Non-esterified Fatty Acid-Induced Reactive Oxygen Species Mediated Granulosa Cells Apoptosis Is Regulated by Nrf2/p53 Signaling Pathway

Negative energy balance (NEB) during the perinatal period can affect dairy cow follicular development and reduce the fecundity. Non-esterified fatty acid (NEFA) concentration is elevated during NEB, and is known to be toxic for multiple cell types. In the ovary, NEB increased NEFA, and may influences follicular growth and development. However, the effect and mechanism of NEFA on granulosa cells (GCs) in vitro remains unknown. In this study, we found that NEFA dose-dependently induced apoptosis in primary cultured granulosa cells. Mechanistically, our data showed that NEFA significantly increased reactive oxygen species (ROS) levels, resulting in the activation of endoplasmic reticulum stress (ERS) and eventually cell apoptosis in GCs. Moreover, NEFA also increased the phosphorylation levels of ERK1/2 and p38MAPK pathways, upregulated the expression of p53 and potentially promoted its translocation to the nuclear, thus transcriptionally activated Bax, a downstream gene of this pathway. NEFA also promoted nuclear factor E2 (Nrf2) expression and its level in the nuclear. To elucidate the mechanism of NEFA action, N-acetyl-l-cysteine (NAC), a ROS scavenger was used to verify the role of ROS in NEFA induced apoptosis of GCs. NAC pretreatment reversed the NEFA-induced ERS-related protein and apoptosis-related protein levels. Meanwhile, NAC pretreatment also blocked the phosphorylation of ERK1/2 and p38 induced by NEFA, and the nucleation of Nrf2 and p53, suggesting that ROS plays a crucial role in regulating the NEFA-induced apoptosis of GCs. Together, these findings provide an improved understanding of the mechanisms underlying GCs apoptosis, which could potentially be useful for improving ovarian function.


Introduction
Due to the integrative outcome of reduced intake and higher demand for maintenance and production, cows in the perinatal period are in the state of negative energy balance (NEB). Non-esterified fatty acid (NEFA) is an important energy metabolite that is used as an indicator of NEB during the perinatal period. The elevation of NEFA is known to result in decreased reproductive potential, especially in high-yielding dairy cows [1]. It was reported that the concentration of NEFA in blood was lower than 0.20 mM in the late lactation and dry milk period, and began to increase in the first two weeks after labor, and the level of NEFA could exceed 0.75 mM at the 10th day of postpartum [2]. In vivo studies have shown that high-yield cows postpartum NEFA level can delay the first ovulation time, increase embryo mortality, reduce the conception rate and pregnancy rate [3]; high-yield cows postpartum NEFA content increase and postpartum first conception rate are negatively correlated, postpartum high-level NEFA

Primary Culture of Bovine Granulosa Cells
The study was approved by the animal protection and utilization committee (Approval number: SYXK (SU) 2017-0027), Nanjing Agricultural University, Nanjing, China. Briefly, bovine ovaries obtained from slaughterhouses (Nanjing, China) were placed in saline at about 37 • C and returned to the laboratory within 2 h. After washing the ovaries with saline, we used a syringe to extract follicular fluid into saline solution in a sterile environment, centrifuged and suspended cells in complete medium. Subsequently, granulosa cells (GCs) were cultured in DMEM/F-12 medium supplemented with 10% FBS, at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 .

Preparation of Cytoplasm and Nuclear Extracts
The Nuclear and Cytoplasm Protein Extraction Kit (Beyotime, China) was used to extra the nuclear and cytoplasm proteins. Briefly, the cells were collected and 200 µL ice-cold cytoplasm extraction buffer A with 1 mM PMSF was added. After incubation with cytoplasm extraction buffer B for 1 min, cell lysates were centrifuged at 12,000× g for 5 min at 4 • C, the supernatant was aspirated as cytoplasmic protein. Next, 50 µL nuclear extraction buffer was added to the sediment. In the next 30 min, vortexing for 30 s every 2 min on the ice took place, then the lysates were centrifuged at 16,000× g for 10 min at 4 • C, the supernatant was aspirated as a nuclear protein.

Immunoprecipitation and Immunoblots
According to the instructions for Pierce Co-Immunoprecipitation Kit (Thermo Fisher Scientific, Waltham, MA, USA), the cells cultured in T75 culture bottle were used for immunoprecipitation. For immunoblots, after incubation with NAC and NEFA, the cells were harvested and lysed with RIPA buffer. The protein content was measured by a BCA assay (Beyotime). Equal amounts of protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to PVDF membranes. Then, 5% skimmed milk was used to block the membranes, the primary antibodies were incubated at 4 • C overnight. The next day, washing membranes three times in TBST for each 10 min and then incubated with the corresponding secondary antibodies for 1 h at room temperature. Antibody detection was accomplished using enhanced chemiluminescence reagent. Densitometry analysis was detected using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Flow-Cytometry Analysis
The cells Annexin V-Alexa Fluor 647/PI Apoptosis Assay Kit was used to measure the extent of apoptosis according to the manufacturer's instructions. Briefly, cells were washed twice with PBS then gently resuspended in binding buffer containing Annexin V/Alexa Fluor 647 (5 uL) and propidium iodide (10 uL) (the cell concentration is 1 × 10 6 /mL). After 15 min in the dark, the apoptosis rates were analyzed by flow cytometry (BD, FACS Calibur, USA). The data analysis was using Flowjo software (Becton, Dickinson and Company, Franklin Lakes, JD, USA).

Reactive Oxygen Species (ROS) Level
The cells were seeded on six-well plates with cover glasses in each well and then treated with NAC for 2 h before treated with NEFA for 24 h. The intracellular levels of ROS were measured by loading the cells with the Dihydroethidium (DHE) (Beyotime Biotechnology, Shanghai, China) [19,20]. The method was based on the DHE, it can enter cells freely through living cell membranes and oxidized by ROS to the product ethidium oxide, which can participate in chromosomal DNA and produce red fluorescence. Briefly, after the treatment, cells were stained with 10 µM DHE in serum-free DMEM-F12 for 30 min at 37 • C in the dark. Then the cells were washed three times with PBS. The fluorescence intensity was measured at 480 nm excitation and 590 nm emission using a fluorescence microscope (Zeiss LSM 700 META (Olympus, Tokyo, Japan)).

Statistical Analysis
All data were expressed as mean ± SEM and the results at least three independent experiments. T-test was used to analyze statistical, using the GraphPad Prism5 software (GraphPad Software Inc., San Diego, CA, USA). P less than 0.05 was considered significant.

Results
3.1. Non-Esterified Fatty Acid (NEFA) Causes Accumulation of ROS, Endoplasmic Reticulum Stress, and Apoptosis in Granulosa Cells (GCs) To determine the correlation between NEFA and the accumulation of ROS in vitro, granulosa cells were cultured in the absence and presence of 0, 0.6, 1.2, 1.8 mM NEFA for 24 h, and DHE staining was performed. The imaging data showed that ROS signals were upregulated NEFA concentration-dependent manner ( Figure 1A). At the same time, the decrease of the SOD2 protein level also suggests the imbalance of redox state ( Figure 1B,C). We next studied if NEFA induce endoplasmic reticulum stress. CHOP, p-PERK, and GRP78 are markers for endoplasmic reticulum stress. Western blot analysis revealed that the NEFA upregulated the p-PERK, GRP78, and CHOP protein expression ( Figure 1B,D-F) in a dose-dependent fashion.

Statistical Analysis
All data were expressed as mean ± SEM and the results at least three independent experiments. T-test was used to analyze statistical, using the GraphPad Prism5 software (GraphPad Software Inc, San Diego, CA, USA). P less than 0.05 was considered significant.

Non-Esterified Fatty Acid (NEFA) Causes Accumulation of ROS, Endoplasmic Reticulum Stress, and Apoptosis in Granulosa Cells (GCs)
To determine the correlation between NEFA and the accumulation of ROS in vitro, granulosa cells were cultured in the absence and presence of 0, 0.6, 1.2, 1.8 mM NEFA for 24 h, and DHE staining was performed. The imaging data showed that ROS signals were upregulated NEFA concentrationdependent manner ( Figure 1A). At the same time, the decrease of the SOD2 protein level also suggests the imbalance of redox state ( Figure 1B,C). We next studied if NEFA induce endoplasmic reticulum stress. CHOP, p-PERK, and GRP78 are markers for endoplasmic reticulum stress. Western blot analysis revealed that the NEFA upregulated the p-PERK, GRP78, and CHOP protein expression ( Figure 1B,D-F) in a dose-dependent fashion.  The incidence of apoptosis was increased in NEFA treated GCs detected via Annexin V-Alexa Fluor 647 staining and flow cytometry (Figure 2A,B). In the meantime, NEFA also prominently increased apoptosis associated Bax/Bcl-2 protein ratio in the GCs ( Figure 2C). Taken together, these observations suggest NEFA induces apoptosis to occur in GCs.
Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 14 expression of SOD2 was decreased under the different concentrate of NEFA. (B,D-F) Protein expression of p-PERK, GRP78, and CHOP were significantly increased, which means the endoplasmic reticulum stress (ERS) was significantly increased under the high level of NEFA. The results are expressed as the mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, not significant.
The incidence of apoptosis was increased in NEFA treated GCs detected via Annexin V-Alexa Fluor 647 staining and flow cytometry (Figure 2A,B). In the meantime, NEFA also prominently increased apoptosis associated Bax/Bcl-2 protein ratio in the GCs ( Figure 2C). Taken together, these observations suggest NEFA induces apoptosis to occur in GCs. The results showed that NEFA-induced apoptosis in GCs. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, and ***p < 0.001 .

Treatment of N-acetyl-L-cysteine (NAC) In Vitro Attenuates ROS Levels, Endoplasmic Reticulum Stress (ERS) Levels and Apoptosis in High-Level NEFA Treated GCs
To determine whether supplementation of NAC can decrease ROS, GCs were cultured in the absence and presence of NEFA, and in addition to minor with NAC (5 mM) for 2 h, the levels of the ROS were assessed. As to be expected, immunostaining revealed that NAC substantially reduced the NEFA induced ROS signals ( Figure 3A). To further determine if endoplasmic reticulum stress (ERS) could be recovered by NAC in high-level NEFA treated GCs, we next analyzed the expression of p-PERK, GRP78, and CHOP at the protein level. It was found that the levels of p-PERK, GRP78, and CHOP were significantly reduced in the NAC-treated group as compared with the NEFA group ( Figure 3B-E). To determine whether supplementation of NAC can decrease ROS, GCs were cultured in the absence and presence of NEFA, and in addition to minor with NAC (5 mM) for 2 h, the levels of the ROS were assessed. As to be expected, immunostaining revealed that NAC substantially reduced the NEFA induced ROS signals ( Figure 3A). To further determine if endoplasmic reticulum stress (ERS) could be recovered by NAC in high-level NEFA treated GCs, we next analyzed the expression of p-PERK, GRP78, and CHOP at the protein level. It was found that the levels of p-PERK, GRP78, and CHOP were significantly reduced in the NAC-treated group as compared with the NEFA group ( Figure 3B-E).
The occurrences of apoptosis in the NAC-treated GCs were also significantly decreased as compared with those in the NEFA groups ( Figure 4A,E). Consistent with this finding, the protein ratio of Bax/Bcl-2 and cleaved caspase-3 also decreased in respond to NAC ( Figure 4B-D). These results suggest that ROS induced apoptosis and ERS may be reversed by NAC.  The occurrences of apoptosis in the NAC-treated GCs were also significantly decreased as compared with those in the NEFA groups ( Figure 4A,E). Consistent with this finding, the protein ratio of Bax/Bcl-2 and cleaved caspase-3 also decreased in respond to NAC ( Figure 4B-D). These results suggest that ROS induced apoptosis and ERS may be reversed by NAC. 3.3. ERK1/2 and p38 Pathways are Involved in NEFA Induced ROS Mediated Apoptosis of GCs ERK1/2 and p38 are important signaling pathways in mitogen-activated protein kinases, which regulate cell proliferation and apoptosis. To investigate the role of these pathways in NEFA induced apoptosis mediated by ROS, cells were treated with NEFA, and Western blots were performed. As shown in Figure 5A-C, suggesting it may activate the ERK1/2 and p38 signaling pathways. Interestingly, the increased phosphorylation of ERK1/2 and p38 were reversed in the presence of NAC ( Figure 5D-F), that ERK1/2 and p38 pathways may be involved in NEFA induced ROS mediated GCs apoptosis.  The results of Bax, Bcl-2, and cleaved caspase-3 protein levels showed that NAC alleviated GCs apoptosis induced by high-level NEFA. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, not significant.

ERK1/2 and p38 Pathways are Involved in NEFA Induced ROS Mediated Apoptosis of GCs
ERK1/2 and p38 are important signaling pathways in mitogen-activated protein kinases, which regulate cell proliferation and apoptosis. To investigate the role of these pathways in NEFA induced apoptosis mediated by ROS, cells were treated with NEFA, and Western blots were performed. As shown in Figure 5A-C, suggesting it may activate the ERK1/2 and p38 signaling pathways. Interestingly, the increased phosphorylation of ERK1/2 and p38 were reversed in the presence of NAC ( Figure 5D-F), that ERK1/2 and p38 pathways may be involved in NEFA induced ROS mediated GCs apoptosis. The results of Bax, Bcl-2, and cleaved caspase-3 protein levels showed that NAC alleviated GCs apoptosis induced by high-level NEFA. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, not significant.

NAC Eliminates ROS via the Nrf2/HO-1 Pathway in GCs
Nrf2 is an important transcription factor in the regulation of oxidative stress and a central regulator in the maintenance of intracellular redox homeostasis. Heme oxygenase-1 (HO-1) is thought to be a major antioxidant enzyme scavenging cellular ROS [21], and its activation is regulated by Nrf2. Immunoblotting analysis showed that at the concentration of 1.2 mM, NEFA increased the Nrf2 and HO-1 expression at the protein level ( Figure 6A-C). To clarify whether the decrease of apoptosis rate by NAC is related to the HO-1 gene, we also examined the protein levels of HO-1 by using immunoblotting. Our results revealed that compare to the NEFA group, NEFA induced HO-1 was significantly reduced by NAC in GCs ( Figure 6E,G). Since oxidative stress can activate Nrf2, a classic antioxidant pathway, we next tested whether NEFA induces Nrf2 translocation into the nucleus. Western blot results revealed that nuclear Nrf2 significantly increased in the presence of NEFA, and then this increase was reversed by NAC ( Figure 7A). Collectively, these observations suggest that ROS induced by NEFA plays an important role in the activation of the Nrf2/HO-1 pathway. (A-C) After treatment with different concentrations of NEFA, p-ERK1/2, ERK1/2, p-p38, and p38 levels were assayed by western blotting. (D-F) After pre-treatment with NAC, protein expression of p-ERK1/2, ERK1/2, p-p38, and p38 were evaluated by western blotting. These results indicated that the ROS induced by NEFA activated the ERK1/2 and p38 pathways. Data are presented as mean ± SEM, * p < 0.05, and ** p < 0.01.

NAC Eliminates ROS via the Nrf2/HO-1 Pathway in GCs
Nrf2 is an important transcription factor in the regulation of oxidative stress and a central regulator in the maintenance of intracellular redox homeostasis. Heme oxygenase-1 (HO-1) is thought to be a major antioxidant enzyme scavenging cellular ROS [21], and its activation is regulated by Nrf2. Immunoblotting analysis showed that at the concentration of 1.2 mM, NEFA increased the Nrf2 and HO-1 expression at the protein level ( Figure 6A-C). To clarify whether the decrease of apoptosis rate by NAC is related to the HO-1 gene, we also examined the protein levels of HO-1 by using immunoblotting. Our results revealed that compare to the NEFA group, NEFA induced HO-1 was significantly reduced by NAC in GCs ( Figure 6E,G). Since oxidative stress can activate Nrf2, a classic antioxidant pathway, we next tested whether NEFA induces Nrf2 translocation into the nucleus. Western blot results revealed that nuclear Nrf2 significantly increased in the presence of NEFA, and then this increase was reversed by NAC ( Figure 7A). Collectively, these observations suggest that ROS induced by NEFA plays an important role in the activation of the Nrf2/HO-1 pathway.    The results showed that Nrf2/HO-1 and p53/Bax signaling pathways were involved in NEFA induced apoptosis. "+", add; "-" without add.

ROS Induced by NEFA Activated p53/Bax Pathway
The tumor suppressor, p53 was an important transcription factor in response to a diverse number of cellular stress including cell death, oxidative stress, and hypoxia [18]. We next examined the effects of NEFA on p53 expression in GCs. It was found that NEFA increased p53 expression dose dependently ( Figure 6A,D). Subsequent study revealed that NEFA induced p53 expression was reversed in the addition of NAC ( Figure 6E,F). It is known that upon stressed, p53 translocated to the nucleus, which in turn controls processes such as cell death and metabolism. We next sought to further clarify if NEFA affects p53 translocation. As shown in Figure 7A, an increase of nuclear p53 protein levels was observed in the presence of NEFA. However, pretreating with NAC decreased NEFA induced nuclear p53 ( Figure 7A). This suggests that the p53 is translocated into the nucleus in response to NEFA, which may have contributed to NEFA-induced apoptosis and NAC could alleviate this partly.
Recent studies had also shown that when p53 is stimulated, it not only activates cell apoptosis but also interacts with Bcl-2 family members in mitochondria [22,23]. Since our data showed that P53 and Bax accumulated in GCs, we evaluated possible interactions between these two key proteins using immune-precipitation. The results showed that the interaction between P53 and Bax in the NEFA group ( Figure 7B,C). In being co-treated with NEFA and NAC group, P53 and Bax interaction was inhibited, suggesting that NEFA, and thus ROS generation may involve in the activation of P53 and Bax interaction.

Discussion
Recent studies have demonstrated that the follicular fluid total NEFA, ammonia, and urea exceeding the physiological concentration are considered as nutrition and metabolic stressors. Refs. [24,25], which can lead to postpartum reproduction problems [1]. Ovarian granulosa cells play an important role in the reproduction of dairy cows. However, no studies have shown the molecular mechanism of the GCs damage with the high-level NEFA in the perinatal period. High levels of NEFA is known to lead to changes in glucose metabolism by an increase of β-oxidation, resulting in an increase in ROS production [26]. Our findings showed that ROS increases with a dose-dependent manner in NEFA treated cells. Oxidative stress can result in production of reactive oxygen species and nucleophilic substances. If not removed in time, the oxidative stress products will cause oxidative damage, such as cell death, inflammatory response. Our finding that ROS accumulation induced by NEFA mediates apoptosis of GCs, which is consistent with this viewpoint.
The apoptosis response of ovarian granulosa cells to NEFA is concentration-dependent. Some studies showed that oxidative stress and endoplasmic reticulum stress are two processes that cause pathological cell death [27,28]. Our data also showed that there was endoplasmic reticulum stress in NEFA induced apoptosis. More interestingly, our data indicated that providing N-acetylcysteine (NAC), a common antioxidant and scavenger of ROS, attenuated NEFA induced ROS, GRP78, CHOP, and p-PERK protein expression and the apoptosis in GCs. These finding suggest that NAC may prevent NEFA-induced apoptosis, at least partly, via inhibiting ROS generation and ER stress.
Mitogen-activated protein kinases (MAPKs) are important signaling pathways in eukaryotic cell biology, which are involved in the regulation of cell proliferation, differentiation, and apoptosis. ERK1/2 and p38 are members of the MAPK family, their roles in regulating cell survival and other actives were well documented [29][30][31]. It has been reported that p38 MAPK was activated by ROS produced in cells [32]. Our finding that NEFA increase the phosphorylated ERK1/2 and p38 and this increase is block by NAC suggest the NEFA action in GCs may involve ROS activated ERK1/2 and p38 pathways.
The signal transduction process is a very complex network. ROS induced by NEFA can activate ERK1/2 and p38 signal pathways in ovarian granulosa cells, but there is still a lack of clear understanding of the specific process of signal transduction and which upstream and downstream signal molecules are involved. Many studies have shown that the MAPK signaling pathway can activate Nrf2 [33][34][35]. Nrf2 is an important transcription factor that contributes to the expression of antioxidant defense genes. Shen et al. showed that the ERK1/2 pathway up regulate Nrf2 transcription activity [36]. Under damaging stimuli, Nrf2 dissociates from Keap1 and translocates into the nucleus, resulting in the transcription activation of phase II enzyme/antioxidant genes, including HO-1 and GST, etc. [37,38]. We found that the expression of Nrf2 protein increased dose-dependently to NEFA. Our results also showed that under the stimulation of NEFA, nuclear Nrf2 and HO-1 expression were increased, is a cell self-protect response ROS, however, apoptosis still occurred, suggesting that the activation of Nrf2 is not enough to resist the damage caused by oxidative stress. Interestingly, we observed that after pretreatment with NAC, the expression of Nrf2 in the nucleus and its downstream protein HO-1 decreased, that is, NAC did not activate the Nrf2/HO-1 signal pathway successfully. We speculate that the possible reason is that NEFA consumes part of NAC after NAC pretreatment, and the weakened oxidative stress can not compensate for the active Nrf2/HO-1 signal pathway. Previous studies have shown that p38 MAPK activation affects the regulation of downstream transcription factors, such as p53 and Nrf2, to control downstream pro-apoptotic and anti-apoptotic gene expression in response to many extracellular stimuli, including oxidative stress [39][40][41]. p53 is a key player in the process of mitochondria-mediated apoptosis; it enhances the expression of Bax [42]. Our data also showed that high-level NEFA significantly upregulated the expression of p53 and Bax protein and promoted the entry of p53 into the nucleus. After adding NAC to inhibit the accumulation of ROS, the expression of p53 and Bax protein, and the entry of p53 into the nucleus were significantly alleviated compared with the 1.2 mM NEFA group. Our data suggests that the p53 response may be induced by ROS, which is consistent to a previous report [43]. Additionally, our finding on NEFA induced p53 and Bax interaction was suppressed by NAC suggest that NEFA induced p53/Bax signaling pathway activation may be mediated by NEFA induced ROS in granulosa cells.
In summary, our study provides further insights on the molecular mechanisms of NEFA-induced apoptosis of GCs. We found that NEFA induced apoptosis and stimulated an increase in ROS production in a dose-dependent manner in GCs. The accumulation of ROS induced by NEFA results in the up-regulation of ERS related proteins such as p-PERK, GRP78, and CHOP. Importantly, the blockage of ROS production by NAC has significantly reversed NEFA-induced ROS, ERS, and cell apoptosis. NEFA also activated ERK1/2 and p38 pathways, and the effects of NEFA were reversed by pretreatment with NAC. In addition, NAC can also partially reverse NEFA-induced Nrf2 and p53 nucleation and the increased expression of downstream genes HO-1 and Bax to regulate cell apoptosis. Based on our findings, a model of how NEFA induces apoptosis through ROS mediated signaling pathway is proposed (Figure 8). The results of this study may be helpful to further reveal the influence of NEFA on the reproductive function of in the ovary, and to further support the importance of reducing perinatal stress in production.