scholarly journals Protective Effect of Fasudil on Hydrogen Peroxide-Induced Oxidative Stress Injury of H9C2 Cardiomyocytes

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Yu Zhang ◽  
Shanxin Liu ◽  
Xiaochun Li ◽  
Jian Ye
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Green Fluorescence ◽  
Rt Pcr ◽  
H2o2 Treatment ◽  
H9c2 Cardiomyocytes

Objective. Oxidative damage is a pathological factor that causes cardiovascular damage in the clinic and is increasingly serious. This study focused on the effect of fasudil on H2O2-induced oxidative damage in cardiomyocytes. Materials and Methods. H9C2 cardiomyocytes were cultured in vitro and divided into three groups: control group (Con group), H2O2 treatment (H2O2 group), and fasudil and H2O2 cotreatment (H2O2+fasudil group). The content levels of LDH and MDA in the supernatant were detected, and the morphology of H9C2 cardiomyocytes was observed by light microscopy. 8-OHdG staining was observed by a fluorescence inversion microscope. Cell Counting Kit (CCK-8), western blotting, real-time polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) were used to investigate the effect of fasudil on the Rho/ROCK signaling pathway. Results. Our results showed that after H2O2 treatment, the H9C2 cardiomyocytes were irregular in shape and elliptical. But the morphology of the H2O2+fasudil group was similar to that of the Con group. The green fluorescence of the H2O2 group was significantly enhancer than that of the Con group, while the green fluorescence of the H2O2+fasudil group was weaker than those of the H2O2 group. By detecting the supernatant, it was found that the contents of LDH were significantly increased, and the contents of SOD and CAT in the H2O2 group were significantly decreased. And the expression of antioxidant indicators in the H2O2 group was significantly decreased by western blotting. The results of RT-PCR showed that SOD1 and SOD2 mRNA in the H2O2 group was significantly reduced, and the contents of GPX1 and GPX3 in the H2O2 group were significantly decreased by enzyme-linked immunosorbent assay (ELISA). The expression of ROCK1, ROCK2, and downstream phosphorylation of myosin phosphatase target subunit-1 (p-MYPT-1) was significantly increased in the H2O2 group, while fasudil inhibited the increase of ROCK1, ROCK2, and p-MYPT-1. Conclusions. Fasudil can inhibit the Rho/ROCK signaling pathway induced by H2O2 and reduce oxidative stress response, inhibit apoptosis, and improve antioxidant enzyme activity in H9C2 cardiomyocytes thereby delaying cell senescence.

scholarly journals Mechanisms Underlying H2O2-Evoked Carbonyl Modification of Cytoskeletal Protein and Axon Injury in PC-12 Cells

2018 ◽  
Vol 48 (3) ◽  
pp. 1088-1098 ◽  
Author(s):  
Xiejun Zhang ◽  
Zongyang Li ◽  
Qiusheng Zhang ◽  
Lei Chen ◽  
Xianjian Huang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Western Blotting ◽  
Axonal Injury ◽  
Cytoskeletal Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
H2o2 Treatment ◽  
Pc 12 Cells ◽  
Axonal Dysfunction ◽  
Β Tubulin

Background/Aims: To investigate the mechanism that enables oxidative stress and cytoskeleton protein carbonylation to contribute to axonal dysfunction in traumatic brain injury (TBI). Methods: We created an in vitro model of neuronal oxidative damage by exposing a neuron-like cell line (PC-12) to different concentrations (100 μM, 200 μM, and 300 μM) of H2O2 for 24 h or 48 h. Carbonyl modification of cytoskeletal proteins (β-actin and β-tubulin) and its impact on β-actin/β-tubulin filament dynamics were determined by enzyme-linked immunosorbent assay, immunostaining, and western blotting. Depolymerization of β-actin/β-tubulin filaments was evaluated using the monomer/polymer ratio of each protein via western blotting. Phosphorylation of the neurofilament heavy chain (P-NFH) was used as an axonal injury marker and detected by immunostaining. Results: Our results showed that H2O2 treatment led to increased oxidative stress in PC-12 cells, as indicated by the increased generation of malondialdehyde and 8-hydroxydeoxyguanosine and decreased intracellular glutathione levels. H2O2 treatment also increased carbonyl modification of total proteins and cytoskeleton proteins β-actin/β-tubulin, which occurred concurrently with the suppression of proteasome activity. Moreover, H2O2 treatment increased the generation of the axonal injury marker P-NFH, and depolymerization of the β-actin/β-tubulin filaments was indicated by increased monomer/polymer ratios of each protein. Lastly, overexpression of the proteasome β5 subunit in PC-12 cells significantly reduced the H2O2-induced accumulation of carbonylated β-actin/ β-tubulin, P-NFH, and β-actin/β-tubulin depolymerization. Conclusions: We concluded that carbonylation of cytoskeleton proteins could lead to depolymerization of their filaments and axonal injury, and proteasome suppression contributes to the accumulation of carbonylated proteins under oxidative conditions.

scholarly journals Fermentation of Panax notoginseng root extract polysaccharides attenuates oxidative stress and promotes type I procollagen synthesis in human dermal fibroblast cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shiquan You ◽  
Xiuqin Shi ◽  
Dan Yu ◽  
Dan Zhao ◽  
Quan An ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Antioxidant Activity ◽  
Oxidative Damage ◽  
Signaling Pathway ◽  
Dermal Fibroblast ◽  
Panax Notoginseng ◽  
Human Dermal Fibroblast ◽  
Down Regulation ◽  
Smad Signaling ◽  
Smad Signaling Pathway

Abstract Background Panax notoginseng is one of the most valuable traditional Chinese medicines. Polysaccharides in P. notoginseng has been shown to significantly reduce the incidence of human diseases. However the application of fermentation technology in Panax notoginseng is not common, and the mechanism of action of P. notoginseng polysaccharides produced by fermentation is still unclear. The specific biological mechanisms of fermented P. notoginseng polysaccharides (FPNP) suppresses H2O2-induced apoptosis in human dermal fibroblast (HDF) and the underlying mechanism are not well understood. Methods In this study, the effects of water extracted and fermentation on concentration of polysaccharides in P. notoginseng extracts were analyzed. After the H2O2-induced HDF model of oxidative damage was established, and then discussed by the expression of cell markers, including ROS, MDA, SOD, CAT, GSH-Px and MMP-1, COL-I, ELN, which were detected by related ELISA kits. The expression of TGF-β/Smad pathway markers were tested by qRT-PCR to determine whether FPNP exerted antioxidant activity through TGF-β signaling in HDF cells. Results The polysaccharide content of Panax notoginseng increased after Saccharomyces cerevisiae CGMCC 17452 fermentation. In the FPNP treatment group, ROS and MDA contents were decreased, reversed the down-regulation of the antioxidant activity and expression of antioxidant enzyme (CAT, GSH-Px and SOD) induced by H2O2. Furthermore, the up-regulation in expression of TGF-β, Smad2/3 and the down-regulation in the expression of Smad7 in FPNP treated groups revealed that FPNP can inhibit H2O2-induced collagen and elastin injury by activating TGF-β/Smad signaling pathway. Conclusion It was shown that FPNP could inhibit the damage of collagen and elastin induced by H2O2 by activating the TGF-β/Smad signaling pathway, thereby protecting against the oxidative damage induced by hydrogen peroxide. FPNP may be an effective attenuating healing agent that protects the skin from oxidative stress and wrinkles.

ALK7 Inhibition Protects Osteoblast Cells Against High Glucose-induced ROS Production via Nrf2/HO-1 Signaling Pathway

2021 ◽  
Vol 21 ◽  
Author(s):  
Zhen Zhao ◽  
Yu Lu ◽  
Huan Wang ◽  
Xiang Gu ◽  
Luting Zhu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Signaling Pathway ◽  
High Glucose ◽  
Collagen I ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heme Oxygenase 1 ◽  
Ros Production ◽  
Alizarin Red

Background: Some studies demonstrated that under high-glucose (HG) condition, osteoblasts develop oxidative stress, which will impair their normal functions. The effects of activin receptor-like kinase 7 (ALK7) silencing on HG-induced osteoblasts remained unclear. Objective: The aim of this study was to explore the effect of ALK7 on HG-induced osteoblasts. Methods: MC3T3-E1 cells were treated with different concentrations of HG (0, 50, 100, 200 and 300mg/dL), and the cell viability was detected using cell counting kit-8 (CCK-8). HG-treated MC3T3-E1 cells were transfected with siALK7 or ALK7 overexpression plasmid or siNrf2, and then the viability and apoptosis were detected by CCK-8 and flow cytometry. The levels of reactive oxygen species (ROS), collagen I and calcification nodule were determined by oxidative stress kits, Enzyme-linked immunosorbent assay and Alizarin red staining. The expressions of NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and osteoblast-associated genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot. Results: Cell viability was reduced with HG treatment. Silencing ALK7 inhibited the effect of HG on increasing cell apoptosis and ROS production, reduced cell viability, mineralized nodules, and downregulated collagen I and osteoblast-associated genes expression in MC3T3-E1 cells. ALK7 silencing activated the Nrf2/HO-1 signaling pathway by affecting expressions of HO-1 and Nrf2. ALK7 overexpression had the opposite effects. In addition, siNrf2 partially reversed the effects of ALK7 silencing on HG-induced MC3T3-E1 cells. Conclusion: ALK7 silencing protected osteoblasts under HG condition possibly through activating the Nrf2/HO-1 pathway.

scholarly journals Lipopolysaccharide exposure induces oxidative damage in Caenorhabditis elegans: protective effects of carnosine

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Jing Ma ◽  
Xiaoyuan Xu ◽  
Ranran Wang ◽  
Haijing Yan ◽  
Huijuan Yao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caenorhabditis Elegans ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Mapk Signaling ◽  
Protective Effects ◽  
Related Gene ◽  
Rt Pcr ◽  
C Elegans ◽  
Apoptosis Related Gene

Abstract Background The present study was designed to investigate the protective effects and mechanisms of carnosine on lipopolysaccharide (LPS)-induced injury in Caenorhabditis elegans. Methods C. elegans individuals were stimulated for 24 h with LPS (100 μg/mL), with or without carnosine (0.1, 1, 10 mM). The survival rates and behaviors were determined. The activities of superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT) and levels of malondialdehyde (MDA) and glutathione (GSH) were determined using the respective kits. Reverse transcription polymerase chain reaction (RT-PCR) was performed to validate the differential expression of sod-1, sod-2, sod-3, daf-16, ced-3, ced-9, sek-1, and pmk-1. Western blotting was used to determine the levels of SEK1, p38 mitogen-activated protein kinase (MAPK), cleaved caspase3, and Bcl-2. C. elegans sek-1 (km2) mutants and pmk-1 (km25) mutants were used to elucidate the role of the p38 MAPK signaling pathway. Results Carnosine improved the survival of LPS-treated C. elegans and rescued behavioral phenotypes. It also restrained oxidative stress by decreasing MDA levels and increasing SOD, GR, CAT, and GSH levels. RT-PCR results showed that carnosine treatment of wild-type C. elegans up-regulated the mRNA expression of the antioxidant-related genes sod-1, sod-2, sod-3, and daf-16. The expression of the anti-apoptosis-related gene ced-9 and apoptosis-related gene ced-3 was reversed by carnosine. In addition, carnosine treatment significantly decreased cleaved caspase3 levels and increased Bcl-2 levels in LPS-treated C. elegans. Apoptosis in the loss-of-function strains of the p38 MAPK signaling pathway was suppressed under LPS stress; however, the apoptotic effects of LPS were blocked in the sek-1 and pmk-1 mutants. The expression levels of sek-1 and pmk-1 mRNAs were up-regulated by LPS and reversed by carnosine. Finally, the expression of p-p38MAPK and SEK1 was significantly increased by LPS, which was reversed by carnosine. Conclusion Carnosine treatment protected against LPS injury by decreasing oxidative stress and inhibiting apoptosis through the p38 MAPK pathway.

A Comparison of an Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting for Detection of Peanut Mottle Virus and Peanut Stripe Virus1

1986 ◽  
Vol 13 (2) ◽  
pp. 64-67 ◽  
Author(s):  
J. L. Sherwood ◽  
H. A. Melouk
Keyword(s):  
Western Blotting ◽  
Immunosorbent Assay ◽  
Protein A ◽  
Mottle Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coat Proteins ◽  
Western Blots ◽  
Peanut Stripe Virus ◽  
Dry Milk ◽  
The Difference

Abstract Western blotting was used to detect infections of peanut cv. Tamnut 74 with peanut mottle virus (PMV) and/or peanut stripe virus (PStV). Leaf samples were ground in electrophoresis sample buffer and heated for 5 min at 95 C prior to electrophoresis in 12% polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose sheets at 100V for 45 min. Western blots were performed by first blocking unbound sites on the nitrocellulose with 5% non-fat dry milk in Tris-buffered saline (TBS), pH 7.4 for 30 min, followed by incubation in a 1/200 dilution of PMV and/or PStV antiserum in TBS (the latter antiserum provided by J. W. Demski, U. of GA) for 45 min. This was followed by incubation in protein-A-peroxidase (2 μg/mL in TBS) for 45 min, followed by 4-chloro-1-napthol plus hydrogen peroxide in TBS. As little as 25 ng of either purified PMV or PStV was detected. This was similar to the limits of detection fo the double sandwich enzyme linked immunosorbent assay (ELISA). Because of the difference in migration of the coat proteins of PMV and PStV, both viruses may be detected in plants infected with PMV and PStV. This assay can be performed in approximately 6 h when mini-gels are used for the initial electrophoretic seperation and does not require the antiserum to be fractionated or bound to an enzyme as is the case with ELISA.

scholarly journals PRDX6 Protects ARPE-19 Cells from Oxidative Damage via PI3K/AKT Signaling

2015 ◽  
Vol 36 (6) ◽  
pp. 2217-2228 ◽  
Author(s):  
Xu Zha ◽  
Guojiu Wu ◽  
Xueying Zhao ◽  
Liqiong Zhou ◽  
Hong Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Blue Light ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Annexin V ◽  
Retinal Disease ◽  
Akt Signaling ◽  
The Body ◽  
H2o2 Treatment

Background/Aims: Oxidative stress that damages cells of the retinal pigment epithelium (RPE) can cause the development of hereditary retinal disease (HRD). PRDX6, which is a member of the PRDX family, is essential for removing metabolic free radicals from the body. However, the effect of PRDX6 on oxidative stress in HRD remains unknown. In this study, we sought to investigate the role of PRDX6 in oxidative stress-induced HRD in ARPE-19 cells and the molecular mechanism involved. Methods: ARPE-19 cells were used in the current study. Intracellular ROS levels were determined by flow cytometry. Lipid peroxidation was measured using a commercial MDA assay kit. Cellular variability was determined by MTT assay. Apoptosis was determined using an Annexin V-FITC Apoptosis Detection Kit. mRNA and protein expression levels were detected by real-time PCR and western blot analysis, respectively. Results: We found that H2O2 and blue light could induce significant oxidative stress damage and cell death in ARPE-19 cells. Furthermore, we found that PRDX6 levels significantly decreased after H2O2 treatment. PRDX6 overexpression protected ARPE-19 cells from H2O2- and blue light-induced oxidative damage, while PRDX6 knockdown enhanced oxidative damage in these cells. Mechanistically, we found that PRDX6 prevented oxidative damage and promoted ARPE-19 cell survival through the PI3K/AKT signaling pathway. Conclusions: Collectively, these results suggest that PRDX6 protects ARPE-19 cells from H2O2-induced oxidative stress and apoptosis and that this protection is mediated at least partially through the PI3K/AKT pathway.

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