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.
Objective To observe and preliminarily explore the effect of mogroside on oxidative stress of retinal pigment epitheliaum (RPE) cells induced by hydrogen peroxide (H2O2) and its possible mechanism. MethodsA experimental study. The RPE cells were divided into control group, H2O2 group, silent information regulator of transcription 1 (SIRT1) inhibitor EX527 group (EX527 group), mogroside group, mogroside+EX527 group. Methyl thiazolete trazolium method was used to detect cell survival rate. Flow cytometry was used to detect cell apoptosis rate. 2',7'-dichlorodihydrofluorescein diacetate fluorescent probe method, xanthine method and enzyme-linked immunosorbent assay method were used to detect the level of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cells respectively. Real-time quantitative polymerase chain reaction and Western blot were used to detect relative expressions of SIRT1, nuclear factor erythroid-2-related actor 2 (Nrf2), heme oxygenase-1 (HO-1) mRNA and protein in cells. One-way ANOVA was used for comparison among groups. The pairwise comparison between groups was tested by the least significant difference t test. Results Compared with the control group, the H2O2 group cell survival rate decreased, the apoptosis rate increased, the ROS level in the cells increased, the SOD activity decreased, the MDA content increased, and the relative expression of SIRT1, Nrf2, HO-1 mRNA and protein decreased (P＜0.05). Compared with H2O2 group, the cell survival rate decreased, apoptosis rate increased, the cell ROS level increased, SOD activity decreased, MDA content increased, SIRT1, Nrf2, HO-1 mRNA and protein expression decreased in EX527 group (P＜0.05); the cell survival rate increased, apoptosis rate decreased, ROS level decreased, SOD activity increased, MDA content decreased, and the relative expression of SIRT1, Nrf2, HO-1 mRNA and protein increased in mogroside group (P＜0.05). Compared with the mogrosides group, the cell survival rate decreased, the apoptosis rate increased, the level of ROS increased, SOD activity decreased, MDA content increased, SIRT1, Nrf2, HO-1 mRNA and protein decreased in mogrosides+EX527 group (P＜0.05). ConclusionsMogrosides can alleviate the oxidative stress response of visual RPE cells induced by H2O2, promote cell proliferation, and reduce cell apoptosis. Mogrosides may exert antioxidant effects by activating the SIRT1/Nrf2 signaling pathway.
Epilepsy is a heterogeneous disease with a very complex etiological mechanism, characterized by recurrent and unpredictable abnormal neuronal discharge. Epilepsy patients mainly rely on oral antiseizure medication (ASMs) the for treatment and control of disease progression. However, about 30% patients are resistance to ASMs, leading to the inability to alleviate and cure seizures, which gradually evolve into refractory epilepsy. The most common type of intractable epilepsy is temporal lobe epilepsy. Therefore, in-depth exploration of the causes and molecular mechanisms of seizures is the key to find new methods for treating refractory epilepsy. Mitochondria are important organelles within cells, providing abundant energy to neurons and continuously driving their activity. Neurons rely on mitochondria for complex neurotransmitter transmission, synaptic plasticity processes, and the establishment of membrane excitability. The process by which the autophagy system degrades and metabolizes damaged mitochondria through lysosomes is called mitophagy. Mitophagy is a specific autophagic pathway that maintains cellular structure and function. Mitochondrial dysfunction can produce harmful reactive oxygen species, damage cell proteins and DNA, or trigger programmed cell death. Mitophagy helps maintain mitochondrial quality control and quantity regulation in various cell types, and is closely related to the occurrence and development of epilepsy. The imbalance of mitophagy regulation is one of the causes of abnormal neuronal discharge and epileptic seizures. Understanding its related mechanisms is crucial for the treatment and control of the progression of epilepsy in patients.
Objective To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs). Methods The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall methodin vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×105 cells/mL); group B, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot. Results The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups (P<0.05); but there was no significant difference between groups D and E (P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance (A) values had significant differences between groups (P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E (P<0.05), but there was no significant difference among groups A, D, and E (P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups (P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups (P<0.05). Conclusions GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.
Objective To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. Methods Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. Results Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). Conclusion The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
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.
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.
To aggressively proliferate and metastasize, cancer cells are in extreme need of energy supply and nutrients. Therefore, a promising cancer therapy strategy is developed to target its hallmark feature of metabolism. Recent findings revealed the regulatory role of caveolin-1 (Cav-1), a structural protein of caveolae, in cancer metabolism. And low Cav-1 expression in tumor stroma was proved to be a central player of cancer malignant phenotype. Here, we summarized the progressions of studies on Cav-1, mitochondria and cancer metabolism to indicate that the altered metabolism induced by Cav-1 and mitochondria association is a major cause of cancer malignant phenotype.
ObjectiveTo investigate the effects of interferon gene stimulating protein (STING) inhibitor (C176) on human retinal microvascular endothelial cells (hRMEC) under oxidative stress. MethodsAn animal experimental study. In vivo experiment: 48 healthy male C57BL/6J mice were randomly divided into wild type mice group (WT group) and diabetes (DM) group, with 24 mice in each group. DM mice were induced by streptozotocin to establish DM model. After successful modeling, DM group was divided into DM+dimethyl sulfoxide (DMSO) group and DM+C176 group, with 12 mice in each group. The mice in the DM+DMSO group were intraperitoneally injected with DMSO at the dose of 50 mg/kg. Mice in DM+C176 group were intraperitoneally injected with STING inhibitor C176 750 nmol at the dose of 50 mg/kg. Four weeks after modeling, immunohistochemical staining, Western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect the expression of STING in the retina of WT and DM mice. The leukocyte adhesion test was used to detect the number of leukocytes adhering to hRMEC in mice with WT, DM+DMSO and DM+C176 groups. In vitro experiment: hRMEC was randomly divided into conventional culture cell group (N group), dimethyl sulfoxide (DMSO) group (with DMSO intervention) and C176 group (with C176 intervention). The cells were induced by 150 μg/ml glycation end products for each group. In vitro leukocyte adhesion test combined with 4', 6-diamino-2-phenylindole staining was used to detect the number of leukocytes adhering to hRMEC. The adherent leukocytes were quantitatively analyzed by flow cytometry; H2DCFDA/reactive oxygen species (ROS) fluorescence probe was used to detect ROS expression in cells; Seahorse XFe96 cell energy metabolism analyzer was used to measure the level of intracellular glycolysis. t-test was used to compare the two groups; single factor analysis of variance was used to compare the three groups. ResultsIn vivo experiment: compared with WT group, the expression level of STING (t=73.248) and the relative expression amount of mRNA (t=67.385) in the retina of DM group mice increased significantly (P＜0.05). Compared with WT group, the number of leukocytes adhering to the retinal vessels of mice in DM+DMSO group was significantly increased, while that in DM+C176 group was significantly decreased (F=84.352, P＜0.01). In vitro: compared with N group and DMSO group, the number of leukocyte adhesion on hRMEC in C176 group decreased significantly (F=35.251, P＜0.01). Compared with N group, the number of leukocytes adhering to hRMEC in DMSO group and C176 group decreased significantly (F=26.374, P＜0.01). The ROS level in hRMEC in C176 group was significantly lower than that in N group and C176 group (F=41.362, P＜0.01). Compared with N group and DMSO group, the glycolysis level of hRMEC in C176 group was significantly reduced, with a statistically significant difference (F=68.741, P＜0.01). ConclusionInhibiting the expression of STING in retinal vascular endothelial cells can improve the progress of DM by inhibiting leukocyte adhesion, ROS production and glycolysis level.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.