| Background:Cardiovascular disease affects human health and quality of life. Acute myocardial infarction (AMI) is a common cardiovascular disease, in particular, the morbidity and mortality due to AMI is increasing gradually. So far, how to prevent AMI injury and to find drug targets has become one research focus. The research of central nerve system pathogenesis and treatment strategies focus on hypothalamic paraventricular nuclei (PVN) and rostral ventrolateral medulla (RVLM), which regulate AMI injury via relative inflammation reaction or oxidative stress signal pathway. Moreover, the regulative mechanism of the PVN on AMI injury is complex and bewildering. In our present study, we demonstrated that P2X7receptor promote proinflammatory cytokines (such as IL-1βand TNF-a) release. Whether central proinflammatory cytokines is involved in the regulative effects on AMI injury? Whether P2X7receptor is involved in the process of proinflammatory cytokines synthesis? And how does the central P2X7receptor modulate sympathetic outflow to regulate the AMI injury? Recently, these detailed molecular mechanisms are still not so clear.Objectives:In our present study, the AMI rat model was adopted to investigate the regulative effects and molecular mechanism of microglial P2X7receptor in the PVN on AMI injury via influencing renal sympathetic nerve activity (RSNA). Based on the morphology and molecular biology, we observed the expressions of P2X7receptor in the PVN between Sham group and AMI group. In order to confirm the paraventricular nuclei P2X7receptor was involved in regulating the AMI injury, we observed the changes of RSNA and cardiac function between Sham group and AMI group. Meanwhile, in order to investigate which kinds of neurons were involved in regulating the AMI injury, we observed the expressions of oxytocin and vasopressin in the PVN between AMI group and AMI+P2X7R-SiRNA group. Finally, in order to investigate furtherly the related signaling pathway mediated by P2X7receptor in regulating AMI injury, we observed the expressions of proinflammatory cytokine (such as IL-1β and TNF-a) and upstream regulatory proteins of proinflammatory cytokines (such as Caspase-1and P65-NF-κB) in the PVN.Methods:The AMI rat model was performed by ligation of the left anterior descending coronary artery. Meanwhile, the MAP, HR, IVP and LVEDP were monitored among Sham, AMI, AMI+P2X7R-siRNA, AMI+BBG groups. In order to detect whether the AMI rat model was performed successly, the ischemic area and the micro-structure of ischemic myocardium were detected by tetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. Confocal laser microscopy together with the double-label immunofluorescence was applied to detect the colocalization between microglial marker Iba-1and P2X7receptor in the PVN. Immunohistochemistry (IHC) and western blot analysis were used to detect the expression of P2X7, IL-1β and TNF-a in the PVN among Sham, AMI, AMI+P2X7R-siRNA, AMI+BBG groups. Meanwhile, the concentration of IL-1β and TNF-a in the PVN tissue was measured by enzyme-linked immunosorbent assay (ELISA). The biological information collected system was used to detect hemodynamic and RSNA, the heart rate variability (HRV) analysis system was used to analyse the changes of cardiac autonomic nervous tones in AMI rats. Immunohistochemistry was used to detect the expressions of oxytocin and vasopressin and upstream regulatory proteins of proinflammatory cytokines (such as Caspase-1and P65-NF-κB) in the PVN between AMI group and AMI+P2X7R-siRNA group.Results:(1) Preparation of AMI modelAfter the AMI rat model was performed, the S-T segment was elevated immediately, the myocardium presenting pale and a poor weakening movement. TTC staining showed the ischemic myocardium remained pale. HE staining showed that AMI injury was aggratated gradually followed by cardiac muscle fibers expressed in a time-dependent manner. These results confirmed the success of duplicating AMI rat model.(2) The colocalization between P2X7receptor and Iba-1Confocal laser microscopy showed that the P2X7receptor was co-expressed with microglial marker Iba-1in the PVN. The presence of P2X7R-IR throughout the confines of the PVN was extensive, and about80%of P2X7R immunoreactivity (IR) was co-localized with microglia. Meanwhile, we found P2X7R-IR was absent from the cell nucleolus.(3) ATP bioluminescence assayCompared with the Sham group, the release of neurotransmitter ATP was increased rapidly in a time dependent manner (P<0.05, n=6). The level of ATP in the PVN was significantly increased during the8h myocardial ischemia period and reached the peak at4h. However, the ATP content was diminished gradually after8h myocardial ischemia time point.(4) The expression of P2X7, IL-1β and TNF-a in the PVNWestern blot analysis showed that the expressions of P2X7receptor in the PVN of AMI rats were significantly increased in a time dependent manner (P<0.05, n=6) and reached the peak at4h myocardial ischemia. After the microinjection of P2X7-siRNA into PVN or BBG (ip.), compared with that of the AMI group, the expression of mRNA and protein of P2X7R, IL-1β and TNF-a were significantly decreased in the PVN of AMI+P2X7-siRNA group rats or AMI+BBG group rats (P <0.05, n=6).(5) Assay of PVN tissue TNF-a and IL-1βCompared with that of the Sham group, the concentrations of IL-1β and TNF-a in the PVN tissue of AMI rats increased significantly (P<0.05, n=6). They were decreased in the AMI+P2X7-siRNA group and AMI+BBG group compared with that in the AMI group (P<0.05, n=6).(6) The expression of OT and VP positive neurons and the expression of Caspase-1and P65-NF-κB in the PVN of each groupThe numbers of OT and VP immunoreactive neurons in the PVN of AMI group were significantly increased compared with that of the sham operated group (P<0.05, n=6). Microinjection of P2X7-siRNA into the PVN could decrease the expression of OT and VP immunoreactive neurons (P<0.05, n=6). Compared with that of the Sham group, the expression of Caspase-1and P65-NF-κB in the PVN of AMI rats increased significantly (P<0.05, n=6). Microinjection of P2X7-siRNA into the PVN could decrease the expression of Caspase-1(P<0.05, n=6). However, microinjection of P2X7-siRNA into the PVN did not significantly decrease the expression of P65-NF-κB in the PVN of AMI rats.(7) Cardiac function and HRV assayCompared with AMI group, microinjection of P2X7-siRNA into the PVN or administration (i.p) of P2X7R antagonist BBG could decrease the MAP, HR, IVP and LVEDP, and also descented the LF/HF ratio (indicates autonomic nerves activity)(P<0.05, n=6).(8) The record of renal sympathetic nerve activity (RSNA)Compared with that of Sham group, the RSNA was enhanced in the AMI rats (P <0.05, n=6). Microinjection of P2X7-siRNA into the PVN or administration (i.p) of BBG attenuated the RSNA (P<0.05, n=6).Conclusions:(1) Li gating left anterior descending coronary artery successfully duplicate the rat model of AMI. Acute myocardial infarction not only induces sympathetic outflows but also induces the overexpression of microglial P2X7receptor in the PVN of rats, which may be related with cardiac afferent reflex. Meanwhile, the P2X7receptor overexpression enhanced RSNA that aggravate AMI injury.(2) Microinjection of P2X7-siRNA into the PVN or administration (i.p) of BBG attenuated the expression of P2X7, IL-1β and TNF-a, decreased the number of OT and AVP positive neurons in the PVN, and also attenuated the RSNA. The activated microglial P2X7receptor could promote the TNF-a and IL-1β release in the PVN, then the TNF-a and IL-1β may activate oxytocinergic and vasopressin neuron that regulate the renal sympathetic nerve activity.(3) Inflammatory cytokines release in the PVN of rats may be mediated by overexpression of the upstream regulatory protein Caspase-1and P65-NF-κB. The inflammatory cytokines sensitize oxytocinergic and vasopressin neuron in the PVN that regulate the renal sympathetic nerve activity, and then influence cardiac function in the AMI rat. |
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