Correlation Between BKca Channels And Preeclampsia In Vascular Smooth Muscle Of Umbilical Artery

Objective:Preeclampsia?PE?is a unique multi-system disease in pregnancy,seriously endangering the health of pregnant women and perinatal infant.The spasm of systemic small vessel is the basic pathological changes[1].Large conductance calcium activated potassium channels?BKca?are the most important ion channels in the regulation of vasomotor function.In this study,To investigate the expression and function changes of BKca in vascular smooth muscle of umbilical artery,and to explore the relationship between preeclampsia and BKca in vascular smooth muscle of umbilical artery,immunohistochemistry,real-time quantitative PCR?QPCR?and Western blotting were performed to detect the positive rate of BKca channel and BKca channel' expression level of mRNA and protein in vascular smooth muscle of umbilical artery.On the basis of this,we further explored the relationship between the fetal growth restriction?FGR?and the changes of BKca in vascular smooth muscle of umbilical artery.This study is to explore the pathogenesis of preeclampsia,and find a potential target to prevent and treat preeclampsia and severe preeclampsia with FGR.Method:?1?HE immunohistochemistry: we selected umbilical arteries form 30 normal pregnant women and 30 women with preeclampsia and divided into normal pregnant group?NP group?and preeclampsia group?PE group?.Part of the umbilical artery was fixed with 10% formaldehyde and embedded with paraffin.At the same time,the rest of the umbilical arteries were divided into different cryopreserved tubes for real-time quantitative PCR and western blot.After that,we followed the steps of HE staining and immunohistochemistry andcompare the expression of BKca in NP group and PE group.?2?Real-time fluorescence quantitative PCR?QPCR?: Total RNA was extracted from vascular smooth muscle of umbilical artery,RNA concentration was measured,cDNA was reverse transcribed into cDNA,and corresponding ?Ct value was obtained by QPCR reaction.Then,we converted the ?Ct value to ??Ct value as the formula that ??Ct value = PE group ?Ct-NP group ?Ct,and we performed ttest analysis after 2-??Ct value was gotten.At last,the differences of BKca ?mRNA and BKca ?₁ mRNA levels between NP group and PE group were compared.?3?Western blotting: At first,we grind umbilical artery tissue to powder with liquid nitrogen,and add 1ml protein lysate to extract sample protein.Then,after electrophoresis,sample protein was transferred to the PVDF membrane,and was incubated with antibody.At last,the result was measured by imaging system and Quantity-One software.The difference of the expression level of BKca ? subunit protein and BKca ?₁ subunit protein in vascular smooth muscle of umbilical artery between NP group and PE group was compared by SPSS17.0 software.The same comparison was performed among normal pregnancy group,preeclampsia group and chronic hypertension combined with preeclampsia group.And the comparison was performed between severe preeclampsia combined with fetal growth restriction group and no fetal growth restriction group as well.The results were expressed as mean difference?x?±s?,in which P <0.05 and P <0.01 were statistically significant.Result:?1??1?HE staining: the selected umbilical artery fitted for the test requirements.Under the microscopy,the umbilical artery vascular smooth muscle cells were with long spindle,slightly thick muscle layer,slightly thick cytoplasm,and single-core nucleus in the centre of the cells,which werestained to blue.Umbilical artery in the preeclampsia disease state might appear vascular smooth muscle disorder,but morphological structure did not change,and no acute atherosclerosis appeared.?2?Immunohistochemistry: we Use qualitative and semi-quantitative analysis.BKca ? subunit and ?₁ subunit were expressed in the vascular smooth muscle of umbilical artery of both NP group and PE group.BKca ? subunit and ?₁ subunit mainly expressed on the cell membrane of the vascular smooth muscle of umbilical artery,but part of them expressed in the cytoplasm.The positive rate of BKca ? subunit had no significant difference in vascular smooth muscle of umbilical artery between NP group and PE group,but the expression of BKca ?₁ subunit in PE group was significantly higher than that in NP group?P<0.05?.?2?QPCR results: The data were analyzed by relative quantitative analysis,which was called 2-??Ctmethod.There was no significant difference in BKca ? subunit mRNA level between the two groups?P=0.808>0.05?.Compared with NP group,the mRNA level of BKca ?₁ subunit in vascular smooth muscle of umbilical artery of PE group was significantly up-regulated,and the difference was statistically significant?P=0.015<0.05?.?3?The results of western blot:?1?The expression level of BKca? subunit in NP group and PE group was not statistically different?P=1.000>0.05?;but compared with NP group,the expression of BKca ?₁subunit in PE group was up-regulated,and the difference was statistically significant?P < 0.01?.?2?Normal pregnancy group,preeclampsia group and chronic hypertension combined with preeclampsia group: compared with the normal pregnancy group,the expression of BKca ? subunit was not statistically different in the three groups?P>0.05?.Compared with normal pregnancy group,the expression of BKca ?₁ subunit was significantly up-regulated inpreeclampsia group,but was significantly down-regulated in chronic hypertension combined with preeclampsia group;moreover,compared with the preeclampsia group,the expression level of BKca ?₁ subunit protein in the patients with chronic hypertension combined with preeclampsia group was significantly down-regulated,and the differences were statistically significant?P1<0.05,P2<0.05,P3<0.01?.?3?Comparison of severe preeclampsia with fetal growth restriction group and non-fetal growth restriction group: There was no significant difference in the expression of BKca ? subunit between the two groups?P>0.05?.The expression level of BKca ?₁ subunit was down-regulated in severe preeclampsia combined with fetal growth restriction,and the difference was statistically significant?*P<0.05?.Conclusion:?1?When umbilical artery is in preeclampsia disease state,there will be a disordered arrangement of vascular smooth muscle without morphological changes and atherosclerosis.?2?Compared with NP group,the expression of BKca ? subunit in umbilical artery smooth muscle was not significantly different?P>0.05?,but the expression of BKca ?₁ subunit was significantly increased?P<0.05?,which suggests that the changes of BKca ?₁ subunit may be related to the development of PE,but BKca ? subunit may not participate in the development of PE.?3?The development of PE caused the change of BKca ?₁ subunit,and the change of ?₁ subunit may be involved in the development of PE.?4?The expression level of BKca ?₁ subunit was down-regulated in severe preeclampsia and fetal growth-restricted vascular smooth muscle of umbilical artery,suggesting that fetal growth restriction may be related to the change of BKca ?₁ subunit.