Pressure Overload, Angiotensin Ⅱ, Norepinephrine Provoke Tumor Necrosis Factor-α Expression In Cardiac Myocytes

Background: Tumor necrosis factor-α(TNF-α) is a pleiotrophic cytokine. It was demonstrated that TNF-αcould participate in the progression of heart failure in the last decade. The inhibiton of TNF-αis an effective method to treat the heart failure, but the mechanism is not very clear. TNF-αcould provoke myocardium hypertrophy, apoptosis and negative inotropic effect, etc, which may be the mechanisms that lead to the damage of cardiac myocytes. TNF-αcan not be detected in normal myocardium, but can be found in myoardium in some pathological conditions without evidence of inflammation. So how to inhibit TNF-αexpression becomes a direction for studying pathology of myocardium. Objectives:1. To investigate whether TNF-αis involved in the process of myocardium hypertrophy induced by pressure overload in vivo;2. To determine whether NE or Ang II is sufficient to upregulate the expression of TNF-αin the cultured cardiac myocytes.3. Chinese traditional medicine 814 may inhibit the inflammation in the lung by reducing the production of TNF-αin macrophage stimulated by LPS. So we would like to explore whether 814 could inhibit the production of TNF-αin cardiac myocytes in the pathological condition.Methods:1. Thirty male, 8 weeks old, SD rats, weighed 180-200g, were selected. Using the abdominal aortic coarctation model, the abdominal aorta above the right renal artery was exposed, and a 0.7mm-silver clamp was placed on the abdominal aorta just above the right renal artery. Sham-operated rats underwent an identical procedure except for the placement of the silver clamp. The operative rats were divided into one-month group and three-month group. The rats in one-month group were breeding for 1 month before test, and the rats in three-month group were breeding for 3 months. The sham operative served as the control of the two models. After breeding for lmonth or 3months respectively, M-mode echocardiography was performed to evaluate cardiac hypertrophy. IVSTd, LVPWd, LVED were measured and LVMI was calculated. After fixed, the heart was made into slides. The left ventricular wall thickness and cross section area of cardiac myocytes were analysised by pathological imagine processing system. Immunohistochemistrical study was performed to detect the synthsis of TNF-αin myocardium.2. The cultured cardiac myocytes were incubated with 10^-5M NE or 10^-7M AngⅡtreated for 24 or 48 hours. Immunohistochemistry was used to detect the expression of TNF-αin cardiac myocytes. RT-PCR was performed to quantify TNF-αmRNA.3. To determine whether NE or AngⅡbe sufficient to activate NF-κ3 in cultured cardiac myocytes, Electrophoretic motif shift assay (EMSA) was performed to detect the binding nuclear protein to oligonucleotides on the upstream of TNF-αgene. The specificity of the binding to the radiolaeled oligonucleotides was demonstrated by cold chase experiments. Super shift assay system was used to declear wheather the binding nuclear proten contained p65-subunit of NF-κB. To determine whether pressure overload was sufficient to activate NF-κB in myocardium, similar procedure was taken.4. The primary cultured cardiac myocytes and the second cultured fibroblast were used to detect the toxicology of the Chinese traditional medicine 814. The cell mortality was measured by calculated the percent of Typanlan staining cells. Immunohistochemical study was performed to observe the production of TNF-αin cardiac myocytes culture and the fibroblast stimulated by 0.1 mg/ml 814 for 24 hours.Results: 1. At the end of the study, 25 rats were enrolled into the study, 12 sham operated rats(6 in one-month control group and 6 in three-month control group), 13 operated rats (7 in one-month group and 6 in three-month group). IVSTd, LVPWd, LVED and LVMI measured by echocardiography, left ventricular wall thicknenss and the cross section area of cardiac myocytes measured by pathological image processing system all revealed that the rats in three-month group develop significantly left ventricular hypertrophy compared with corresponding sham operated, but there was no significant difference in left ventricular wall thickness and the cross section area of cardiac myocytes between one-month group rats and sham operated rats. The TNF-αimmunohistochemistry staining was negative in myocardium of the two control groups, but it was persistently positive in pressure overload rat hearts despite of hypertrophy or not.2. TNF-αimmunohistochemistry staining was negative in control cultured cardiac myocytes, but it was positive in cardiac myocytes treated with NE or AngⅡ. With the prolongation of the treating time, the staining granule was more obvious. TNF-αmRNA level was detected by RT-PCR, the optical dense ratio of TNF-αPCR product was obviously increased in NE or AngⅡtreated cardiac myocytes, and the quantity was elevated in a time depended manner.3. EMSA showed the intensity of the binding band between the oligonucleotides and nuclear protein in control cardiac myocytes was very weak, but it was obviously strong in cardiac myocytes treated by NE or AngⅡ, super shift assay stsytem made it disapear. Similar binding tendency was also observed in pressure overload myocardium compared with correspongding control.4. The mortality of the cultured cardiac myocytes incubated with 814 increased in a dose dependent manner, the mean value was 94.5％, 74.2％, 54.9％, 37.6％, 21.6％corresponding to 1mg/ml, 0.5mg/ml, 0.1mg/ml, 0.05mg/ml, 0.01mg/ml 814. After rejecting the effect of the solvent (alcohol), there is still a dose dependent effect on cell mortality; The toxicology of 814 on fibroblast was also dose dependent, the mean value of mortality of cardiac fibroblast stimulated by 1mg/ml,0.5mg/ml,0.1mg/ml,0.05mg/ml,0.01mg/ml 814 was 78.1％, 56.4％, 40.6％, 25.4％, 15.9％, respectively. There was brown granule in the cytoplasm of cardiac myocytes and fibroblast incubated with 0.1 mg/ml 814, but it is negative in control cardiac myocytes and fibroblast.Conclusions: NE, AngⅡand the ventricular pressure overload are all known suggestive factors of cardiac hypertrophy. NE or AngⅡsignificantly increase the protein and mRNA level of TNF-αin the cultured cardiac myocytes. Similar upregulation of the expression of TNF-αis observed in pressure overloaded myocardium induced by aortic coarctation, which is earlier than the present of morphorism hypertrophy and persistently exists in the hypertrophied myocardium, which suggests that TNF-αmay participate in the remodeling process. The intensified binding of nuclear protein and the potential NF-κB binding sequencese suggests that NF-κB translocate to the nuclear. NF-κB activity is increased in NE or AngⅡtreated cardiac myocytes and pressure overload myocardium, which may be the same pathway leading to the expression of TNF-αin different pathological condition. -619～-591 (cat tcc ctc tg g ggc tgc ccc art ctc ctc) and -508～-481 (ctc aga caa ggg ggc ttt ccc tcc tca ac) on the upstream of TNF-αare the potential promoters. Chinese traditional medicine 814 had toxicology on cultured cardiac myocytes and fibroblast in a dose dependent manner. 0.1 mg/ml 814 can make cultured cardiac myocytes and fibroblast synthesis TNF-α.