The Study Of Transient Outward Potassium Channel Protein In The PVN On RSNA In Rats With CHF And The Intervention Effects Of Qiliqiangxin

Objective: Guided by the theory of TCM and based on “brain for medullary sea, the gathering of the true qi”, this study is to discuss the central mechanism of compound Chinese medicine on chronic heart failure. The left anterior descending branch ligation of coronary of SD rats was banded to establish the chronic congestive heart failure（CHF）animal model.The purpose is to discuss the relationship among the transient outward potassium channel protein expression in the paraventricular nucleus（PVN）,enhancement of the sympathetic activity, cardiac function changes and ventricular remodeling, and to observe the effects of AngⅡ/AT₁R/MAPK_S pathway on Kv4.2 and Kv4.3 expression, at the same time to discuss the intervention effect of Qiliqiangxin on the expression of transient outward potassium channel protein and RAS system,to reveal the central mechanism of compound Chinese medicine in chronic heart failure by inhibiting the activation of sympathetic.Methods: The study includes three sections:1 Study on relationship between the expression of transient outward potassium channel protein in the PVN and RSNA in rats with CHFThe left anterior descending branch ligation of coronary of SD rats was banded to establish the CHF animal model. After 4 weeks, left ventricular ejection fraction（LVEF） examined by ultrasonic cardiogram to assess the heart failure model. SD rats were divided into the Sham group and CHF group. the heart weight index, lung weight index were texted. Enzyme linked immunosorbent assay was used to measure the level of plasma NE and serum NT-proBNP, the expression of ransient outward potassium channel protein Kv4.2 and Kv4.3 were determined by immunohistochemical, RT-PCR and western blot. The MAP, HR and RSNA were measured by Power Lab in anesthetized rats with PVN microinjection of potassium channel blocker 4-AP. The purpose was to evaluate the effects of transient outward potassium channel in PVN on sympathetic nerve.2 The effects of AngⅡ/AT₁R/MAPK_S pathway on Kv4.2 and Kv4.3 expressionThe mold method was same with the first part. Four weeks after the administration, the SD rats were divided into Sham+VEH, CHF+VEH, CHF + Losartan, CHF + PD98059, CHF+ SB203580.All animals were continuously administered via a syringe pump injector connected to lateral ventricle.Four weeks after drug delivery, echocardiogram was used to evaluate the cardiac function and the heart weight index, lung weight index were texted. Enzyme linked immunosorbent assay was used to measure plasma NE and serum NT-proBNP.The expression of ERK, P-ERK, P38, P-P38 at protein levels in the PVN in Sham+VEH and CHF+VEH and CHF + Losartan groups were determined by western blot.The changes of P-CREB, Kv4.2, Kv4.3 at protein levels in all groups were determined by western blot after given the ERK inhibitor and p38 inhibitor.The purpose is to evaluate the function of AngⅡ/AT₁R/MAPK_S pathway regulating the expression of transient outward potassium channel.3 The intervention effects of Qiliqiangxin administered through the lateral ventricle on Kv4.2, Kv4.3 expression and RAS systemThe mold method and administration method were same with the second part. Fours weeks after the administration, the SD rats were divided into Nomal, Sham+VEH, CHF+VEH, CHF + Losartan, CHF+QLQX-L and CHF+QLQX-H. After four weeks, Echocardiogram was used to evaluate the cardiac function and the heart weight index, lung weight index were texted. HE was used to observe myocardial tissue morphology and enzyme linked immunosorbent assay was used to measure plasma NE and serum NT-proBNP.The RSNA was measured by PowerLab in anesthetized rats and the expression of Kv4.2, Kv4.3, ACE, AT₁ R at protein levels in the paraventricular nucleus were determined by western blot.The purpose is to reveal the compound Chinese medicine inhibiting sympathetic activation central mechanism in chronic heart failure.Results: 1 Study on relationship between the expression of transient outward potassium channel protein in the PVN and RSNA in rats with CHF 1.1 The changes of cardiac function in two groupsCompared with Sham group, LVDd, LVDs, LVVd, LVVs of the CHF group were significantly increased and EF, FS were significantly decreased（P<0.01）. 1.2 Results of heart weight index and lung weight index in two groupsCompared with Sham group, HW/BW and LW/BW of the CHF group were obviously increased（P<0.01）. 1.3 The determination results of NE, NT-proBNP in two groupsCompared with Sham group, NE and NT-proBNP of the CHF group were remarkably increased（P<0.01）. 1.4 Immunohistochemical staining was performed to examine Kv4.2 and Kv4.3 productionCompared with Sham group, the production of Kv4.2 and Kv4.3 in the CHF group were decreased. 1.5 The changes of the expression of Kv4.2 and Kv4.3 at mRNA and protein levels in the PVNCompared with Sham group, Kv4.2 and Kv4.3 at mRNA and protein levels of the CHF group were significantly decreased（P<0.05, P<0.01）. 1.6 The changes of MAP, HR and RSNA in anesthetized rats with PVN microinjection of potassium channel blocker 4-APCompared with microinjection of ACSF, microinjection of 4-AP（0.4, 0.8, 1.6 nmol/200nl） into PVN induced a increase in MAP, HR and RSNA in both Sham and CHF rats（P<0.05, P<0.01）, while the CHF rats exhibited smaller responses to 4-AP than did in Sham rats（P<0.01）. 2 The effects of Ang Ⅱ /AT₁R/MAPK_S pathway on Kv4.2 and Kv4.3 expression 2.1 The changes of cardiac function in each groupCompared with Sham+VEH group, LVDd, LVDs, LVVd, LVVs of the CHF+VEH group were significantly increased and EF, FS were remarkably decreased（P<0.01）;Compared with CHF+VEH group, LVDd, LVDs, LVVd, LVVs of all the administration groups were decreased and EF, FS were increased to varying degrees（P<0.01）. 2.2 Results of heart weight index and lung weight index in each groupCompared with Sham+VEH group, HW/BW and LW/BW of the CHF +VEH group were obviously increased（P<0.01）;Compared with CHF+VEH group, all the indexes of the administration group can be reduced to varying degrees（P<0.05, P<0.01）. 2.3 The determination results of NE, NT-proBNP in each groupCompared with Sham+VEH group, NE and NT-proBNP of the CHF+VEH group were significantly increased（P<0.01）; Compared with CHF+VEH group, NE and NT-proBNP of all the administration groups were decreased to varying degrees（P<0.05, P<0.01）. 2.4 The changes of the expression of ERK, P-ERK, P38, P-P38 at protein levels in the PVN of Sham+VEH group, CHF+VEH group and CHF + Losartan groupThe expression of ERK, P38 of Sham+VEH group, CHF+VEH group and CHF + Losartan group had no differences; Compared with Sham +VEH group, P-ERK and P-P38 at protein level of the CHF+VEH group was significantly increased（P<0.05, P<0.01）;Compared with CHF +VEH group,P-ERK and P-P38 at protein level of the CHF+Losartan group was decreased（P<0.05, P<0.01）. 2.5 The changes of the expression of P-CREB, Kv4.2, Kv4.3 at protein level in the PVN in each groupCompared with Sham+VEH group, the expression of P-CREB at protein level in CHF+VEH group was increased, Kv4.2 and Kv4.3 was decreased（P<0.01）; Compared with CHF+VEH group, the expression of P-CREB at protein level was decreased, Kv4.2 and Kv4.3 was increased in all the administration groups（P<0.01）. 3 The intervention effects of Qiliqiangxin administered through the lateral ventricle on Kv4.2, Kv4.3 expression and RAS system 3.1 The changes of cardiac function in each groupCompared with Sham+VEH group, LVDd, LVDs, LVVd, LVVs of the CHF+VEH group were increased and EF, FS were decreased（P<0.01）;Compared with CHF+VEH group,LVDd, LVDs, LVVd, LVVs of the CHF+ Losartan group and CHF+QLQX-H group were decreased and EF, FS were increased（P<0.05, P<0.01） and LVDs, LVVs of CHF+QLQX-L group were decreased and EF, FS were increased（P<0.01） and there is no statistical difference between CHF+ Losartan group and CHF+QLQX-H group（P>0.05）. 3.2 Results of heart weight index and lung weight index in each groupCompared with Sham+VEH group, HW/BW and LW/BW of the CHF +VEH group were obviously increased（P<0.01）; Compared with CHF+VEH group, all the indexes of the CHF+ Losartan group and CHF+QLQX-H group were decreased（P<0.01） and the LW/BW of CHF+QLQX-L group was decreased（P<0.01） and there is no statistical difference between CHF+ Losartan group and CHF+QLQX-H group（P>0.05）. 3.3 Morphological observation of cardiac muscle tissueThe Normal group and Sham+VEH group had normal cardiac muscle tissue. Myocardial tissue cell nucleus were dyed into blue and cardiac muscle bundle was full.The cardiac muscle fibrosis and inflammatory cell infiltration were not seen. The CHF+VEH group myocardial infarction area organization was dyed lightly and had severe cardiac muscle fibrosis and a large number of inflammatory cell infiltration. The number of myocardial cells were decreased. Each administration group had different degrees of inhibition of cardiac muscle fibrosis and inflammatory cell infiltration was relieved. 3.4 The determination results of NE, NT-proBNP in each groupCompared with Sham+VEH group, NE and NT-proBNP of the CHF+VEH group were significantly increased（P<0.01）; Compared with CHF+VEH group, NE and NT-proBNP of all the administration groups were decreased（P<0.05, P<0.01） and there is no statistical difference among these treatment groups（P>0.05）. 3.5 The changes of RSNA in each groupCompared with Sham+VEH group, the RSNA of the CHF +VEH group was increased（P<0.01）;Compared with CHF+VEH group, the RSNA of the administration group was reduced（P<0.05, P<0.01） and there is no statistical difference between CHF+ Losartan group and CHF+QLQX-H group（P>0.05）. 3.6 The changes of the expression of Kv4.2, Kv4.3, ACE, AT₁ R at protein level in the PVN in each groupCompared with Sham+VEH group, the expression of ACE and AT₁ R at protein level in CHF+VEH group were increased, Kv4.2 and Kv4.3 were decreased（P<0.01）; Compared with CHF+VEH group, the expression of ACE and AT₁ R were decreased, Kv4.2 and Kv4.3 were increased in all the administration groups（P<0.05, P<0.01） and there is no statistical difference among these treatment groups（P>0.05）.Conclusion:1 Downregulation of Kv4.2 and Kv4.3 expression in the PVN may be a potential mechanism for sympathoexciation in rats that involved in the progression of ventricular remodeling.2 Research showed that Losartan and MAPKs inhibitor by intraventricular administration could significantly inhibit the downregulation of Kv4.2 and Kv4.3 expression.These results revealed that Ang Ⅱ/AT₁R/MAPK_S pathway involved in the regulation of the reconstruction of transient outward potassium channel.3 Research showed that Chinese medicine Qiliqiangxin capsule by intraventricular administration could significantly inhibit the downregulation of Kv4.2 and Kv4.3 expression, decrease the activation of the RAS system, reduce the renal sympathetic nerve activity,decrease the ventricular remodeling and improve cardiac function.