ABSTRACT
Objective:To evaluate the relationship between silence information regulator 1 (SIRT1) and signal transducers and activators of transcription 3 (STAT3) acetylation during high glucose-induced cardiac microvascular endothelial cell injury.Methods:Cardiac microvascular endothelial cells of Sprague-Dawley rats were cultured.The cells at the logarithmic growth phase were selected and divided into 3 groups ( n=24 each) using a random number table method: control group (C group), high glucose group (HG group) and high glucose+ SIRT1 agonist SRT1720 group (HG+ SRT group). The cardiac microvascular endothelial cells were seeded in a 6- or 96-well cell culture plate at a density of 2×10 5 cells/ml.When the cell density reached 50%, the culture medium was then replaced with high-glucose (glucose 33 mmol/L) DMEM culture medium containing with 10% fetal bovine serum and 1% double antibody in HG and HG+ SRT groups.In group HG+ SRT, 20 μmol/L SRT1720 was added simultaneously, and the cells were cultured at 37 ℃ in an incubator with 5% CO 2 for 24 h. The cell viability was determined by CCK-8 assay, the activity of superoxide dismutase (SOD) was detected using a spectrophotometer, the levels of lactic dehydrogenase (LDH), interleukin-6 (IL-6) and tumor necrosis factor-β (TNF-β) in the supernatant were detected by enzyme-linked immunosorbent assay, and the expression of SIRT1, acetylated STAT3 (ac-STAT3) and phosphorylated STAT3 (p-STAT3) was determined by Western blot. Results:Compared with C group, the cell viability and SOD activity were significantly decreased, levels of LDH, IL-6 and TNF-β in the supernatant were increased, expression of SIRT1 was down-regulated, and expression of ac-STAT3 and p-STAT3 was up-regulated in group HG and group HG+ SRT ( P<0.05). Compared with group HG, the cell viability and SOD activity were significantly increased, levels of LDH, IL-6 and TNF-β in the supernatant were decreased, expression of SIRT1 was up-regulated, and expression of ac-STAT3 and p-STAT3 was down-regulated in group HG+ SRT ( P<0.05). Conclusion:SIRT1 can alleviate high glucose-induced cardiac microvascular endothelial cell injury by promoting STAT3 deacetylation.
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Objective:To evaluate the relationship between protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway-mediated endoplasmic reticulum stress and the reduction of cerebral ischemia-reperfusion (I/R) injury by dexmedetomidine in mice by the in vivo experiment and the cell experiment. Methods:In the in vivo experiment, 20 healthy clean-grade male mice, aged 6-8 weeks, weighing 20-30 g, were divided into 4 groups ( n=5 each) using a random number table method: sham operation group (group S), sham operation+ dexmedetomidine group (group SD), cerebral I/R group (group IR) and cerebral I/R+ dexmedetomidine group (group IRD). Cerebral I/R was established by two-vessel occlusion plus hypotension.Dexmedetomidine 25 μg/kg was intraperitoneally injected at 10 min of ischemia in group IRD and at the corresponding time point in group SD.Neurological function was assessed using modified neurological severity score at 1 h of reperfusion.The animals were then sacrificed and brain tissues were taken for determination of the expression of endoplasmic reticulum stress-related proteins such as immunoglobulin heavy chain-binding protein (BIP), eukaryotic translation initiation factor 2α (EIF-2α), phosphorylated EIF-2α (p-EIF-2α), PERK and phosphorylated PERK (p-PERK) (by Wester blot). In the cell experiment, a mouse hippocampal neuronal cell line was selected and divided into 4 groups ( n=12 each) using a random number table method: control group (group C), oxygen-glucose deprivation/restoration (OGD/R) group (group OGD/R), OGD/R+ dexmedetomidine group (group OGD/R+ D) and OGD/R+ ISRIB (PERK pathway inhibitor) group (group OGD/R+ ISRIB). Cells were exposed to 94%N 2-5%CO 2-1%O 2 and incubated in a low-glucose DMEM medium for 6 h followed by restoration to establish OGD/R model.At 30 min before OGD, dexmedetomidine (final concentration 5 mmol/L) was added in group OGD/R+ D, and ISRIB (final concentration 10 mmol/L) was added in group OGD/R+ ISRIB.After 12-h restoration was completed, the cell survival rate was detected by CCK-8 assay.At 24 of restoration, the expression of endoplasmic reticulum stress-related proteins was determined by Wester blot. Results:In the in vivo experiment, compared with group S, neurobehavioral score was significantly increased and the expression of BIP, p-EIF-2α and p-PERK in brain tissues was up-regulated in group IR ( P<0.05). Compared with group IR, neurobehavioral score was significantly decreased and the expression of BIP, p-EIF-2α and p-PERK in brain tissues was down-regulated in group IRD ( P<0.05). In the cell experiment, compared with group C, the expression of BIP, p-EIF-2α, PERK and p-PERK was significantly up-regulated, and the cell survival rate was decreased in group OGD/R ( P<0.05). Compared with group OGD/R, the expression of BIP, p-EIF-2α, PERK and p-PERK was significantly down-regulated, and the cell survival rate was increased in OGD/R+ D, OGD/R+ ISRIB groups ( P<0.05). Compared with group OGD/R+ ISRIB, the expression of PERK was significantly down-regulated ( P<0.05) and no significant change was found in the other parameters in group OGD/R+ D ( P>0.05). Conclusion:The mechanism by which dexmedetomidine reduces cerebral I/R injury may be related to inhibiting PERK pathway-mediated endoplasmic reticulum stress in mice.
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Objective:To evaluate the relationship between silence information regulator 1 (SIRT1) and NOD-like receptor protein 3 (NLRP3) inflammasomes during myocardial ischemia-reperfusion (I/R) in diabetic rats.Methods:SPF-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-220 g, were used in this study.Type 1 diabetes mellitus was induced by intraperitoneal 1% streptozotocin diluted in citrate buffer solution 60 mg/kg.The rats were continuously fed for 8 weeks after successful establishment of the model.Forty-two rats with type 1 diabetes mellitus were divided into 4 groups by a random number table method: diabetic sham operation group (DS group, n=6), diabetic myocardial I/R group (DIR group, n=12), diabetic myocardial I/R plus SIRT1 agonist SRT1720 group (DIR+ SR group, n=12) and diabetic myocardial I/R plus SIRT1 inhibitor EX-527 group (DIR+ EX group, n=12). Eighteen non-diabetic rats were divided into 2 groups by a random number table method: non-diabetic sham operation group (NS group, n=6) and non-diabetic myocardial I/R group (NIR group, n=12). The myocardial I/R model was established by ligation of the left anterior descending coronary artery for 30 min followed by 120-min reperfusion in anesthetized rats.Blood samples were collected from the carotid artery immediately after the end of reperfusion for determination of serum troponin I (cTnI), creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) levels (by enzyme-linked immunosorbent assay). Then the rats were sacrificed, and myocardial tissues were obtained for determination of myocardial infarct size (by TTC method) and expression of SIRT1, NLRP3 and IL-1β (by Western blot) and for microscopic examination of pathological changes of myocardial tissues (by HE staining). The percentage of myocardial infarct size was calculated. Results:Compared with group NS, the serum levels of cTnI, CK-MB and LDH were significantly increased, the expression of SIRT1 in myocardial tissues was down-regulated, and the expression of NLRP3 and IL-1β was up-regulated in group NIR ( P<0.05). Compared with group DS, the serum levels of cTnI, CK-MB and LDH were significantly increased, the expression of SIRT1 in myocardial tissues was down-regulated, and the expression of NLRP3 and IL-1β was up-regulated in group DIR ( P<0.05). Compared with group NIR, the percentage of myocardial infarct size and serum levels of cTnI, CK-MB and LDH were significantly increased, the expression of SIRT1 in myocardial tissues was down-regulated, the expression of NLRP3 and IL-1β was up-regulated ( P<0.05), and the pathological changes were accentuated in group DIR.Compared with group DIR, the percentage of myocardial infarct size and serum levels of cTnI, CK-MB and LDH were significantly decreased, the expression of SIRT1 in myocardial tissues was up-regulated, the expression of NLRP3 and IL-1β was down-regulated ( P<0.05), and the pathological changes were significantly attenuated in group DIR+ SR, and the percentage of myocardial infarct size and serum levels of cTnI, CK-MB and LDH were significantly increased, the expression of SIRT1 in myocardial tissues was down-regulated, the expression of NLRP3 and IL-1β was up-regulated ( P<0.05), and the pathological changes were accentuated in group DIR+ EX. Conclusion:The up-regulated expression of SIRT1 can inhibit the activation of NLRP3 inflammasomes and produces endogenous protection during myocardial I/R in diabetic rats.
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Objective:To evaluate the role of c-Abl in myocardial ischemia-reperfusion (I/R) injury in diabetic rats.Methods:Sixty male SPF-grade healthy Sprague-Dawley rats, aged 7-9 weeks, weighing 210-230 g, were divided into 6 groups by a random number table method: sham operation group (S group, n=6), myocardial I/R group (IR group, n=12), diabetic sham operation group (DS group, n=6), diabetic myocardial I/R group (DIR group, n=12), diabetic myocardial I/R plus AVV9-siRNA-c-Abl group (DIR+ c-Abl group, n=12), and diabetic myocardial I/R plus AVV9-GFP group (DIR+ GFP group, n=12). One percent streptozotocin 60 mg/kg was intraperitoneally injected to induce type 1 diabetes mellitus.AVV9-siRNA-c-Abl and AVV9-GFP 1×10 12 mg/kg were slowly injected at 4 weeks after establishing the model in DIR+ c-Abl and DIR+ GFP groups.Myocardial ischemia was induced by 30 min occlusion of left anterior descending branch (LAD) of coronary artery followed by 2 h reperfusion at 8 weeks after establishing the model.At the end of reperfusion, left ventricular systolic pressure (LVSP) and the maximum rate of increase and decrease of left ventricular systolic pressure (±dp/dt max) were monitored, and blood samples were collected for determination of the concentrations of serum lactic dehydrogenase (LDH) and creatine kinase-MB (CK-MB) (by enzyme-linked immunosorbent assay)and myocardial infarct size (except group S and group DS). Myocardial tissues were obtained for determination of the apoptosis index of cardiomyocytes(by TUNEL staining), expression of c-Abl, p53 and activated caspase-3 (by Western blot), and binding of c-Abl and p53 (c-Abl/p53) (by co-immunoprecipitation method). Results:LVSP and ±dp/dt max were significantly decreased, serum LDH and CK-MB concentrations were increased, apoptosis index and c-Abl/p53 were increased, and the expression of c-Abl, p53 and activated caspase-3 was up-regulated in group IR when compared with group S and in group DIR as compared with group DS ( P<0.05). Compared with group DIR, the LVSP and ± dp/dt max were significantly increased, serum LDH and CK-MB concentrations were decreased, myocardial infarct size was decreased, apoptosis index and c-Abl/p53 were decreased, and the expression of c-Abl, p53 and activated caspase-3 was down-regulated in group DIR+ c-Abl ( P<0.05), and no significant change was found in the parameters mentioned above in group DIR+ GFP ( P>0.05). Conclusion:c-Abl is involved in the pathophysiological process of myocardial I/R injury probably by activating p53 signaling pathway in diabetic rats.