| Backround and Objective:Coronary heart disease and myocardial infarction is clinically common diseases. During myocardial ischemia, a number of cell types release large quantities of ATP in the extracellular or interstitial space, which activates cardiac afferent nerve, characterized by an increase in blood pressure and sympathetic nerve activity. The strengthen sympathoexcitatory reflex may increase the blood pressure and heart rates, which enhanced ischemic myocardiaol injury. ATP activates P2X receptors to produce functions. Previous research works in our laboratory showed that P2X3 and P2X2/3 receptors of cervical sympathetic ganglia contributed to the myocardial ischemic nociceptive response. Baicalin is the major flavonoid components of the root of Scutellaria baicalensis and known for its strong anti-inflammatory properties, anti-oxidant properties, anti-viral properties, anti-cancer properties. It was extensively researched for utility in a number of therapeutic areas as an important medical agent. It is not clear for the role of baicalin in the sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X2/3 receptor in rat cervical sympathetic ganglia. The present study was aimed to observe the changes of rat systolic blood pressure, heart rate in the myocardial ischemic rats and the changes of P2X2/3 receptor expression value in superior cervical ganglion (SCG), stellate ganglion (SG) neurons and heart after treated with baicalin and to explore the effect mechanisms of baicalin on sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X2/3 receptor in rat SCG and SG neurons.Methods:(1)A rat model of myocardial ischemia injury was established by ligating the left anterior descending coronary artery. Electrocardiogram (ECG) and H-E stain of cardiac tissue were used to observe the effect of baicalin on heart function and cardiac tissue in rat myocardial ischemia injury. (2)The blood pressure and heart rates were measured by non-invasive blood pressure determinator (the indirect tail-cuff plethysmography) to observe the effect of baicalin on the blood pressure and heart rates in rat myocardial ischemia injury. (3)CK-MB and cTn-T(cardiac muscle enzymes) were measured cardiac muscle enzymed to observe the effect of baicalin on myocardial ischemia injury. (4)The coexpressions of P2X3 and P2X2 receptors in rat SCG, SG neurons and heart of different groups were detected by immunofluorescence. (5)The expression of P2X3 and P2X2 receptors protein in rat SCG, SG neurons and heart were analyzed by western blotting and immunohistochemistry to observe the effect of baicalin on myocardial ischemia injury. (6)The expression of P2X3 and P2X2 receptors mRNA in rat SCG, SG neurons and heart were analyzed by RT-PCR to observe the effect of baicalin on myocardial ischemia injury. (7)Epinephrine was measured by ELISA. (8)ATP was measured by CellTiter-Glo.Results:(1)After ligating the left anterior descending coronary artery, ST segment in the ECG was high upward. After 14 days, the abnormal Q wave appeared obviously in myocardial ischemic rats. HE stain showed significant bleeding, myocardial atrophy, fibrosis and necrosis. Myocardial tissues in myocardial ischemic group showed severe atrophy, hypertrophy and proliferation of fiber connective conditions. After treated with baicalin, abnormal Q wave and myocardial issue changes induced by myocardial ischemia were improved. (2)Compared with control rats systolic blood pressure, diastolic blood pressure and heart rate in the myocardial ischemic rats were increased (n=5)(p<0.05). After the treatment with baicalin in the myocardial ischemic rats, systolic blood pressure, diastolic blood pressure and heart rate were lower than those in the myocardial ischemic rats (n=5) (p<0.05). There is no significant differences between normal group, sham group and baicalin control group (n=5)(p>0.05). Systolic blood pressure, diastolic blood pressure and heart rate in the myocardial ischemic rats treated baicalin were higher than those in control groups(n=5)(p<0.05). (3)CK-MB and cTn-T of blood serum in the myocardial ischemic rats were increased compared with those in control rats (n=5)(p<0.05). CK-MB and cTn-T of blood serum in the ischemic rats treated baicalin were lower than those in ischemic rats(n=5)(p<0.05). There is no significant differences between normal group, sham group and baicalin control group (n=5)(p>0.05). (4)Double-label immunofluorescence results showed that P2X3 and P2X2 receptors were co-expressed in SCG, SG neurons and myocardial tissues. The co-expressed cell of P2X3 and P2X2 receptors in myocardial ischemic injury group exhibited more intense staining than those in control groups. The co-expressed value in the ischemic rats treated baicalin were lower than those in ischemic rats(n=5)(p<0.05). (5)The expression value of P2X3 and P2X2 imunoreactivity in SCG, SG neurons and myocardial tissues was studied by immunohistochemistry. In the SCG, SG neurons and myocardial tissues, the average optical density (ODs) of P2X3 receptor expression in myocardial ischemic group was significantly higher than those in myocardial ischemic injury treated with baicalin and control group(p<0.05). The data in myocardial ischemic injury treated with baicalin group were higher than those in control groups(p<0.05). No difference was found in the intensity of P2X3 receptor between normal group, sham group and baicalin control group(p>0.05). In the SCG, SG neurons and myocardial tissues, the average optical density (ODs) of P2X2 receptor expression in myocardial ischemic group was significantly higher than those in myocardial ischemic injury treated with baicalin and control groups(p<0.05). The data in myocardial ischemic injury treated with baicalin group were higher than those in normal group, sham group and baicalin control group (p<0.05). No difference was found in the intensity of P2X2 receptor between normal group, sham group and baicalin control group (p>0.05). (6)The expression value of P2X3 and P2X2 mRNA in SCG, SG neurons and myocardial tissues was studied by RT-PCR. The average OD of P2X3 mRNA in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury treated with baicalin, normal group, sham group and baicalin control group (p<0.05). The average OD of P2X2 mRNA in myocardial ischemic injury group was significantly higher than those in myocardial ischemic injury treated with baicalin and control groups (p<0.05).(7)The P2X3 and P2X2 protein level was analyzed by western blotting. The average ODs of P2X3 and P2X2 protein expression (normalized to each (3-actin internal control) in SCG, SG neurons and myocardial tissues of myocardial ischemic group was significantly higher than those in myocardial ischemic injury treated with baicalin group and control group (p<0.05). The average ODs of P2X3 and P2X2 protein expression in myocardial ischemic injury treated with baicalin group is lower than those in myocardial ischemic group. (8)The concentration of epinephrine in rat serm is tested by ELISA. The results show that the concentration of epinephrine in myocardial ischemic group was significantly higher than those in myocardial ischemic injury treated with baicalin group and control group (p<0.05, F=367.943). The concentration of epinephrine in myocardial ischemic injury treated with baicalin was lower than that in myocardial ischemic group (p<0.05). No difference was found in the concentration of epinephrine between normal group, sham group and baicalin control group (p>0.05). (9)The concentration of ATP in rat serm is tested by CellTiter-Glo. The concentration of ATP in myocardial ischemic group was significantly higher than those in myocardial ischemic injury treated with baicalin group and control group (p<0.05,F=8.954). The concentration of epinephrine in myocardial ischemic injury treated with baicalin was lower than that those in myocardial ischemic group (p<0.05). No difference was found in the concentration of ATP between normal group, sham group and baicalin control group (p>0.05).Conclusions:Baicalin could decrease the expressions of P2X3 and P2X2 receptor mRNA and protein in cervical sympathetic ganglia and myocardial sympathetic nerves of myocardial ischemia rats. These results suggest that baicalin may decrease sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X2/3 receptor in rat cervical sympathetic ganglia to depress blood pressure and heart rate and produce the protection of heart. |
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