Dataset of effect of Wogonin, a natural flavonoid, on the viability and activation of NF-κB and MAPKs in IL-1β-stimulated human OA chondrocytes

Abstract

This article contains data related to the article "Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" (Khan et al. 2017) [1]. The data are related to effects of Wogonin on the viability and IL-1β-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et al. 2017) [1].

Keywords: MAPK; Mass-spectrometry; NF-κB; Nrf2; Osteoarthritis; Wogonin.

Fig. 1

Expression of COL2A1 and COL10A1 in monolayer culture of human OA chondrocytes: Human OA chondrocytes were cultured in monolayer and total RNA were isolated and mRNA expression of COL2A1, and COL10A1 were quantified using TaqMan assay. Gene expression was determined by the ΔCt method and results were normalized to β-actin expression.

Fig. 2

Effect of Wogonin (WG) on viability of human OA chondrocytes. (A) Human OA chondrocytes (0.02×10⁶/ well of 96-well plate) were treated with Wogonin (10–50 µM) for 24 h and cell viability was measured by MTT assays. Chondrocytes treated with 0.1% DMSO served as control. Viability was expressed relative to control cells. (B) Flow cytometric histograms of OA chondrocytes treated in the presence or absence of Wogonin and stained with PI. Cells were harvested at 24 h after treatment with Wogonin and stained with PI. Twenty thousand cells in each group were acquired using a flowcytometer. The Sub-G1 region represents the percentage of cells undergoing apoptosis. Data points represent mean±SD from two subjects.

Fig. 3

Effect of Wogonin (WG) on IL-1 β induced activation of MAPKs and NF-κB in OA chondrocytes. Human OA chondrocytes were pre-treated with Wogonin (50 µM) for 2 h followed by treatment with IL-1β (10 ng/ml) for 15 or 30 min. Cell lysate were prepared from harvested chondrocytes using RIPA lysis buffer. (A) Activation of MAPKs was investigated by immunoblotting using primary antibodies specific for phospho-ERK1/2, phospho-JNK, phospho-p38. Expression of total ERK1/2, total JNK, or total-p38 was used as control. (B) Degradation of Iκβα was investigated by immunoblotting in cell lysate prepared as above. β-actin was used as a control for equal loading. Immunoblot are representatives of two blots performed on samples obtained from two individuals.

Fig. 4

Genetic ablation of Nrf2 using specific siRNA in chondrocytes: OA chondrocytes were transfected with siRNA specific for Nrf2 (50, 100 nM) using nucleofection. Forty-eight hours after the transfections, RNA was isolated form OA chondrocytes and gene expression of Nrf2 was measured by quantitative PCR using the SYBR® green assay (Life Technologies). Bar graph represents mean±SD from two subjects. *p≤0.05, as compared to control siRNA.

Fig. 5

Mass-spectrometric calibration curve of Wogonin: Four point calibration curve was prepared using purified Wogonin dissolved in methanol at the concentrations of 0.001, 0.01, 0.1 and 1 mg/ml using multiple reaction monitoring and negative ion mode (MRM-) by monitoring transition pairs m/z 283.00 (precursor ion)/162.9 (product ion).