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Background The exposure to diesel particulate matter (DPM) and its polycyclic aromatic hydrocarbons (PAH) is closely related to the morbidity and mortality of ischemic heart disease (IHD). However, it is unclear what key components and targets of DPM exposure involve in myocardial ischemia-hypoxia injury and associated mechanisms. Objective To identify key PAH components of DPM that act on myocardial hypoxic injury, andclarify the role of oxygen sensors-regulated anaerobic metabolism in DPM and key components-induced hypoxic injury and the targets of the key PAH components. Methods Human cardiomyocyte cell line AC16 cells were exposed to 0, 1, 5, and 10 μg·mL−1 DPM in a high glucose DMEM medium with 10% fetal bovine serum (FBS) (HGM) or low FBS (0.5%) in high glucose DMEM medium (LFM), for 12 h under 2% O2, and expression of hypoxia-inducible factor-1α (HIF-1α), Bax, and Cleaved-caspase3 was determined by Western blotting. Under normal condition, the cell viability was detected after PAH exposure for 12 h. Under the condition of ischemia-hypoxia model, cells were exposed to 0, 0.005, 0.5, and 5 µg·mL−1 PAH for 12 h, and the protein expression of HIF-1α, Bax, and Cleaved-caspase3 was determined. After exposure to DPM or PAH for 12 h, the contents of pyruvate and lactate in cells were detected. Pretreatment with glycolysis inhibitor GSK2837808A was used to explore the role of glycolysis in DPM and benzo[a]pyrene (BaP)-induced hypoxia injury. A molecular docking technique was used to analyze the binding affinity between PAH and oxygen sensors (prolyl hydroxylase domain-containing protein 2, PHD2, and factor-inhibiting hypoxia-inducible factor 1, FIH1), and the protein levels of PHD2, FIH1, and hydroxyl-HIF-1-alpha (OH-HIF-1α) after the DPM or BaP treatment were further determined. Results Under hypoxia, DPM exposure in the LFM induced the expression of HIF-1α, Bax, and Cleaved-caspase3 (P<0.01). Therefore, hypoxia and LFM were selected as the basic ischemia and hypoxia condition. Except for anthracene (Ant) (P>0.05), other PAH decreased cell viability when the concentration was above 1 μg·mL−1 (P<0.05). All concentrations of BaP induced the expression of HIF-1α protein (P<0.05), and the protein levels of Bax and Cleaved-caspase3 were up-regulated after the 0.5 and 5 µg·mL−1 BaP exposure (P<0.01). After exposure to DPM (1, 5 and 10 μg·mL−1) or BaP (0.5 and 5 μg·mL−1), the intracellular pyruvate and lactate contents increased (P<0.05). The glycolysis inhibitor co-treatment decreased the levels of HIF-1α, Bax, and Cleaved-caspase3 proteins compared with the DPM or BaP exposure group for 12 h (P＜0.05). The binding abilities of the five PAHs to the oxygen sensors PHD2 and FIH1 were strong, and BaP was the strongest. Although the DPM or BaP exposure had no effects on the protein levels of PHD2 and FIH1 in AC16 cells (P＜0.05), the protein level of OH-HIF-1α was decreased (P＜0.01). Conclusion BaP exposure can promote hypoxia and injury of myocardial cells and is the key PAH component of DPM that induces myocardial ischemia and hypoxia injury. BaP exposure inhibits the hydroxylation function of PHD2 on HIF-1α by combining with PHD2, decreases the level of OH-HIF-1α and induces HIF-1α accumulation. And then HIF-1α promotes anaerobic metabolism and accelerates ischemia and hypoxia injury of myocardial cells.
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Aim: To investigate the neuroprotective effect of salidroside on CoCl2 -induced hypoxia injury in PC 12 cells, and further explore the mechanism of salidroside on inhibiting complement and neuroprotection. Methods: The model of CoCl2-induced hypoxia injury in PC 12 cells was established. The expressions of Egrl, Egr4, C3 and the activity of caspase-3 were assessed with different concentrations of salidroside treatment. The expressions of Egrl, Egr4 and the activity of caspase-3 were tested with C3ar inhibitor and salidroside treatment. The expressions of Bax, Bcl-xl, C3 and the activity of caspase-3 were determined with Egr4 siRNA and salidroside treatment. Results: Salidroside significantly reduced the caspase-3 activity and the protein expression of Bax and C3, and improved the expression of Bcl-xl, Egrl, and Egr4 proteins in CoCl2-induced PC12 cells. Salidroside reduced the caspase-3 activity and the expression of Egrl, Egr4 with C3ar inhibitor treatment. With siRNA interference with the expression of Egr4, salidroside reversed the activity of caspase-3, Bax and Bcl-xl, but the effect on C3 was not obvious. Conclusions: Salidroside has neuroprotective effect on the model of CoCl2 -induced hypoxia injury in PC12 cells, which is related to the inhibition of complement component C3 and the improvement of the expression of Egr4 by salidroside.
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Myocardial infarction (MI) is a common presentation for ischemic heart disease, which is a leading cause of death. Emodin is a Chinese herbal anthraquinone used in several diseases. However, the effect of emodin in hypoxia-induced injury in cardiomyocytes has not been clearly elucidated. Our study aimed to clarify the functions of emodin in hypoxia-induced injury in rat cardiomyocytes H9c2 and explore the underlying mechanism. The effects of emodin on cell viability and apoptosis were analyzed by the Cell counting kit-8 assay and flow cytometry assay, respectively. The cell proliferation- and cell apoptosis-related proteins were detected by western blot. qRT-PCR was used to determine the relative expression of miR-138. Cell transfection was performed to alter miR-138 and MLK3 expression. miR-138 target was performed by dual luciferase activity assay. Sirt1/AKT and Wnt/β-catenin pathways-related factors phosphorylation were analyzed by western blot. Emodin inhibited hypoxia-induced injury in H9c2 cells by promoting cell viability and reducing cell apoptosis. miR-138 was down-regulated by hypoxia treatment but up-regulated by emodin. Up-regulation of miR-138 alleviated hypoxia-induced cell injury. Down-regulation of miR-138 attenuated the growth-promoting effect of emodin on hypoxia-induced injury, whereas up-regulation of miR-138 enhanced the growth-promoting effects of emodin. The underlying mechanism might be by inactivating Sirt1/AKT and Wnt/β-catenin pathways. MLK3 was negatively regulated by miR-138 expression and inactivated Sirt1/AKT and Wnt/β-catenin pathways. Emodin alleviated hypoxia-induced injury in H9c2 cells via up-regulation of miR-138 modulated by MLK3, as well as by activating Sirt1/AKT and Wnt/β-catenin pathways.
Subject(s)
Animals , Rats , Cell Hypoxia/drug effects , Cell Survival/drug effects , Emodin/therapeutic use , Myocytes, Cardiac/pathology , Cell Proliferation/drug effects , Hypoxia/complications , Signal Transduction , Up-Regulation , Cell Line , Myocytes, Cardiac/drug effects , MicroRNAsABSTRACT
Objective To investigate the cytoprotection and mechanism of carbachol(CCH)to stimulate M3mus-carinic acetylcholine receptor(M3-mAChR) against hypoxia injury induced by cobaltous chloride hexahydrate (CoCl2) in rat cardiomyocyte line H9c2. Methods Select the normal rat cardiomyocyte line H9c2 as the control group, rat cardiomyocyte line H9c2 was managed with CoCl2to develop hypoxia injury model, M3-mAChR spe-cific agonist CCH and antagonist 4-diphenyl-acetoxy-N-methyl-piperidine methiodide(4-DAMP) were used for in-tervention. The cell viability was tested by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT); The apoptosis in cardiomyocyte was detected by flow cytomery(FCM); The expression of M3-mAChR,caspase-3, HIF-1α and HO-1 proteins was measured by Western blot assay. Results In hypoxia group,the ap-optosis rate was significantly increased while cell proliferation decreased, the expression of HIF-1α, caspase-3 and HO-1 proteins were up-regulated obviously;After treatment with CCH,the apoptosis and cell proliferation of cardiomyocytes were significantly decreased, while the proliferation of cells increased, and the expression of M3-mAChR, HIF-1α and HO-1 proteins increased, the expression of caspase-3 protein was significantly decreased. Moreover, when applying 4-DAMP as intervention, these effects mediated by CCH was abolished.Conclusions CCH stimulates M3-mAChR against hypoxia injury induced by CoCl2in rat cardiomyocyte strain H9c2, and the mechanism may be related to down-regulation of caspase-3 expression and up-regulation of HIF-1α and HO-1 protein expression.
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Objective To explore the role of c-Jun N-terminal protein kinase(JNK)pathway in renal tubular epithelial cells(RTEC)transdifferentiation by detecting the expression of JNK pathway in the injury of hypoxic RTEC. Methods In vitro cultured rat RTEC were randomly divided into four groups of normal control, hypoxia,inhibitors,dimethyl sulfoxide(DMSO). The groups of hypoxia,inhibitor and DMSO were placed into a vacuum tank to establish hypoxia model. The cells were harvested 6,12 and 24 h after hypoxia started. RT-PCR and Western blot test were used to detect the mRNA expressions of alpha smooth muscle actin(α- SMA)and protein expressions of JNK,pJNK and alpha SMA,respectively,in all the groups. Results Hypoxia induced significantly increased expressions of α-SMA mRNA and proteins,and JNK and pJNK proteins as well in RTEC (all P<0.05).After addition of inhibitors,the expression of α-SMA mRNA and proteins,the protein expression of pJNK in RTEC were significantly decreased(all P < 0.05)and the expression of JNK protein was significantly increased(P < 0.05). There was no significant difference in the expressions of JNK,pJNK,α-SMA in RTECs between hypoxia group and DMSO group(all P>0.05).There was a significant positive correlation between pJNK protein and α-SMA protein expression(P < 0.01). Conclusion In hypoxic RETC injury in rats,JNK pathway may be involved in the RTEC phenotype transdifferentiation.
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Objective To investigate the effect of metformin on rat adrenal pheochromocytoma PC12 cell injury induced by hypoxia and its possible molecular mechanisms.Methods PC12 Cells were cultured under hypoxia conditions (0.3％ O2).The cell survival and apoptosis were detected by methylrosanilinium chloride (crystal violet) staining and Annexin-V/propidium iodide (PI) staining,the cell viability measured by CCK8 assay,and the signaling activity and protein levels were detected by Western blotting.Results Physical hypoxia at an O2 concentration of 0.3％ resulted in time-dependent injury to PC12 cells,while metformin pretreatment had protective effect on PC12 cell injury induced by hypoxia.AMP-activated protein kinase (AMPK) signaling was activated by metformin treatment,and AMPK kinase inhibitor compound C blocked the protective effect of metformin on hypoxia-induced PC12 cell injury.Conclusion These results demonstrate that metformin pretreatment has protective effect on PC12 cell injury induced by hypoxia,and AMPK signaling plays an important role in the protective effect of metformin.
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Objective To study in vitro the inhibitory effects and mechanisms of N-butanol extract of Potentilla anserine L.(NP)against hypoxia-induced nitric oxide(NO)in hippocampus neuron of rats. Methods The models of hippocampus neurons hypoxia injury of Sprague-Dawley(SD)neonatal rats were cultured in vitro. The cultured hippocampus neurons were divided randomly into blank control group, hypoxia injury model group, nimodipine group(2 μmol/L)and NP high(250.0 mg/L),middle(62.5 mg/L),low(15.6 mg/L)dose groups. The activities of hippocampus neurons were examined by methyl thiazolyl tetrazolium(MTT)assay,and meanwhile their contents of nitrogen monoxidum(NO)were detected. Half quantity reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting were used to detect neuronal nitric oxide synthetase(nNOS)mRNA and protein expression levels respectively in each group,immunocytochemistry stain was used to detect protein positive rate. Results Compared with blank control group,the activity of neuron〔absorbance(A)value〕was significantly decreased(0.0826±0.0095 vs. 0.3315±0.0105),content of NO(μmol/g:0.0509±0.0027 vs. 0.0291±0.0032), the expression levels of nNOS mRNA (0.1463±0.0081 vs. 0.0801±0.0058), the positive rate of nNOS〔(74.4238±3.9423)%vs.(28.3714±4.1361)%〕,the expression levels of nNOS protein(A value:1.9130±0.0471 vs. 0.5068±0.0368)were all significantly increased in the hypoxia injury model group(all P0.05). Conclusions NP can ameliorate the injury of rat hippocampus neurons induced by hypoxia in vitro. The possible mechanisms might be related to the effective inhibition of the synthesis of nNOS and NO excessive generation.
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Objective:To investigate the direct effect of different concentrations of hydrogen sulfide(H_2S)on neonatal rat myocardial cells by hypoxia injury during different time point,and the indirect effect of reoxygenation injury in order to analyze the protective role of H_2S on myocardial cells subjected to hypoxia/reoxygenation injury. Methods:Primary cultured myocardial cells from neonatal rats were randomly divided into 9 groups:Normal control group,Hypoxia NaHS 0 μmol/L group,100 μmol/L group,200 μmol/L group,400 μmol/L group and those groups subjected to hypoxia for 24 h,48 h and 72 h. Hypoxia/reoxygenation NaHS 0 μmol/L group,100 μmol/L group,200 μmol/L group,400 μmol/L group and those groups subjected to hypoxia for 24 h,48 h,72 h and then reoxygenation for 2 h. The number of survival cells were counted and the activity of lactate dehydrogenase(LDH)were measured respectively at each time point. Results:At the same time point of 24 h,48 h,72 h hypoxia,the number of survival cells increased and the activity of LDH decreased in hypoxia NaHS treatment groups as compared with Hypoxia NaHS 0 μmol/L group(P<0.01). The protective role of Hypoxia NaHS 200 μand 400 μmol/L groups were better than NaHS 100 μmol/L group at 24 h of time poit(P<0.05～0.01). The number of survival cells by hypoxia/reoxygenation increased and the leakage of LDH from reoxygenation cells decreased in hypoxia/reoxygenation NaHS treatment groups as compared with hypoxia/reoxygenation NaHS 0 μmol/L group(P<0.01).Conclusion:NaHS 100 to 400 μmol/L had the protective effects on myocardial cells by hypoxia/reoxygenation injury. NaHS 200 to 400 μmol/L might offer the best protective effects on myocardial cells by hypoxia injury at 24 h.