The Investigation Of The Underlying Mechanism Of Qiliqiangxin Capsules Ameliorating Pressure Overload-Induced Cardiac Hypertrophy In Mice

Cardiac hypertrophy is an adaptive response of the heart when it is subjected to various pathologic stimulus, The pressure overload induced by different pathologic stimulus(such as hypertension, aortic stenosis) can cause compensatory cardiac hypertrophy in heart at the very beginning, characterized by the incrassate ventricular wall and enhanced ventricular systolic function. The existence of the continuing stilumus will, however, result in the excessive activation of the Renin-Angiotensin-Aldosterone System and other Neuroendocrine Systems, including the release of AngiotensinII and Catecholamines,which will lead to the increasing oxygen consumption of the cardiomyocytes; If the coronary arterial blood supply can not satisfy with the needs of the cardiac hypertrophy, the heart will degenerate into the irreversible phase of chronic heart failure. Therefore, The suppression of cardiac hypertrophy at the early phase will slow down the process of developing into heart failure, as well as reducing the mortality of it. In the clinical practice, angiotensin converting enzyme inhibitors(ACEI) and AT1receptor blocker(ARB) are wildly prescribed since they can protect heart from cardiac hypertrophy by exactly inhibiting the excessive activation of RAAS and weakening the activation of AT1receptor.ErbB receptors are amongst one of classic Tyrosine Kinase Receptors family, which have been wildly studied in the tumor biology. ErbB2and ErbB4are the principal ErbB receptors in the postnatal heart, Recently it is reported that ErbB2, ErbB4play a critical role in cardiovascular development and maintenance of heart function. Meanwhile, the role of these receptors in promoting the proliferation of cardiomyocytes under some physiological and pathological conditions has been speculated. C/EBPP is a member of the bHLH family of gene DNA-binding transcription factors and has pivotal roles in cell proliferation; Moreover, it has been reported that the increase in cardiomyocyte proliferation, which is regulated by C/EBPβ and subsequent elevation of CITED4, could contribute to chronic exercise-induced physiological cardiac hypertrophy in mice. More importantly, mice with reduced C/EBPβ were also resistant to the development of cardiac dysfunction during pressure overload. Therefore, it is insightful to promote cardiomyocyte growth through regulation of C/EBPβ/CITED4in the treatment of pathological cardiac hypertrophy.Qiliqiangxin capsules(QL), a kind of Chinese traditional medicine compounds extracted from a group of herbal medicine, have been wildly prescribed for the trearment of chronic heart failure and shown to be effective through increasing cardiac contractibility and kidney blood flow as well as regulating inflammatory responses. However, it is unknown whether QL has a role in preventing myocardial remodeling and subsequent cardiac dysfunctions in hypertensive subjects, Therefore, the present study was carried out to firstly test whether QL has benefical effects in a mouse model of chronic pressure overload and secondly the underlying mechanism which are involved in the effects of QL. The effects of QL were also compared to Lorsartan,a common ARB, which is known to have cardio-protective effects in the treatment of hypertension.We firstly performed in vivo studies by establishing cardiac hypertrophy model in C57BL/6mice which were subjected to transverse aorta constriction (TAC) for2or4weeks. Meanwhile, QL and Losartan were administrated into mice in different group by gastric tube respectively. At2weeks after TAC, The mice treated with QL and Losartan showed a less cardiac hypertrophy, characterized by abolished upregutation of AT1, p-ERK and hypertrophic genes. At4weeks after TAC, QL, but not Losartan, significantly upregulated the phosphorylation of ErbB2and ErbB4compared to vehicle treatment. Moreover, QL treatment did significantly increase the number of Ki-67-positive cardiomyocytes which is harnessed by the expression of C/EBPβ/CITED4, while Losartan only induced a slight increase in these cell. Meanwhile, in the heart of mice with QL treatment,the expression of C/EBPβ both at gene and protein levels was downregulated and CITED4gene was upregulated. However, the cardio-protective effects can be abolished by either inhibiting ErbB receptors or blocking the CITED4gene expression. Therefore, we are prone to assume that the underlying mechanism of QL ameliorating pressure overload-induced cardiac hypertrophy may be related with the weakening the cardiac hypertrophy signal transduction and cardiomyocytes proliferation, which is quite different from that of Losartan. Furthermore, we used Angiotensinogen-deficient (ATG-/-) male mice which were subjected to TAC operation to investigate whether QL, like Losartan can directly inhibit the activation of AT1receptor without AngII in the model of pressure overload-induced cardiac hypertrophy. Interestingly, The results indicate that The inhibition effect of QL on cardiac hypertrophy was abolished without the existence of Ang II. Thus, QL, different from Losartan, ameliorate the cardiac hypertrophy via abating activation of AT1receptor by reducing the concentration of AngiotensinII, not via directly suppressing the AT1receptor like Losartan.Above all, the underlying mechanism of Qiliqiangxin Capsules ameliorating pressure overload induced cardiac hypertrophy in mice may be attributed to firstly reducing he concentration of Angll and attenuating the activation of AT1receptor, which will result in the weakening the cardiac hypertrophy cell signal transductio4n. Secondly, At4weeks after TAC, The activation of ErbB receptors and the expression change of C/EBPβ/CITED4can induce more cardiomyocytes proliferation which will contribute to, at least a part, the preventionn of cardiac remodeling and dysfunction induced by pressure overload.PART1AT1and ErbB receptors are involved in the effects of Qiliqiangxin Capsules on ameliorating cardiac hypertrophy after TAC in miceObjective:To elucidate whether the AT1and ErbB receptors are involved in the effects of Qiliqiangxin Capsules on ameliorating cardiac hypertrophy after TAC in mice.Methods:8-10weeks old C57BL/6mice were randomly grouped into4groups: Sham-operated group, TAC plus Saline group, TAC plus Qiliqiangxin Capsules group, TAC plus Losartan group. All the mice were subjected to sham or TAC operation. Qiliqiangxin Capsules(0.6mg/kg.d) and Losartan(13.4mg/kg.d) were administrated into mice by gastric tube, Echocardiography and hemodynamic measurement were carried out at2or4weeks after TAC, The expression of AT1receptor and phosphorylation of ErbB2and ErbB4,as well as p-ERK were detected by western blot methods.Results:At2or4weeks after TAC, hemodynamic datum show that BP and LVESP were similarly elevated in all examined TAC mice while LVEDP was changed in saline treatment group at4weeks after operation. At2weeks after TAC, however, the two drugs significantly ameliorating cardiac hypertrophy characterized by incrassate left ventricular anterior and posterior wall and increasing CSA and fibrotic areas,as well as the upregulation of AT1and p-ERK((P<0.05). There is no significantly changes between the two drugs. At4weeks after TAC, Interestingly, The results turn out to be similar but for the expression of p-ErbB2与p-ErbB4was strikingly elevated in QL group (P<0.05)Conclusion:Qiliqiangxin Capsules can ameliorate cardiac hypertrophy induced by pressure overload in mice like Losartan through downregulating the activation of AT1receptor. However, the difference is that QL, not Loartan,can activates the ErbB receptors which will contributes to the effects on the heart protection. All indicats that AT1and ErbB receptors are involved in the effects of Qiliqiangxin Capsules on ameliorating cardiac hypertrophy after TAC in mice PART2Qiliqiangxin Capsules ameliorate Pressure Overload-Induced Cardiac Hypertrophy in mice through regulating the expression of C/EBPβ/CITED4which promotes cardiomyocytes proliferationObjective:To investigate whether Qiliqiangxin Capsules promotes cardiomyocytes proliferation which is regulated by the expression of C/EBPβ/CITED4in the process of ameliorating Pressure Overload-Induced cardiac hypertrophy in mice.Methods:Western blotting methods and Real-time PCR methods were carried out to examine the expression of C/EBPβ and CITED4both at protein and mRNA levels in the myocardium which is preserved in the PART1experiments. Ki-67positive cardiomyocytes were detected by Immunofluorescence methods. CITED4siRNA which shows the best inhibit effect on target gene was constructed.8-10weeks old C57BL/6mice were randomly grouped into5groups:Sham-operated group, TAC plus Saline group, TAC plus Qiliqiangxin Capsules group, TAC plus Qiliqiangxin Capsules plus ErbB receptor inhibitor group, TAC plus Qiliqiangxin Capsules plus CITED4siRNA group. All the mice were subjected to sham or TAC operation. Qiliqiangxin Capsules(0.6mg/kg.d) or saline were administrated into mice by gastric tube, ErbB receptor inhibitor(CI-1033) was administrated into mice subcutaneously at dosage of2mg/kg.day. CITED4siRNA was injected into myocardium through LV cavity during the operation of TAC. At4weeks after TAC, Echocardiography, hemodynamic and histopathology measurements were carried out respectively to detect the extent of cardiac hypertrophy in mice.Results:At4weeks after TAC, The downregulation of C/EBPβ and upregulation of CITED4both at protein and mRNA levels were found in the Qiliqiangxin Capsules treatment group. Meanwhile, Ki-67positive cardiomyocytes were found in the same group. Moreover,4weeks after TAC, ErbB receptor inhibitor or CITED4siRNA abolished the protective effects of Qiliqiangxin Capsules on cardiac hypertrophy characterized by significant elevation of LVEDP, CSA and fibrotic areas as well as the reduction in the LVFS.Conclusion:Qiliqiangxin Capsules promotes cardiomyocytes proliferation which is regulated by the expression of C/EBPβ/CITED4in the process of ameliorating Pressure Overload-Induced cardiac hypertrophy in mice, which can be abolished by the ErbB receptor inhibitor and CITED4siRNA. PART3Qiliqiangxin Capsules ameliorate Pressure Overload-Induced Cardiac Hypertrophy in mice via abating activation of AT1receptor by reducing the concentration of AngiotensinⅡObjective:To investigate whether AngiotensinⅡ plays pivotal roles in the progress of Qiliqiangxin Capsules ameliorating Pressure Overload-Induced Cardiac Hypertrophy in mice and its pontential molecular mechanism.Methods:To estalish cardiac hypertrophy model, Transverse Aorta Constricting(TAC) surgery were carried out in mice.8-10weeks old Wild Type (WT) and Angiotensinogen-deficient (ATG-/-) male mice were divided into three different groups by random:Sham-operated group, TAC plus Saline group, TAC plus Qiliqiangxin Capsules group.After operation, Qiliqiangxin Capsules(0.6mg/(kg.d),dissolved in saline) was administrated into mice by gastric tube, the rest two group mice were treated with identical volume saline.2weeks After TAC, all the mice were sacrified, Echocardiography and hemodynamic measurement were performed at2weeks after TAC. And cardiac histology and hypertrophic gene expression was analyzed. AngⅡ levels in serum and myocardium were measured by ELISA. The expression of AT1receptor and phosphorylation of ERK were detected by western blot method.Results:As regards to WT mice, Compared with Sham group, The concentration of AngⅡ and other indices related with cardiac hypertrophy in saline group varied significantly (P<0.05), While most of indices in Qiliqiangxin Capsules group improves compared with that in saline group (P<0.05); As regards to ATG-/-mice, The concentration of AngⅡ were extremly low and showed no significant change between the three groups(P>0.05), However,Compared with Sham Group, The indices related with cardiac hypertrophy in the other two groups changed obviously (P<0.05) but between the two groups, there is no statistically difference (P>0.05)Conclusion:The potential molecular mechanism of Qiliqiangxin Capsules ameliorating Pressure Overload-Induced cardiac hypertrophy is reducing the concentration of AngⅡ, attenuating the intensity of celluar signal transduction when it binds to AngiotensinⅡ type1receptor(AT1R), not directly suppressing the activation of AT1receptor induced by mechanical stretch such as pressure overload.