| Objective:To investigate the effects of epirapril on the changes of voltage-dependent kvl.3 channel currents in CD4+ T lymphocytes and the expression of mRNA and protein in kvl.3 channels in patients with chronic heart failure,and to provide an immunological mechanism for the treatment of heart failure with epril.METHOD:Peripheral blood samples of patients with chronic heart failure(conformance to the inclusion criteria,stageⅡ,Ⅲ)and normal volunteers were collected.Separation of CD4+ T lymphocytesfrom Peripheral Blood by immunomagnetic beads,Detection of cell purity by flow cytometry,Effect of different concentrations of epl on the activity of CD4+ T lymphocytes by CCK-8,The changes and effects of CD4+ T lymphocytes kvl.3 channel currents in nomal group,chronic heart failure group(CHF)and in vitro intervention with epirone were recorded by whole-cell patch clamp technique,Expression of kvl.3,kCa3.1 and CRAC channel mrna in CD4+ T lymphocytesin Ctrl group and CHF+EPL group were detected by RT-PCR method,Detection of kvl.3 channel protein expression on CD4+T lymphocytes membrane by in-cell western blotting,Detection of intracellular Ca2+fluorescence intensity by fluorescent dye flou-3,Detection of the secretion of IL-2 and TGF-β inflammatory factors in CD4+ T lymphocytes incubation fluid in three groups by elisa method and the results of EPL intervention.RESULT:The purity of CD4+ T lymphocytes obtained by the isolation of immunomagnetic beads was 98.7%;The optimal inhibitory concentration of EPL is 30 μm;The peak current density of Kvl.3 potassium channel in CD4+ T lymphocytes in patients with chronic heart failure was 217.34 ± 11.74 pA/pF,which was significantly higher than that in Ctrl group(44.63 ± 7.43)pA/pF(P<0.01).;The peak current density of kvl.3 channel decreased to(72.13 ± 10.73)pA/pF after treatment with epirapril,which was significantly lower than that of chf group(P<0.01).There was no significant difference in the difference of cell membrane capacitance between the three groups.The expression of mRNA and protein in kvl.3 channel in CHF group was 10.74 times and 1.20 times higher than that in Ctrl group(P<0.01).The expression of mRNA and protein in kvl.3 channel of CHF group was 64.80%and 39.62%(p<0.01)lower than that of chf group,respectively.The expression of kca3.1 and crac channel mrna in chf group was 4.73 times and 3.77 times higher than that in ctrl group(P<0.01).The expression of KCa3.1 and CRAC channel mRNA in chf group was 19.30%and 37.14%respectively lower than that in CHF group(P<0.01).The levels of i1-2 in CD4 T lymphocyte culture medium were(27.84 ± 0.98)pg/ml,CHF:(153.42 ± 2.02)pg/ml,CHF+EPL:(84.3 ± 2.91)pg/ml,(p<0.01).TGF-β:Ctrl(21.25 ±0.80)pg/ml,CHF:(37.78 ±0.84)pg/ml,CHF4+EPL:(21.99±1.25)pg/ml,respectively.The intracellular calcium fluoresce nee intensity(CHF)of CD4 T was(140.95 ± 6.56),which was significantly higher than that of Ctrl(14.70±4.94)(P<0.01),and decreased significantly compared with CHF group(85.05 ± 2.60).CONCLISION:The peak current density of kvl.3 channel on the membrane of CD4 T lymphocytes in CHF group was significantly higher than that in Ctrl group,It is suggested that during the development of chronic heart failure,the immune system is activated and the differentiation function of CD4 T lymphocytes into lower level cells is enhanced,and the corresponding cytokine secretion is increased;the mRNA and protein of the three ion channels of CD4 T lymphocytes were highly expressed in chronic heart failure.Eplerenone could significantly down-regulate the relative expression of mRNA and Kvl.3 channel proteins in Kv 1.3 KCa3.1 and CRAC ion channels.The changes of kvl.3 channel were especially prominent,and Epridone could significantly reduce the content of Ca2+ in the cells.It is suggested that aldosterone receptor antagonists may reduce the activation/proliferation of t lymphocytes by inhibiting the kvl.3 channel of CD4+T lymphocytes.Reduce the release of cytokines,which is beneficial to heart failure. |
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