The Scutellarin Protects Mouse Ovarian Granulosa Cells from ZEA-Induced Injury

Background The zearalenone (ZEA) contained in the animal grain feeds is produced by Fusarium fungi and this toxin targets ovarian granulosa cells (GCs) to cause reproductive disorders in female animals. Current research on drugs that can rescue ZEA-induced GCs damage is limited. The purpose of this study was to explore the effect of scutellarin (Scu) on ZEA-induced apoptosis of mouse ovarian GCs and its mechanism. In one set of experiments, the primary cultured mouse ovarian GCs were co-treated with ZEA and scutellarin for 24 h. The results showed that Scu signicantly alleviated ZEA-induced cell damage, restored cell cycle arrest, and inhibited apoptosis by reducing the ratio of cleaved-caspase-3, cleaved-PARP, and Bax/Bcl-2. In other set of experiments, six weeks old mice were intragastrical administered with 40 mg/kg ZEA for 2 h, followed by 100 mg/kg Scu for 3 d. It was shown that Scu inhibited ZEA-induced apoptosis and positive signal expression of cleared-caspase-3 in the ovarian granulosa layer, with the involvement of mitochondrial apoptotic pathway. treating the cells with 2000, 1000 and 500 μg/mL of Scu for 24 h, and the cell cycle distribution was detected using ow cytometry. The results showed that all these different doses of Scu reduced the number of cells in the S phase, and the difference between 2000 and 1000 μg/mL group was signicant. 1000 μg/mL Scu signicantly increased the number of cells in the G2/M phase as shown in Fig.3B, and C. These results revealed that Scu could affect the cell cycle via promoting the transformation of cells from S-phase to G2/M-phase.


Abstract Background
The zearalenone (ZEA) contained in the animal grain feeds is produced by Fusarium fungi and this toxin targets ovarian granulosa cells (GCs) to cause reproductive disorders in female animals. Current research on drugs that can rescue ZEA-induced GCs damage is limited. The purpose of this study was to explore the effect of scutellarin (Scu) on ZEA-induced apoptosis of mouse ovarian GCs and its mechanism.

Results
In one set of experiments, the primary cultured mouse ovarian GCs were co-treated with ZEA and scutellarin for 24 h. The results showed that Scu signi cantly alleviated ZEA-induced cell damage, restored cell cycle arrest, and inhibited apoptosis by reducing the ratio of cleaved-caspase-3, cleaved-PARP, and Bax/Bcl-2. In other set of experiments, six weeks old mice were intragastrical administered with 40 mg/kg ZEA for 2 h, followed by 100 mg/kg Scu for 3 d. It was shown that Scu inhibited ZEA-induced apoptosis and positive signal expression of cleared-caspase-3 in the ovarian granulosa layer, with the involvement of mitochondrial apoptotic pathway.

Conclusion
Scu attenuated ZEA-induced reproductive toxicity by targeting mouse ovarian GCs, mainly affecting cell cycle phase distribution and apoptosis via mitochondrial apoptotic pathway in vitro and in vivo. These data provide strong evidence that Scu can be further developed as potential new therapeutic drug for preventing or treating reproductive toxicity caused by animal exposure to ZEA found in the grains of animal feeds. Background Zearalenone (ZEA) is a secondary metabolite produced by some Fusarium species, naturally exists in cereals, such as corn, barley, and sorghum 1 . Due to its high resistance to heat and widespread existence, ZEA contained in the grains is di cult to be eliminated 2 . Exposure of humans and animals to ZEA poses a great threat to humans and animals health as the ZEA can cause severe systemic toxicity in many organs 3,4 . The main target organs are those in the female reproductive system and ZEA induced toxicity can cause reproductive disorders in pigs, cattle, horses, and mice. The disorders include follicular development problem, ovulation abnormalities, and follicular atresia, leading to great economic losses to livestock and poultry industry [5][6][7] .
Granulosa cells (GCs) being one type of the important somatic cells of ovarian follicles play a key role in the growth and development of the follicles. GCs secrete steroid hormones and provide nutritional support for oocyte development [8][9][10] Studies have shown that apoptosis occurred in 10% or more of GCs population will cause follicle atresia, which results in reproductive disorders [11][12][13] ZEA is shown to cause ovarian GCs apoptosis as well as steroid hormone secretion disorders and reproductive disorders 14,15 .
ZEA inhibited the proliferation of GCs in a dose-dependent manner, induce apoptosis and necrosis of GCs through the mitochondrial apoptosis pathway 16 The ER stress and autophagy were also involved in the mechanism of the action of ZEA on GCs 17 . Therefore, inhibiting GCs apoptosis may alleviate the reproductive toxicity caused by ZEA.
The adsorption of ZEA using physical and chemical methods and its degradation using microbial enzyme are used to eliminate ZEA from Fusarium contaminated grains 18 . However, these methods have only achieved limited results. Thus, ZEA can still reach animal feed and human diets, and it is becoming important to explore therapies including drugs to protect tissue cells from their injuries caused by ZEA toxicities. Developing a drug for a therapy would be a pragmatic step to take in this end. Speci cally, the ZEA being a mycotoxin induces ovarian GCs apoptosis and affect the secretion of steroid hormones.
Therefore, it is of great signi cance to develop drugs that could ameliorate ZEA-induced GCs apoptosis.
Reports have shown that proanthocyanidins, saffron, lycopene can protect tissue cells from ZEA-induced damage [19][20][21] . Scutellarin (Scu) is a avonoid compound derived from plants such as Erigeron breviscapus, Scutellaria barbata, and Scutellaria baicalensis. These herbs are widely used in the treating cardiovascular and cerebrovascular diseases, diabetes, metabolic disorders, and other related diseases in clinical practice 22,23 . The pharmacological studies have shown that Scu has anti-in ammatory, antioxidant, anti-apoptotic, and antibacterial properties. Studies have reported that Scu can protect kidney from damaged due to cisplatin by inhibiting the expression of in ammatory factors and pro-apoptosis related proteins 24 . Based on these ndings, we hypothesize that the Scu may protect tissue cells against ZEA-induced reproductive toxicity and does so via modulating apoptotic pathway.
In this study, we aimed to test this hypothesis by observing the effect of Scu on ZEA-induced ovarian GCs apoptosis in vitro and in vivo through the mitochondrial apoptotic pathway. Furthermore, the effect of Scu to relieve ZEA-induced GCs S-phase arrest was also checked. The ndings of this study suggested that Scu could be further developed as a new effective drug for the treatment of reproductive toxicity caused by ZEA. The mouse GCs were collected and cultured according to our previously describe study 25 . Brie y, threeweek-old female Kunming mice were intraperitoneally injected with 5 IU PMSG, and were euthanized 46 h later. Bilateral ovaries were collected and punctured with 26 gauged# needles under a stereomicroscope to isolate GCs and passed through 0.074 mm sieve and centrifuged (1000 rpm, 5 min). After washing three times with PBS, the cells were resuspended in DMEM/F12 medium supplemented with 10% FBS and 1% streptomycin-penicillin, and were incubated at 37°C with 5% CO 2 . Cell cycle distribution analysis Cells were seeded in 6-well plates at a density of 1×10 6 cells/mL. After reaching 80-90% con uency, the cells were treated with the medium containing different concentrations of Scu (2000, 1000 and 500 μg/mL) with or without ZEA (60 μM) for 24 h. Cells were collected and xed with cold 70% ethanol for 2 h. After being washed with PBS, cells were incubated with PI/RNase A in dark for 30 min, then the cell cycle distribution was detected using ow cytometry.

Animals and Treatments
Five-week-old female Kunming mice were provided by Charles River (Beijing, China), and were housed under standard laboratory conditions of room temperature (22-24°C) and relative humidity (50-60%), with a 12 h light-dark cycle. The mice were allowed free access to full rodent food and water. The overview of the experimental protocol is shown in Fig 2. The mice were allowed to acclimatize for 1 week. The mice were then randomly divided into control, model, scutellarin groups with eight mice in each group. The model and scutellarin groups were both treated with a single intragastric administration of ZEA dissolved in corn oil at 40 mg/kg. After 2 h, Scu group was intragastrically given 100 mg/kg Scu in PBS (PH=7.4) for 3 days. The control group was intragastric administered with corn oil and then 2 h later with PBS (Fig.2). All mice were weighed and sacri ced post 72 h of ZEA administration, and the ovaries were collected for further study. These procedures in the protocol was performed by conforming the regulations and guidelines of ethical committee of Shanxi Agricultural University (Taigu, China).
In situ TUNEL uorescence staining assay Apoptotic cells were detected using deadend TM uorometric TUNEL system according to the manufacturer's protocol (Promega, Germany). Brie y, after treated with medium containing different concentrations of Scu (2000, 1000, and 500 μg/mL) with ZEA (60 μM) for 24 h, the cells were xed with 4% paraformaldehyde at 4℃ for 25 min. After two washes with PBS, the cells were incubated with 0.2% Triton X-100 for 5 min. After washing, cells were equilibrated with 100 µL equilibration buffers at room temperature for 8 min. The equilibration buffer was discarded and 50 µL rTdT incubation buffer was added to the cells on a 5cm 2 area on a tissue slide, and incubated at 37°C for 60 min. The cells were then incubated with 2×SSC for 15 min. After being washed, the slides were sealed with mounting medium with DAPI, and analyzed under a uorescence microscope.
Apoptosis in the ovarian tissues was also investigated by TUNEL staining. Brie y, 4% paraformaldehydexed, para n-embedded sections were depara nized, rehydrated, treated with 20 μg/mL proteinase K for 8 min at room temperature, and then re-xed with 4% paraformaldehyde for 5 min. After washing, the sections were treated with 100 µL equilibration buffers at room temperature for 8 min, and then incubated with rTdT incubation buffer at 37°C for 1 h in a humidi ed chamber away from light. After reaction, the sections were washed with 2×SSC for 15 min. Finally, the sections were sealed with mounting medium with DAPI, and analyzed under a uorescence microscope.

Western blotting analysis
Total proteins were extracted from cells or tissues using a total protein extraction kit (KeyGen, China), and protein concentrations were measured using BCA assay. Proteins were separated via SDS-PAGE and then transferred to a PVDF membrane. After being blocked with 5% non-fat dry milk for 2 h at room temperature, the membranes were subsequently incubated with primary antibodies to β-actin, Bax, Bcl-2, cleaved-caspase-3 or cleaved-PARP) 4°C overnight. After three washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After three nal washes with TBST, the protein bands were observed using an ECL kit (CWBIO, China), and the band intensities were analyzed using Image J software.

Immunohistochemistry
Immunohistochemical staining was conducted according to the manufacturers' instructions. Brie y, 4% paraformaldehyde-xed, para n-embedded sections were depara nized, rehydrated, and treated with H 2 O 2 for 10 min. After washing with PBS, the sections were blocked with 5% BSA for 10 min. Then 40 μL cleaved-caspase-3 antibody (1:50) was added to each section and incubated at 4°C overnight. After rinsing with PBS, the sections were incubated with a biotinylated secondary antibody for 10 min at room temperature. After being washed, the sections were added the streptavidin labeled with catalase. Subsequently, the DAB was used for color development. The sections were stained with hematoxylin for tissue morphology. After being dehydrated with gradient ethanol and cleared with xylene, the sections were sealed with neutral resin. The signals were observed under a uorescence microscope and photographed.

Statistical analysis
The data were presented as mean ±standard deviation (SD). All statistical analysis procedures were performed in GraphPad Prism TM 5 software (GraphPad Software Inc., La Jolla, CA, USA), and one-way analysis of variance (ANOVA) was used to determine signi cant differences among groups. *p<0.05; **p<0.01; ***p<0.001.

ZEA reduces GCs viability
As shown in Fig. 4A, starting from 10 µM of ZEA, the cell viability was decreased in a dose-dependent manner, and decreased signi cantly from 30 µM. At 60 µM, 50% of cell viability was remained. To further evaluate that the suppressive effect was time-dependent, the cells were treated with 60 µM ZEA for 6, 12, 24, 36 and 48 h. The results con rmed that the inhibitory effect was time-dependent with the longer time of treatment producing more severe inhibitory effects (Fig. 4B). Based on these two results, 60 µM concentration of ZEA with 24 h duration treatment was therefore selected as the positive control to establish a GCs damaged model for subsequent experiments.

Scu promotes GCs proliferation
Scu with concentrations ranged from 31.25 to 2000 μg/mL showed no cytotoxic effect on GCs. It promoted cell proliferation in a dose-dependent manner (Fig.3A). Therefore, high concentrations (2000, 1000 and 500 μg/mL) of Scu were chosen for the following studies. The mechanism of Scu on GCs proliferation was explored by treating the cells with 2000, 1000 and 500 μg/mL of Scu for 24 h, and the cell cycle distribution was detected using ow cytometry. The results showed that all these different doses of Scu reduced the number of cells in the S phase, and the difference between 2000 and 1000 μg/mL group was signi cant. 1000 μg/mL Scu signi cantly increased the number of cells in the G2/M phase as shown in Fig.3B, and C. These results revealed that Scu could affect the cell cycle via promoting the transformation of cells from S-phase to G2/M-phase.

Scu rescues ZEA-induced GCs injury
In order to investigate whether Scu protects GCs from ZEA-induced cytotoxicity, GCs were cultured with medium containing Scu and ZEA. The results demonstrated that ZEA markedly decreased the cell viability, which was signi cantly reversed by Scu treatment (Fig. 5A). The cell morphology of ZEA treated cells became rounded with destroyed of cells adhesion (Fig. 5B). These changes were restored by Scu treatment as shown in Fig. 5B. These results demonstrated that Scu could signi cantly rescue ZEAinduced GCs injury as indicated by the restoration of cell viability and morphology.
Scu prevents ZEA-induced S-phase arrest As shown in Fig.6, compared with control group, ZEA signi cantly decreased the number of cells in G0/G1 phase, and increased the number of cells in the S phase and no change in the number of cells in G2/M phase. These results revealed that ZEA induced S phase arrest. The co-treatment of Scu (2000,1000 and 500 μg/mL) and ZEA signi cantly increased the number of cells in G0/G1 phase while decreased the number of cells in S phase compared to the model. These data indicated that Scu prevented ZEA-induced S-phase arrest.

Scu rescues ZEA-induced GCs injuries via mitochondrial apoptotic pathway
In order to explore the protective effect of Scu on ZEA-induced GCs injuries, the cells were co-treated with different doses (2000,1000 and 500 μg/mL) of Scu and ZEA (60 μM) for 24 h. The results of TUNEL assay demonstrated that ZEA markedly increased the apoptotic rate (35%) comparing to control group (Fig.7A, B). In contrast, Scu signi cantly reversed the effect of ZEA. These data showed that Scu could inhibit the apoptosis of GCs induced by ZEA.
To investigate the anti-apoptosis mechanism of Scu in GCs under ZEA treatment, western blot analysis was used to detect the protein expressions of Bax, Bcl-2, cleaved-caspase-3 in apoptotic pathway and the apoptosis hallmark protein, the cleaved-PARP. As shown in Fig.7C, ZEA exposure remarkedly increased the level of cleaved-caspase-3, cleaved-PARP, and the ratio of Bax/Bcl-2. All these levels were decreased by Scu treatment. These results demonstrated that Scu had anti-apoptotic effect against the ZEA-induced GCs apoptosis through regulating mitochondrial apoptotic pathway.

Scu rescues ZEA-induced the apoptosis of mouse ovary in vivo
To investigate the effect of ZEA and the protective effect of Scu on ZEA-induced apoptosis of ovary in mice, apoptosis in ovarian tissues of each groups was detected by TUNEL staining. The results demonstrated that ZEA exposure has increased the ovarian tissues apoptosis. The apoptosis occurred predominantly in follicle granulosa layers (white arrows, Fig.8). These ndings con rmed that 40 mg/kg ZEA induced granulosa cells apoptosis in mice. The numbers of apoptotic cells in Scu treated group was signi cantly decreased. This result indicated that Scu is capable of preventing or rescuing ZEA-induced granulosa cells apoptosis.
Anti-apoptotic mechanism of Scu in vivo To investigate whether Scu exerts a protective role in vivo through the mitochondrial apoptotic pathway, the localization and expression of cleaved-caspase-3 was measured by immunohistochemistry and western blot analyses. The immunohistochemistry study in Fig.9A and B showed that, compared with control group, the expression of cleaved-caspase-3 was markedly increased in model. And the positive cells were mainly located in follicle granulosa layers and to a lesser extent in theca layer (black arrow) and ovary stroma (thick white arrow). The expression of cleaved-caspase-3 was signi cantly decreased in Scu group, especially in follicle granulosa layer, and positive tissue cells of cleaved-caspase-3 in theca layer and ovary stroma were also decreased.
The apoptosis related proteins were measured using western blot showed that ZEA treatment signi cantly increased the ratio of Bax/Bcl-2 and the expression of cleaved-PARP, while Scu treatment signi cantly reversed these changes (Fig.9B). These data suggested that Scu attenuated ZEA-induced apoptosis in granulosa layer via mitochondrial apoptotic pathway in vivo.

Discussion
Present study was aimed to assess if Scu can protect the ovarian GCs from ZEA-induced cytotoxicity. It was found that Scu can rescue ZEA-induced apoptosis in the GCs.
Using in vitro cell culture methods, we established a cellular model of ZEA-induced injuries on GCs isolated from mouse. It was found that 60 µM ZEA for 24 h led to 50% viability reduction of GCs in the cell culture model. This is a new model in addition to porcine GCs model where the 90 µM ZEA for 24 h caused 50% viability reduction 16 . Yang reported that the 25 µM β-zearalenol treatment for 24 h reduced 50% viability to build the bovine GCs model. We further found that ZEA inhibited the proliferation of GCs in a dose-and time-dependent manner that is consistent to the ndings by Chen 17 . At the same time, we established that Scu promoted GCs proliferation and affected cell cycle distribution. These ndings suggested that Scu may play an important role on female egg growth.
Ben Salem and Yang used a dose of ZEA that could inhibit 50% of cells viability as a treatment condition for subsequent experiments 15,26 . Hence, using the newly established ZEA toxicity mode, i.e.-60 µM ZEA treatment for 24 h in the present study, we found that different dose of Scu signi cantly alleviated the ZEA-induced cell damage.
In the ensuing experiment we assessed the protective effect of Scu against ZEA-induced injuries from cellular model to a mouse model. First, a ZEA-induced mouse ovarian damage model was established by intragastric administration of 40 mg/kg ZEA. Researches have reported that ZEA exposure caused abnormal and hypertrophy of female ovaries, as well as an obvious toxicity on the gametogenesis and embryonic development of female or male mice 5,27,28 . After treated with a single intragastric administration of 40 mg/kg ZEA, obvious damage was observed in the testis of male mice 19 . Our results showed that 40 mg/kg ZEA markedly induced the ovarian tissues apoptosis, indicated the model was built successfully to meet our objective. According to dose of the reference 29 , we chose 100 mg/kg Scu for continuous intragastric administration for 3 days, and the results showed that Scu attenuated ZEAinduced TUNEL-positive cells in ovary. All data demonstrated that Scu has protection against ZEAinduced ovary injury in vivo and in vitro. It could be used as a candidate drug for follow-up research. And more dose of Scu is suggested to be used in the further research animal study to provide full evidence for the clinical application of Scu.
We then explored the molecular mechanism as how Scu exerts the protection of GCs against the ZEA induced injures on follicles from the perspective of cell cycle distribution and apoptosis. We found that ZEA facilitated cell cycle progression from the G0/G1 phase to S phase. Li reported that ZEA has promoted the transition from G0/G1 phase to S cell cycle phase, and cells are arrested in S phase 30 . This is consistent with our results. However, there was no obvious change in cells number at G2/M phase. The data suggested that ZEA exerted its cytotoxic effect via redistribution of cell cycle phases with a speculation of DNA damage in G0/G1 phase.
Mitochondria are an important organelle in cells which are considered as the main cellular location of the apoptosis pathway. The pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 play a critical role in the regulation of apoptosis. For example, Bcl-2 family proteins are involved in the regulation of the apoptosis by controlling membrane permeability. Caspase-3 is a major executor of apoptosis 31 . Poly (ADP-ribose) polymerase (PARP) which is involved in DNA repair and genome stability is a major target of caspases [32][33] . We found that ZEA signi cantly increased the ratio of Bax/Bcl-2, the expression of cleaved-caspase-3 and cleaved-PARP. This nding is supported by previous studies that ZEA could induce GCs apoptosis via mitochondrial pathway with an increase in Bax and caspase-3 [34][35][36] .
Scu has signi cantly alleviated the injury induced by ZEA, and has affected the cell cycle phase distribution of normal GCs. Hence, we speculated that Scu could attenuate the cytotoxicity caused by ZEA via affecting cell cycle distribution. Our results of reliving ZEA-induced S phase arrest by Scu showed that it could play a protection role in this end, but the mechanism still needs to be further studied.
Researches have shown that ZEA induces apoptosis in different types of cells such as bovine GCs, primary Leydig cells 15,[37][38][39] . With these preceding results, we suspected that the protective mechanism of Scu against ZEA-induced GCs injury could be through apoptosis. Indeed, we found that Scu treatment alleviated apoptosis induced by ZEA both in vitro and in vivo models. These results showed that Scu signi cantly alleviated ZEA-induced cell apoptosis via decreasing the ratio of Bax/Bcl-2, and the expression of cleaved-caspase-3 and cleaved-PARP in vivo and in vitro. And it is worth noting that the positive signal of cleaved-caspase-3 in ZEA group was signi cant in follicle granulosa layers, theca layer and ovary stroma. While the protein expression levels of cleaved-caspase-3, especially in follicle granulosa layers, were decreased in Scu treated mouse. These ndings indicated that Scu could block ZEA-induced GCs apoptosis in mice via mediating mitochondrial related proteins in vivo and in vitro. The apoptosis in theca layer and ovary stroma induced by ZEA and rescued by Scu is a new found in the current study and still need to further explore as our mainly research was focused on the apoptosis of GCs.
Conclusion our data demonstrated that 1), in vitro and in vivo animal models of ZEA-induced GCs injuries were established successfully. 2), Scu attenuated ZEA-induced reproductive toxicity by targeting mouse ovarian GCs, mainly affecting cell cycle phase distribution and apoptosis via mitochondrial apoptotic pathway in vitro and in vivo. 3), the Scu can be further developed as potential new therapeutic drug for preventing or treating reproductive toxicity caused animals exposure to ZEA found in the grains of animal feed.

Declarations
Ethics approval and consent participate The experimental protocol was approved by conforming the regulations and guidelines of ethical committee of Shanxi Agricultural University.

Consent for publication
All authors critically revised the manuscript for important intellectual contents and approved the nal manuscript.

Availability of data and material
The datasets analyzed in the present study are available from the corresponding author on reasonable request.