HMGB-1 Increases Proinflammatory Reaction via TLR4 in Human Granulosa Cells of Endometriosis

All experiments were performed using hGL5 cell line. The research protocol was approved by the Institutional Review Board of the Gachon University of Medicine and Science (GAIRB 2017-250). The immortalized human granulosa cell line hGL5 was kindly provided by Professor Casarini (University of Modena and Reggio Emilia, Moderna, Italy). It was cultured in medium supplemented with 10% fetal bovine serum (FBS), 2% ultrasonic G, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin and maintained in an incubator at 37 °C and 5% CO 2 . hGL5 cells were seeded at density of 1 × 10 5 cells/well in six-well tissue culture plate. After 24 h, the culture medium was replaced with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) containing 2% FBS. The cells were treated with 100 or 200 μM of H 2 O 2 for 2 h to induce oxidative stress. Then 100 μL of CCK-8 reagent from Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was added to each well, and the cells were incubated at 37 °C for 24 h to evaluate cell proliferation. The supernatants were transferred to 96-well plate, and optical density at 450 nm was determined using VersaMax reader (Molecular Devices, Sunnyvale, CA, USA). The observance at 450 nm optical density was obtained. After treatment with 100 or 200 μM of H 2 O 2 for 2 h, 1 × 10 5 cells/well of hGL5 cells in 6-well plates were incubated in serum-free medium for 24 h. The cells were harvested and washed once with cold phosphate-buffered saline (PBS) and once with binding buffer (10 mM Hydroxyethyl piperazine Ethane Sulfonicacid, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ) from Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BD Pharmingen, BD Biosciences, San Jose, CA, USA). They were resuspended (1 × 10 6 cells/mL) in the binding buffer. Then, 100 µL of the cell suspension (1 × 10 5 cells) was transferred to 5 mL culture tube and incubated with five μL FITC-conjugated annexin V (AV) and 10 μL propidium iodide (PI) for 15 min at 25 °C in the dark. Next, the apoptosis was measured within 1 h using FACScan analyzer (Becton Dickinson and Company, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The apoptotic cells were defined as those stained positive for AV. The percentage of apoptotic cells, or the sum of cells in the early and late stages of apoptosis out of the entire cells, was calculated. For Western blots for apoptosis-related proteins and HMGB-1, hGL5 cells were treated with different concentrations of H 2 O 2 for 2 h and incubated for 24 h in serum-free DMEM. For Western blot for NF-κB pathway-related proteins and TLR4, hGL5 cells were treated with 200 μM of H 2 O 2 for 2 h and incubated for 0 min, 15 min, 30 min, and 60 min in serum-free DMEM. For both experiments, hGL5 cells were harvested and lysed using radioimmunoprecipitation analysis buffer (RIPA buffer; iNtRON Biotechnology, Seongnam, Republic of Korea) with protease inhibitor cocktail (Cell Signaling Technology, Beverly, MA, USA). After mixing, the cell lysates were distributed in 40 μL aliquots. The lysates were then centrifuged for 30 min at 13,000 rpm at 4 °C. Proteins were collected from the supernatant, and the protein concentration was determined using bicinchoninic acid protein analysis kit (Thermo Scientific, Hudson, NH, USA). Western blot was performed as follows: 30 μg of each cell lysate were added to 5× buffer and boiled. After centrifugation, the supernatant was filtered, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The samples were loaded onto 8% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed as follows. The membranes were subjected to bovine serum albumin treatment for 1 h at room temperature, and the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies were cleaved caspase-3 (monoclonal anti-rabbit antibody; ab32042, Abcam, Cambridge, UK), Bcl-2 (monoclonal anti-rabbit antibody; ab32124, Abcam, Cambridge, UK), Bax (monoclonal anti-rabbit antibody; ab205822, Abcam, Cambridge, UK), beta-actin (monoclonal anti-mouse antibody; 66009, Proteintech, Rosemont, IL, USA), HMGB-1 (polyclonal anti-rabbit antibody; 6893, Cell Signaling Technology, Danvers, MA, USA), TLR4 (polyclonal anti-rabbit antibody; GTX21436, GeneTex, Irvine, CA, USA), NF-κB (p65) (polyclonal anti-rabbit antibody; ab16502, Abcam, Cambridge, UK), IκBα (monoclonal anti-mouse antibody; 4814, Cell Signaling Technology, Danvers, MA, USA), and pIκBα (mono-clonal anti-rabbit antibody; 2859, Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies used were anti-mouse antibody (IgG antibody; 7076, Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit antibody (IgG antibody; 7074, Cell Signaling Technology, Danvers, MA, USA). After using enhanced chemiluminescence solution (Advansta, San Francisco, CA, USA), bands were quantified using ImageJ software (version 1.53 q; National Institute of Health, Betheda, MD, USA). After treatment with 100 or 200 μM of H 2 O 2 for 2 h, 1 × 10 5 cells/well of hGL5 cells in 6-well plates were incubated in serum-free DMEM for 24 h. The cell supernatant was harvested, and the collected supernatant was analyzed for estradiol levels using an Estradiol Parameter Assay Kit (Bio-Techne, Minneapolis, MN, USA). According to the manufacturer’s instructions, total RNA was isolated from hGL5 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration and purity were measured with spectrophotometer at A260 and A260/280, respectively. Moreover, according to the manufacturer’s instructions, RNA was reverse-transcribed into cDNA using Primescript™ RT reagent kit (Takara Bio, Kusatsu, Japan). The sequences of primers used were as follows: for GAPDH: forward, 5′-TCGACAGTCAGCCGCATCTTCTTT-3′ and reverse 5′-ACCAAATCCGTTGACTCCGACCTT-3′; for HMGB-1: forward, 5′-CAGGGCCAAACCGATAGGAAA-3′ and reverse, 5′-TCGTGCACCGAAAGTTTCAA-3′; for TLR4: forward, 5′-CAGAGTTTCCTGCAATGGATCA-3′ and reverse, 5′-GCTTATCTGAAGGTGTTGCACAT-3′. GAPDH was used as an internal control for evaluating the relative expressions of HMGB-1 and TLR4. qRT-PCR was performed on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the following thermocycling conditions: Initial denaturation at 95 °C for 30 s, followed by 32 cycles at 55 °C for 30 s and 72 °C for 30 s. A mixture of 10 µL SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used. After treatment with 500 to 2000μM of H 2 O 2 for 2 h, 3 × 10 5 cells/well of hGL5 cells in 12-well plates were incubated in serum-free DMEM for 24 h. The collected supernatant was analyzed for IL-1β and IL-6 levels using Human Interleukin-1β (IL-1β) ELISA kit (ab214025, ABCAM, Cambridge, UK) and Human Interleukin-6 (IL-6) ELISA kit (ab178013, ABCAM, Cambridge, UK). Student t -tests with Bonferroni adjustment were performed to determine the differences in the cell count, mRNA, protein expression levels, estradiol secretion levels, and inflammatory molecule release levels. Linear regression analyses were used to investigate the linear association between cell proliferation, mRNA expression, estradiol secretion, inflammatory molecule release, and the concentration or duration of H 2 O 2 treatment, allowing assessment of dose- and time-dependent effects. SPSS Statistics (version 28; IBM, New York, NY, USA) and R software (version 4.1.2; www.r-project.org (accessed on 10 April 2023); R Foundation for Statistical Computing, Vienna, Austria) were used for all statistical analyses. Statistical significance was set at p -value < 0.05.

Endometriosis (EMS) is an inflammatory disease characterized by endometrial tissue outside the endometrium and myometrium. As with other gynecologic diseases, diagnosis of EMS is made by integrating symptoms with various imaging studies including ultrasound, CT, and MRI, along with blood test results [ 1 ]. Up to 50% of the women with pelvic pain or infertility are affected by EMS [ 2 , 3 ]. Both primary and secondary infertility are highly prevalent in EMS patients [ 4 ]. Elevated levels of inflammatory cytokines in the pelvic cavity of EMS patients suggest that inflammation and immune dysregulation are related to the establishment of EMS [ 5 ]. Oxidative stress results from an imbalance between reactive oxygen species and antioxidants. It has been widely accepted that oxidative stress contributes to the pathophysiology and progression of EMS by promoting local inflammation within the pelvic cavity [ 6 ]. Activated macrophages and endometrial tissue transplanted into the pelvic cavity induce oxidative stress [ 7 ]. The increased oxidative stress also alters the follicular microenvironment and is suspected to negatively affect the entire process of oocyte development [ 8 ]. Granulosa cells, which form multiple layers surrounding oocytes in follicles, influence the oocyte through direct gap junctions and play crucial roles in steroid hormone synthesis. As a result, they determine the oocyte competence and the infertility [ 9 ]. To understand the function and underlying mechanism of granulosa cells, selecting the appropriate ovarian granulosa cell line is important. The in vitro system lacks confounding factors such as paracrine factors and, therefore, is optimal to observe receptor functions at the molecular level. The immortalized human granulosa cell line (hGL5), a well-established human granulosa cell line, demonstrates a consistent steroidogenic pathway and exhibits high proliferation rates, making it an optimal model for investigating cell survival, death, and steroidogenic activity [ 10 ]. The study with the hGL5 cell line can contribute to investigating infertility [ 11 ]. To explore infertility in EMS patients, it is crucial to understand the impact of oxidative stress on granulosa cells due to EMS. Kolesarova et al. used hGL5 cells as an in vitro model to study oxidative stress in ovarian granulosa cells [ 12 ]. Isoquercitrin, a dietary flavonoid, was shown to reduce H 2 O 2 -induced reactive oxygen species in hGL5 cells, demonstrating the model’s relevance for oxidative damage research. Additionally, Yun et al. reported that H 2 O 2 treatment increases HMGB-1 and TLR4 expression in endometrial cells [ 13 ]. High-mobility group box-1 (HMGB-1) is a member of the chromosomal protein superfamily and acts as an inflammatory cytokine that can bind to DNA [ 14 ]. When passively released into the extracellular space, HMGB-1 functions as a damage-associated molecular pattern (DAMP) [ 15 ]. It has been reported that HMGB-1 expression is increased by oxidative stress-induced cell death in human endometrial stromal cells (HESCs) from patients with EMS [ 13 ]. HMGB-1 interacts with toll-like receptor 4 (TLR4) and activates inflammatory pathways such as the NF-κB pathway, thereby inducing alterations in eutopic endometrium and contributing to the development of EMS [ 16 ]. In the present study, we aimed to determine whether oxidative stress decreases the granulosa cell proliferation by inducing apoptosis and increases the release of HMGB-1, which in turn promotes inflammation via TLR4 and NF-κB pathways, ultimately lowering the quality of follicle in patients with EMS.

To determine the effect of oxidative stress on the cell viability of granulosa cells, the change in viable cell count was examined by varying concentrations of H 2 O 2 treatment to hGL5 cells using the CCK-8 assay. With H 2 O 2 treatment of 100 μM and 200 μM, the cell count decreased compared to that of the control treated with PBS. Linear regression analysis showed significant an inverse correlation between the concentration of H 2 O 2 and cell proliferation ( Figure 1 ). To gain insight into the effect of oxidative stress on the decreased proliferation of hGL5 cells, an apoptosis assay was performed after H 2 O 2 treatment of 100 μM and 200 μM for 2 h. The proportion of live, early apoptotic, late apoptotic, and necrotic cells is expressed in Figure 2 A. Early and late apoptotic cells increased as the concentration of H 2 O 2 treatment increased. Because hGL5 cells were incubated with serum-free media for 24 h, the control also showed few apoptotic cells as a baseline. In flow cytometry analysis, the cell population shifted from live to early and late apoptotic after H 2 O 2 treatment with 100 μM and 200 μM compared to the control ( Figure 2 B). A Western blot was performed to detect the expression of apoptosis-related proteins at the same time as cell death increased ( Figure 3 ). Differing concentrations of H 2 O 2 -treated hGL5 cells were treated for 2 h and harvested after 24 h of incubation. After H 2 O 2 treatment of various concentrations, the expression of caspase-3 decreased, but the expression of cleaved caspase-3 increased with low intensity after H 2 O 2 treatment. The expression of pro-apoptotic protein Bax increased, whereas that of the anti-apoptotic protein Bcl-2 decreased. Because one of the main functions of granulosa cells is steroidogenesis, increased cell death may decrease estradiol production and secretion. After inducing oxidative stress with H 2 O 2 , ELISA for estradiol was performed with the supernatant. The estradiol level after 200 µM of H 2 O 2 treatment was significantly lower than that of the PBS-treated control ( Figure 4 ). Although 100 µM of H 2 O 2 treatment did not show a significant decrease, linear regression analysis showed a decreasing tendency of estradiol secretion in the supernatant after H 2 O 2 in a dose-dependent manner. To examine HMGB-1 changes in hGL5 cells after adding oxidative stress, qRT-PCR and Western blotting were performed. First, 200 µM H 2 O 2 was treated for 1 and 2 h. The mRNA level of HMGB-1 in cell lyses increased after H 2 O 2 treatment in a time-dependent manner ( Figure 5 A). Western blot for HMGB-1 was performed in the supernatant of hGL5 cells harvested after 2 h of H 2 O 2 treatment and 24 h of incubation. Western blot showed increased extracellular HMGB-1 release in the supernatant according to the increased cell death induced by the increased dose of H 2 O 2 treatment ( Figure 5 B). TLR4, a receptor of HMGB-1, was examined in hGL5 cells after oxidative stress. The mRNA level of TLR4 was analyzed with qRT-PCR after treatment with different concentrations of H 2 O 2 . Compared to the PBS-treated control, the mRNA level was significantly increased after H 2 O 2 treatment in a dose-dependent manner ( Figure 6 ). To examine the relationship between oxidative stress and NF-κB pathway activation in hGL5 cells, we treated H 2 O 2 at a fixed dose of 200 µM, increasing the treatment time. We assessed the protein synthesis of TLR4 in hGL5 cell lyses immediately after media change as a baseline to 60 min after to catch the beginning of the signaling. In hGL5 cell lyses, phosphorylated IκBα (pIκBα) showed a peak increase 15 min after H 2 O 2 treatment. Phosphorylation of IκBα liberates NF-κB proteins to translocate to the nucleus, activating the NF-κB pathway. Because the experiment was performed with whole lysates, only the gradual decrease in NF-κB (p65) as time passed due to degradation after its activation was observed ( Figure 7 ). We measured proinflammatory cytokines in the supernatant of H 2 O 2 -treated hGL5 cells to determine the final outcome of the chain reaction resulting from oxidative stress-induced NF-κB pathway activation. It was difficult to detect the cytokine secretion under the same conditions of our experiment as before. Therefore, we increased the hGL5 cell concentration and the dose of H 2 O 2 . IL-1β was significantly increased after treatment with 2000 µM H 2 O 2 for 2 h. Even though the significance was shown at 2000 µM only, there was a significant increasing tendency of IL-1β in a dose-dependent manner analyzed by linear correlation analysis ( Figure 8 ). IL-6’s average concentration after H 2 O 2 treatment was not significantly increased compared to that of the PBS-treated control. However, it showed a dose-dependent trend as the dose of H 2 O 2 treatment increased to 2000 µM ( Figure 9 ). Secretion levels of IL-1β and IL-6 in the hGL5 cell line increased in a dose-dependent manner with increasing H 2 O 2 concentrations, and these results are summarized in Table 1 . To summarize, due to EMS, increased oxidative stress affects the viability and increases the apoptosis of granulosa cells. HMGB-1 release increases, contributing to a positive feedback loop amplifying oxidative stress. The oxidative stress also leads to increased expression of TLR4 sequentially and decreased steroidogenesis of granulosa cells. The interaction of HMGB-1 and its receptor, TLR4, activates the NF-κB pathway, and the release of inflammatory molecules IL-1β and IL-6.

In the context of in vitro fertilization (IVF), EMS is associated with reduced oocyte yield, lower implantation rates, and decreased pregnancy success. By affecting oocyte yield and quality, EMS exerts a detrimental impact on the follicular environment, thereby impairing fertility. HMGB-1, a well-known danger signal, has been implicated in inflammation, cell proliferation, immunity, metabolism, cancer, and oxidative stress [ 17 , 18 , 19 , 20 ]. Studies on the pathophysiology of EMS have indicated that HMGB-1 participates in activating the NF-κB pathway and regulating inflammatory responses and autophagy in HESCs [ 16 , 21 ]. Similarly, Gonzalez-Ramos et al. demonstrated the activation of the NF-κB pathway by proinflammatory cytokines contributes to the progression of EMS [ 22 ]. Despite these insights, the role of HMGB-1 in infertility is unknown. To the best of our knowledge, the present study is the first to report the role of HMGB-1 associated with granulosa cells. hGL5 cell proliferation was reduced due to apoptosis, which also resulted in decreased estradiol secretion. In assisted reproductive technology (ART), the quality of oocytes and embryos is one of the most critical parameters determining pregnancy outcomes. Since oocytes are surrounded by granulosa cells, these cells play an essential role in follicle development, fertility, and oocyte viability. Not only the quantity but also the function of granulosa cells, particularly in terms of steroidogenesis, is important [ 23 , 24 ]. Decreases in estradiol levels in EMS patients are associated with reduced IVF outcomes, including pregnancy and live birth rates [ 25 , 26 ]. Therefore, diminished steroidogenesis due to granulosa cell apoptosis may negatively affect fertilization. HMGB-1 release increased after oxidative stress in the hGL5 cell line as HMGB-1 release increased in HESCs by oxidative stress-induced cell death [ 16 ]. Also, increased TLR4 expression was observed. The binding of HMGB-1 to TLR4 may lead to the worsening of oxidative stress in EMS. It is rather well known that HMGB-1-TLR-4 interaction stimulates inflammatory reaction in various cells [ 27 , 28 ]. Clinical studies have demonstrated that women with EMS exhibit altered serum and local cytokine profiles characterized by increased levels of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α, as well as elevated anti-inflammatory cytokines including IL-4 and IL-10, reflecting a dysregulated inflammatory environment [ 29 , 30 ]. The expression of IL-1β and IL-6 after H 2 O 2 treatment was also increased in the hGL5 cell line in this study. Huang et al. demonstrated that increased HMGB-1 levels are associated with the enhanced release of inflammatory cytokines, including IL-1β and IL-6 [ 21 ]. Activation of the NF-κB pathway following oxidative stress may represent the link between HMGB-1 and inflammatory cytokine production, highlighting the pivotal role of the NF-κB pathway in EMS [ 31 , 32 ]. In Western blot analysis of NF-κB pathway-related proteins, IκBα levels remained unchanged over time, whereas its phosphorylated form peaked at 15 min post-treatment and gradually decreased thereafter. This is likely due to cytoplasmic localization of IκBα and subsequent NF-κB nuclear translocation resulting from ubiquitination of IκB [ 33 ]. Along with decreased steroidogenesis, increased release of IL-1β and IL-6 may partly explain the low quality of oocytes in EMS. Further studies are warranted to explore this possibility. Our study has shown that oxidative stress due to EMS affects the follicular environment via the apoptosis of granulosa cells, decrease in estradiol secretion, the cascade involving HMGB-1, TLR4, the NF-κB pathway, and the release of inflammatory molecules IL-1β and IL-6. The limitation of this study is that the results were only from an in vitro cell-level perspective. Therefore, it may not fully capture the complex environment of ovarian follicles in vivo. Confirming the involvement of the HMGB-1-TLR4-NF-κB axis through in vivo experiments with actual follicles is necessary. Also, the variability and heterogeneity of EMS in patients were not reflected in our cell-based model. Clinical differences could influence the observed molecular pathways differently. We focused on major inflammatory cytokines such as IL-1β and IL-6 in relatively higher concentrations of H 2 O 2 . However, many other factors may contribute to EMS-related infertility. Future research with human clinical samples is essential to validate the clinical relevance of the HMGB-1/TLR4/NF-κB axis in EMS.

This study showed increased HMGB-1 release, decreased steroidogenesis, and increased inflammation in granulosa cells after oxidative stress to explain the follicular environmental changes in patients with EMS and infertility. These findings can be suggested as a new research topic and therapeutic target for such patients.