ABSTRACT
Objective:To evaluate the effect of superior cervical ganglion block (SCGB) on cardiac function and nucleotide like receptor protein 3 (NLRP3) signaling pathway in a rat model of myocardial ischemia-reperfusion (I/R).Methods:Sixty healthy SPF male Sprague-Dawley rats, weighing 250-300 g, aged 2-3 months, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (sham group), myocardial I/R group (IR group), myocardial I/R + normal saline group (IR+ NS group), and myocardial I/R + SCGB group (IR+ SCGB group). Myocardial I/R model was developed by ligation of the left anterior descending branch of the coronary artery for 45 min followed by restoration of blood flow in anesthetized aninals. IR+ SCGB group received SCGB (0.25% ropivacaine 0.1 ml) at 10 min before reperfusion once a day for 2 consecutive weeks, while 0.9% sodium chloride was given instead of ropivacaine in IR+ NS group. Blood samples were collected at 24 h and 14 days of reperfusion for determination of serum concentrations of norepinephrine (NE), troponin T (TnT), tumor necrosis factor-alpha (TNF-α), interleukin-18 (IL-18) and IL-1β by enzyme-linked immunosorbent assay. Echocardiography was performed before ischemia and at 14 days of reperfusion, and left ventricular short axis shortening rate (FS), ejection fraction (EF), and cardiac output (CO) were measured. The rats were sacrificed at 14 days of reperfusion and the hearts were taken for determination of the contents of norepinephrine (NE) in myocardial tissues in the infarction area (by enzyme-linked immunosorbent assay), percentage of myocardial fibrosis area (by Masson staining), M1 macrophage marker CD68 + cell count in the infarction area (by immunohistochemical method), and expression of NLRP3 and gasdermin D (GSDMD) in myocardial tissues (by Western blot). Results:Compared with Sham group, the serum concentrations of TnT, TNF-α, IL-18 and IL-1β, percentage of myocardial fibrosis area, and NE levels in serum and myocardial tissues were significantly increased, the expression of NLRP3 and GSDMD in myocardial tissues was up-regulated, CD68 + cell count was increased, and EF, CO and FS were decreased in IR group ( P<0.05). Compared with IR group, the serum concentrations of TnT, TNF-α, IL-18 and IL-1β, percentage of myocardial fibrosis area, and NE levels in serum and myocardial tissues were significantly decreased, the expression of NLRP3 and GSDMD in myocardial tissues was down-regulated, CD68 + cell count was decreased, and EF, CO and FS were increased in IR+ SCGB group ( P<0.05), and no statistically significant changes were found in the parameters mentioned above in IR+ NS group ( P>0.05). Conclusions:SCGB can improve the cardiac function in a rat model of myocardial I/R, and the mechanism may be related to the inhibition of NLRP3 signaling pathway.
ABSTRACT
Objective:To evaluate the role of NIMA-related kinase 7 (NEK7)/Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) signaling pathway in sepsis-associated encephalopathy in mice.Methods:A total of 150 healthy adult male C57BL/6 mice, aged 8-12 weeks, weighing 20-25 g, were divided into 5 groups ( n=30 each) by a random number table method: sham operation group (Sham group), sepsis group (CLP group), sepsis+ NLRP3 inhibitor MCC950 group (CLP+ MCC950 group), sepsis+ NEK7 siRNA group (CLP+ NEK7 siRNA group), and sepsis+ NC siRNA group (CLP+ NC siRNA group). Sepsis was induced by classic cecal ligation and puncture (CLP) in anesthetized animals.MCC950 10 mg/kg was intraperitoneally injected for 3 consecutive days after operation in CLP+ MCC950 group, while the equal volume of normal saline was given instead in Sham group.Immediately after operation and on 3rd day after operation, NEK7 siRNA 3 nmol/20 g was injected into the ventricle in CLP+ NEK7 siRNA group, and the equal dose of NC siRNA was injected into the ventricle instead in Sham group.The survival of mice was recorded on 4th postoperative day.On 4th and 7th days after operation, 10 mice in each group were selected for Y maze space recognition experiment.On 7th day after operation, 5 mice in each group were randomly sacrificed and hippocampal tissues were taken for determination of the contents of interleukin-1beta (IL-1β), interleukin-18 (IL-18) and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay), and 6 mice in each group were sacrificed and hippocampal tissues were taken for determination of the expression of NEK7, NLRP3, cleaved-caspase-1 and apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) (by Western blot). Results:The survival rates were 100%, 50%, 73%, 60% and 53% in Sham, CLP, CLP+ MCC950, CLP+ NEK7 siRNA, and CLP+ NC siRNA groups, respectively, on day 4 after surgery.Compared with Sham group, the frequency of entries into novel arm was significantly reduced, and the time spent in the novel arm was shortened at 4th and 7th days after operation, and the contents of IL-1β, IL-18 and TNF-α in hippocampus were increased, and the expression of NEK7, NLRP3, cleaved-caspase-1 and ASC was up-regulated at 7th day after operation in CLP group ( P<0.05). Compared with CLP group, the frequency of entries into novel arm was significantly increased, and the time spent in the novel arm was prolonged at 4th and 7th days after operation, and the contents of IL-1β, IL-18 and TNF-α in hippocampus were decreased at 7th day after operation in CLP+ MCC950 and CLP+ NEK7 siRNA groups, the expression of NLRP3, cleaved-caspase-1 and ASC was significantly down-regulated at 7th day after operation in CLP+ MCC950 group, the expression of NEK7, NLRP3, cleaved-caspase-1 and ASC was significantly down-regulated in CLP+ NEK7 siRNA group ( P<0.05), and no significant change was found in the parameters mentioned above in CLP+ NC siRNA group ( P>0.05). Conclusions:NEK7/NLRP3 signaling pathway is involved in SAE in mice, and the underlying mechanism may be related to promotion of inflammatory responses.