BW373U86 upregulates autophagy by inhibiting the PI3K/Akt pathway and regulating the mTOR pathway to protect cardiomyocytes from hypoxia–reoxygenation injury

2020 ◽  
Vol 98 (10) ◽  
pp. 684-690
Author(s):  
Qianyi Liang ◽  
Xiaoling Huang ◽  
Chaokun Zeng ◽  
Dewei Li ◽  
Yongyong Shi ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Viability ◽  
Ischemia Reperfusion ◽  
Underlying Mechanism ◽  
Rat Cardiomyocytes ◽  
Expression Levels ◽  
Test Kit ◽  
Reoxygenation Injury ◽  
Δ Opioid Receptor ◽  
Hypoxia Reoxygenation

The purpose of this study was to explore the protective effect of BW373U86 (a δ-opioid receptor (DOR) agonist) on ischemia–reperfusion (I/R) injury in rat cardiomyocytes and its underlying mechanism. Primary rat cardiomyocytes were cultured and pretreated with BW373U86 for intervention. The cardiomyocytes were cultured under the condition of 94% N2 and 5% CO2 for 24 h to perform hypoxia culture and conventionally cultured for 12 h to perform reoxygenation culture. The cell viability of cardiomyocytes was detected by an MTT assay (Sigma–Aldrich). The autophagy lysosome levels in cardiomyocytes were evaluated by acidic vesicular organelles with dansylcadaverine (MDC) staining (autophagy test kit, Kaiji Biology, kgatg001). The protein expression levels of LC3, p62, and factors in the PI3K/Akt/mTOR signaling pathway were detected by Western blot. Pretreatment with BW373U86 could improve the cell viability of cardiomyocytes with hypoxia–reoxygenation (H/R) injury (p < 0.05). Interestingly, after coculture of BW373U86 and PI3K inhibitor (3-methyladenine), the protein expression levels of p-Akt in cardiomyocytes were markedly increased in comparison with those in the BW373U86 group (p < 0.05). However, there were no significant differences in the protein expression levels of mTOR between the coculture group and the BW373U86 group (p > 0.05). BW373U86 upregulated autophagy to protect cardiomyocytes from H/R injury, which may be related to the PI3K/Akt/m TOR pathway.

Effects of Gingko biloba Extract on Gap Junction Changes Induced by Reperfusion/Reoxygenation After Ischemia/Hypoxia in Rat Brain

2005 ◽  
Vol 33 (06) ◽  
pp. 923-934 ◽  
Author(s):  
Zhen Li ◽  
Xian-Ming Lin ◽  
Pei-Li Gong ◽  
Fan-Dian Zeng ◽  
Guan-Hua Du
Keyword(s):  
Protein Expression ◽  
Gap Junction ◽  
Neurological Deficit ◽  
Ischemia Reperfusion ◽  
Cx43 Expression ◽  
Expression Levels ◽  
Gingko Biloba ◽  
Mrna And Protein Expression ◽  
Cx43 Mrna ◽  
Hypoxia Reoxygenation

Gap junction communication between astrocytes plays an important role in the brain. The purpose of this study was to investigate the effects of Gingko biloba extract (GBE) on the changes of connexin 43 (Cx43) mRNA and protein expression levels of rat cortex and hippocampus induced by ischemia-reperfusion and astrocyte gap junction intercellular communication (GJIC) induced by hypoxia-reoxygenation. After 2 hours of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion, there was obvious neurological deficit in rats. Cx43 mRNA and protein expression levels of rat cortex and hippocampus in the ischemia hemisphere were decreased significantly. When GBE at doses of 50 and 100 mg/kg body weight was administrated by p.o. daily for 7 days, the neurological deficit was improved, and lower Cx43 mRNA and protein expression levels induced by ischemia-reperfusion were recovered to normal. The i.p. injection of nimodipine (0.7 mg/kg weight body) also showed improvement on neurological deficit and Cx43 expression levels. Astrocyte GJIC was measured by the fluorescence recovery after photobleaching (FRAP). Hypoxia-reoxygenation induced a significant decrease in GJIC. Pretreatment with GBE (100 mg/l) and nimodipine (1.6 mg/l) significantly prevented the hypoxia-reoxygenation inhibition of GJIC. These results suggest that GBE could exert its neuroprotective effects by improvement of Cx43 expression and GJIC induced by hypoxia/ischemia-reoxygenation/ reperfusion injury.

scholarly journals Dexmedetomidine Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury by Suppressing TLR4-MyD88-NF-κB Signaling

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Jin-meng Gao ◽  
Xiao-wen Meng ◽  
Juan Zhang ◽  
Wei-rong Chen ◽  
Fan Xia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Ischemia Reperfusion Injury ◽  
Cell Damage ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Mrna Levels ◽  
Rat Cardiomyocytes ◽  
Knock Down ◽  
Ldh Activity ◽  
Hypoxia Reoxygenation

Objective. We previously reported that dexmedetomidine (DEX) offers cardioprotection against ischemia/reperfusion injury in rats. Here, we evaluated the role of toll-like receptors 4- (TLR4-) myeloid differentiation primary response 88- (MyD88-) nuclear factor-kappa B (NF-κB) signaling in DEX-mediated protection of cardiomyocytes usingin vitromodels of hypoxia/reoxygenation (H/R).Methods. The experiments were carried out in H9C2 cells and in primary neonatal rat cardiomyocytes. Cells pretreated with vehicle or DEX were exposed to hypoxia for 1 h followed by reoxygenation for 12 h. We analyzed cell viability and lactate dehydrogenase (LDH) activity and measured tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), and IL-1βmRNA levels, TLR4, MyD88, and nuclear NF-κB p65 protein expression and NF-κB p65 nuclear localization. TLR4 knock-down by TLR4 siRNA transfection and overexpression by TLR4 DNA transfection were used to further confirm our findings.Results. DEX protected against H/R-induced cell damage and inflammation, as evidenced by increased cell survival rates, decreased LDH activity, and decreased TNF-α, IL-6, and IL-1βmRNA levels, as well as TLR4 and NF-κB protein expression. TLR4 knock-down partially prevented cell damage following H/R injury, while overexpression of TLR4 abolished the DEX-mediated protective effects.Conclusions. DEX pretreatment protects rat cardiomyocytes against H/R injury. This effect is partly mediated by TLR4 suppression via TLR4-MyD88-NF-κB signaling.

scholarly journals N-n-Butyl Haloperidol Iodide Ameliorates Cardiomyocytes Hypoxia/Reoxygenation Injury by Extracellular Calcium-Dependent and -Independent Mechanisms

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Yanmei Zhang ◽  
Gaoyong Chen ◽  
Shuping Zhong ◽  
Fuchun Zheng ◽  
Fenfei Gao ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Extracellular Calcium ◽  
Camp Content ◽  
Calcium Dependent ◽  
Reoxygenation Injury ◽  
Myocardial Ischemia Reperfusion ◽  
Pka Pathway ◽  
Hypoxia Reoxygenation

N-n-butyl haloperidol iodide (F2) has been shown to antagonize myocardial ischemia/reperfusion injury by blocking calcium channels. This study explores the biological functions of ERK pathway in cardiomyocytes hypoxia/reoxygenation injury and clarifies the mechanisms by which F2ameliorates cardiomyocytes hypoxia/reoxygenation injury through the extracellular-calcium-dependent and -independent ERK1/2-related pathways. In extracellularcalcium-containing hypoxia/reoxygenation cardiomyocytes, PKCαand ERK1/2 were activated, Egr-1 protein level and cTnI leakage increased, and cell viability decreased. The ERK1/2 inhibitors suppressed extracellular-calcium-containing-hypoxia/reoxygenation-induced Egr-1 overexpression and cardiomyocytes injury. PKCαinhibitor downregulated extracellularcalcium-containing-hypoxia/reoxygenation-induced increase in p-ERK1/2 and Egr-1 expression. F2downregulated hypoxia/reoxygenation-induced elevation of p-PKCα, p-ERK1/2, and Egr-1 expression and inhibited cardiomyocytes damage. The ERK1/2 and PKCαactivators antagonized F2’s effects. In extracellular-calcium-free-hypoxia/reoxygenation cardiomyocytes, ERK1/2 was activated, LDH and cTnI leakage increased, and cell viability decreased. F2and ERK1/2 inhibitors antagonized extracellular-calcium-free-hypoxia/reoxygenation-induced ERK1/2 activation and suppressed cardiomyocytes damage. The ERK1/2 activator antagonized F2’s above effects. F2had no effect on cardiomyocyte cAMP content or PKA and Egr-1 expression. Altogether, ERK activation in extracellular-calcium-containing and extracellular-calcium-free hypoxia/reoxygenation leads to cardiomyocytes damage. F2may ameliorate cardiomyocytes hypoxia/reoxygenation injury by regulating the extracellular-calcium-dependent PKCα/ERK1/2/Egr-1 pathway and through the extracellular-calcium-independent ERK1/2 activation independently of the cAMP/PKA pathway or Egr-1 overexpression.

scholarly journals Propofol Suppressed Hypoxia/Reoxygenation-Induced Apoptosis in HBVSMC by Regulation of the Expression of Bcl-2, Bax, Caspase3, Kir6.1, and p-JNK

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Jianhai Zhang ◽  
Yunfei Xia ◽  
Zifeng Xu ◽  
Xiaoming Deng
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Underlying Mechanism ◽  
Sod Activity ◽  
Expression Levels ◽  
Mda Content ◽  
Cerebral Ischemic ◽  
Induced Apoptosis ◽  
Cerebral Ischemic Reperfusion ◽  
Hypoxia Reoxygenation

Recent studies have found that propofol may protect brain from cerebral ischemic-reperfusion injury. However, the underlying mechanism remains unclear. The effects of propofol were evaluated in HBVSMC after hypoxia/reoxygenation (H/R). Cell viability and levels of SOD, LDH, and MDA were measured. Apoptosis was detected by flow cytometry. The levels of Bax, Bcl-2, Caspase3, Sur2b, Kir6.1, JNK, p-JNK, mTOR, and p-mTOR proteins were measured by western blotting. H/R decreased cell viability and SOD activity and increased LDH leakage and MDA content in HBVSMC, all of which were significantly reversed by propofol. Propofol suppressed the levels of H/R-induced apoptosis. The expression of Bcl-2 and p-mTOR was significantly downregulated and the expression levels of Bax, Caspase3, Kir6.1, and p-JNK were upregulated following H/R injury. The ratio of p-JNK/JNK was increased; however, that of p-mTOR/mTOR decreased correspondingly. The effects on the expression of these proteins were reversed by propofol treatment. SP600125 enhanced and Everolimus attenuated the effect of propofol. These findings suggested that the protective effect of propofol against H/R injury in the HBVSMC was through the inhibition of apoptosis by inducing the expression of Bcl-2 and p-mTOR as well as inhibiting the expression levels of Bax, Caspase3, Kir6.1, and p-JNK.

Overexpression of miR-431 attenuates hypoxia/reoxygenation-induced myocardial damage via autophagy-related 3

2020 ◽  
Author(s):  
Kang Zhou ◽  
Yan Xu ◽  
Qiong Wang ◽  
Lini Dong
Keyword(s):  
Cell Viability ◽  
Cell Apoptosis ◽  
Myocardial Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Myocardial Damage ◽  
Protective Role ◽  
Human Cardiomyocytes ◽  
Hypoxia Reoxygenation

Abstract Myocardial injury is still a serious condition damaging the public health. Clinically, myocardial injury often leads to cardiac dysfunction and, in severe cases, death. Reperfusion of the ischemic myocardial tissues can minimize acute myocardial infarction (AMI)-induced damage. MicroRNAs are commonly recognized in diverse diseases and are often involved in the development of myocardial ischemia/reperfusion injury. However, the role of miR-431 remains unclear in myocardial injury. In this study, we investigated the underlying mechanisms of miR-431 in the cell apoptosis and autophagy of human cardiomyocytes in hypoxia/reoxygenation (H/R). H/R treatment reduced cell viability, promoted cell apoptotic rate, and down-regulated the expression of miR-431 in human cardiomyocytes. The down-regulation of miR-431 by its inhibitor reduced cell viability and induced cell apoptosis in the human cardiomyocytes. Moreover, miR-431 down-regulated the expression of autophagy-related 3 (ATG3) via targeting the 3ʹ-untranslated region of ATG3. Up-regulated expression of ATG3 by pcDNA3.1-ATG3 reversed the protective role of the overexpression of miR-431 on cell viability and cell apoptosis in H/R-treated human cardiomyocytes. More importantly, H/R treatments promoted autophagy in the human cardiomyocytes, and this effect was greatly alleviated via miR-431-mimic transfection. Our results suggested that miR-431 overexpression attenuated the H/R-induced myocardial damage at least partly through regulating the expression of ATG3.

Abstract 172: Thrombopoietin Receptor Agonists Protect Human Cardiac Myocytes from Injury by Activation of Cell Survival Pathways

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
John E Baker ◽  
Jidong Su ◽  
Stacy Koprowski ◽  
Anuradha Dhanasekaran ◽  
Tom P Aufderheide ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Survival Pathways ◽  
Thrombopoietin Receptor ◽  
Reoxygenation Injury ◽  
Hypoxia Reoxygenation

Thrombopoietin confers immediate protection against injury caused by ischemia/reperfusion in the rat heart at a dose that does not increase platelet levels. Eltrombopag is a small molecule agonist of the thrombopoietin receptor; the physiological target of thrombopoietin. Administration of thrombopoietin and eltrombopag result in a dose- and time-dependent increase in platelet counts in patients with thrombocytopenia. However, the ability of eltrombopag and thrombopoietin to immediately protect human cardiac myocytes against injury and the mechanisms underlying myocyte protection are not known. Human cardiac myocytes (7500 cells, n=10/group) were treated with eltrombopag (0.1- 30.0 μM) or thrombopoietin ( 0.1 - 30.0 ng/ml) and then subjected to 5 hours of hypoxia (95% N 2 /5%CO 2 ) and 16 hours of reoxygenation to determine their ability to confer resistance to necrotic and apoptotic myocardial injury . The thrombopoietin receptor (c-Mpl) was detected in unstimulated human cardiac myocytes by western blotting. Eltrombopag and thrombopoietin confer immediate protection to human cardiac myocytes against injury from hypoxia/reoxygenation by decreasing necrotic and apoptotic cell death in a concentration-dependent manner with an optimal concentration of 3 μM for eltrombopag and 1.0 ng/ml for thrombopoietin. The extent of protection conferred to cardiac myocytes with eltrombopag is equivalent to that of thrombopoietin. Eltrombopag and thrombopoietin activate multiple pro-survival pathways; inhibition of JAK-2 (AG-490, 10 μM), p38 MAPK (SB203580, 10 μM), p44/42 MAPK (PD98059, 10 μM), Akt/PI 3 kinase (Wortmannin, 100 nM), and src kinase (PP1, 20 μM) prior to and during hypoxia abolished cardiac myocyte protection by eltrombopag and thrombopoietin. These inhibitors had no effect on hypoxia/reoxygenation injury in myocytes when used alone. Eltrombopag and thrombopoietin may represent important and potent agents for immediately and substantially increasing protection of human cardiac myocytes, and may offer long-lasting benefit through activation of pro-survival pathways during ischemia.

Changes of Blood Pressure Rhythm and Clock Protein Expression Levels in Spontaneously Hypertensive Rats After Ischemia-Reperfusion

2021 ◽  
Author(s):  
Jing Jin ◽  
Yumeng Liu ◽  
Jing Huang ◽  
Dong Zhang ◽  
Jian Ge ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Protein Expression ◽  
Circadian Clock ◽  
Spontaneously Hypertensive Rats ◽  
Ischemia Reperfusion ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Expression Levels ◽  
The Relationship ◽  
Clock Protein

Abstract Objective A variety of circadian patterns of blood pressure after ischemic stroke in patients with essential hypertension appear to be a potential risk of stroke recurrence, but the mechanism is still unclear. This study intends to reveal the changes in blood pressure rhythm and circadian clock protein expression levels in spontaneously hypertensive rats (SHR) after ischemia-reperfusion, and the relationship between the two. Methods Using the SHR middle cerebral artery occlusion experimental model, the systolic blood pressure was continuously monitored for 24 hours after the operation to observe the blood pressure rhythm. The rat tail vein blood was taken every 3h, and the serum CLOCK, BMAL1, PER1 and CRY1 protein expression levels were detected by Elisa. Pearson correlation analysis counted the relationship between SHR blood pressure rhythm and circadian clock protein fluctuation after ischemia-reperfusion. Results The proportion of abnormal blood pressure patterns in the SHR + tMCAO group was significantly higher than that in the SHR group, the serum CLOCK expression was relatively constant, and the circadian rhythm of BMAL1, PER1 and CRY1 protein expression changed significantly. Pearson analysis showed that PER1 protein level was negatively correlated with dipper (r = -0.565, P = 0.002) and extreme-dipper (r = -0.531, P = 0.001) blood pressure, and was significantly positively correlated with non-dipper blood pressure (r = 0.620, P < 0.001). Conclusion The rhythm pattern of blood pressure after ischemia-reperfusion in SHR is obviously disordered, and it is closely related to the regulation of Per1 gene.

scholarly journals Pituitary Adenylate Cyclase Activating Polypeptide Elicits Neuroprotection Against Acute Ischemic Neuronal Cell Death Associated with NMDA Receptors

2018 ◽  
Vol 51 (4) ◽  
pp. 1982-1995 ◽  
Author(s):  
Yuji Kaneko ◽  
Julian P. Tuazon ◽  
Xunming Ji ◽  
Cesario V. Borlongan
Keyword(s):  
Cell Death ◽  
Adenylate Cyclase ◽  
Protein Expression ◽  
Cell Viability ◽  
Caspase 3 ◽  
High Mobility ◽  
High Mobility Group ◽  
Expression Levels ◽  
Pituitary Adenylate Cyclase ◽  
Nmdar Subunits

Background/Aims: The endogenous neurotrophic peptides pituitary adenylate cyclase-activating polypeptides (PACAP-27/38) protect against stroke, but the molecular mechanism remains unknown. Methods: Primary rat neural cells were exposed to PACAP-27 or PACAP-38 before induction of experimental acute ischemic stroke via oxygen-glucose deprivation-reperfusion (OGD/R) injury. To reveal PACAP’s role in neuroprotection, we employed fluorescent live/dead cell viability and caspase 3 assays, optical densitometry of mitochondrial dehydrogenase and cell growth, glutathione disulfide luciferase activity, ELISA for high mobility group box1 extracellular concentration, ATP bioluminescence, Western blot analysis of PACAP, NMDA subunits, apoptosis regulator Bcl-2, social interaction hormone oxytocin, and trophic factor BDNF, and immunocytochemical analysis of PACAP. Results: Both PACAP-27 and PACAP-38 (PACAP-27/38) increased cell viability, decreased oxidative stress-induced cell damage, maintained mitochondrial activity, prevented the release of high mobility group box1, and reduced cytochrome c/caspase 3-induced apoptosis. PACAP-27/38 increased the protein expression levels of BDNF, Bcl-2, oxytocin, and precursor PACAP. N-methyl-D-aspartate receptor (NMDAR)-induced excitotoxicity contributes to the cell death associated with stroke. PACAP-27/38 modulated the protein expression levels of NMDAR subunits. PACAP-27/38 increased the protein expression levels of the GluN1 subunit, and decreased that of the GluN2B and GluN2D subunits. PACAP-27, but not PACAP-38, increased the expression level of the GluN2C subunit. Conclusion: This study provides evidence that PACAP regulated NMDAR subunits, affording neuroprotection after OGD/R injury.

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