scholarly journals Overexpression of CRABP2 inhibits dexamethasone-induced apoptosis in human osteoblast cells

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Haiping Zhang ◽  
Ziliang Yu ◽  
Farui Sun ◽  
Jin Jin
Keyword(s):  
Protein Expression ◽  
Underlying Mechanism ◽  
R Package ◽  
Treated Group ◽  
Gene Expression Omnibus ◽  
Human Osteoblast ◽  
Osteoblast Cells ◽  
Gene And Protein Expression ◽  
Osteoblast Apoptosis ◽  
Induced Apoptosis

Abstract Background The purpose of the current study was to explore the role and underlying mechanism of cellular retinoic acid binding protein 2 (CRABP2) in dexamethasone (DEX)-induced apoptosis in human osteoblast cells. Methods GSE10311 was downloaded from the Gene Expression Omnibus (GEO) database to identify the differentially expressed genes (DEGs) by the limma/R package. Primary human osteoblast was isolated and treated with different concentration of DEX (0, 10-8, 10-7, 10-6, 10-5, and 10-4 mol/L), and cell viability and flow cytometry were used to detect cell proliferation and apoptosis. A CRABP2 overexpression plasmid (oe-CRABP2) was used to overexpress CRABP2, and western blotting was conducted to detect protein expression. Results We found that CRABP2 was downregulated in the DEX-treated group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that DEGs were associated with PI3K/Akt signaling pathway. DEX downregulated CRABP2 gene and protein expression, inhibited viability, and induced human osteoblast apoptosis. Overexpression of CRABP2 reversed DEX-induced apoptosis in human osteoblast. Moreover, overexpression of CRABP2 delayed the progression of DEX-induced osteonecrosis of the femoral head (ONFH) animal model. Conclusion In conclusion, CRABP2 is effective at inhibiting DEX-induced human osteoblast apoptosis and delayed ONFH progression.

scholarly journals Overexpression of FGF2 delays the progression of osteonecrosis of the femoral head activating the PI3K/Akt signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Pei Lu ◽  
Yi-min Shen ◽  
Ting Hua ◽  
Ting Pan ◽  
Gang Chen ◽  
...  
Keyword(s):  
Femoral Head ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Underlying Mechanism ◽  
Treated Group ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Gene And Protein Expression ◽  
Induced Apoptosis

Abstract Background The purpose of the current study was to explore the role and underlying mechanism of FGF-2 in dexamethasone (DEX)-induced apoptosis in MC3T3-E1 cells. Methods GSE21727 was downloaded from the Gene Expression Omnibus (GEO) database to identify the differentially expressed genes (DEGs) by the limma/R package. MC3T3-E1 cells were exposed to DEX at different concentrations (0, 10−8, 10−7, 10−6, 10−5 and 10−4 mol/L), and cell viability, flow cytometry and TUNEL assay were used to detect cell proliferation and apoptosis. An FGF-2-pcDNA3 plasmid (oe-FGF-2) was used to overexpress FGF-2, and western blotting was conducted to detect protein expression. Results We found that FGF-2 was downregulated in the DEX-treated group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that DEGs were associated with PI3K/Akt signaling pathway. DEX downregulated FGF-2 gene and protein expression, inhibited viability and induced MC3T3-E1 cell apoptosis. Overexpression of FGF-2 reversed DEX-induced apoptosis in MC3T3-E1 cells. FGF-2-mediated anti-apoptosis was impaired by inactivating the PI3K/AKT pathway with LY294002. Moreover, overexpression of FGF2 delayed the progression of DEX-induced osteonecrosis of the femoral head (ONFH) animal model by regulation PI3K/Akt signaling pathway. Conclusion In conclusion, FGF-2 is effective at inhibiting DEX-induced MC3T3-E1 cell apoptosis through regulating PI3K/Akt signaling pathway.

Icariin Stimulates hFOB 1.19 Osteoblast Proliferation and Differentiation via OPG/RANKL Mediated by the Estrogen Receptor

2020 ◽  
Vol 22 (1) ◽  
pp. 168-175 ◽  
Author(s):  
Lin-Jun Sun ◽  
Chong Li ◽  
Xiang-hao Wen ◽  
Lu Guo ◽  
Zi-Fen Guo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Protein Expression ◽  
Chinese Herb ◽  
Human Osteoblast ◽  
Osteoblast Cells ◽  
Expression Ratio ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Mrna And Protein Expression

Background:: Icariin (ICA), one of the main effective components isolated from the traditional Chinese herb Epimedium brevicornu Maxim., has been reported to possess extensive pharmacological actions, including enhanced sexual function, immune regulation, anti-inflammation, and antiosteoporosis. Methods:: Our study was designed to investigate the effect of ICA on cell proliferation and differentiation and the molecular mechanism of OPG/RANKL mediated by the Estrogen Receptor (ER) in hFOB1.19 human osteoblast cells. Results:: The experimental results show that ICA can stimulate cell proliferation and increase the activity of Alkaline Phosphatase (ALP), Osteocalcin (BGP) and I Collagen (Col I) and a number of calcified nodules. Furthermore, the mRNA and protein expression of OPG and RANKL and the OPG/ RANKL mRNA and protein expression ratios were upregulated by ICA. The above-mentioned results indicated that the optimal concentration of ICA for stimulating osteogenesis was 50ng/mL. Subsequent mechanistic studies comparing 50ng/mL ICA with an estrogen receptor antagonist demonstrated that the effect of the upregulated expression is connected with the estrogen receptor. In conclusion, ICA can regulate bone formation by promoting cell proliferation and differentiation and upregulating the OPG/RANKL expression ratio by the ER in hFOB1.19 human osteoblast cells.

scholarly journals Effects of Four Compounds from Gentianella acuta (Michx.) Hulten on Hydrogen Peroxide-Induced Injury in H9c2 Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Kai Ren ◽  
He Su ◽  
Li-juan Lv ◽  
Le-tai Yi ◽  
Xue Gong ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Expression ◽  
Treated Group ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Protective Effects ◽  
H9c2 Cells ◽  
Mtt Method ◽  
Bioassay Guided Fractionation ◽  
Induced Apoptosis

In previous studies, Gentianella acuta (Michx.) Hulten was reported to contain xanthones, iridoids, terpenoids, and sterols and is mainly used to cure hepatitis, jaundice, fever, headache, and angina pectoris. In this study, we used bioassay guided fractionation to identify compounds from G. acuta and investigated their activity against hydrogen peroxide (H2O2)-induced apoptosis of H9c2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic (GCLC) expression were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated using western blot. The results showed that all four compounds had protective effects on H9c2 cells. The transcription levels of HO-1 and GCLC significantly increased in H9c2 cells pretreated with norswertianolin (1), swetrianolin (2), demethylbellidifolin (3), and bellidifolin (4). However, compared to the model group, the transcription levels of Nrf2 were not enhanced by pretreatment with compounds 1, 2, and 4. The protein expression levels of HO-1 and GCLC in H9c2 cells were greater than that in the H2O2-treated group, and the expression of Nrf2 was not significantly changed except by swetrianolin treatment; inhibitors can reverse the protective effect by ZnPP (15 μM), BSO (10 μM), and brusatol (10 μM). The results indicated that the four compounds isolated from G. acuta inhibited the oxidative injury induced by H2O2 by activating the Nrf2/ARE pathway in H9c2 cells and provide evidence that G. acuta may be a potential therapeutic agent for the treatment of cardiovascular diseases.

Immunomodulatory Drugs Inhibit H2O2 Decomposition in Multiple Myeloma Cells and Its Mediated Cytotoxicity Is Determined By Cellular Antioxidative Capacity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2475-2475
Author(s):  
Sinto Sebastian Chirackal ◽  
Yuan Xiao Zhu ◽  
Esteban Braggio ◽  
Chang-Xin Shi ◽  
Sonali Panchabhai ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Lines ◽  
Oxidative Capacity ◽  
Antioxidative Capacity ◽  
H2o2 Decomposition ◽  
Gene And Protein Expression ◽  
Induced Apoptosis

Abstract Introduction Lenalidomide is an immunomodulatory drug (IMID) used to treat Multiple Myeloma (MM). Although a role for cereblon (CRBN)-mediated degradation of Ikaros proteins (IKZF1 and IKZF3) has been shown, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated anti-MM activity and/or resistance are undiscovered. Therefore, we aimed to analyze whether IMIDs (thalidomide, lenalidomide, and pomalidomide) are inducing oxidative stress in MM and what determines these drugs varying sensitivity and/or resistance. Methodology Amplex Red Assay has been performed to analyze IMIDs-mediated inhibition of H2O2 decomposition in both, in-vitro and in-vivo assays. Lentiviruses were prepared in 293T cells for CRBN, IgL-λ & IgL-k, and Bim knockdown experiment. Quantification of MM cellular anti-oxidative capacity for determining IMID sensitivity was standardized with H2O2-mediated oxidation of FADH2 and NAD(P)H. To measure apoptosis and gene expression analysis 106 cells were incubated with lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. Results We discovered that IMIDs inhibit peroxidase-mediated decomposition of H2O2 in both, in vitro horseradish peroxidase (HRP) assays and in human MM cell lines (HMCLs). Of the IMIDs analyzed, pomalidomide was the more potent inhibitor. H2O2 treatment effectively degraded IKZF1 and IKZF3 in HMCLs. To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, we used CRBN knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line. We treated both sets of isogenic cell lines with lenalidomide and H2O2 for 3 hours, and we showed that H2O2 similarly mediates IKZF1 and IKZF3 degradation in a CRBN-dependent fashion. Next, we tested viability of CRBN present and absent cell lines with increasing concentrations of lenalidomide and H2O2 for 3 days. Lenalidomide-induced cytotoxicity was CRBN dependent, but H2O2 was not after 3 days, as shown by MTT assays. The capacity of MM cells to decompose H2O2 was measured via a biochemical test that quantitatively measured cellular anti-oxidative capacity. IMID sensitivity was well correlated with cellular anti-oxidative capacity, likely, cells more efficiently decompose H2O2was resistant and cells were not sensitive to IMID. This result shows that antioxidant capacity determines lenalidomide sensitivity among HMCLs with similar CRBN protein expression. We discovered that lenalidomide-mediated cytotoxicity in MM was attributable to oxidative damage of intracellular immunoglobulin proteins. By using several sets of isogenic cells lines with and without CRBN expression, we confirmed that lenalidomide treatment caused accumulation of IgL dimers only in CRBN-positive cells. Lenalidomide-induced IgL dimerization lead to decreased secretion and consequent intracellular accumulation of IgL, as evidenced by unchanged IgL mRNA expression, increased total intracellular IgL protein, and decreased secretion of IgL. After 72 hours of lenalidomide treatment we found decreased XBP-1u, increased XBP-1s, and over-expressed GRP78/BiP endoplasmic reticulum stress (ERS) marker proteins in CRBN positive cells but not in CRBN knock-down cells. We observed Bim requirement, especially BimEL, after lenalidomide treatment in CRBN-positive lenalidomide-sensitive cells. Our data reveals that lenalidomide-mediated; progressive ERS can positively enhance bortezomib-induced apoptosis in an in-vitro MM model. We pretreated MM cells with lenalidomide and then treated them with bortezomib. OPM2 cells pretreated with lenalidomide for 2 days clearly showed increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated. Conclusion IMIDs inhibit H2O2 decomposition. Ikaros protein degradation is a consequence of H2O2 mediated oxidative stress. Therefore, cells producing high H2O2 and with less antioxidative capacity are more sensitive to IMIDs. On the basis of this discovery, we would be able to predict which patients will benefit from IMIDs-mediated therapy and develop new drugs other than IMIDs that can inhibit intracellular H2O2 decomposition in MM. At present, CRBN may be required for IMIDs to effectively inhibit H2O2decomposition. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity.

Cellular alterations and modulation of protein expression in bitumen-challenged human osteoblast cells

2012 ◽  
Vol 19 (9) ◽  
pp. 4030-4041 ◽  
Author(s):  
Alka Dhondge ◽  
Subin Surendran ◽  
Muhil Vannan Seralathan ◽  
Pravin K. Naoghare ◽  
Kannan Krishnamurthi ◽  
...  
Keyword(s):  
Protein Expression ◽  
Human Osteoblast ◽  
Osteoblast Cells ◽  
Cellular Alterations

scholarly journals COL3A1 and MMP9 Serve as Potential Diagnostic Biomarkers of Osteoarthritis and Are Associated With Immune Cell Infiltration

2021 ◽  
Vol 12 ◽  
Author(s):  
Shushan Li ◽  
Haitao Wang ◽  
Yi Zhang ◽  
Renqiu Qiao ◽  
Peige Xia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Immune Cell ◽  
Gene Expression Omnibus ◽  
Pathological Process ◽  
Future Research ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Age Related ◽  
Gene And Protein Expression

BackgroundOsteoarthritis (OA) is one of the most common age-related degenerative diseases. In recent years, some studies have shown that pathological changes in the synovial membrane occur earlier than those in the cartilage in OA. However, the molecular mechanism of synovitis in the pathological process of OA has not been elucidated. This study aimed to identify novel biomarkers associated with OA and to emphasize the role of immune cells in the pathogenesis of OA.MethodsMicroarray datasets were obtained from the Gene Expression Omnibus (GEO) and ArrayExpress databases and were then analyzed using R software. To determine differential immune cell subtype infiltration, the CIBERSORT deconvolution algorithm was used. Quantitative reverse transcription PCR (qRT-PCR) was used to determine the relative expressions of selected genes. Besides, Western blotting was used to assess the protein expression levels in osteoarthritic chondrocytes.ResultsAfter analyzing the database profiles, two potential biomarkers, collagen type 3 alpha 1 chain (COL3A1), and matrix metalloproteinase 9 (MMP9), associated with OA were discovered, which were confirmed by qRT-PCR and Western blotting. Specifically, the results revealed that, as the concentration of IL-1β increased, so did the gene and protein expression levels of COL3A1 and MMP9.ConclusionThe findings provide valuable information and direction for future research into novel targets for OA immunotherapy and diagnosis and aids in the discovery of the underlying biological mechanisms of OA pathogenesis.

scholarly journals Shenqiwan Ameliorates Renal Fibrosis in Rats by Inhibiting TGF-β1/Smads Signaling Pathway

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Hongshu Chen ◽  
Yiqing Xu ◽  
Yuanxiao Yang ◽  
Xiaojie Zhou ◽  
Shijie Dai ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Renal Fibrosis ◽  
Epithelial Mesenchymal Transition ◽  
Interstitial Fibrosis ◽  
Underlying Mechanism ◽  
Positive Control ◽  
Control Group ◽  
Mesenchymal Transition ◽  
Gene And Protein Expression

Epithelial-mesenchymal transition (EMT) refers to the transition of epithelial cells into mesenchymal cells. Emerging evidence suggests that EMT is a key point in renal interstitial fibrosis (RIF). Traditional Chinese Medicine Shenqiwan (SQW) is widely used in clinical treatment of chronic kidney disease, but the underlying mechanism remains unclear. The purpose of this study is to investigate the effect of SQW on renal fibrosis and its association with TGF-β1/Smads signaling pathway. A rat model of adenine (150 mg/kg) was established and intragastrically treated with various concentrations of SQW at dose of 1.5 g/kg, 3 g/kg, and 6 g/kg. Control group and model group were given the same volume of saline. Meanwhile, the positive control group was treated with Enalapril (4 mg/kg). Animals were sacrificed on 21st day after administration. The results showed that SQW could significantly relieve renal pathological damage caused by adenine, increase gene and protein expression of E-cadherin, and decrease the expression of Vimentin in kidney samples. In addition, SQW efficiently inhibited the mRNA and protein expression of p-Smad2/3 by upregulating Smad7. These results suggest that SQW could slow down the progression of renal fibrosis, possibly by inhibiting TGF-β1/Smads signaling pathway.

scholarly journals THE EFFECT OF ADMINISTRATION OF AN EQUAL DOSE OF DIFFERENT CLASSES OF PHYTOCHEMICALS ON HEME OXYGENASE-1 GENE AND PROTEIN EXPRESSION IN MICE LIVER

2019 ◽  
pp. 256-260
Author(s):  
AZMAN ABDULLAH ◽  
NADIA SALEM ALRAWAIQ ◽  
AHMED ATIA
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Heme Oxygenase ◽  
Corn Oil ◽  
Treated Group ◽  
Control Group ◽  
Heme Oxygenase 1 ◽  
Gene And Protein Expression ◽  
Beneficial Response ◽  
Mice Liver

Objective: Heme oxygenase-1 (HO-1) is enzyme that possesses antioxidant, anti-inflammatory, and cytoprotective functions. Induction of HO-1 occurs as an adaptive and beneficial response to various injurious stimuli such as oxidative stress. This study is aimed at monitoring the effects of administration of equal doses (50 mg/kg) of sulforaphane (SFN), curcumin, quercetin, indole-3-carbinol, and butylated hydroxyanisole (BHA) for 14 days on the levels of liver HO-1 gene and protein expression in mice. Method: A total of 48 adult male ICR white mice (25–30 g) were divided into eight groups: Normal control group (n=6), SFN-treated group (n=6), quercetin-treated group (n=6), curcumin-treated group (n=6), BHA-treated group (n=6), indole-3-carbinol treated group (n=6), vehicle 1 control group (n=6), and vehicle 2 control group (n=6). All chemicals were administered intraperitoneally at a dose of 50 mg/kg for 14 days. Vehicle 1 (dimethyl sulfoxide, TweenTM 20, and normal saline at a ratio of 0.05:0.1:0.85) was used to dissolve SFN, quercetin, and curcumin. Vehicle 2 (corn oil) was used to dissolve indole-3-carbinol and BHA. At day 15, the animals were sacrificed and their livers were isolated. From the liver, total RNA was extracted, reverse transcribed and subjected to quantitative real‐time polymerase chain reaction to detect HO-1 gene expression. Agarose gel electrophoresis was also performed to verify the specificity of the amplification. HO-1 protein expression was determined by Western blotting. Results: HO-1 gene expression showed significant increase of 4.6±0.3, 3.6±0.2, 3.6±0.4, 3.3±0.3, and 3.0±0.4-fold and HO-1 protein expression showed significant increase of 2.3±0.2, 2.2±0.2, 2.2±0.1, 1.8±0.1, and 1.7±0.2-fold following treatment with 50 mg/kg of SFN, indole-3-carbinol, BHA, curcumin, and quercetin, respectively, compared to controls (p<0.05). Conclusion: At a dose of 50 mg/kg, SFN administration for 14 days resulted in the highest induction of HO-1 gene and protein expression level in mice liver, and quercetin the lowest.

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