摘要
Objective:To evaluate the effect of verbascoside on cold ischemia-reperfusion injury following heterotopic heart transplantation in mice and the relationship with nuclear factor kappa B (NF-κB)/nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) signaling pathway.Methods:This experiment was performed in two parts. Part Ⅰ animal experiment Eighteen SPF healthy male C57BL/6 mice, aged 6-8 weeks, weighing 24-28 g, were divided into 3 groups ( n=6 each) by the random number table method: control group (C group), cold ischemia-reperfusion (I/R) group (I/R group) and cold I/R + verbascoside group (I/R+ VB group). Cold I/R injury model of mouse heart transplantation was prepared by neck heterotopic heart transplantation using the modified non-suture cuff technique. The donor heart in group C was immediately transplanted to the recipient after removal, while the donor heart in group I/R was stored in the 4 ℃ University of Wisconsin solution for 8 h before transplantation to the recipient, and verbascoside 20 mg/kg was intraperitoneally injected at 3 days before surgery in donor and recipient mice in I/R+ VB group. At the end of reperfusion, the myocardial tissue of the transplanted heart was obtained after assessing the beating score for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity by enzyme-linked immunosorbent assay. Part of the donor myocardium was taken for examination of the pathological results. The expression of NF-κB, p-NF-κB, NOD-like receptor protein 3 (NLRP3), ACS and caspase-1 was detected by Western blot. The expression of IL-1β, TNF-α and IL-6 mRNA was detected by real-time polymerase chain reaction. Part Ⅱ cell experiment Rat cardiomyocyte H9c2 cold hypoxia-reoxygenation model was developed, and the cells were divided into 3 groups ( n=24 each) by the random number table method: control group (C group), cold hypoxia-reoxygenation group (H/R group), and cold hypoxia-reoxygenation+ verbascoside group (H/R+ VB group). The cells were exposed to hypoxia for 18 h at 10 ℃ followed by restoration of reoxygenation for 24 h at 37 ℃ to develop the cold hypoxia-reoxygenation model. The cell viability and LDH activity were determined. The expression of NF-κB and NLRP3 was detected by Western blot, and the expression of phosphorylated NF-κB (p-NF-κB) was detected by immunofluorescence staining. Results:Part Ⅰanimal experiment Compared with C group, the MDA content was significantly increased, the beating score of grafts and SOD activity were decreased, and the expression of p-NF-κB, NLRP3, ACS and caspase-1 and IL-1β, TNF-α and IL-6 mRNA was up-regulated ( P<0.05), and myocardial histopathological injury was aggravated in I/R group. Compared with I/R group, the content of MDA was significantly decreased, the beating score of grafts and SOD activity were increased, the expression of p-NF-κB, NLRP3, ACS and caspase-1 and IL-1β, TNF-α and IL-6 mRNA was down-regulated ( P<0.05), and the myocardial histopathological injury was alleviated in I/R+ VB group. Part Ⅱ cell experiment Compared with C group, the cell viability was significantly decreased, the activity of LDH was increased, and the expression of p-NF-κB, NLRP3, ACS, caspase-1 and p-NF-κB was up-regulated in H/R group ( P<0.05). Compared with H/R group, the cell viability was significantly increased, the activity of LDH was decreased, and the expression of p-NF-κB, NLRP3, ACS, caspase-1 and p-NF-κB was down-regulated in H/R+ VB group ( P<0.05). Conclusions:Verbascoside can alleviate cold I/R injury following heterotopic heart transplantation in mice, and the mechanism may be related to inhibition of activation of NF-κB/NLRP3 signaling pathway.
摘要
Objective:To evaluate the role of bilateral superior cervical sympathetic ganglia (SCG) in myocardial ischemia-reperfusion (I/R) injury in mice and the relationship with NOD-like receptor protein 3 (NLRP3) inflammasomes.Methods:Thirty-two healthy SPF male C57BL mice, aged 8-10 weeks, weighing 25-30 g, were divided into 4 groups ( n=8 each) by the random number table method: sham operation group (NS group), myocardial I/R group (NIR group), bilateral SCG excision group (SCGx group) and bilateral SCG excision + myocardial I/R group (SCGx+ IR group). The myocardial I/R injury model was prepared by ligating the anterior descending branch of the left coronary artery for 30 min followed by 24 h reperfusion in isoflurane-anesthetized mice. Bilateral superior cervical sympathectomy was performed at 3 days before reperfusion. Blood samples were collected from the inferior vena cava at 24 h of reperfusion for examination of pathological changes (by HE and WGA staining) and for measurement of serum creatine kinase isoenzymes (CK-MB) activity, cardiac troponin I (cTnI) concentration, norepinephrine (NE) concentration and lactic dehydrogenase (LDH) activity (by enzyme-linked immunosorbent assay), superoxide dismutase (SOD) activity (by colorimetric method), myocardial reactive oxygen species (ROS) level (by DHE method), myocardial infarct size(by TTC method), and expression of interleukin-1beta (IL-1β), IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), NLRP3 mRNA (by quantitativepolymerase chain reaction ), and expression of tyrosine hydroxylase (TH), IL-1β, TNF-α, NLRP3, atrial natriuretic peptide (ANP)and brain natriuretic peptide (BNP) (by Western blot). Results:Compared with NS group, the NE concentration was significantly decreased, and TH expression was down-regulated in SCGx group, and the serum CK-MB activity, concentrations of cTnI and NE, LDH activity and myocardial ROS level were significantly increased, SOD activity was decreased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was up-regulated, and the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was up-regulated in NIR group ( P<0.05). Compared with SCGx group, the serum CK-MB activity, concentrations of cTnI and NE, LDH activity and myocardial ROS levels were significamtly increased, SOD activity was decreased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was up-regulated, and the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was up-regulated in SCGx+ NIR group ( P<0.05). Compared with NIR group, the serum CK-MB activity, cTnI concentration, LDH activity and myocardial ROS level were significantly decreased, SOD activity was increased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was down-regulated, the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was down-regulated, and myocardial infarct size was decreased in SCGx+ NIR group ( P<0.05). Conclusions:The mechanism by which bilateral SCG excision attenuates myocardial I/R injury is associated with decreased NLRP3 inflammatory inflammasome activation and inhibition of inflammatory responses in mice.
摘要
Objective:To evaluate the role of ferroptosis in hypoxia-reoxygenation (H/R) injury in cardiomyocytes cultured in high-fat high-glucose (HFHG) medium.Methods:Cardiomyocytes H9c2 cells were commonly cultured and divided into 3 groups ( n=20 each) using a random number table method: control group (C group), HFHG-H/R group and Ferrostatin-1 (Fer-1) plus HFHG-H/R group (Fer-1+ HFHG+ H/R group). H9c2 cells were cultured in a HFHG medium for 12 h and then exposed to 1%O 2-5%CO 2-94%N 2 for 4 h, followed by 2 h reoxygenation in a cell incubator.Fer-1 at a final concentration of 10 μmol/L was added while the cells were cultured in the HFHG medium in group Fer-1+ HFHG+ H/R.At 2 h of reoxygenation, the cell viability was measured using CCK-8 assay, the activity of lactate dehydrogenase (LDH) in the supernatant was measured using 2, 4-dinitrophenylhydrazine color method, the activity of reactive oxygen species (ROS) was measured by fluorescent probe DCFH-DA flow cytometry, and the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), nuclear receptor coactivator 4 (NCOA4), and glutathione peroxidase 4 (GPX4) was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the activities of LDH release and ROS were increased, and the expression of ACSL4 and NCOA4 was up-regulated ( P<0.05), and no significant change was found in the expression of GPX4 in group HFHG+ H/R ( P>0.05). Compared with group HFHG+ H/R, the cell activity was significantly increased, the activities of LDH and ROS were decreased, and the expression of ACSL4 and NCOA4 was down-regulated ( P<0.05), and no significant change was found in the expression of GPX4 in Fer-1+ HFHG+ H/R group ( P>0.05). Conclusions:Ferroptosis is involved in the process of H/R injury in cardiomyocytes cultured in HFHG medium.