| IntroductionThe renin -angiotensin system (RAS) is upregulated during myocardial ischemia , infarction and ischemia - reperfusion injury' . Ang II, the major effector molecule of the RAS, binds to two main receptor subtypes , the Ang II type 1 ( AT, ) and the type 2 ( AT2) receptor to exert its physiological effects. The type 1 receptor medicates the major cardiovascular effects of Ang II, several of which enhance ischemia - reperfusion injury. The role of the type 2 receptor and the relation between RAS and other proiflammatory cytokine are not yet well defined. Several studies have suggested that Ang - converting enzyme inhibitors protecting hearts from ischemia - reperfusion injury . The effects of the type 1 receptor blockade on ischemia - reperfusion injury are debated. The purposes of this study were to investigate the protecting machanism of valsartan on myocardial ischemia - reperfusion injury using Langendorff isolated heart and myocytes anoxia - reoxygenation modle.Methods1. To investigate bradykinin mechanism of protection of reperfused ische-mic heart by valsartanThe hearts of 40 SD rats were isolated, linked to Langendorff perfusion apparatus, and randomly divided into 5 equal groups: control group, to be perfused with modified Kreb - Henseleit (K - H) buffer for 110 minutes;ischemi-a/reperfusion (I/R) group, to be perfused with K - H buffer for 20 min, ex-posed to ischemia for 30 min, and then reperfused with K - H buffer for 60 min;HOE140 group, to be perfused with K - H buffer with HOE140, a bradykinin B2receptor antagonist for 20 min, exposed to ischemia for 30 min, and then reperfused with K - H buffer with HOE140 for 60 min;valsartan group, perfused with K - H buffer with valsartan for 20 min, exposed to ischemia for 30 min, and then reperfused with K - H buffer with valsartan for 60 min;and valsartan + HOE140 group, perfused with K - H buffer with valsartan + HOE140 for 20 min, exposed to ischemia for 30 min, and then reperfused with K - H buffer with valsartan + HOE140 for 60 min. The left ventricular systolic pressure (LVSP) and maximal uprising velocity of left ventricular pressure ( + dp/ dtmax) were measured 20 minutes after the stabilization of perfusion, and 20, 40, and 60 minutes after reperfusion. After reperfusion Evans blue was infused via the aorta and the no - flow area was measured. The isoenzyme of creatine ki-nase (CK - MB) in the effluent liquid from the heart was measured 20 minutes after the stabilization of perfusion, and 1 , 30, and 60 minutes after reperfusion.2. To determine the effects of valsartan on AT, and AT, receptor expression during ischemia reperfusionThe hearts of 24 SD rats were isolated, linked to Langendorff perfusion apparatus, and randomly divided into 3 equal groups;control group, I/R group and valsartan group. The coronary effluent was measured. The structure was observed using light microscope and electron microscope. The AT, and AT2 receptor mRNA expression were examined by Northern blot. The AT, and AT: receptor protein expression were examed by Northern blot.3. To explore the effect of valsartan on TNF - alpha during myocardial ischemia reperfusion in isolated rat hearts and myocytesIn vitro The hearts of 24 SD rats were isolated, linked to Langendorff perfusion apparatus, and randomly divided into 3 equal groups;control group, I/R group and valsartan group. The levels of TNF - alpha in the coronary effluent were determined by ELISA. TNF - alpha protein expression was examed by immunohistochemistry stain and Western blot. TNF - alpha mRNA expression was examined by Northern blot. In myocytes Single myocytes isolated from neonatal rat hearts were subjected to the conditions mimicking ischemia andreperfusion. Ischemic condition was produced by superfusing myocytes with hy-poxic solutions containing elevated concentrations of K+ , H + , and lactate as described by Koyama et al. The myocytes were devided into 5 groups: control group, the myocytes treated with serum - free DMEM medium for 3 hours;hy-poxia/reoxygenation( H/R) group, hypoxia for 1 hour , reoxygenation for 2 hours;valsartan group, hypoxic and reoxygenation solutions containing valsartan ljxmol/L;angiotensinll group, serum - free DMEM medium containing angio-tensin II 1 jxmol/L for 3 hours;angiotensin II + valsartan group, serum - free DMEM medium containing valsartan 1 jxmol/L for 1 hours, valsartanl jxmol/L + angiotensinll 1 jx mol/L for 3 hours. The extent of cellular damage was accessed by the amount of released lactate dehydrogenase ( LDH) . The levels of TNF - alpha in supernatants of incubated cardiac myocytes were determined by ELISA.Results1. Bradykinin mechanism of protection of reperfused ischemic heart by valsartanThe LVSP and + dp/dtmax of the control group were significantly higher than the other 4 groups (all P < 0.01). The LVSP and + dp/dtmax of the valsartan group were significantly higher than those of the I/R group (both P < 0. 01). The LVSP and + dp/dtmax of the valsartan + HOE140 group were significantly lower than those of the valsartan group (P < 0. 01 and P < 0. 05). The no -flow area of the HOE 140 group was not significantly different from that of the 1/ R group (P > 0. 05 ) . The no - flow area of the valsartan group was significantly smaller than that of the I/R group, almost half of that of the latter (P < 0. 01). The CK - MB levels after I/R at different time points were all significantly higher than that of the control group (all P < 0. 01). The CK - MB levels of the HOE140 group were not significantly different from those of the I/R group (all P > 0. 05). The CK - MB levels at different time - pints of the valsartan group were all significantly lower than those of the I/R group (allP < 0.01). The CK - MB levels at different time - pints of reperfusion of the valsartan + HOE 140 group were all significantly higher than those of the valsartan group (allP < 0. 05). Linear correlation analysis showed that the CK - MB level 60 minutes after reperfusion was positively correlated with the no - flow area with a correlation coefficient of 0. 85 (P < 0.01).2. The effects of valsartan on AT, and AT2 receptor expression during ischemia reperfusion in isolated rat heartsCompared with control, I/R dcreased coronary effluent (3. 3 ml. min"1 ± 0.5 ml. min"1 vs5.9 ml. min'1 ±0.8 ml. min"1 ,P <0. 01). I/R damaged the myocardial structure. AT, receptor mRNA and protein expression were increased, but AT2 receptor mRNA and protein expression were decreased during ischemia - reperfusion. Valsartan improved left ventricular function ( + dp/dtmax 1337 mmHg. s"1 ±226 mmHg. s'J;- dp/dtmax -871 mmHg. s"1 ±208mmHg. s" ,compared with I/R group, all P <0. 01) ,increased coronary effluent(4. 2 ml. min ± 0. 7 ml. min" , compared with I/Rgroup, P < 0. 01). Increased AT2 receptor mRNA and protein expression with no changes in AT, receptor mRNA and protein expression.3. The effect of valsartan on TNF - alpha during myocardial ischemia reperfusion in isolated rat hearts and myocytesIn vitro After ischemia — reperfusion, TNF - alpha expressed in the myocardial cytoplasm. TNF -alpha mRNA expression intensified from 0. 25 ±0.01 (control group) to 1. 28 ± 0. 13 ( P < 0. 01 ) , TNF - alpha protein expression intensified from 0. 36 ±0.03 (control group) to 0.9 ±0. 08(P < 0.01). The TNF - alpha mRNA and protein expression declined to 0. 8 ±0. 04 and 0.47 ± 0.02 in valsartan - treated hearts, respectively (all P< 0.01). The effluent TNF - alpha levels were 14. 5 ng/L ±1.9 ng/L in control group, 99. 8 ng/L ± 18. 0 ng/L in I/R group(P < 0. 01) , and 59. 6 ng/L 38. 3 ng/L in valsartan -treated group(P < 0.01). In myocytes Concentrations of TNF - alpha in su-pernatants of incubated cardiac myocytes in H/R group and treated with 1 jxmol/ L of Ang - II were 55. 3 ng/L ±8.8 ng/L and 58.7 ng/L ± 15. 6 ng/L, respectively . When pretreated with 1 fimol/L of valsartan, TNF - alpha was declined. A parallel changes of LDH release in supematants of incubated cardiac myocytes were observed.Conclusions1. AT, receptor blockade valsartan induces short - term cardioprotection partially associated with increasing the level of bradykinin during myocardial ischemia reperfusion .2. Valsartan protect myocardium from ischemia - reperfusion injury associated with enhanced AT2 receptor mRNA and protein expression.3. Ischemia reperfusion increased TNF - alpha levels, the same as angio-tensin II. Valsartan protect myocardium from ischemia - reperfusion injury by reducing cardiac tumor necrosis factor - alpha expression and content. |
PDF Full Text Request