Genistein Protects Genioglossus Myoblast Against Hypoxia-induced Injury through PI3K-Akt and ERK MAPK Pathways

Obstructive sleep apnea and hypopnea syndrome (OSAHS) is a clinical syndrome characterized by recurrent episodes of obstruction of the upper airway during sleep that leads to a hypoxic condition. Genioglossus, an important pharyngeal muscle, plays an important role in maintaining an open upper airway for effective breathing. Our previous study found that genistein (a kind of phytoestrogen) protects genioglossus muscle from hypoxia-induced oxidative injury. However, the underlying mechanism is still unknown. In the present study, we examined the effects of hypoxia on genioglossus myoblast proliferation, viability and apoptosis, and the protective effect of genistein and its relationship with the PI3K/Akt and ERK MAPK pathways. Cell viability and Bcl-2 were reduced under hypoxic condition, while ROS generation, caspase-3, MDA, and DNA damage were increased following a hypoxia exposure. However, the effects of hypoxia were partially reversed by genistein in an Akt- and ERK- (but not estrogen receptor) dependent manner. In conclusion, genistein protects genioglossus myoblasts against hypoxia-induced oxidative injury and apoptosis independent of estrogen receptor. The PI3K-Akt and ERK1/2 MAPK signaling pathways are involved in the antioxidant and anti-apoptosis effect of genistein on genioglossus myoblasts.


Materials. Sprague-Dawley (SD) rats were obtained from the Experimental Animal Center of Second
Military Medical University (Shanghai, China). All animal care and use were conducted according to the National Research Council guidelines and approved by the Animal Care and Use Committee of Zhejiang University. The cell culture medium, class II collagenase, trypsin and fetal bovine serum (FBS) were from Life Technologies (Grand Island, NY, USA). The 2′,7′-dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probes (Eugene, Oregon, USA). The genistein, ICI 182780, SB 203580, and SP 600125 were obtained from Sigma Chemical Company (St Louis, MO, USA). The wortmannin, U0126, and antibodies of phospho-Akt, phospho-ERK1/2, and Bcl-2 were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-rabbit I horseradish peroxidase (HRP)-conjugated goat antibodies were obtained from Santa Cruz Biotechnologies (Heidelberg, Germany). The MTT, DAPI, MDA and caspase-3 detecting kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The BrdU detection kit was from obtained Boehringer Mannheim Biochemica (Mannheim, Germany).
Primary culture of genioglossus myoblast and hypoxia exposure. The genioglossus muscle tissues were obtained from newborn (3-5 d) SD rats. They were trimmed of excessive connective and fat tissues, hand-minced with a sterile scissor into approximately 1 mm 3 , and washed with PBS for three times. Enzymatic dissociation was performed by two-step digestion of minced muscles at 37 °C in 0.5% collagenase type II for 20 min and 0.05% trypsin for 10 min. The digestion was terminated by growing medium (GM, high glucose DMEM containing 10% FBS and 1% penicillin-streptomycin), and cells were separated from muscle fiber fragments and tissue debris by 120 mesh screen cloth. The cells were centrifuged, and plated in tissue culture dishes coated with poly-lysine in GM. Cells were incubated in normoxic condition with 37 °C and 5% CO 2 . However, hypoxic conditions were created in the hypoxia chamber with 0.5% O 2 and 5% CO 2 .
Detection of cell proliferation. The cells were seeded into 96-well cell culture plates at a density of 2 × 10 4 cells/well. After 24 h culture at 37 °C and 5% CO 2 , 10 μM of genistein (the concentration was decided by our previous study) were added to the plates . After pretreated with genistien for 1 h, the cells were incubated under normoxic or hypoxic conditions for another 24 h, 48 h, and 72 h. MTT assay was performed following the well-described procedure 25 . Briefly, the wells were washed three times with DMEM. Then, 180 μl aliquots of DMEM and 20 μl aliquots of MTT solution (5 mg/ml of PBS) were added to each well at the established time. After 4 h of incubation at 37 °C and 5% CO 2 , the media were removed and formazan crystals were solubilized with 150 μl DMSO. The plates were read on ELx800 absorbance microplate reader (Bio-Tec Instruments INC, USA) at 550 nm wavelength. The results of MTT assay were verified by BrdU DNA incorporation assay via detection of DNA synthesis 26 . The incorporated BrdU was measured using a BrdU detection kit according to the manufacturer's instructions. The absorbance of the extract was read at a wavelength of 450 nm with a reference wavelength of 630 nm. DAPI staining. Cells were seeded onto 6-well tissue culture plates at a concentration of 1 × 10 5 cells/ well and incubated under normoxia or hypoxia conditions at the presence or absence of genistein for 48 h. At the end of incubation, cells were fixed with ice-cold 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton x-100 for 25 min and washed with ice-cold PBS three times. At last the cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI, 5 μg/ml) for 25 min and observed under a fluorescence microscope with a peak excitation wave length of 340 nm. Nuclei were identified as normal, fragmented, or condensed. Fragmented or condensed nuclei were classified as apoptotic. The results were expressed as a percentage of apoptotic cells. A minimum of 500 cells were counted for each treatment from at least three independent experiments. Flow cytometry analysis. Cells were seeded onto 6-well tissue culture plates at a concentration of 1 × 10 5 cells/well and incubated under normoxia or hypoxia conditions at the presence or absence of genistein for 48 h. At the end of incubation, trypsin digestion was performed to detach the cells from culture plates. We resuspended the cells in 1x binding buffer and adjusted the concentration to 1 × 10 6 cells/mL. Cell suspensions were added to 5 mL Falcon tubes containing 5 μL of FITC-Annexin V and 5 μL of PI dye and then gently mixed. Following incubation in the dark at room temperature (~25 °C) for 15 min, 400 μL of 1x binding buffer was added to each tube, and the samples were analyzed within 1 h using a flow cytometer (BD Biosciences). Caspase fluorometric assay. Measurement of caspase-3 activity was performed as previously described 28 .
Western blot analysis. Cells were harvested into a lysis buffer consisting of 2.5 mM HEPES, pH 7.5, 10% glycerol, 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 100 mM Na pyrophosphate, 50 mM NaF, 0.1 mM NaVO4, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 2 μg/ml pepstatin. Equal protein amounts (50 μg) of genioglossus homogenates were electrophoresed through 12% SDS-polyacrylamide gel and electroblotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The blots were then washed in PBST (PBS with 0.05% Tween-20), blocked with 5% fat-free milk in PBST for 1 h, and incubated overnight with an appropriate primary antibody at the dilutions recommended by the supplier (1:1000). The detection was made with HRP-conjugated secondary antibodies (1:10000) and ECL System by Kodak IMAGE STATION 2000 MM (Kodak, Rochester, NY, USA). Statistical analysis. Statistical analyses were performed using the SPSS Statistical Analysis Software, version 13.0. All data are expressed as the means ± the standard deviation from three independent experiments. Statistical significance of differences was calculated using ANOVA followed by Tukey's HSD post hoc test. Statistical significance was established at P < 0.05.

Results
Oxidative stress and genioglossus myoblast injury can be induced by Hypoxia. Genioglossus myoblast were incubated in hypoxic conditions of 0.5% O 2 and 5% CO 2 for 24 h, 48 h, and 72 h. Cell viability unchanged following 24 h hypoxic exposure, while it was significantly decreased after 48 and 72 h (Fig. 1A). The levels of H 2 O 2 was measured using DCF-DA to determine whether hypoxia stimulates the generation of ROS in genioglossus myoblast. The data showed that the intracellular H 2 O 2 was significantly increased in a time-dependent manner (0-72 h) (Fig. 1B). Caspase 3 plays an important role in apoptosis and is responsible for chromatin condensation and DNA fragmentation 29 . Our data showed that the level of caspase 3 expression was increased as the extension of the time under the hypoxic conditions (0-72 h) (Fig. 1C). Besides, the index of lipid peroxidation, MDA, was increased following 72 h hypoxia exposure (Fig. 1D). B-cell lymphoma protein-2 (Bcl-2), as a regulator of permeabilization of the mitochondrial outer membrane and the consequent release of cytochrome c, prevents apoptosis and hypoxia-induced injury 30,31 . The level of Bcl-2 was reduced at 48 h, but it was increased at 72 h. However, the level of Bcl-2 remained less than normal value (Fig. 1E,F).

Effects of genistein on hypoxia-induced apoptosis of genioglossus myoblast. DAPI staining and
Annexin V-FITC/PI flow cytometry analysis were used to detect the effects of hypoxia on the nuclear changes and apoptosis of genioglossus myoblast. As shown in Fig. 2A and B, nuclei had the normal phenotype demonstrating bright and homogenously at normoxic or 24 h hypoxic condition. But we found that this process was inhibited by genistein (Fig. 2C). Nuclei that emitted bright fluorescence, fragmented, or condensed were classified as apoptotic. The results from flow cytometry analyses indicated that the early-stage and late-stage apoptosis rate were significantly increased after 48 h hypoxia exposure. Genistein significantly decreased the early-stage apoptosis of genioglossus myoblasts (Fig. 2D,E).

Relationship between hypoxia-induced cell injury, genistein and estrogen receptor (ER).
In order to observe the relationship between ER and genistein on hypoxia-induced genioglossus myoblast, cells were treated with genistein and ICI 182780 (ER antagonist, 100 nM). The inhibition of hypoxia on cell viability was attenuated after cells were treated by genistein with or without ICI 182780. Results showed genistein attenuated hypoxia-induced H 2 O 2 generation and its effect on cell viability was not influenced by ICI 182780 (Fig. 3A,B). Exogenous H 2 O 2 (100 μM) was used as a positive control for assessing the effect of hypoxia-induced H 2 O 2 generation on the cell viability (Fig. 3C).

Relationship among the ERK1/2-MAPK and PI3K-Akt pathways, cell injury, and apoptosis.
The signaling molecules associated with hypoxia-induced cell injury and apoptosis, p38, ERK1/2, JNK MAPK, and PI3K-Akt were evaluated. No changes were detected in p38 and JNK MAPK proteins under hypoxic conditions (data not shown). Meanwhile, we found phospho-Akt and phospho-ERK1/2 were remarkably reduced after 24 h and 48 h of hypoxia, but they were recovered at 72 h (Fig. 4A-D).

Discussion
OSAS is associated with oxidative stress and UA muscle fatigue 12 . Furthermore, it is reported that oxidative-stress contributes to hypoxia-induced respiratory muscle impairment in an animal model of OSAS 32 . However, the underlying mechanism is still unknown. Genioglossus is a very important UA dilating muscle, which protrudes the tongue, increases the oropharyngeal size, and decreases airway collapsibility. Therefore, genioglossus plays a significant role in maintaining an open UA for effective breathing 9 . We established a genioglossus myoblast culture model under hypoxic conditions to detect the underlying mechanism of oxidative stress and muscle injury. The results of this study demonstrate that genistein partially protects genioglossus myoblast against hypoxia-induced oxidative stress injury. This protective effect is mediated by the regulation of ROS, lipid peroxidation, Bcl-2, and caspase-3 through the PI3K-Akt and ERK1/2 MAPK signaling pathways, which suggests that genistein is effective against oxidative stress through multiple ways in genioglossus myoblast.
This study found that hypoxic exposure stimulates the generation of ROS, which is coincident with OSAS and the animal model of the disorder 33,34 . ROS includes superoxide anion, hydrogen peroxide and hydroxyl radical, and plays a physiological role in the function of a number of cellular signaling pathways 35 . However, excessive ROS induces cell injury including DNA damage, lipid peroxidation, and apoptosis. In this study, it was detected that hypoxic exposure significantly reduces the viability of genioglossus myoblasts. Apoptotic nuclei were increased by hypoxic exposure, indicating that hypoxia induces apoptosis of genioglossus myoblasts. In addition, caspase-3, an apoptosis marker, was also increased under hypoxic conditions. Bcl-2, an anti-apoptosis protein, was reduced under a hypoxic condition though it recovered partially with a longer exposure. MDA, the index of lipid peroxidation, was increased after 72 h of hypoxia. Therefore, we conclude that hypoxia induces oxidative cell injury in a ROS-dependent manner, leading to apoptosis through down-regulation of Bcl-2 and up-regulation of caspase-3, consistent with previous studies 36 . Genistein, an isoflavone phytoestrogen, is a biologically active plant substance with a chemical structure similar to that of endogenous estrogen. In this study, we found that genistein attenuates the deleterious effects of hypoxia on cell viability, which is ROS-dependent but not ER-dependent. This discovery is different from the finding of a previous study which determined that genistien exerts protective effect on oxidative stress-induced human endothelial cells (HUVECs) injury through ERs 37 . The reason might be that genistein has a relatively high binding affinity for ER β. ER β is highly expressed in HUVECs, but lowly expressed in skeletal muscles [38][39][40] . In addition, genistein increased Bcl-2 levels and attenuated caspase-3 activity and lipid peroxidation independent of ERs. This suggests that anti-apoptotic and antioxidant actions represent two of the most important mechanisms by which genistein protect genioglossus myoblasts against a hypoxic injury. The antioxidant property of genistein has been demonstrated in several studies and is reported to be more effective than that of antioxidant vitamins and estrodiol in scavenging ROS and lipid peroxidation [41][42][43] . However, other research indicates that genistein stimulates the expression of apoptotic markers such as caspase-3. Taken together, these observations implicate that variation in the dosage of genistein and cell type used in experiments can result in a totally distinct outcome. The antioxidant property probably accounts for the protective effect of genistein on genioglossus fatigue which was found in a previous study 44 . Furthermore, the result is consistent with the finding that superoxide scavengers attenuate rat pharyngeal dilator muscle fatigue in hypoxia 45 .
It has been determined that the MAPK and PI3K/Akt signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death 46,47 . The pathways play a central role in orchestrating growth, proliferation, and anti-apoptotic mechanisms to promote cell cycle and survival. These cellular processes are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. The mammalian family of MAPK includes extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun NH 2 -terminal kinase (JNK). In this study, we found that p38 and JNK MAPK were not involved in hypoxia-induced genioglossus myoblast injury. This finding is different from previous research showing that p38 and JNK are involved in hypoxia-induced hepatocyte injury, which suggests that the signaling pathways related to hypoxia are cell-specific 36 . Hypoxia decreased the expression of the phospho-Akt and phospho-ERK1/2, and Akt and ERK inhibitor (wortmannin and U0126) partially reversed the protection of Gen on the hypoxia-induced injury. This result suggests that the PI3K/Akt and ERK MAPK pathways are involved in the hypoxia injury and protective effect of genistein on genioglossus myoblasts. The conclusion can be verified by the result that genistein increased the expression of phospho-Akt and phospho-ERK1/2, and that wortmannin and U0126 reversed the effect of genistein on Bcl-2, caspase-3, and MDA. Therefore, the effects of genistein on hypoxia-induced genioglossus myoblast injury are presumably through non-genomic pathways rather than ER-mediated genomic pathways. Signaling pathways including the ERK1/2 and PI3K/Akt cascades permit non-genomic actions of genistein.
In conclusion, genistein protects primary cultured genioglossus myoblasts against hypoxia-induced oxidative injury and cell apoptosis independent of ER. The PI3K-Akt and ERK1/2-MAPK signaling pathways are involved in the antioxidant and anti-apoptotic effects of genistein on genioglossus myoblasts under hypoxic conditions.