Anti-inflammatory Effect of Apigenin on Microglia after Oxygen Glucose Deprivation and Reperfusion
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Objective To study the neuroprotective effect of apigenin on microglia which was exposed to oxygen glucose deprivation and reperfusion (OGD/R), which was characterized by its influence on IL-1β and TNF-α expression. Methods Primary microglial cultures were prepared from newborn rat brain. The purity of isolated cells were identified by GSA-IB4. The cells were randomized into 5 groups:normal group, DMSO group and apigenin-treated groups (10, 25, 50 μmol/L). The cells of DMSO group and apigenin-treated group were exposed to 8 h of OGD and 24 h of reperfusion in the presence or absence of apigenin at a range of concentrations. Culture supernatants were collected and IL-1β and TNF-α were detected by ELISA assay. Results The expression of IL-1β and TNF-α were significantly higher in DMSO group (P0.01), however, apigenin concentration-dependently suppressed IL-1β and TNF-α production of microglial. Conclusion Apigenin may play a neuroprotective effect which was related with depression of IL-1β and TNF-α production in OGD/R microglia.Cite
Objective
To evaluate the effect of hydrogen preconditioning during cold ischemia phase on the activity of nuclear factor erythroid 2-related factor 2 (Nrf2) in rat pulmonary microvascular endothelial cells (PMVECs) subjected to hypoxia-reoxygenation(H/R).
Methods
PMVECs were isolated from clean-grade male Sprague-Dawley rats, aged 2-3 weeks, using the tissue block adherence method and divided into 4 groups (n=25 each) using a random number table method: control group (group C), H/R group, oxygen group (O group) and hydrogen group (H group). Cells were incubated for 4 h with 4 ℃ low potassium dextransolution(LPD) pre-equilibrated with 95% oxygen and 5% carbondioxide to simulate the cold ischemia phase.LPD pre-balanced with 95% oxygen and 5% carbon dioxide was replaced with LPD, and then cells were incubated for 1 h at room temperature to simulate the lung transplantation period.LPD was rapidly replaced with 37 ℃ M199 complete culture solution, and cells were incubated in the mixture of 40% oxygen-5% carbondioxide-55% nitrogen to simulate the reperfusion period.In O and H groups, the cells were exposed to 40% oxygen-60% nitrogen and 3% hydrogen-40% oxygen-57% nitrogen during the cold ischemia period, respectively, and the gas mixture was replaced every 20 min.The cell culture fluid was collected 4 h later for determination of interleukin(IL)-6, IL-10 and tumor necrosis factor-alpha (TNF-α) concentrations (by enzyme-linked immunosorbent assay) and malondialdehyde (MDA) concentrations (by thiobarbituric acid method). The cytoplasm and nucleoproteins were extracted for measurement of Nrf2 expression(by Western blot) and cell apoptosis (by flow cytometry and TUNEL assay). The cell apoptosis rate was calculated.
Results
Compared with C group, the IL-6, TNF-α and MDA levels were significantly increased, the IL-10 level was decreased, the apoptosis rate was increased, and the expression of Nrf2 in nucleus was up-regulated in H/R group (P>0.05). Compared with H/R group, the IL-6, TNF-α and MDA levels were significantly decreased, the IL-10 level was increased, the apoptosis rate was decreased, and the Nrf2 expression in cytoplasm was down-regulated in O and H groups (P 0.05). Compared with O group, the IL-6, TNF-α and MDA levels were significantly decreased, the IL-10 level was increased, the apoptosis rate was decreased, the expression of Nrf2 in nucleus was up-regulated, and the expression of Nrf2 protein in cytoplasm was down-regulated in H group (P<0.05).
Conclusion
The mechanism by which hydrogen preconditioning during cold ischemia phase reduces H/R injury to rat PMVECs is related to activating Nrf2 and thus inhibiting oxidative stress.
Key words:
Hydrogen; Cold ischemia; Microvessels; Endothelial cells; Lung; NF-E2-related factor 2
Malondialdehyde
Thiobarbituric acid
Hypoxia
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Objective
To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape，function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion injury and the action mechanism of AMPK inhibition on neuroprotective effects.
Methods
One hundred and twenty male Kunming mice were randomly divided into sham group，ischemia-reperfusion group，ischemia-reperfusion therapy group (all n=40).Each mouse in ischemia-reperfusion therapy group was given an intraperitoneal inject of compound C (20 mg/kg) after occlusion.A mode of middle cerebral artery occlusion was made by insertion of a thread through internal carotid artery.After 24 hours of reperfusion，neurological deficit scores，infarct volume and expression of specific marker for microglia of ionized calcium-binding adaptor molecule 1 (Iba1) were investigated，and the expressions of inducible nitric oxide synthase (iNOS)，nicotinamide adenine dinucleotide phosphate oxidase 2 (gp91phox)，tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined.
Results
After cerebral ischemia-reperfusion injury，neurological deficit scores and infarct volume of ischemia-reperfusion therapy group were significantly reduced compared with ischemia-reperfusion group.Small amount of Iba1 ((5.97±1.26) /HP)，iNOS ((6.25±2.02) /HP)，gp91phoxmRNA (0.010±0.007)，TNF-α ((249.62±48.37) pg/mg) and IL-1β ((107.41±11.77) pg/ mg) expressions were observed in sham group.The expressions of Iba1，iNOS，gp91phoxmRNA，TNF-αand IL-1βin ischemia-reperfusion group increased significantly to (11.36±1.18) /HP (P=0.000)，(16.38±2.43) /HP (P=0.000)，0.240±0.067 (P=0.000)，(442.92±97.59) pg/mg (P=0.002) and (209.09±24.69) pg/mg (P=0.000) respectively.Compared with ischemia-reperfusion group，the expressions of Iba1，iNOS，TNF-α and IL-1βin ischemia-reperfusion therapy group decreased significantly to (7.60±1.62) /HP (P=0.000)，(9.32±2.20) /HP (P=0.000)，(290.60±61.40) pg/mg (P=0.007) and (142.61±20.60) pg/mg (P=0.000) respectively，and the expression of gp91phoxmRNA declined to 0.170±0.055 (P=0.052) without statistical significance.
Conclusions
Inhibition of AMPK activity has neuroprotective effects.Its mechanism may be related to the inhibition of microglia activation and reduction of expression of iNOS，gp91phox，TNF-αand IL-1β.
Key words:
Brain ischemia; Reperfusion injury; AMP-activated protein kinases; Microglia; Nitric oxide synthase; Tumor necrosis factor-alpha
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Objective
To evaluate the effects of different concentrations of PNU282987 on anoxia/reoxygenation(A/R)injury to cardiomyocytes of rats.
Methods
After being cultured for 72 h, cardiomyocytes obtained from neonatal rats were seeded in 6-well plates(2 ml/well, a total of 108 wells)at a density of 5×105 cells/ml and randomly divided into 6 groups according to a random number table(n=18 each): control group(C group); A/R group; 3, 10, 30 and 60 μmol/L PNU282987 groups(P3, P10, P30 and P60 groups). The cells were exposed to anoxia for 6 h followed by reoxygenation with 95% O2 -5% CO2 at 37 ℃ for 6 h. The corresponding concentrations of PNU282987 were added to the culture medium during reoxygenation in P3, P10, P30 and P60 groups.The amount of lactic dehydrogenase(LDH)released was measured using colorimetric method.Apoptosis in cardiomyocytes was detected by flow cytometry with Annexin Ⅴ/propidium iodide staining.The concentrations of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)in the supernatant were determined using enzyme-linked immunosorbent assay.
Results
Compared to C group, the LDH release rate, early and late apoptosis rates, and the concentrations of IL-6 and TNF-α in the supernatant were significantly increased in A/R, P3, P10, P30 and P60 groups.Compared to A/R group, the LDH release rate and early apoptosis rate were significantly decreased(especially in group P30), and the concentrations of IL-6 and TNF-α in the supernatant were decreased in a concentration-dependent manner in P3, P10, P30 and P60 groups.Compared to A/R group, no significant changes were found in late apoptosis rate in P3, P10, P30 and P60 groups.
Conclusion
PNU282987 3, 10, 30 and 60 μmol/L can reduce A/R injury to cardiomyocytes of rats, especially 30 μmol/L.
Key words:
Receptors, cholinergic; Myocytes, cardiac; Cell hypoxia; Oxygen
Propidium iodide
Optical density
Lactic dehydrogenase
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Objective To explore whether hypertonic saline (HS) could affect the microglial activation and release of monocyte chemotactic protein 1 (MCP-1) in vitro experiments,and to study the mechanism.Methods Microglia isolated from primary mixed glia cells by shaking method were divided into four groups:control group,hypoxia + glucose free medium group (hypoxia group),100 mmol/L HS + hypoxia + glucose free medium group (HS group) and SB203580 (a specific inhibitor of p38 MAPK) group.Oxygen-glucose deprivation (OGD) was performed in all experiment groups but control group.Double immunofluorescence and ELISA were used to determine whether HS could decrease the production of MCP-1 after OGD,and western blot was employed to determine the effect of HS on the level of phosphorylated p38 MAPK.Results Double immunofluorescence showed that 100 mmol/L HS attenuated MCP-1 level in HS group after treatment with 100 mmol/L HS compared with hypoxia group,and the level of MCP-1 in cultured medium released by microglia was significantly decreased after 100 mmol/L HS or SB203580 treatment for 4 h.There was no significant difference in MCP-1 between HS group and SB203580 group (P ＞ 0.05),but compared with control group (107.956 ± 8.921),there were higher levels of MCP-1 in hypoxia group (173.467 ±9.027),HS group:(145.957 ± 10.952),and SB203580 group (136.279 ± 15.63),P ＜0.05.At 30 min,1 h,2 h and 4 h after hypoxia,western blot showed that the levels of phosphorylated p38 MAPK markedly decreased in HS group as compared to the hypoxia group at corresponding intervals (control group:0.590±0.105.Hypoxia group:30 min,1.553 ±0.215; 1 h,1.212±0.161; 2 h,1.179 ± 0.105; 4 h,1.178 ±0.122.HS group:30 min,1.028 ±0.168; 1 h,0.852 ±0.112; 2 h,0.927 ±0.121; 4 h,0.815 ± 0.107; P ＜ 0.05).Conclusions HS decreasing MCP-1 level in microglia after hypoxia may be attributed to the inhibition of p38 MAPK signaling pathway.
Key words:
Hypertonic saline; Cerebral edema; Hypoxia; Microglia; monocyte chemotactic protein-1; p38; SB203580; Inflammatory response
Hypoxia
Hypertonic saline
Monocyte
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Objective To investigate the effect of hypoxic preconditioning on anti-inflammatory responses of bone marrow mesenchymal stem cells (BMSCs) in rats through in vitro and in vivo experiments.Methods In vitro experiment The isolated rat BMSCs were cultured by whole bone marrow adherence method.The cells at passage 3 were seeded in 24-well plates at a density of 1 × 106 cells/ml and randomly divided into 5 groups (n =8 wells each) using a random number table:control group (group C),normoxia-incubated group (group N),hypoxic preconditioning group (group H),hypoxia preconditioning + STAT3 inhibitor Stattic group (group HS) and hypoxia preconditioning + anti-IL-10 monoclonal antibody group (group HA).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to21％ O2-74％ N2-5.0％ CO2 for48 h.In group H,BMSCs were exposed to 0.5％ O2-94.5％ N2-5.0％ CO2 for 24 h followed by 24 h exposure to normoxia.In HS and HA groups,500 μg/ml Stattic and 100 μg/rnl anti-IL-10 monoclonal antibody were added to the culture medium before hypoxia preconditioning,respectively.The expression of phosphorylated STAT3 (p-STAT3) and IL-10 was determined by Western blot.In vivo experiment Healthy male Sprague-Dawley rats,weighing 300-350 g,in which intrathecal catheters were successfully implanted without complications,underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Three hundred rats with spinal cord I/R injury were randomly divided into C,N,H,HS and HA groups (n =60 each) using a random number table.Immediately after onset of reperfusion,DMEM medium 300 μl was injected intrathecally in group C,and BMSC suspension 300 μl (1 × 106 cells/ml) was injected intrathecally in N,H,HS and HA groups.Neurological function was scored before ischemia and at 4,12,24 and 48 h of reperfusion (T0-,).The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the content of IL-10,TNF-α,IL-1β,IL-6,monocyte chemotactic protein-1 (MCP-1),and macrophage inflammatory protein-1 α (MIP-1 α) (by ELISA) and the number of activated microglia (by immuno-histochemistry).Results Compared with C and N groups,the expression of pSTATβ and IL-10 was significantly up-regulated,the neurological function score and IL-10 content were increased,the content of TNF-α,IL-1β,IL-6,MCP-1 and MIP-1α and the number of activated microglia were decreased in group H (P ＜ 0.05).Compared with group H,the expression of p-STAT3 and IL-10 in group HS and expression of IL-10 in group HA was significantly down-regulated,and the neurological function score and IL-10 content were decreased,and the content of TNF-α,IL-13,IL-6,MCP-1 and MIP-1α and the number of activated microglia were increased in HS and HA groups (P ＜ 0.05).Conclusion Hypoxic preconditioning can enhance anti-inflammatory effects of BMSCs,thus increasing BMSCs-induced reduction of spinal cord ischemia-reperfusion injury in rats.
Key words:
Cell hypoxia; Ischemica preconditioning; Mesenchymal stem cells transplantation ; Spinal cord ; Inflammation
Hypoxia
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Objective
To evaluate the effect of Tanshinonne ⅡA preconditioning on nuclear factor kappa B (NF-κB) signaling pathway in N9 cells subjected to oxygen-glucose deprivation and restoration (OGD/R).
Methods
N9 cells cultured in vitro were divided into 4 groups (n=16 each) by using a random number table method: control group (C group), OGD/R group, vehicle group (V group) and Tanshinone IIA preconditioning group (TP group). N9 cells were subjected to O2-glucose deprivation for 4 h followed by restoration of O2-glucose supply for 24 h to establish the OGD/R model.Group C receive no treatment.In group TP, N9 cells were cultured in medium containing Tanshinone ⅡA (final concentration 5 mg/L) for 24 h before the model was established.Dimethyl sulfoxide was added to the culture medium in group V. At the 24 h of reoxygenation, the level of lactate dehydrogenase (LDH) and nitric oxide(NO)in culture medium was detected by spectrophotometry, cell apoptosis was assessed by using TUNEL, and the expression of NF-κB in nucleus was detected by Western blot.The apoptosis rate was calculated.
Results
Compared with group C, the levels of LDH, IL-β, TNF-α and NO in culture medium and apoptosis rate were significantly increased, and the expression of NF-κB in nucleus was up-regulated in OGD/R, V and TP groups (P 0.05).
Conclusion
The mechanism by which Tanshinonne ⅡA preconditioning reduces OGD/R injury to N9 cells is related to inhibiting NF-κB signaling pathway.
Key words:
TANSHINONE; Hypoxia-ischemia, brain; NF-kappa B
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Objective
To explore the anti-inflammatory effect of dexmedetomidine(Dex) in attenuating hypoxia/reoxgenation(H/R) injury of H9C2 cells.
Methods
Rat cardiomyocyte cell line H9C2 cells were cultured in vitro, and Na2S2O4 was used to establish the hypoxia and reoxygenation injury model on H9C2 cells. Cultured H9C2 cells were divided into three groups: normal control group (group C), the cells were cultured as usual, group H/R, the cells were subjected to 1 h hypoxia induced by 4 mmol/L Na2S2O4, then followed by 12 h reoxygenation by replaced the normal media, Dex preconditioning group (group D), Dex at various concentrations(0.1, 1.0, 10.0 μmol/L) was performed prior to Na2S2O4-induced H/R injury, the rest of the steps are essentially the same as the group H/R. The cell survival rate 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide(MTT) and lactate dehydrogenase(LDH) activity were detected after treatment, the cellular morphology was observed by inverted miscroscope, the TNF-α, IL-6, IL-1β mRNA expression were detected by reverse transcription-polymerase chain reaction(RT-PCR).
Results
Compared with the group C, the cell survival of group H/R decreased to(44±12)%(P<0.01), LDH activity increased significantly(P<0.01), compared with the group H/R, the cell survival rate and LDH activity of the group D were improved, Dex at 1 μmol/L concentration preconditioning significantly restored cell viability to(75±7)%(P<0.05) and reduced the activity of LDH significantly(P<0.05). The condition of cell necrosis, irregular cell arrangement, and the decline of cell membrane refractive index caused by H/R injury were partly reversed by Dex. Compared with the group C, the TNF-α, IL-6, IL-1β mRNA expression level of the group D and the group H/R increased significantly. Compared with group H/R, the TNF-α, IL-6, IL-1β mRNA expression levels of group D decreased significantly(P<0.05).
Conclusions
Dex preconditioning attenuates hypoxia and reoxygenation injury of H9C2 cells. The mechanism may be related to inhibiting inflammatory reaction.
Key words:
Dexmedetomidine; Proconditioning; Hypoxia/reoxygenation injury; Inflammatory response
Hypoxia
Viability assay
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Objective
To investigate the protective roles of Epigallocatechin gallate (EGCG) on li-ver ischemia reperfusion injury (IRI) in rats.
Methods
30 healthy male SD rats were selected and equally and randomly divided into 3 groups. Sham group, IRI group and IRI-EGCG group were established to construct 70% liver IRI rat model. Drinking water with 0.4 mg/ml EGCG was administered for 2 weeks before the experiment in IRI-EGCG group. HE staining was performed to evaluate the injury. Transaminases in serum were investigated to assess liver injury. p-p85 and p-AKT was detected by Western-blot assay. qPCR was carried out to study the mRNA expression of TNF-α, IL-6 and IL-1β in liver tissue. The secretion of TNF-α, IL-6 and IL-1β in serum was examined with ELISA assay.
Results
EGCG pretreatment reduced AST and ALT in serum [AST: (550.0±66.5) IU/L vs. (220.0±63.5) IU/L; ALT: (376.0±25.7) IU/L vs. (158.0±33.1) IU/L, all P<0.05] and mitigated liver tissue damage. p-p85 and p-AKT increased due to liver IRI, and IRI-EGCG group showed higher expression of p85 and AKT. The proinflammatory cytokines of TNF-α, IL-6 and IL-1β exhibited a relatively lower mRNA expression in IRI-EGCG group comparing with IRI group. IRI-EGCG group also revealed a decreased secretion of TNF-α, IL-6 and IL-1β in serum [TNF-α: (398.0±33.4) ng/L vs. (211.0±23.6) ng/L; IL-6: (341.0±27.3) ng/L vs. (187.0±19.6) ng/L; IL-1β: (486.0±43.7) ng/L vs. (352.0±31.5) ng/L; all P<0.05].
Conclusion
EGCG pretreatment can enhance IRI-induced activation of PI3K/AKT signaling and reduce the release of proinflammatory cytokines to exert liver protective effects.
Key words:
Liver; Ischemia reperfusion injury (IRI); Epigallocatechin gallate (EGCG); Signal transduction, PI3K/AKT; Inflammatory cytokine
Proinflammatory cytokine
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To investigate the protective effect of high-density lipoprotein (HDL) on the mice cardiac myocytes induced by oxygen and glucose deprivation (OGD).Cardiac cells of primary scavenger receptor-B1 knockout mice (SR-B1-/-) and normal C57 mice (SR-B1+/+) were obtained by protease digestion and differential adhesion method. (1) The two kinds of cells were divided into normal control group (Con group), OGD group, OGD+HDL group. Propidium iodide (PI) staining were used to determine the necrosis of cardiac myocytes. (2) SR-B1+/+ cardiac cells were divided into Con group, OGD group, OGD+HDL group, and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) inhibitor LY294002 group. PI staining were used to determine the necrosis of cardiac myocytes. TUNEL staining was used to determine the cell apoptosis. The kit was used to determine the contents of MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) in the culture medium supernatant. The expressions of SR-B1 and Akt protein were determined by Western Blot.(1) In SR-B1+/+ cardiomyocytes, HDL could inhibit cell necrosis induced by OGD. There was no protective effect of HDL on OGD in the SR-B1-/- cardiomyocytes. (2) The study of SR-B1+/+ cells was showed that compared with Con group, necrotic cells were significantly increased and cell activity were significantly decreased, the cell viability were significantly decreased, the contents of LDH and CK-MB in supernatant were significantly increased, the expressions of phosphorylated Akt (p-Akt) and SR-B1 were significantly decreased in OGD group. Compared with OGD group, the number of necrotic cells in the OGD+HDL group was significantly decreased [PI positive cells rate: (26.71±5.94)% vs. (64.24±18.34)%], the cell activity was significantly increased [(63.84±6.95)% vs. (26.71±5.13)%], the contents of LDH and CK-MB in supernatant were significantly decreased [LDH (U/L): 896.3±161.5 vs. 1 568.3±243.5, CK-MB (U/L): 304.3±72.9 vs. 583.6±81.6], the expressions of p-Akt and SR-B1 were significantly increased (p-Akt/t-Akt: 0.84±0.13 vs. 0.18±0.06, SR-B1/β-actin: 1.23±0.19 vs. 0.09±0.02), with statistically significant differences (all P < 0.05). Compared with OGD+HDL group, necrotic cells in LY294002 group were increased, cell activity was decreased, LDH and CK-MB contents in supernatant were increased, p-Akt and SR-B1 expressions were decreased; there was no statistical difference between LY294002 group and OGD group. There was no significant difference in cell apoptosis among the 4 groups.HDL has protective effect on the mice myocardial cells. The mechanism may be related with the up regulation of the expression of SR-B1 protein by the activation of PI3K/Akt pathway.
Propidium iodide
Creatine kinase
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Objective
To explore the changes of tumor necrosis factor-α(TNF-α), nuclear factor kappa B(NF-κB), free radical in brain tissue in newborn rats with hypoxic-ischemic brain damage(HIBD), to clarify the role of TNF-α, NF-κB, free radical in HIBD.
Methods
The 56 SD neonatal rats were randomly divided into control group and hypoxic-ischemic 6 h, 12 h, 24 h, 48 h, 72 h, 7 d groups. The conventional method was used to establish HIBD model. The level of TNF-α was measured by enzyme-linked immumosorbent assay, NF-κB was detected by electrophoretic mo-bility shift assay. The neuron apoptosis was identified by flow cytometry. Superoxide dismutase(SOD), molondialdehyde(MDA), glutathione peroxidase(GSH-Px) were identified by spectrophotography.
Results
The rate of neuron apoptosis, the contents of TNF-α, NF-κB, MDA in brain tissue were significantly higher in 6 h, 12 h, 24 h, 48 h, 72 h groups than those in contral group(all P<0.05), and the apoptosis, MDA reached the peak at the 24 h after hypoxia, TNF-α reached the peak at the 12 h after hypoxia, NF-κB reached the peak at the 48 h after hypoxia. But the content of SOD and the activity of GSH-Px in hypoxic-ischemic group were significantly lower than those in control group at 6 h, 12 h, 24 h, 48 h, 72 h after hypoxia. And the rate of neuronal apoptosis was positively correlated with TNF-α, NF-κB and MAD(all P<0.05), negatively correlated with GSH-Px and SOD(all P<0.05), the contents of TNF-α, NF-κB were positively correlated with MAD(all P<0.05), negatively correlated with the content of SOD and the activity of GSH-Px(all P<0.05).
Conclusions
TNF-α, NF-κB, free radical are early changed after HIBD, So they play an important role in HIBD, inflammation and free radical can promote brain damage.
Key words:
hypoxia-ischemia; tumor necrosis factor-α; nuclear factor-kappa B; inflammation; rat, newborn
Hypoxia
Brain damage
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