Salidroside alleviates oxidative stress and apoptosis via AMPK/Nrf2 pathway in DHT-induced human granulosa cell line KGN — Source link

AMPK, Salidroside, Polycystic ovary, Oxidative stress and Signal transduction Induction of autophagy by salidroside through the AMPK-mTOR pathway protects vascular endothelial cells from oxidative stress-induced apoptosis. Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway. Endogenous and Exogenous Modulation of Nrf2 Mediated Oxidative Stress Response in Bovine Granulosa Cells: Potential Implication for Ovarian Function. Role of NRF2 in MiR-181c/AMP-Activated Protein Kinase Signaling in Cancer Botrysphin D arsenic-induced oxidative stress in human lung epithelial cells via activating Nrf2/ARE signaling pathways. Abstract Background: In the past few years, emerging evidence established persistent oxidative stress to be a key player in the pathogenesis of polycystic ovary syndrome (PCOS). Particularly, it damages the function of granulosa cells, and thus hinders the development of follicles. The present study aimed to explore and establish the protective effects of salidroside on dihydrotestosterone (DHT) cell line (KGN), mediated via antioxidant mechanisms. Methods: KGN cells were treated with DHT as a PCOS cell model, and then incubated with salidroside in different concentrations. Apoptosis and reactive oxygen species (ROS) accumulation were assessed by ow cytometry, mitochondrial membrane potential depolarization and the nuclear translocation of Nrf2 were detected by immunouorescence staining, and the level of apoptosis-related proteins and antioxidant proteins was assessed by western blotting. Results: Salidroside partly reversed DHT mediated effects, via stimulation of nuclear factor erythroid 2 ‐ related factor 2 (Nrf2) signaling pathway and the downstream antioxidant proteins heme oxygenase ‐ 1(HO ‐ 1) and quinine oxidoreductase 1(NQO1). Additionally, knockdown of Nrf2 resulted in a deterioration in DHT ‐ induced oxidative stress and apoptosis. It partly moderated the protective effects of salidroside as well. Mechanistically, AMPK was identied to be the upstream signaling involved in salidroside ‐ induced Nrf2 activation, as silencing of AMPK partly prevented the upregulation of Nrf2 and the downstream proteins HO ‐ 1 and NQO1. Conclusion: The present study is the rst to effectively demonstrate the inhibitory effect of salidroside on DHT ‐ stimulated oxidative stress and apoptosis in KGN cells, which was dependent on Nrf2 activation that involved AMPK.


Introduction
Granulosa cells constitute the cell layer present in the follicles that interact with oocytes and mediate the metabolism, transport of materials, and signal transduction in oocytes. Additionally, these cells play a crucial role in follicular growth, proliferation, differentiation, atresia/ovulation, and formation of the corpus luteum 1 . Several female reproductive endocrine diseases related to hormonal changes and follicular development, such as premature ovarian failure and polycystic ovary syndrome (PCOS), are associated with abnormal changes in granulosa cells, which are known to be involved in the disease developmental process 2,3 . PCOS is the most common female reproductive endocrine disease; however, the pathogenesis of the disease remains unclear. Excessive androgen has been proved to play a vital role in the pathogenesis of PCOS. In particular, higher androgen levels result in insulin resistance and obesity and ultimately affect the function of granulosa cells and follicle development via complex mechanisms 4 .
Granulosa-like tumor cell line (KGN) possesses steroidogenesis capability and is considered to be a reliable cell model to gain mechanistic insights into human granulosa cells 5 . In recent times, DHTinduced KGN cells have been widely used for in vitro studies involving PCOS 6, 7 .
In general, oxidative stress refers to a series of adaptive reactions caused by an imbalance between the oxidants and antioxidants. During this process, excessive reactive oxygen species (ROS) cannot be eliminated in time, which get accumulated and interfere with the normal redox state of the cells. Several previous studies have reported the existence of oxidative stress in PCOS. Interestingly, the levels of oxidative stress markers, such as 8-OHDG and MDA, have been reported to be elevated in the serum and follicular uid of PCOS patients 8,9 . An increase in oxidative stress has been proved to be related to the progression of PCOS and the associated complications, such as T2DM and cardiovascular diseases 10 . Besides this, oxidative stress also leads to the apoptosis of granulosa cells, thereby obstructing the development and maturation of oocytes 11 . The antioxidant systems respond to the oxidative damage, incurred by the inevitable generation of ROS, via a series of signaling pathways. Nuclear factor (erythroidderived 2)-like 2 (NRF2) has been identi ed as one of the main transcription factors associated with the oxidative stress response 12 . It belongs to the Cap'n'Collar (CNC) subfamily of basic leucine zipper (bZIP) transcription factors. In particular, Nrf2 consists of seven conserved NRF2-ECH homology (Neh) domains with different functions, which modulate the stability of Nrf2 and contribute to its central role in the redox system. Nrf2 activity is precisely regulated by Kelch-like-ECH-associated protein 1 (Keap1). In the steadystate, Nrf2 is anchored in the cytoplasm at a low basal protein level by Keap1, which binds NRF2 via its Cterminal domain, resulting in Keap1-Cullin (CUL) 3-RING-box protein-mediated ubiquitination of NRF2 and degradation by the 26S proteasome 13 . Under oxidative stress, Keap1 is inactivated, and the newly synthesized Nrf2 escapes from Keap1 and translocate quickly into the nuclear region, where it induces the transcription of an array of antioxidant genes, such as heme oxygenase 1 (HO-1), quinine oxidoreductase 1 (NQO1), and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) 14 .
However, the underlying mechanism involved in the activation of Nrf2 and disassociation from the Keap1-Nrf2 complex remains elusive. Several previous studies have testi ed that Nrf2 and its downstream protein like HO-1 could be enhanced by the activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt-dependent phosphorylation 15,16 . AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that plays a pivotal role in the maintenance of redox homeostasis 17 . A recent study reported that AMPK promoted the detachment of Nrf2-Keap1 complex and nuclear transcription in sodium uoride (NaF)-stimulated microglia 18 .
Salidroside is a phenylpropanoid glycoside compound, extracted from the dried roots or whole plant of Rhodiola rosea, which possesses antioxidant properties (Fig. 1A). In fact, several studies have previously established that salidroside alleviated oxidative stress injury in the cardiovascular system, endocrine disease, neurodegenerative disease, [19][20][21] etc. Moreover, a recent study reported that salidroside alleviated HUVECs cell injury, induced by oxidative stress, via activation of the Nrf2 signaling pathway 22 . However, very limited information is available regarding the role of salidroside on oxidative stress in PCOS.
The present study aimed to explore whether salidroside can incur a protective effect on suppressing the oxidative stress and apoptosis in DHT-induced KGN, and thus unravel the underlying mechanism responsible for its functions in KGN cells

Cell culture and treatment
The KGN cells were provided by the RIKEN BioResource Research Centre (Tsukuba, #RCB1154, Japan).
The KGN cells were then treated with DHT (500 nM) as a PCOS cell model, which been used in reveral studies 3,7 . To detect the effects of salidroside, the KGN cells were incubated with salidroside at different concentrations for 24 h.

Cell viability assay
Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology, #C0037, China). The KGN cells were seeded in 96-well at-bottomed plates at 3 × 10 3 per well , treated with DHT, low or high doses of salidroside and incubated for 24 h or 48 h, followed by incubation with cck8 (10 µL/well) for 1.5 h, and the absorbance was read at 450 nm on a microplate reader (Perkin Elmer, USA).

Detection of cell apoptosis
Apoptosis of the KGN cells was detected with the Annexin V-PE Apoptosis Detection Kit (Beyotime Biotechnology, #C1065L). After treatment, the KGN cells were collected with 2.5% trypsin (Gibco Life Technologies, #15090046, USA) and washed twice with PBS. Next, 0.5 mL of the binding buffer was added to resuspend the KGN cells. Then, the resuspended cells were incubated with 5 μL of annexin V-FITC and 10 μL of propidium iodide (PI) in the dark at 37℃ for 15 min. The percentage of apoptotic KGN cells was then calculated by ow cytometry.

Cell immuno uorescence staining
The KGN cells were seeded into a 6-well plate and xed with 4% paraformaldehyde for 5 min, after which 0.2% Triton X-100 in PBS was used for permeation and 10% normal goat serum was used to block the cells. Then, the cells were incubated with an anti-Nrf2 antibody (1:200; Abcam, #ab137550, UK) overnight at 4℃. The cells were washed with PBS and incubated with a secondary antibody Cy3 conjugated goat anti-rabbit IgG (H+L) for 1 h. 4',6-Diamidino-2-phenylindole (DAPI) was used to label the nuclei. Images were visualized by using a uorescence microscope (Olympus BX6 with DP72 Camera, Japan).

Mitochondrial Membrane Potential Detection
The Mitochondrial Membrane Potential (MMP) Assay Kit with JC-1 (Beyotime Biotechnology, #C2006, China) was used to analyze the mitochondrial depolarization according to the manufacturer's protocol. The cells were incubated with JC-1 solution for 20 min and then washed with the JC-1 staining buffer. Fluorescence microscopy (Olympus IX73P2F, Japan) was used to analyze the MMP and the corresponding images were assessed by the Image-J software. The red/green uorescence intensity ratio was used to calculate the changes in the MMP.

Statistical Analysis
All data were analyzed with the GraphPad Prism 8 software. All quantitative data were presented as mean ± standard deviation (SD). Multiple comparisons were used by one-way ANOVA, followed by Tukey's posthoc test. P 0.05 was considered to be statistically signi cant.

Salidroside improves the survival of DHT-induced KGN cells
To assess the effects of salidroside on KGN cells and determine the appropriate concentration required for its activity, the cytotoxicity of salidroside was evaluated using a cck8 assay. Brie y, KGN cells were  (Figure 2A and B). Following this, JC-1 uorescent staining was used to detect the changes in the mitochondrial membrane potential (MMP). As shown in Figures 2C and D, the JC-1 uorescent probe mainly accumulated in the mitochondrial matrix of the normal cells and emitted red uorescence. In the case of DHT-treated KGN cells, the JC-1 probe failed to accumulate in the mitochondrial matrix and emitted green uorescence along with low red uorescence, which indicated a decrease in MPP. In comparison to these, mitochondrial depolarization was found to be signi cantly improved in the case of salidroside-treated KGN cells. The results for apoptosis analysis further indicated that DHT aggravated apoptosis in KGN, however, 30 μM and 100 μM salidroside signi cantly reversed the adverse effects of DHT ( Figure 2E and F).
In general, oxidative stress and other cellular stressors initiate mitochondrial apoptosis via mitochondrial outer membrane permeabilization (MOMP), which involves the release of cytochrome C into the cytoplasm 23 . In the cytoplasm, cytochrome C activates apoptosis mediator caspase-9, which in turn cleaves and activates the executor caspase, caspase-3 24 . This eventually disintegrates cells, followed by cell death. Furthermore, this process is tightly regulated by anti-apoptotic protein Bcl-2 and pro-apoptotic effector protein Bax 25 . Thus, to further determine the effects of salidroside on KGN cells, the levels of these apoptosis-related proteins were assessed. Interestingly, salidroside treatment resulted in a signi cant decrease in the levels of Bax, cleaved caspase-9, and cleaved caspase-3 in KGN cells as compared to DHT-induced cells. In comparison to these, Bcl-2 levels were found to be reversed upon salidroside treatment ( Figure 2G and H). SOD2 is a mitochondrial antioxidant enzyme that removes intracellular ROS and plays a vital role in the maintenance of redox balance 26 . As shown in Figure 2G and H, DHT treatment resulted in a slight increase in SOD2 protein levels, whereas the protein expression levels of SOD2 were signi cantly enhanced by salidroside ( Figure 2G and H).

Salidroside activates Nrf2 pathway in KGN cells
Keap1-Nrf2-antioxidant response element (ARE) signaling pathway plays a pivotal role in the biological response to oxidative stress, via regulation of the transcription of a variety of antioxidant genes 27 . Thus, the effects of salidroside on the Keap1/Nrf2 pathway and its downstream antioxidant proteins in DHTinduced KGN cells were evaluated. The results for western blot analysis indicated that DHT treatment slightly increased the expression of Nrf2, whereas salidroside treatment resulted in signi cant stimulation of the total Nrf2 protein expression and promoted its nuclear translocation. In concordance with these results, the expression of Keap1 was also restrained after salidroside treatment ( Figure 3A-C). Moreover, the immuno uorescence staining results further con rmed the nuclear translocation of Nrf2 ( Figure 3D). To evaluate the levels of Nrf2-targeted antioxidant proteins, the protein expression of NQO1 and HO-1 was assessed. As shown in Figures 3E and F, HO-1 and NQO1 were slightly upregulated upon DHT exposure, whereas salidrodide treatment resulted in signi cant upregulation of these proteins ( Figure 3E and F). The aforementioned results indicated that salidroside initiated Nrf2 signaling in KGN cells.

Protective effects of salidroside treatment were blocked by Nrf2 knockdown
To further prove the role of Nrf2 pathway activation in the protective role of salidroside in KGN cells, the cells were transfected with Nrf2 siRNA. The knockdown e ciency of Nrf2 siRNA-3 was found to be ~75% and thus was selected to silence the target gene in the following studies ( Figure 4A and B). As shown in Figures 4C and D, the Nrf2 siRNA decreased the total protein levels of Nrf2 and partially prevented the nuclear translocation of Nrf2. Besides this, the silencing of Nrf2 nulli ed the upregulation of antioxidant protein NQO1 and HO-1 ( Figure 4C and D). In addition to this, the levels of the apoptosis-related proteins were also assessed. The results for western blotting analysis showed that the attenuation of Bax, cleaved caspase-3, and cleaved caspase-9 and upregulation of Bcl-2 and SOD2 were prevented by Nrf2 siRNA supplementation ( Figure 4E and F). In concordance with these results, ROS production evaluation by ow cytometry and JC-1 probe staining showed that the protective effects of salidroside on ROS elimination and mitochondrial depolarization in KGN cells were partly reversed by Nrf2 silencing (Figure 4G-J). Treatment with salidroside (60 μM) resulted in a decrease in apoptosis in DHT-exposed KGN cells, while knockdown of Nrf2 nulli ed this protection ( Figure 4K and L).

Effects of salidroside on MAPK and AMPK pathways in DHT-treated KGN cells
Recent studies have reported that AMPK, phosphatidylinositol 3-kinase (PI3K), c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated protein kinase (ERK) promote the separation of the Keap-Nrf2 complex and assist in its nuclear translocation 28-31 . To explore the regulatory signaling pathway involved in Nrf2 nuclear translocation, the expression levels of proteins in the MAPK and AMPK pathways were assessed in KGN cells. As shown in Figures 5A and B, salidroside treatment conferred no effect on p-ERK and p-Akt, and a slight increase observed for p-JNK and p-p38. However, these changes were not statistically signi cant. Importantly, salidroside resulted in a signi cant increase in the phosphorylation of AMPK in DHT-treated KGN cells ( Figure 5A and C). Next, the effect of salidroside on AMPK was examined under different concentration conditions (0, 30, 60, 80, and 100μM ). Interestingly, salidroside mediated AMPK activation in a dose-dependent manner ( Figure 5D and E).

Involvement of AMPK in salidroside-induced Nrf2 nuclear translocation
To further investigate the role of AMPK in salidroside-induced Nrf2 activation, siRNA molecules speci c to AMPK were utilized for a knockdown. The knockdown e ciency of AMPK siRNA-2 was found to be ~65% ( Figure 6A and B), and it was selected to silence the target gene in the following studies ( Figure   6C-G). The results for western blotting analysis demonstrated that AMPK siRNA prevented the phosphorylation of AMPK and the nuclear translocation of Nrf2 ( Figure 6C-E). Besides this, the expression of the downstream antioxidant proteins HO-1 and NQO-1 was found to be inhibited ( Figure 6F and G). Next, an AMPK speci c inhibitor, Compound C, was used to revalidate the expression of AMPK at the concentration of 10μM as several studies 32 . The obtained results were consistent with the effects of AMPK knockdown ( Figure 6H-L). Altogether, the aforementioned data suggested that AMPK is the upstream signaling molecule in the nuclear translocation of Nrf2.

Discussion
Over the past two decades, comprehensive studies have demonstrated that oxidative stress contributes to the initiation and development of pathological processes that impair female reproduction 33 . PCOS is the most common reproductive endocrine disorder in women under reproductive age; however, the pathological mechanism for the same remains unclear. Interestingly, the intrinsic association of PCOS and redox imbalance has been increasingly detected and established in several studies 34 . KGN cell line is considered to be consistent with the immature granulosa cells obtained from smaller antral follicles, in terms of physiological characteristics 5 . In fact, DHT-induced KGN cells have been widely used as a PCOS model for in vitro studies 7 . Salidroside, the main bioactive compound extracted from Rhodiola, has been shown to possess a variety of pharmacological activities, including anti-in ammatory, antidepressive, anti-oxidant, and anti-cancerous activities 19,[35][36][37] . Recent accumulation of evidence proved that salidroside could alleviate oxidative stress levels. The present study is the rst to demonstrate that salidroside could ameliorate apoptosis and oxidative stress in KGN cells, PCOS cell model, via activation of the Nrf2 signaling pathway.
In general, ovarian granulosa cells grow around oocytes and deliver metabolites required for the growth and development of oocytes. Additionally, these cells also participate in the production of estrogen and play a key role in the growth and development of follicles 38 . The accumulation of ROS could possibly cause oxidative stress that might lead to apoptosis in oocytesdeteriorating the quality of oocytes, which further affects the fertility outcomes 39 . Recent studies have reported that increased apoptosis and ROS levels in thin granulosa cells participate in the pathogenesis of PCOS, and result in abnormal follicle development and clinical pregnancy 40 . Hence, the present study utilized DHT-induced KGN cells to assess the protective effects of salidroside on the PCOS cell model. First, the survival of KGN cells was tested in the presence of salidroside. Both low and high concentrations of salidroside showed no side effects on the viability of KGN cells and improved the survival inhibition post DHT treatment. In concordance with these results, further functional analyses also showed that salidroside alleviated ROS accumulation, mitochondrial depolarization, and increased apoptosis in KGN cells. Cell apoptosis mediated by mitochondria is initiated by intracellular apoptotic stimulators, like DNA damages and anoxic damage, and regulated mainly by Bcl-2 family proteins, comprising of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. Bcl-2 family proteins control cellular apoptosis by regulating MOMP and promoting the release of proteins from intermembrane space into the cytoplasm, which further activates the caspase cascade, resulting in cell death 41 . In the present study, the changes of expression levels of apoptosis-related proteins, namely Bax, Bcl-2, cleaved caspase-3, and cleaved caspase-9 were suggestive of activation of mitochondrial apoptosis in DHT-induced KGN cells and the protective effects of salidroside.
The transcription factor Nrf2 is well established key regulator involved in cellular oxidative stress 42 . Recent studies have reported the protective effects of Nrf2 in various biological processes, including in ammation, autophagy, and unfolded protein response [43][44][45] . In steady conditions, Nrf2 is mainly located in the cytoplasm at a low basal protein level, mainly due to Keap1 mediated proteasomal degradation. During oxidative stress, electrophiles and ROS inactivate Keap1, resulting in the release of Nrf2 from the Nrf2-Keap1 complex, followed by its nuclear translocation. Post its nuclear accumulation, Nrf2 binds to the antioxidant response element (ARE) and stimulates downstream anti-oxidant proteins, like HO-1 and NQO1 42 . Several studies have previously revealed that salidroside treatment activated Nrf2 in different disease models 22,46 . In the present study, DHT treatment resulted in a slight increase in Nrf2 protein levels. In comparison to this, salidroside decreased the levels of Keap1 protein, and thus induced a signi cant increase in Nrf2 levels, further promoting its nuclear translocation. Besides this, the antioxidant proteins HO-1 and NQO1 were also found to be upregulated after salidroside treatment. Additionally, silencing of Nrf2 resulted in the deterioration of oxidative stress and apoptosis and a partial reversal of the protective effects conferred by salidroside in KGN cells. Altogether, these results demonstrated that salidroside alleviates apoptosis and oxidative stress via upregulation of Nrf2 mediated antioxidant proteins.
It remains unclear which signaling pathway participated in the stimulation of Nrf2. MAPK and AMPK signal pathways have been previously reported to be involved in the upstream signal of Nrf2 47,48 . AMPK is known as an evolutionarily conserved energy sensor in cellular energy homeostasis. Under oxidative stress, AMPK is actived by the phosphorylation at Thr172 and exerts an antioxidant effect through promoting the transcription of downstream genes such as FoxO factors, DAF-16, Nrf2 and SIRT1 49 . In the present study, phosphorylation of AMPK was found to be upregulated in the case of salidroside treatment. Additionally, AMPK siRNA supplementation partly restrained the stimulation and nuclear translocation of Nrf2. Similarly, the use of an inhibitor of AMPK, Compound C, showed consistent inhibitory effects on Nrf2 activation. Thus, these results indicated that AMPK activated Nrf2 as an upstream signal in salidroside-treated KGN cells. However, Nrf2 activation could be mediated by different pathways. Several studies have previously shown that there are alternative mechanisms for Nrf2 stimulation, independent of Keap1 50 . Besides this, the regulatory factors at the post-transcriptional level like RNA-binding proteins (RBPs) can bind to the 30-UTR of NFE2L2mRNA to increase the Nrf2 activation 42,51 . Thus, other modes of Nrf2 stimulation might be involved in the protective effects conferred by salidroside treatment in KGN cells, which need to be explored in the future.
In summary, the present study for the rst time demonstrated that salidroside could restrain apoptosis and oxidative stress in KGN cells under DHT exposure, via AMPK mediated Nrf2 activation. Besides this, these ndings are indicative of the therapeutic potential of salidroside for PCOS treatment that should be explored in appropriate in vivo models.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Con ict of interest
The authors declare no con ict of interest.    Effects of salidroside on MAPK and AMPK pathway in DHT-treated KGN cells (A-C) Representative western blotting bands and the relative expression levels of ERK, p-ERK, p38, p-p38, JNK, p-JNK, Akt, p-Akt, AMPK and p-AMPK, quantitative data of the relative expression levels were normalized to GAPDH.