically significant<0.05);5.The expression of iNOS was significantly decreased in maternal plasm of ICP patients than that of normal pregnancies(p<0.05);The expression of iNOS was significantly decreased in umbilical plasm of ICP patients than that of normal pregnancies(p<0.05);6.The fluorescence intensity of mitochondria in ICP group was significantly higher than that in the control group(p<0.05);7.Fluorescence microscopy was used to observe fluorescein-labeled placental apoptotic cells.The apoptosis rate of placental cells in ICP group was significantly higher than that of the control group(p<0.05).The apoptosis rate of placental cells in ICP group is significantly higher than the control group,the difference was statistically significant(p<0.05).CONCLUSIONS:1.Abnormal expression of LncRNA-DPT and iNOS in ICP patients may be related to the pathogenesis of ICP.2.Our study showed that LncRNA-DPT may reduce the expression of iNOS,reduce the synthesis of nitric oxide(NO),simultaneously activate tissue cell apoptosis,and participate in the pathogenesis of ICP.Part ? The molecular mechanism of LncRNA-DPT up-regulates NF-?B involved in intrahepatic cholestasis of pregnancyOBJECTIVE:To construct a rat model of ICP and detect the expression of LncRNA-DPT and NF-?B signaling pathway-related proteins in ICP rats.To investigate the effects of LncRNA-DPT on NF-?B signaling pathway,iNOS expression and tissue apoptosis.METHOD:1.The rats model of ICP was constructed by using estradiol benzoate,and the general data of the two groups were analyzed.2.Reverse transcription real-time PCR(RT-qPCR)was used to detect the expression of LncRNA-DPT in the maternal-fetal interface of the two groups.3.RT-qPCR was used to detect the expression of iNOS in the placenta and umbilical tissue of the two groups.4.The pathological sections of HE in ICP rats were observed under microscope and the ICP rat model was successfully constructed.The expression of iNOS in placenta and umbilical cord were detected by immunohistochemistry(IH).Western blotting was used to detect the expression of iNOS in placental tissues.The expression of iNOS in pregnant rats plasma was detected by enzyme-linked immunosorbent assay(ELISA).5.The apoptosis in placenta and umbilical tissue of the two groups were detected by mitochondrial ros detection.6.Regulate the expression of LncRNA-DPT in human cell HTR-8/SVneo,draw the cell growth curve to observe the changes of cell proliferation ability,and detect its effect on p65 protein,iNOS expression and apoptosis.RESULTS:1.Both the two groups of rats were first pregnant in the study.Before ICP model was established,there were no significant differences in age,weight,TBA,CG,ALT and AST between the two groups(p>0.05).After ICP model was successfully constructed,the TBA,CG,ALT and AST of rats in ICP group were significantly higher than those in control group(p<0.05).2.RT-q PCR showed that the expression of LncRNA-DPT in placenta of ICP rats was significantly lower than that of normal rats,the difference was statistically significant(p<0.05).3.Hepatic intracellular cholestasis was observed in HE sections of ICP rat liver.iNOS was mainly expressed in placental syncytiotrophoblast cells,and its expression in placenta of ICP rats was significantly reduced(p<0.05).iNOS was mainly expressed in umbilical cord endothelial cells,and its expression in umbilical tissue of ICP rat was significantly reduced(p<0.05).4.The expression of iNOS was significantly decreased in plasm of ICP rats(p<0.05);5.The expression of mitochondria ROS in ICP group was significantly higher than that of control group,the difference was statistically significant(p<0.05).6.After transfection,the expression of LncRNA-DPT in si-DPT group was significantly lower than that in control group,the difference was statistically significant(p<0.05).7.According to the cell growth curve drawn by MTT method,the proliferation ability of cells transfected by si-DPT was significantly weakened,and the difference was statistically significant(p<0.05).8.The apoptotic rate of HTR-8/Svneo cells was detected by flow cytometry,compared with the control group,the apoptotic rate of cells in the si-DPT group increased significantly(p<0.05).9.The expression of p65 protein in the cytoplasm of was significantly higher than that of the nucleus in the control group(p<0.05);the expression of p65 protein in the cytoplasm of was significantly lower than that of the nucleus in the si-DPT group(p<0.05).CONCLUSIONS:1.The ICP pregnant rat model was successfully constructed by estradiol benzoate,which can be used for ICP research.2.The NF-?B signaling pathway can be activated by downregulation of LncRNA-DPT,the expression of iNOS and nitric oxide(NO)was reduced,the apoptosis of tissues was activated,which participate in the pathogenesis and progression of ICP. |
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