Normal human HaCaT keratinocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco^®, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and antibiotics. Oridonin and H₂O₂ were purchased from Sigma-Aldrich and Merck (Darmstadt, Germany), respectively. For toxicity and cell viability assays, 4×10⁴ HaCaT cells per well were seeded into 96-well plates and 7×10⁵ cells were seeded into 60-mm culture plates, respectively. Oridonin and H₂O₂ were diluted into DMSO (Sigma-Aldrich) and deionized water, respectively. The cells were treated with various doses of oridonin and a fixed dose (800 μM) of H₂O₂ for 3–24 h. Propidium iodide (PI) and Triton X-100 were purchased from Sigma-Aldrich.

The effects of oridonin on the growth of HaCaT cells treated with or without H₂O₂ were assessed using water-soluble tetrazolium salt (WST-1) assays. After treatment, the HaCaT cells were mixed with 100 μl of WST-1 solution followed by incubation at 37°C for 1 h. Cell viability was determined after measuring the absorbance at 450 nm using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). All results are expressed as the mean percentage ± standard deviation (SD) of 3 independent experiments. When comparing treated and untreated cells, p-values <0.05 as determined by the Student’s t-test were considered to indicate statistically significant differences.

Intracellular ROS scavenging assays were performed by measuring the fluorescence intensity of the 2′7′-dichlorofluroescein diacetate (DCF-DA) probe, which was proportional to the amount of ROS formed. The cells pre-treated with and without oridonin were incubated with H₂O₂ for 3 h prior to harvest. The cells were then mixed with DCF-DA solution and incubated at 37°C for 1 h. Fluorescence intensity was measured using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Cell cycle and death were estimated by assessing the incorporation of the fluorescent dye, PI. Cells treated with and without oridonin and/or H₂O₂ were harvested, resuspended, and then incubated with PI staining solution (50 μg/ml PI, 0.5% Triton X-100, and 100 μg/ml RNase) at 37°C for 1 h. Fluorescence intensity was detected using a BD FACSCalibur flow cytometer.

Total RNA was isolated using TRIzol reagent (Invitrogen, Life Technologies) according to the manufacturer’s instructions. RNA integrity, concentration and purity were estimated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and MaestroNano (Maestrogen, Las Vegas, NV, USA). RNA samples that exhibited A_260/280 and A_260/A230 values >1.8, as well as an RNA integrity number (RIN) >8.0, were subjected to microarray analysis, which was performed using SurePrint G3 Human V16 miRNA 8×60 K arrays (Agilent Technologies) according to a previously described protocol (16). Derived data were analyzed using GeneSpring GX software, version 11.5 (Agilent Technologies). The raw data were filtered using Flag and t-tests. miRNA expression was evaluated by assessing the fluorescence ratio between 2 samples. Those displaying >1.5-fold increase or decrease were selected for further bioinformatics analysis.

To investigate the biological functions of miRNAs that exhibited significant changes in expression, we identified their putative target genes using MicroCosm Targets, version 5 (www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/). The cellular functions of the target genes were then determined using AmiGO, a Gene Ontology (GO)-based analysis and categorization tool (amigo.geneontology.org/cgi-bin/amigo/browse.cgi).

Prior to investigating the protective effects of oridonin on H₂O₂-induced cellular stress, we first determined the dose range of oridonin that causes cytotoxicity in HaCaT cells. The HaCaT cells were treated with various concentrations of oridonin (1–20 μM) for 24 h, and cytotoxicity was estimated using the WST-1 assay (Fig. 1A). Cell viability was maintained at doses between 1 to 5 μM; however, higher concentrations of oridonin (10 and 20 μM) decreased HaCaT cell viability. We also determined that HaCaT cell viability decreased following treatment with H₂O₂ in a dose-dependent manner (Fig. 1B). As we were concerned about the combined effects of H₂O₂ and oridonin on HaCaT cytotoxicity, the cells were pre-treated with various doses of oridonin for different periods of time prior to the induction of oxidative stress with H₂O₂. Cell viability was analyzed using the WST-1 assay. Surprisingly, the oridonin-pre-treated HaCaT cells exhibited a marked resistance to H₂O₂-mediated cytotoxicity (Fig. 1C–F). In fact, this effect increased in a concentration and time-dependent manner, suggesting that oridonin exerts a protective effect against H₂O₂-induced oxidative stress in HaCaT cells.

We subsequently investigated the biological mechanisms involved in the protective effects of oridonin against H₂O₂-induced oxidative stress. Changes in cell viability can be physiologically related to cell cycle arrest and cell death. Therefore, we examined cell cycle progression by PI staining and flow cytometry. The HaCaT cells treated as indicated in Fig. 2 were collected, fixed, stained with PI solution, and subsequently analyzed by flow cytometry. Our data demonstrated that the distribution of cells across the various stages of the cell cycle was similar between the oridonin-treated and the control (DMSO-treated) cells, confirming that treatment with 5 μM oridonin was non-cytotoxic (Fig. 2A and B). By contrast, the percentage of cells in the sub-G1 phase was much higher in the H₂O₂-treated HaCaT cells compared with the untreated and oridonin-treated cells (Fig. 2C). Nevertheless, this increase was not observed in the HaCaT cells pre-treated with oridonin prior to the induction of oxidative stress (Fig. 2D). These results suggest that treatment with oridonin maintains cell viability by reducing HaCaT cell death in H₂O₂-induced oxidative stress.

H₂O₂ is well established as a strong inducer of ROS, which, if present at high levels, promote cell death. Since oridonin is a diterpenoid compound, and some diterpenoid compounds have been shown to have antioxidant properties, we examined the possibility that oridonin acts as a ROS scavenger. HaCaT cells grown in oridonin-containing medium were treated with H₂O₂ for 3 h. Following exposure to H₂O₂, the cells were stained with DCF-DA solution and the levels of ROS were then analyzed by flow cytometry. Unlike treatment with H₂O₂, oridonin alone did not induce significant ROS production in the HaCaT cells (Fig. 3A and C). Of note, our results demonstrated that the increased ROS production induced by H₂O₂ was reduced to the levels of the controls following treatment with oridonin (Fig. 3D), indicating that oridonin has a scavenging effect on ROS produced in response to H₂O₂ in HaCaT cells.

Since miRNAs have been reported to regulate almost every biological process, including development, differentiation, proliferation and apoptosis (17–19), we sought to determine the effects of oridonin on miRNA expression in HaCaT cells treated with H₂O₂ for 24 h. A total of 21 miRNAs were differentially expressed following treatment with oridonin (Fig. 4 and Table I). More specifically, 6 miRNAs were upregulated while 15 miRNAs were downregulated. These results indicate that, although the majority of miRNAs did not exhibit significant changes in expression, treatment with oridonin still affected the miRNA expression levels in the HaCaT cells exposed to H₂O₂.

The miRNAs that exhibited altered expression levels following treatment with oridonin are likely involved in the the cellular mechanisms responsible for the protective effects of oridonin against H₂O₂-induced oxidative stress in HaCaT cells. Therefore, we used the miRbase Target Database tool, MicroCosm, to identify the putative target genes of these miRNAs. We then determined the biological functions associated with the target genes by GO analysis using AmiGO. Finally, the target genes were grouped according to biological processes. Our data demonstrated that the target genes of the differentially expressed miRNAs could be categorized into 4 groups, namely aging, skin development, apoptosis and cell proliferation (Tables II and III).

The GO terms contained bi-directional processes for each term. For example, ‘apoptosis’ included both anti-apoptotic and pro-apoptotic processes. Therefore, we further categorized the target genes into subsets of GO terms, such as anti-apoptosis and positive or negative regulation of the cell cycle, cell growth and cell proliferation (Fig. 5). A greater number of target genes of the upregulated miRNAs was associated with the negative regulation of the cell cycle, growth and proliferation than with the positive regulation of these processes. Conversely, the target genes of the downregulated miRNAs were more biased towards anti-apoptosis and positive regulation of the cell cycle, growth and proliferation. These results suggest that the upregulated miRNAs may potentially target genes involved in cell death, whereas the downregulated miRNAs may regulate genes critical for cell survival. Further categorization of the target genes demonstrated that the upregulated miRNAs may be linked to the positive regulation of p53 pathways and the activation of MAPKK activity, while those targeted by the downregulated miRNAs are associated with antioxidant activity and the positive regulation of DNA repair (Fig. 6). Collectively, these results suggest that the oridonin-mediated protective effects against H₂O₂-induced damage in HaCaT cells involve changes in the expression of specific miRNAs that regulate cell proliferation and apoptosis.