ObjectiveTo investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.MethodsThe skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.ResultsAfter identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased (P<0.05), and the level of ROS also significantly increased (P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased (P<0.05), the relative expression of Bcl-2 protein significantly increased (P<0.05), the cell apoptosis rate and level of ROS also significantly decreased (P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.ConclusionInhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.
ObjectiveTo review the recent research progress about the pathogenesis and prevention of reactive oxygen species (ROS) in the hepatic ischemia-reperfusion injury (HIRI). MethodsSearched the related literatures in recent years from the databases such as CNKI, PubMed and so on, summarized the recent research progress about the generation mechanism of ROS, the damage mechanism of ROS, and the prevention method of ROS. ResultsA mass of ROS originated from polymorphonuclear leukocytes, Kupffer cells, mitochondria, and the enzymes in hepatic tissue in HIRI. It mainly destroyed sugar molecules of oligosaccharide chains on the cell membrane, unsaturated fatty acid, protein molecules, mitochondrial, and genetic material. This mechanism lead to cell injuried or even death. The main method of prevention and cure to HIRI is eliminating ROS by using enzymes, vitamins, Chinese herbal medicines etc. ConclusionsThe research about ROS in HIRI has advanced. Aiming at the damage resulted from ROS in the liver, Scholars have came up with a variety of control methods which is feasible. However, many issues need to be further investigated.
Objective Glucocorticoid is the main cause of non-traumatic avascular necrosis of femoral head. To explore the changes of reactive oxygen species (ROS) in the bone microvascular endothel ial cells treated with glucocorticoid so as to investigate the pathogenesis of steroid-induced avascular necrosis of femoral head. Methods The cancellous bone of femoral head was harvested from voluntary donators undergoing total hip arthroplasty, and then the bone microvascular endothel ial cells were isolated by enzyme digestion. The cells at passage 3 were cocultured with different concentrations of hydrocortisone (0, 0.03, 0.10, 0.30, and 1.00 mg/mL) for 24 hours. MTT assay was used for the inhibitory rate of cell prol iferation, flow cytometry for apoptosis rate, and fluorescence probe for the production of ROS and xanthine oxidase (XOD). Results At 2-3 days primary culture, the cells were spindle and arranged l ike cobbles and they reached confluence after 1 week. The inhibitory rates of cell prol iferation in 0.03, 0.10, 0.30, and 1.00 mg/mL groups were 20.22% ± 2.97%, 22.94% ± 4.52%, 43.98% ± 3.35%, and 78.29% ± 3.85%, respectively; and 2 high-concentration groups (0.30 and 1.00 mg/mL groups) were significantly higher (P lt; 0.05) than 2 low-concentration groups (0.03 and 0.10 mg/mL groups). The apoptosis rates in 0, 0.03, 0.10, 0.30, and 1.00 mg/mL groups were 0.10% ± 0.01%, 0.23% ± 0.02%, 1.83% ± 0.04%, 6.34% ± 0.11%, and 15.33% ± 0.53%, respectively; 2 high-concentration groups (0.30 and 1.00 mg/mL groups) were significantly higher (P lt; 0.05) than 0 mg/mL group. In 0, 0.30, and 1.00 mg/ mL groups, the ROS levels were 57.35 ± 7.11, 120.47 ± 15.68, and 166.15 ± 11.57, respectively, and the XOD levels were 0.017 9 ± 0.000 9, 0.028 3 ± 0.001 7, and 0.067 7 ± 0.004 1, respectively; there were significant differences in the levels of ROS and XOD among 3 groups (P lt; 0.05). Conclusion Increasing of ROS production in bone microvascular endothel ial cells can be induced by high concentration glucocorticoid, and it can result in cell injury
ObjectiveTo investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4 (BMP4) on Müller cell migration, reactive oxygen species (ROS) generation and vascular endothelial growth factor (VEGF) expression. MethodsMüller cells cultured in vitro were divided into normal control group, BMP4 group, BMP4+ no-load plasmid group (BMP4+NC group) and BMP4+SMAD9 small interference plasmid group (BMP4+siSMAD9). Cells in BMP4 group, BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h. Subsequently, BMP4+NC group was transfected with empty plasmid. BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h. The effect of BMP4 on Müller cell migration was determined by cell scratch test. The effect of BMP4 on the production of ROS in Müller cells was detected by flow cytometry. Western blots and real-time quantitative fluorescence polymerase chain reaction (qPCR) were used to detect the relative mRNA expression levels of glutamine synthetase (GS) and glial fibrinoacidic protein (GFAP) in Müller cells. VEGF expression in Müller cells was detected by immunofluorescence. One-way analysis of variance was used to compare groups. ResultsThe results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group, and the difference was statistically significant (F=68.319, P＜0.001). Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group, and the difference was statistically significant (F=52.158, P＜0.001). Western blot and qPCR results showed that the protein levels of GS and GFAP (F=42.715, 36.618) and mRNA relative expression levels (F=45.164, 43.165) in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group. The difference was statistically significant (P＜0.01). The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was significantly higher than that in BMP4+siSMAD9 group, and the difference was statistically significant (F=46.384, P＜0.05). ConclusionTargeted regulation of SMAD9 expression by BMP4 can up-regulate VEGF expression and promote the migration and ROS production of Müller cells.
ObjectiveGelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia. Methods Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively. Islets were cultured under hypoxia (1%O2) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability. Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O2) and hypoxia (1%O2) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O2) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O2) conditions, respectively. Results GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group (P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C (P<0.05). Conclusion GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.
Objective To explore the potential protective effect in vivo of Edaravone, a free radical scavenger on model of acute lung injury in rats with sepsis. Methods Twenty-four male Wistar rats were randomly divided into three groups, ie. a control group( NS group) , a model group( LPS group) , a Edaravone treatment group( ED group) . ALI was induced by injecting LPS intravenously( 10 mg/ kg) in the LPS group and the ED group. Meanwhile the ED group was intravenously injected with Edaravone( 3 mg/ kg) . The NS group was injected with normal saline as control. The lung tissue samples were collected at 6 h after intravenous injection. The wet / dry ( W/D) weight ratio of lung tissue was measured. The levels of myeloperoxidase ( MPO) , malondialdehyde ( MDA ) and superoxide dismutase ( SOD) in lung tissue homogenate were assayed. The pathological changes and expression of nuclear factor-kappa B( NF-κB) in lung tissue were also studied. Results Compared with the NS group, The W/D, pathological scores, NF-κB expression, MPO and MDA levels in the LPS group were significantly higher( all P lt; 0. 01) , and the level of SOD was apparently lower( P lt; 0. 01) . The W/D, pathological scores, NF-κB expression, MPO and MDA levels in the ED group were significantly lower than those in the LPS group( all P lt; 0. 01) and higher than those in the NS group( all P lt; 0. 01) . And the level of SOD in lung tissue of the ED group was higher than that in the LPS group and lower than that in the NS group ( P lt; 0. 01) . Conclusions Edaravone has protective effect on ALI rat model. The mechanismmay be related to its ability of clearing the reactive oxygen species, inhibiting the activation of the signal pathway of NF-κB and inflammatory cascade.
目的 观察黄芪三七合剂（Aamp;R）对肾缺血再灌注损伤（IRI）大鼠血液活性氧（ROS）变化的影响，探讨其抗IRI损伤的机制。 方法 雄性Sprague-Dawley（SD）大鼠30只，随机分为正常组（n=5）、假手术组（SG）（n=5）和IRI 24 h组（n=10），Aamp;R组（n=10）。造模：采用微血管夹夹闭双侧肾蒂，22 min后松开动脉夹，用5/0尼龙缝合线缝合腹部。再灌注24 h后将小鼠行麻醉处死。Aamp;R组给予Aamp;R（3 mL/d），假手术组及IRI 24 h组给予同等体积的生理盐水。采用全自动生化分析仪检测各组大鼠的肾功能，苏木精-伊红染色了解肾脏病理损害，流式细胞仪检测红细胞ROS。 结果 IRI 24 h组和Aamp;R组肾小管出现不同程度的管腔扩张、变性与坏死，间质炎性细胞浸润、充血水肿等变化。IRI后24 h时，IRI 24 h组、Aamp;R组血清尿素氮（BUN）和肌酐（Cr）均高于假手术组、正常组，差异有统计学意义（P＜0.05）；Aamp;R组ROS荧光强度阳性率显著低于IRI 24 h组，差异有统计学意义（P＜0.05）。Aamp;R组肾小管损伤评分明显低IRI 24 h组（P＜0.05）。相关性分析发现，红细胞ROS荧光强度阳性率与肾小管损伤评分、肌酐、尿素氮水平成正相关（r=0.917，P＜0.01；r=0.897，P＜0.01；r=0.896，P＜0.01）。 结论 Aamp;R对肾脏缺血再灌注损伤具有明显的保护作用，其机制可能为抑制血液中ROS的活性，从而抑制氧化应激对肾脏的损伤。
Objective To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism. MethodsThe HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. ResultsThe number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups (t=12.311, 15.643, 17.346; P＜0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups (t=16.311, 14.833, 18.442; P＜0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences (t=17.342, 16.813, 18.794; P＜0.001). ConclusionPSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.
Reactive oxygen species (ROS) play an important role in the pathogenesis of various cardiovascular diseases, by leading to cell apoptosis and thus causing organic injuries. Anti-ROS therapy is highly anticipated, but currently, there is still no appropriate prevention method. Studies have shown that thioredoxin (Trx), being a kind of significant endogenous antioxidant system, has excellent antioxidant capacity. Promotion of Trx can reduce key biomolecules to eliminate ROS or regulate many signaling pathways, thus resisting ROS injuries, which may be a new anti-ROS strategy. Therefore, we reviewed the research progress of Trx in cardiac antioxidant therapy to discuss its potential and possibility to be a target for prevention of heart-related ROS injury.
ObjectiveTo investigate the molecular mechanism by which metastasis-associated protein 3 (MTA3) participates in glioma resistance through reactive oxygen species. Methods Protein expression in glioma stem cells (GSCs) and non-GSCs was detected using Western blotting. GSCs included U87 and SHG44 cells, while non-GSCs included U87s and SU-2 cells. After overexpressing MTA3, U87 and SHG44 cells were divided into Lv-scr and Lv-MTA3 groups. The self-renewal capacity of glioma cells was assessed through a neurosphere formation assay. Cell survival fractions were examined following exposure to 0, 2, 4, 6, 8, and 10 Gy X-ray irradiation under normoxic or hypoxic conditions. Apoptosis and reactive oxygen species expression were analyzed using flow cytometry. Immunofluorescence staining was performed to detect the stem cell markers CD133 and nestin, as well as the differentiation markers glial fibrillary acidic protein (GFAP, for astrocytes) and neuronal class Ⅲ β-tubulin. Results In GSCs, MTA3 expression was lower in the U87s and SU-2 groups. After MTA3 overexpression, Lv-MTA3 expression was higher in U87s and SU-2 compared to the Lv-scr group. Under normoxic or hypoxic conditions, U87 and SU-2 showed greater radioresistance compared to glioma cell lines U87 and SHG44. Compared to non-GSCs, basal reactive oxygen species formation was reduced in GSCs, while reactive oxygen species generation was increased in non-GSCs. Following exposure to different doses of X-rays under normoxic or hypoxic conditions, GSCs with MTA3 overexpression exhibited greater radiosensitivity than those with stable integration. Additionally, MTA3 overexpression slightly increased the oxygen enhancement ratio (OER) in GSCs. MTA3 overexpression reduced the immunoreactivity of CD133 and nestin in both stem cell lines, and increased immunofluorescence staining of GFAP and neuronal class Ⅲ β-tubulin, with statistically significant differences (P<0.05). Conclusions MTA3 is downregulated in GSCs. Overexpression of MTA3 reduces the radioresistance and stemness of GSCs both in vitro and in vivo. MTA3 plays a crucial role in regulating the radiosensitivity and stemness of GSCs through reactive oxygen species.