Geraniin (purity ≥98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), and was dissolved in dimethyl sulfoxide (DMSO) and stored at −80°C. The stock concentration of geraniin was 10 mM. DMSO and H₂O₂ were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM)/F12 and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The conjugated antibodies used to identify MSCs: Fluorescein isothiocyanate (FITC)-labeled anti-CD29 (555005) and anti-CD44 (561859), and phycoerythrin-labeled anti-CD45 (553091) and anti-CD90 (551401), as well as the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit, were all purchased from BD Biosciences (Franklin Lakes, NJ, USA). Cell Counting kit-8 (CCK-8) was from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Hoechst 33342, JC-1 dye, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, glutathione (GSH) kit (S0053), malondialdehyde (MDA) kit (S0131), ROS scavenger N-acetyl-L-cysteine (NAC), cell mitochondrial protein isolation kit (C3601), radioimmunoprecipitation assay (RIPA) lysis buffer, bicinchoninic acid (BCA) protein assay kit and mouse polyclonal anti-β-actin (AA128) were all purchased from Beyotime Institute of Biotechnology (Beijing, China). Rabbit antibodies against phosphorylated-protein kinase B [p-Akt (Ser473); 4060s], Akt (9272s), caspase-3 (9662s), B-cell lymphoma 2 (Bcl-2; 2876s), Bcl-2-associated X protein (Bax; 2772s) and cytochrome c (Cyt C; 4272s), and LY294002 [phosphoinositide 3-kinase (PI3K) specific inhibitor], were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit (ZB-2301) and anti-mouse (ZB-2305) secondary antibodies were obtained from OriGene Technologies, Inc., (Beijing, China).

MSCs were isolated and harvested from male Sprague-Dawley rats (age, 3 weeks old; weight, 60–80 g), as previously described with minor modifications (18). A total of 10 SPF Sprague-Dawley rats were purchased from the Laboratory Animal Science Department of the Second Affiliated Hospital of Harbin Medical University (Harbin, China). They were kept under standard animal housing conditions (temperature, 21±1°C; humidity, 55±5%), at a 12-h dark/light cycle and had access to unlimited food and water. The present study was approved by the Local Ethics Committee on Animal Care and Use of Harbin Medical University. Briefly, total bone marrow was flushed from the tibias and femurs of the rats with 10 ml DMEM/F12 using a sterile syringe. After centrifugation at 300 × g for 5 min, the remaining pellets were resuspended in 5 ml DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin, and were then seeded into cell culture flasks at 37°C in an atmosphere containing 5% CO₂. Following incubation for 48 h, the culture medium and non-adherent cells were discarded and fresh medium was added. The medium was replaced every 72 h thereafter. Cells were cultured until they reached 80% confluence, after which they were passaged. Cells were split using 0.25% trypsin and were expanded at a 1:2 or 1:3 dilution. Cells from passages 3–5 were used in the subsequent experiments. The MSC population was characterized according to positive (CD44, CD29 and CD90) and negative (CD45) cell surface markers by flow cytometry, as reported in our previous studies (18,19).

All treatments were conducted at 37°C in the incubator. MSCs were seeded into six-well plates or a 25 cm² culture flask. Once cell density reached 60–70%, H₂O₂ (100, 200, 300, 400 and 500 µM), mixed with serum-free DMEM/ F12 for 4 h, was used to establish an in vitro oxidative stress model. Geraniin, at 1, 5, 10 and 20 µM, was separately preincubated in DMEM/F12 for 24 h. The inhibitor of PI3K, LY294002 (25 µM), or the ROS scavenger, NAC (500 µM), was added 1 h prior to H₂O₂ treatment without geraniin co-treatment. Cells cultured in complete medium without any specific treatment comprised the control group.

MSC viability was assessed using the CCK-8 assay. Briefly, cells were plated into 96-well plates (3×10³ cells/well). Following cell adhesion to the plates, appropriate treatments were administered. Subsequently, the medium was removed and replaced with 100 µl fresh DMEM/F12 and 10 µl CCK-8 solution in each well. The plates were maintained at 37°C for 1 h. Finally, absorbance was detected at 450 nm using a Tecan Infinite 200 PRO microplate reader (Tecan Austria GmbH, Grödig, Austria).

Apoptosis of MSCs was determined using the Annexin V-FITC/PI staining method. Following treatment, the cells were harvested, washed with ice-cold PBS and resuspended in 400 µl binding buffer. The cell suspension was then incubated with 5 µl Annexin V solution for 15 min at room temperature in the dark, followed by incubation with 5 µl PI for an additional 5 min. The cells were immediately analyzed by flow cytometry using BD FACSCanto II (BD Biosciences). Approximately 1×10⁵ cells were detected in each sample. According to the reaction principles, Annexin V⁻/PI⁻ staining signified viable cells, Annexin V⁺/PI⁻ indicated early apoptotic cells, and Annexin V⁺/PI⁺ represented late apoptotic or necrotic cells.

MSCs were treated with geraniin and H₂O₂ in six-well plates. Hoechst 33342 was used to detect cell nuclear condensation and fragmentation. After fixing in 4% paraformaldehyde for 15 min at room temperature, cells were washed twice with PBS, stained with 5 µg/ml Hoechst 33342 for 5 min and washed a further two times with PBS. Finally, cells were assessed by fluorescence microscopy. Apoptotic cells were identified by condensed or fragmented nuclei.

Intracellular ROS levels were determined using a ROS assay kit. Briefly, cells were incubated with the diluted fluorescent probe, DCFH-DA, for 20 min at 37°C. Cells were then washed three times with serum-free DMEM/F12, collected and analyzed using a flow cytometer. Due to the important roles of GSH and MDA in ROS-associated oxidative stress, their concentrations were also measured using commercial kits according to the manufacturer's protocols.

JC-1 was used to measure alterations in Ψm. Briefly, cells were washed with PBS, stained with 5 µM JC-1 and maintained for 20 min at 37°C. Subsequently, cells were washed twice with ice-cold JC-1 staining buffer and were then directly observed under a fluorescence microscope, or were collected and analyzed by flow cytometry.

Cells were washed with ice-cold PBS, lysed with RIPA lysis buffer and centrifuged at 12,000 × g for 15 min at 4°C. The extraction of cytoplasmic and mitochondrial proteins was conducted in accordance with the manufacturer's protocol. BCA protein assay was used to quantify protein concentrations. Equal amounts of total protein (50 µg/ lane) were separated by 8–12% SDS-PAGE and were transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk diluted in Tris-buffered saline containing 0.05% Tween-20 (TBST) at 37°C for 1 h, and were then incubated with diluted primary antibodies against p-Akt (S473) (1:1,000), total-Akt (1:1,000), cleaved caspase-3 (1:1,000), Bax (1:1,000), Bcl-2 (1:1,000), Cyt C (1:1,000) and β-actin (1:800) overnight at 4°C. After washing three times with TBST, membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. The images of the immune complexes were developed by ECL in the dark, and images were captured using a Tanon-5200 (Tanon Science and Technology Co., Ltd., Shanghai, China). Band density was determined using ImageJ (1.48u; National Institutes of Health, Bethesda, MD, USA).

All data are presented as the mean ± standard deviation and were analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Differences between groups were analyzed by one-way ANOVA with a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Initially, the effects of geraniin on MSC viability were examined using the CCK-8 assay. Treatment with geraniin for 24 h had little influence on cell viability compared with the 0 µM geraniin group (Fig. 1B). This result indicated that geraniin did not exert toxic effects on MSCs.

H₂O₂ has been reported to induce apoptosis of MSCs at various concentrations and time-points (20,21). To establish a successful in vitro oxidative stress model, the present study investigated the proapoptotic effects of H₂O₂ on MSCs. Following treatment with various concentrations of H₂O₂ (100–500 µM) in serum-free medium for 4 h, MSCs were stained with Annexin V-FITC/PI. The proportion of early apoptotic cells (Annexin V⁺/PI⁻) was significantly increased following treatment with ≥200 µM H₂O₂, whereas the proportion of late apoptotic or necrotic MSCs (Annexin V⁺/PI⁺) was markedly increased following treatment with ≥300 µM (Fig. 2A and B). In addition, the expression levels of cleaved caspase-3 were significantly increased at 200 µM and continued to increase to 500 µM (Fig. 2C). We then selected treatment with H₂O₂ at 300 µM for 4 h as the condition to induce effective apoptosis, since this concentration generated moderate apoptotic cells.

To determine whether geraniin rescues MSCs from H₂O₂-induced apoptosis, MSCs were pretreated with increasing concentrations of geraniin (1, 5, 10 and 20 µM) for 24 h. MSCs were then co-treated with geraniin and H₂O₂. A significant reversal in the percentage of H₂O₂-induced Annexin V⁺/PI⁻ cells, in response to geraniin, was observed by flow cytometry (geraniin 1 µM, 20.53±1.25%; 5 µM, 18.67±1.89%; 10 µM, 11.57±1.01%; 20 µM, 7.23±0.31%; P<0.05 vs. H₂O₂, 23.83±1.32%) (Fig. 3A and B). However, 1 µM geraniin had no effect on the proportion of Annexin V⁺/PI⁺ cells, whereas the other concentrations significantly reduced the percentage of late apoptotic or necrotic cells (geraniin 5 µM, 7.88±2.58%; 10 µM, 7.05±1.72%; 20 µM, 4.40±0.48%; P<0.05 vs. H₂O_2, 12.97±1.65%) (Fig. 3A and B). Furthermore, in apoptotic cells, nuclei become condensed or fragmented, whereas in normal cells, nuclei are circular or oval. Hoechst 33342 staining was used to confirm the presence of nuclear morphological alterations. Cells treated with H₂O₂ appeared to possess shrunken and fragmented nuclei; however, those pretreated with geraniin exhibited marked amelioration of H₂O₂-induced nuclear impairment. These data indicated that geraniin may effectively attenuate H₂O₂-induced MSC apoptosis (Fig. 3C).

Since ROS serves a pivotal role in proapoptotic signaling cascades (22), the present study examined the effects of geraniin on ROS generation using flow cytometry. The results demonstrated that H₂O₂ induced a 7.6-fold increase in ROS production compared with the control group. However, pretreatment with geraniin markedly suppressed ROS generation in a concentration-dependent manner (Fig. 4A and B). Following treatment with NAC, a general ROS scavenger, similar results were recorded compared with 20 µM geraniin (mean fluorescence intensity: Geraniin 20 µM, 476.33±46.65; NAC, 443.80±53.15, P>0.05) (Fig. 4B). Furthermore, alterations in intracellular GSH and MDA contents were investigated; cells pretreated with geraniin had significantly increased GSH levels, whereas MDA production was suppressed by geraniin (Fig. 4C and D). These findings indicated that geraniin is able to enhance the cellular antioxidant system and remove redundant ROS.

Mitochondria in eukaryotic cells are the primary components of respiration, and are critical in the defense against oxidative stress-induced damage (23). Maintaining the Ψm is essential to ensure the scavenging efficiency of ROS, and to prevent cell apoptosis or other stress-associated events induced by excessive ROS (24). JC-1 is a Ψm-sensitive dye, which aggregates in the mitochondrial matrix and exhibits red fluorescence in normal cells. However, when the Ψm is reduced, JC-1 is converted to its monomer state, which exhibits green fluorescence. H₂O₂ resulted in a marked reduction in Ψm within MSCs, whereas geraniin markedly upregulated the Ψm, as identified by fluorescence microscopy (Fig. 5A). In addition, the ratio of red/green fluorescence intensity was significantly downregulated under H₂O₂ exposure compared with in the control group; however, this effect was reversed by geraniin in a concentration-dependent manner (Fig. 5B and C). These findings suggested that geraniin may exert beneficial effects on mitochondrial function.

It has previously been reported that geraniin exerts cytoprotective effects on HepG2 cells via activation of the extracellular signal-regulated kinase 1/2 and PI3K/Akt pathways (25). Due to the importance of the PI3K/Akt pathway on classical survival signals (26), the present study aimed to determine the association between geraniin and the PI3K/Akt pathway in MSCs. Cells were treated with geraniin (20 µM) for the indicated periods of time; the protein expression levels of p-Akt (Ser473) were transiently upregulated at 15 min and peaked at 60 min, prior to subsequent downregulation (Fig. 6A). In addition, the effects of 1 h treatment with various concentrations of geraniin on p-Akt (Ser473) expression in MSCs was investigated; p-Akt expression was upregulated in a dose-dependent manner. Conversely, p-Akt expression was markedly inhibited following pretreatment with LY294002, a PI3K-specific inhibitor (Fig. 6B). These findings indicated that geraniin may activate the PI3K/Akt signaling pathway in a time- and dose-dependent manner.

To gain further insight into the role of the PI3K/Akt pathway in the protective effects of geraniin on MSCs, cells were preconditioned with LY294002 and were then exposed to 20 µM geraniin and H₂O₂. PI3K inhibition significantly attenuated the anti-apoptotic effects of geraniin on MSCs under H₂O₂ treatment, as evidenced by a decrease in cell survival rate using CCK-8 assay (Fig. 6C). Since reductions in the Ψm and increased cleaved caspase-3 expression are initiating and amplifying factors of the mitochondrial apoptosis pathway, it may be suggested that H₂O₂ induces apoptosis of MSCs through regulating the mitochondrial apoptosis pathway. Therefore, the expression levels of Cyt C, cleaved caspase-3, Bax and Bcl-2 were investigated. Treatment with geraniin induced a marked increase in the expression levels of mitochondrial Cyt C and Bcl-2, and a decrease in the expression levels of cytoplasmic Cyt C, cleaved caspase-3 and Bax (Fig. 6D–F). These effects were reversed by LY294002. These data indicated that the PI3K/Akt signaling pathway may contribute to the prosurvival role of geraniin in MSCs under H₂O₂ treatment.