{"id":616780,"date":"2024-06-17T20:00:00","date_gmt":"2024-06-18T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/exploring-the-effects-of-naringin-on-oxidative-stress-impaired-osteogenic-differentiation-via-the-wnt-%ce%b2-catenin-and-pi3k-akt-pathways-scientific-reports\/"},"modified":"2024-06-18T14:44:37","modified_gmt":"2024-06-18T18:44:37","slug":"exploring-the-effects-of-naringin-on-oxidative-stress-impaired-osteogenic-differentiation-via-the-wnt-%ce%b2-catenin-and-pi3k-akt-pathways-scientific-reports","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/exploring-the-effects-of-naringin-on-oxidative-stress-impaired-osteogenic-differentiation-via-the-wnt-%ce%b2-catenin-and-pi3k-akt-pathways-scientific-reports\/","title":{"rendered":"Exploring the effects of naringin on oxidative stress-impaired osteogenic differentiation via the Wnt\/\u03b2-catenin and PI3K\/Akt pathways – Scientific Reports","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
<\/div>\n

Data collection and processing<\/h3>\n

Using the Gene Expression Omnibus (GEO) database (https:\/\/www.ncbi.nlm.nih.gov\/GEO\/<\/a>) with the keyword \u2018osteoporosis\u2019 for retrieval, we downloaded the GSE3595839<\/a><\/sup> dataset for OP and non-OP samples. Using a standardized GEO2R gene microarray (http:\/\/www.ncbi.nlm.nih.Gov\/geo\/geo2r<\/a>), we compared the DEGs between both samples and defined those that had a |logFC (fold change)|>\u20091 and false discovery rate \u2212\u2009log10(P<\/i> value)\u2009<\u20091.3 (FDR\u2009<\u20090.05) as OP-related DEGs. The data were visualized using R 4.3.2\u2019s ggplot2 3.5.0 and pheatmap 1.0.12 packages (R Core Team, Austria).<\/p>\n

Enrichment analysis of DEGs<\/h3>\n

The General Database for Annotation, Visualisation, and Integrated Discovery40<\/a><\/sup> (DAVID; https:\/\/david.ncifcrf.gov<\/a>) was used for Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes41<\/a><\/sup> (KEGG) functional enrichment analysis. The GO analysis consisted of three components: biological process (BP), molecular function (MF), and cellular component (CC). The data were visualised using the ggplot2 3.5.0 package. P<\/i>-values\u2009<\u20090.05 were considered statistically significant.<\/p>\n

Screening of Nar core targets<\/h3>\n

\u201cNaringin\u201d was used as a keyword in PubChem (https:\/\/pubchem.ncbi.nlm.nih.gov\/<\/a>) to retrieve the Simplified Molecular Input Line Entry System (SMILES) chemical formula. Then, using this formula, we screened the TargetNet database (http:\/\/targetnet.scbdd.com\/home\/index\/<\/a>) and only included models with an area under the curve (AUC) cut-off value of 0.7. The potential targets of Nar were identified using extended-connectivity (ECFP4) fingerprint retrieval, and a protein\u2013protein interaction (PPI) network was drawn using the Search Tool for the Retrieval of Interacting Genes\/Proteins (STRING) database. The degree value of each node in the PPI network was sorted using Cytoscape_v3.10.0-SNAPSHOT42<\/a><\/sup> (Cytoscape Team, USA). The top 10 targets with the highest scores were selected and defined as the core targets.<\/p>\n

Screening of the potential OP-associated drug targets<\/h3>\n

OP-related proteins were searched in Comparative Toxicogenomics Database (CTD), DisGeNET, and GeneCards using \u201costeoporosis\u201d as the keyword. The core target intersection was determined using an online Venn diagram tool (http:\/\/bioinformatics.psb.ugent.be\/webtools\/Venn\/<\/a>), and the potential Nar action targets associated with OP were obtained.<\/p>\n

Molecular docking<\/h3>\n

Oestrogen receptor 1 (ESR1) and heat shock protein 90 alpha family class A member 1 (HSP90AA1) proteins obtained from the Protein Data Bank (PDB) (https:\/\/www.rcsb.org\/<\/a>) were pre-treated for structural optimization using molecular operating environment software (MOE; Chemical Computing Group, Canada) and the sitefinder function was used to identify the binding pockets For molecular docking, the receptor was set as ESR1 or HSP90AA1, site as site 1, and the ligand as Nar. The results of the analysis were visualized using PyMOL 2.6.0 software43<\/a><\/sup> (Schr\u00f6dinger, Inc., USA).<\/p>\n

Cell culture<\/h3>\n

Resuscitated MC3T3-E1 cells (EK-Bioscience, China) were cultured in minimum essential medium alpha (\u03b1-MEM; Shanghai BasalMedia Technologies Co., Ltd., China) medium containing 10% fetal bovine serum (Nanjing SenBeiJia Biological Technology Co., Ltd., China) and 1% penicillin\u2013streptomycin solution (Nanjing SenBeiJia Biological Technology Co., Ltd., China) at 37\u2103 in a 5% CO2<\/sub> incubator (Sanyo, Japan). After the cells reached 80%\u201390% confluency, they were treated with 0.25% trypsin\/0.02% ethylenediaminetetraacetic acid solution (Sigma, USA) and re-plated at a dilution of 1:3.<\/p>\n

CCK-8 assay<\/h3>\n

MC3T3-E1 cells in the logarithmic growth phase were seeded in 96-well plates with 1\u2009\u00d7\u2009104<\/sup> cells\/well and cultured for 24 h. Cells were treated with a low-serum medium containing various Nar concentrations (Sigma, USA) or H2<\/sub>O2<\/sub> (Sigma, USA) for 24 h or with Nar following 24 h of H2<\/sub>O2<\/sub> treatment for 24, 48, and 72 h. Cell counting kit-8 (CCK-8) reagent (Meilun Biological, China) was added to the cells and incubated for 2 h. Absorbances were measured at 450 nm using a microplate reader (Molecular Devices, USA). Cell survival rate was calculated using the optical density (OD) values as follows:<\/p>\n

\n

$$begin{gathered} left[ {left( {OD; value; of; experimental; wells} right.} right. hfill – left. {OD; value; of; blank; wells} right)\/left( {OD; value; of; control; wells} right. hfill – left. {left. {OD; value ;of ;blank; wells} right)} right] times 100% . hfill end{gathered}$$<\/span><\/p>\n<\/div>\n

Cytotoxicity assay<\/h3>\n

MC3T3-E1 cells were subjected to 0.3 mM H2<\/sub>O2<\/sub> and 0.1 \u00b5M Nar for 24 h to detect lactate dehydrogenase (LDH) (Elabscience, China) production. The MC3T3-E1 cells were seeded in 96-well plates and cultured for 24 h. Cells were treated according to the following groups: control, induction, H2<\/sub>O2<\/sub>, Nar, and H2<\/sub>O2<\/sub>\u2009+\u2009Nar. Then, 10 \u03bcL of lysate was added to the control well with maximum enzyme activity, and the cells were cultured for another 1 h after blowing and mixing. After centrifugation at 400\u2009\u00d7\u2009g<\/i> for 5 min, 50 \u03bcL of supernatant to be measured and 50 \u03bcL of reaction working solution was added to each well, and the microplate was shaken. After incubating at 37 \u00b0C for 10 min, the reaction termination solution was added, and the absorbance was measured at 450 nm using a microplate reader. Cytotoxicity was calculated as follows:<\/p>\n

\n

$$begin{gathered} left[ {left( {D; value; of; sample; test; well} right.} right. hfill – left. {OD; value; of; sample; control; well} right) hfill \/left( {OD; value; of; maximum; enzyme; activity; control; well;} right. hfill – left. {left. {OD; value; of; sample; control; well} right)} right] times 100% hfill end{gathered}$$<\/span><\/p>\n<\/div>\n

ROS assay<\/h3>\n

After cleaning with phosphate-buffered saline (PBS), a 2′-7’dichlorofluorescin diacetate (DCFH-DA) (Applygen Technologies, China) probe diluted with \u03b1-MEM (final concentration of 10 \u03bcM) was added to the MC3T3-E1 cells in 6-well plates and incubated at 37 \u00b0C for 30 min in the dark. During this period, the probe was gently shaken every 5 min to ensure full contact with the cells. The cells were washed three times and then re-suspended with \u03b1-MEM. ROS levels were recorded under a fluorescence microscope within 30 min at 525 nm to determine the establishment of the oxidative stress model. Fluorescence staining used ImageJ 13.0.6 software44<\/a><\/sup> to detect fluorescence brightness as ROS expression. The cell suspension was detected by fluorescent enzyme, the absorbance was measured at 520 nm, and the results were expressed by fluorescence value.<\/p>\n

Nitric oxide assay<\/h3>\n

Dilute the standard in the kit to 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.57 \u03bcM, the OD values of each group were detected by microplate reader, and the concentration calculation formula was obtained by making a standard curve. In 96-well plate, 50 \u03bcL control(10%FBS-\u03b1-MEM) and 50 \u03bcL Griess reagent were added and incubated at room temperature in the dark for 5 min. Then, the absorbance was detected using at 540 nm. A standard curve was drawn, the OD values of the sample group were substituted into the formula to calculate the NO expression in the group, and the concentration of NO was calculated to confirm the establishment of the oxidative stress model.<\/p>\n

Alkaline phosphatase staining<\/h3>\n

Osteogenesis was induced in the MC3T3-E1 cells seeded in 6-well plates according to the different groups. Cell culture was performed with \u03b1-MEM in the control group, with basic osteogenic differentiation medium (\u03b1-MEM, 10% FBS, 50 \u03bcg\/mL ascorbic acid, and 10 mM sodium \u03b2-glycerophosphate) in the induction group, and in the hydrogen peroxide group, basic osteogenic differentiation medium was added after 24 h of H2<\/sub>O2<\/sub> action. For the Nar group, we added Nar to the basal osteogenic differentiation medium, while for the H2<\/sub>O2<\/sub>\u2009+\u2009Nar group, we added the above Nar-containing osteogenic differentiation medium after 24 h of H2<\/sub>O2<\/sub> action. and stained using the BCIP\/NBT alkaline phosphatase chromogenic kit (Beyotime, China). After 2 weeks, the medium was removed, and the cells were washed three times with PBS for 3\u20135 min each, fixed with 4% paraformaldehyde for 15 min, and washed a further three times with PBS. Then, 1 mL of the staining working solution was added to each well and incubated at room temperature for 20 min in the dark. After removing the working solution, the staining solution was washed three times with double distilled H2<\/sub>O and imaged under an inverted microscope.<\/p>\n

Alizarin red S staining<\/h3>\n

Osteogenesis was induced in the MC3T3-E1 cells seeded in 6-well plates according to different groups and then stained with the osteoblast mineralized nodule staining kit (Beyotime, China). After 21 days, the medium was discarded, and the cell surface was lightly washed three times with PBS for 3\u20135 min each, fixed with 4% paraformaldehyde for 15 min, and washed a further three times with double distilled H2<\/sub>O. Alizarin Red S staining working solution (1 mL) was added to each well and the cells were stained for 30 min. After discarding the solution, the cells were washed three times with ddH2<\/sub>O and imaged under an inverted microscope.<\/p>\n

Alkaline phosphatase activity assay<\/h3>\n

Osteogenesis was induced in MC3T3-E1 cells in 6-well plates for 5, 7, and 9 days, according to different groups. The cells were harvested and lysed, and the remaining supernatant was collected. The alkaline phosphatase (ALP) detection kit (Beyotime, China) was used following the manufacturer\u2019s instructions. Absorbances were measured at 405 nm, and the activity of ALP was calculated (Table S1<\/a>).<\/p>\n

Western blot<\/h3>\n

Osteogenesis was induced in the MC3T3-E1 cells for 14 days, and they were treated according to different groups. Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, China), quantified using the bicinchoninic acid method (Applygen Technologies Inc., China), and denatured by boiling after adding loading buffer. Whole-cell protein extracts were separated by sodium dodecyl sulphate\u2013polyacrylamide gel electrophoresis and analyzed using Western blot (WB). The amount of protein was loaded with 10 \u03bcg\/well. The PVDF membranes (0.22 \u03bcm) were then blocked with 5% BSA for 2 h. The following primary antibodies were prepared at 1:1000 after washing the membrane and incubated overnight at 4 \u00b0C: anti-GAPDH (Affinity, USA), anti-PI3K (Affinity, China), anti-p-PI3K (Affinity, China), anti-Akt (Affinity, China), anti-p-Akt (Affinity, China), anti-GSK-3\u03b2 (Affinity, China), anti-p-GSK-3\u03b2 (Affinity, China), anti-LRP-5 (Abcam, China), anti-\u03b2-catenin (Cell Signalling Technology, USA), anti-Runx2 (Affinity, China), and anti-OPN (Beyotime, China). The membranes were then washed three times with PBS-Tween, incubated with a secondary antibody for 2 h at room temperature, and washed three more times. Enhanced chemiluminescence solution (Applygen Technologies, China) was then applied, and the relative image intensity was calculated using ImageJ software (Table S1<\/a>).<\/p>\n

Statistical analysis<\/h3>\n

Data are expressed as the mean\u2009\u00b1\u2009standard deviation. All statistical analyses were performed using SPSS 25.0 (IBM Corp., USA) and GraphPad Prism 8.0 (GraphPad Software, Inc., USA). The independent sample t-test was used for comparison between two groups. A one-way analysis of variance (ANOVA) followed by Tukey\u2019s post-hoc test was used to compare multiple groups. P<\/i>-values\u2009<\u20090.05 were considered statistically significant.<\/p>\n

Ethics approval and consent to participate<\/h3>\n

The data for the bioinformatics analyses in this study were obtained from online databases, and the cell lines were purchased by the company, so no ethical approvals were involved.<\/p>\n