| Backround and Objective: Myocardial ischemia activates cardiac sympathetic afferent nerve endings and elicits chest pain, which is often associated with a sympathoexcitatory reflex characterized by an increase in blood pressure and sympathetic nerve activity. Extracellular adenosine 5'-triphosphate (ATP) which is released from ischemic myocardial tissues interacts with purinergic receptors to play an important role in nociceptive transmission. P2X3 receptors are expressed selectively at high levels in nociceptive sensory neurons. P2X3 receptors are localized in sympathetic ganglion neurons, including superior cervical ganglion (SCG) and stellate ganglion (SG) and activated by extracellular ATP. Study in our laboratory has shown that P2X3 receptors in the SCG neurons and SG neurons activated by ATP were involved in the nociceptive transmission of myocardial ischemic injury. Puerarin(Pue) is an active principle extracted from Chinese herbal medicine Ge Gen and has many functions, including expanding coronary, reducing myocardial oxygen consumption, improving myocardial contractile function, promoting blood circulation and protecting central nervous system.The previous reserch has shown that puerarin can reduce currents activated by P2X receptor agonist in nodose ganglia and dorsal root ganglia and then supress nociceptive transmission. Litter is known about the role of puerarin in the sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X3 receptor in rat SCG and SG neurons. The present study was aimed to observe the effect of puerarin on the P2X3 and TH in myocardial ischemic rat SCG and SG neurons,and to explore the effect of puerarin on sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X3 receptor in rat SCG and SG neurons.This will help people gain a better understanding of puerarin in the treatment of myocardial ischemia and its possible role in target, and for treatment of myocardial ischemia, myocardial infarction, to provide a new experimental evidence.Methods:(1)Establishment of rat myocardial ischemia model.Electrocardiogram (ECG) and H–E stain of cardiac tissue to explore the effect of puerarin on ischemia cardiac tissue in rat myocardial ischemia injury. (2) Systolic blood pressure, heart rate and respiration were measured by non-invasive blood pressure determinator with different transducer.(3)The expressions of P2X3 receptors and TH in rat SCG and SG neurons and myocardial tissues were detected by immunofluorescence. (4)The expression of P2X3 protein in rat SCG and SG neurons were analyzed by western blotting and immunohistochemistry. (5)The expression of P2X3 mRNA in SCG and SG neurons was analyzed by in situ hybridization.Results: (1) Compared with the control, the abnormal Q wave or ST-segment displacement appeared obviously in myocardial ischemic rats. H–E stain results show that the myocardial tissues from myocardial ischemic group showed severe atrophy, hypertrophy and proliferation of fiber connective tissue. Puerarin can improve abnormal Q wave, ST-segment and myocardial issue changes induced by myocardial ischemia. (2) Systolic blood pressure(73.35±2.51mmHg), heart rate(445.67±16.58 times/min) and respiration(92.33±5.78times/min) in the myocardial ischemic rats(n=6) were increased,compared with those (69.13±0.55 mmHg, 366.17±8.44 times/min and 72.83±2.63 times/min, respectively) in control rats(n=6)(p<0.05). After the treatment with puerarin in the myocardial ischemic rats (n=6), systolic blood pressure (69.85±1.61 mmHg), heart rate (374.00±6.89 times/min) and respiration (76.33±2.16 times/min) were lower than those in the myocardial ischemic rats (p<0.05). (3)Immunofluorescence results showed that TH and P2X3 receptors were co-expressed in SCG, SG neurons and myocardial tissues. The TH and P2X3 receptors in myocardial ischemic injury group exhibited more intense staining than those in control group. After myocardial ischemic injury in rats treated with puerarin, the expression of TH and P2X3 immunoreactivity was lower than that in myocardial ischemic rats. (4) The expression of P2X3 in SCG and SG neurons was studied by immunohistochemistry. The average optical density (ODs) of P2X3 receptor expression in the SCG neurons in saline control group, puerarin control group, myocardial ischemic injury group and myocardial ischemic injury treated with puerarin were 0.10±0.03,0.08±0.01,0.15±0.03,0.07±0.04 (n=10 for each group) , respectively. The average OD of P2X3 receptor expression in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury treated with puerarin (p<0.05), saline control group (p<0.05) and puerarin control group (p<0.01; F (3, 36) =11.768). No difference was found in the intensity of P2X3 receptor of SCG among saline control group, puerarin control group and myocardial ischemic injury treated with puerarin (p>0.05; F (2, 27) =1.292). The average ODs of P2X3 receptor expression in the SG in saline control group,puerarin control group,myocardial ischemic injury group and myocardial ischemic injury treated with puerarin were 0.16±0.02,0.15±0.01, 0.20±0.02, 0.15±0.01 (n=10 for each group), respectively. The average OD of P2X3 receptor expression in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury rats treated with puerarin group(p<0.05), saline control group (p<0.05) and puerarin control group(p<0.01; F(3,36)=9.544). No difference was found in the intensity of P2X3 receptor expression of SG among saline control group, puerarin control group and myocardial ischemic injury treated with puerarin(p>0.05; F(2,27)=0.895).(5)The levels of P2X3 mRNA were studied by ISH. The average ODs of P2X3 mRNA in the SCG in saline control group,puerarin control group,myocardial ischemic injury group and myocardial ischemic injury rats treated with puerarin group were 0.13±0.02, 0.12±0.01, 0.23±0.03, 0.12±0.01 (n=10 for each group), respectively. The average OD of P2X3 mRNA in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury treated with puerarin (p<0.05), saline control group (p<0.05) and puerarin control group (p<0.01; F (3, 36) =42.589). No difference was found in the intensity of P2X3 mRNA of SCG among saline control group, puerarin control group and myocardial ischemic injury rats treated with puerarin group(p>0.05; F(2,27)=1.253).The average ODs of P2X3 mRNA in the SG in saline control group, puerarin control group, myocardial ischemic injury group and myocardial ischemic injury rats treated with puerarin group were 0.08±0.01,0.09±0.01,0.12±0.02,0.09±0.02 (n=10 for each group), respectively. The average OD of P2X3 mRNA in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury rats treated with puerarin group (p<0.05), saline control group (p<0.05) and puerarin control group (p<0.01; F (3, 36) =11.345). No difference was found in the intensity of P2X3 receptor expression of SG among saline control group, puerarin control group and myocardial ischemic injury treated with puerarin(p>0.05; F(2,27)=0.906).(6) P2X3 protein level was analyzed by western blotting. The average ODs of P2X3 protein expression(normalized to eachβ-actin internal control) in the SCG in saline control group,puerarin control group,myocardial ischemic injury group and myocardial ischemic injury rats treated with puerarin group were 0.97±0.02,0.98±0.04,1.15±0.04,0.99±0.03 (n=10 for each group), respectively. The average OD of P2X3 protein expression in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury rats treated with puerarin group(p<0.05), saline control group (p<0.05), puerarin control group(p<0.01; F(3, 36)=52.418). No difference was found in the intensity of P2X3 protein expression of SCG among saline control group, puerarin control group and myocardial ischemic injury rats treated with puerarin group (p>0.05;F(2,27)=0.552). The average ODs of P2X3 protein expression in the SG in saline control group, puerarin control group, myocardial ischemic injury group and myocardial ischemic injury rats treated with puerarin group were 0.99±0.02,0.98±0.02,1.11±0.05,0.99±0.03 (n=10 for each group), respectively. The average OD of P2X3 protein expression in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury rats treated with puerarin group(p<0.05), saline control group (p<0.05), puerarin control group(p<0.01; F(3,36)=26.192). No difference was found in the intensity of P2X3 protein expression of SG among saline control group, puerarin control group and myocardial ischemic injury rats treated with puerarin group(p>0.05;F(2,27)=1.043).Conclusions: Puerarin could decrease the expressions of P2X3 receptor in SCG, SG neurons and myocardial sympathetic nerves in myocardial ischemia rats, and then reduce systolic blood pressure, slow down heart rate and respiration in myocardial ischemia injury rats. These results suggest that puerarin may depress sympathoe- xcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X3 receptor in rat superior cervical and stellate ganglia.
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