Mast cell tryptase-PAR2 axis promotes ovarian fibrosis through RNF152-mediated stabilization of Bcl-xL

doi:10.21203/rs.3.rs-4721528/v2

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Mast cell tryptase-PAR2 axis promotes ovarian fibrosis through RNF152-mediated stabilization of Bcl-xL

https://doi.org/10.21203/rs.3.rs-4721528/v2

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Ovarian fibrosis is a basic histologic feature of premature ovarian insufficiency(POI), which poses a great threat to women of reproductive age. Collagen abundance in ovarian theca-stroma cells contributes to ovarian fibrogenesis. However, the role and mechanism of mast cells in the development of POI have not yet been determined. Our study for the first time found that tryptase secreted by activated mast cells induced COL1A1 and COL1A2 production, two subunits of collagen I in mouse theca–stroma cells by protease-activated receptor-2 signaling. Inhibition of PAR2 or Bcl-xL attenuated the increases of COL1A1 and COL1A2 caused by tryptase. In addition, knockdown of RNF152 reversed the downregulation of collagen production caused by si-Bcl-xL. Clinically, tryptase levels in serum and follicular fluid were higher in both bPOI and POI patients than in controls. Tryptase concentrations in serum and follicular fluid were positively associated with follicle stimulating hormone(FSH) and negatively associated with anti-Müllerian hormone (AMH). Cromolyn sodium, a mast cell stabilizer, reduces collagen I production, but has no effect on hormone synthesis and follicle number in a cisplatin-induced POI mouse model. Our findings shed new insights into the biological function of tryptase in ovarian fibrosis and POI.

Health sciences/Endocrinology/Endocrine system and metabolic diseases/Endocrine reproductive disorders

Biological sciences/Cell biology/Mechanisms of disease

Biological sciences/Physiology/Reproductive biology/Reproductive disorders/Infertility

The prevalence of infertility ranges from 8–12%¹. Premature ovarian insufficiency (POI) is a common cause of infertility among women of reproductive age. POI refers to premature exhaustion of the resting pool of primordial follicles and the cessation of ovarian function in women under the age of 40 years. The core features of POI include high follicle stimulating hormone (FSH) and increased risks of severe health disturbances, including cognitive impairment, cardiovascular disease, sleep disorders, vaginal dryness and osteoporosis^2,3. Three stages of the disorder have been demonstrated, namely, occult or subfertility, biochemical POI (bPOI) with a high level of FSH, and overt POI with irregular menstruation⁴. The etiological factors for POI can be categorized as genetic, autoimmune, or iatrogenic. However, the pathophysiological mechanisms of most cases still remain unclear, and effective strategies to rescue ovarian function rarely exists in overt POI patients. Hence, unraveling the fundamental pathogenesis of POI is an important challenge.

Several known gynecologic diseases are characterized by ovarian fibrosis such as polycystic ovarian syndrome, ovarian chocolate cyst and premature ovarian failure, all of which are more susceptible to infertility^5–7. Fibrosis is a common characteristic feature that occurs in the progression of chronic diseases into advanced stages, which can be commonly seen in various organs, including liver, heart, lung, and kidney. The pathological characteristics of fibrosis are chronic inflammation and deposition of extracellular matrix (ECM)⁸. Ovarian inflammation is essential for oocyte quality, ovulation and ovulatory wound repair, while the sustained inflammation and constant remodeling throughout a reproductive lifetime are thought to be persistent stimulation that promote ovarian fibrosis⁹. The major mechanism of POI is follicle depletion, which is correlated with invasion of the ovarian cortex by fibrous tissues. By secreting large amounts of ECM, ovarian theca–stroma cells play an important role in the initiation and development of ovarian fibrosis during POI¹⁰. Collagen I is the most plentiful component of ECM and widely expressed in the ovary. Our previous study also showed that the high expression of COL1A1 and COL1A2, two subunits of collagen I in mouse theca-stroma cells, were associated with ovarian fibrosis and POI¹¹.

Mast cells act as crucial cellular sensors and regulators of inflammation and fibrosis^12–14. In addition to playing a critical role in allergic inflammation, mast cells can sense various "danger signals" by the receptors expressed both on the cell surface and within the cells. Under various types of stress, mast cells serve as important sources of several mediators, including tryptase and histamine¹⁵, which can alter and interact with the local environment leading to tissue fibrosis, repair, and remodeling^{12–14,16−18}. However, the exact effect of mast cells on ovarian theca-stroma cells and its underlying mechanism remains poorly defined. Given this background, we hypothesized that mast cells were responsible for inducing ovarian fibrosis, thus causing the occurrence and development of POI.

In this study, we comprehensively elucidated for the first time the effect of tryptase on disruption of ovarian function in samples from POI patients and in an experimental POI mouse model.

Recruitment of patients

A total of 60 women with diverse ovarian reserves were enrolled from the Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University from May 2019 to June 2022, consisting of 20 patients with bPOI, 20 patients with POI and 20 control women. The research was approved by the Ethics Committee of Renji Hospital with written informed consent from all participants. Women under 40 years with an FSH level higher than 25 IU/L on two occasions more than 4 weeks apart are diagnosed as POI. The diagnosis of bPOI was based on FSH level ≥10 IU/L but <25 IU/L and anti-Müllerian hormone (AMH) <1.1 ng/ml. The control group included women who had undergone the first cycle of in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment with tubal or male factor-related infertility. Women under 40 years with FSH < 10 IU/L and AMH ≥ 1.1 ng/ml are recognized with normal ovarian reserve. They received oocyte retrieval on the same day as the bPOI patients with similar age and BMI. Women with hyperthyroidism, PCOS, hyperprolactinemia, and history of radiotherapy, chemotherapy, or ovarian operation were excluded.

Sample collection and laboratory tests

Peripheral blood was obtained from 20 POI patients, 20 bPOI patients and 20 control women on the third day of the menstrual cycle before their first cycle of IVF or ICSI treatment. The levels of FSH, luteinizing hormone (LH), progesterone (P4), estradiol (E2), and AMH were tested by electrochemiluminescence immunoassay kits (Roche, Germany).

The antral follicle count (AFC) of 60 women with diverse ovarian reserves in the early follicular phase was detected by transvaginal ultrasonography.

Follicular fluid was collected from 20 patients with bPOI and 20 control women. After ovarian stimulation, women with bPOI and controls underwent oocyte retrieval guided by transvaginal B-scan ultrasound. Ovarian stimulation and oocyte retrieval were based on the conventional protocols of the Center for Reproductive Medicine, Renji Hospital. Follicular fluid from the

first large follicle (diameter ≥ 14 mm) was collected and then centrifuged at 2,500 rpm for 15 min. All samples were stored at −80°C until used.

Animal care

The experimental procedures were approved by the Institutional Animal Care and Use Committee of Fudan University, and conducted in accordance with institutional guidelines on the care and use of laboratory animals. Female C57BL/6 mice aged 8 weeks were housed under specific pathogen-free conditions (ambient temperature: 22.0±1.0°C; humidity: 40%) during breeding and experimentation, with ad libitum access to normal food and water.

Animal studies

Cisplatin was used to induce POI mouse model as previous study¹⁹. C57BL/6 female mice were injected with cisplatin (1.5 mg/kg) or saline intraperitoneally for 10 consecutive days. Saline-injected mice were used as WT control (Normal group) and cisplatin-injected mice were used as the POI model.

In order to assess the effect of cromolyn sodium on ovarian function and fibrosis, we intraperitoneally injected mice with 10mg/kg cromolyn (n=20 per group) or sterile saline (n=20 per group) at two days before cisplatin treatment. The dose of cromolyn sodium was based on previous studies^20-22. The two groups were then administered with cisplatin (1.5 mg/kg) or saline intraperitoneally for 10 consecutive days. Therefore, female C57BL/6 mice were divided into four groups (n=10 per group): (1)saline-injected mice (control group); (2) cromolyn-treated mice (Cro group); (3) mice treated with sterile saline only before administration of cisplatin (Cis group); and (4) mice treated with 10mg/kg cromolyn before administration of cisplatin (Cis+Cro group).

Hematoxylin and eosin staining and follicle counting

Mice were euthanized at the end of the study, ovaries were collected and fixed with 4% paraformaldehyde for 24 h. After the paraffin embedding and sectioning (5 mm in thickness) process, ovary tissues were used for histopathology examination by hematoxylin and eosin (H&E) staining. The follicles with an oocyte containing a clearly visible nucleus were counted. The follicles were categorized as primordial, primary, secondary, and atresia follicles as previously described¹¹. Five slides were selected randomly for each group and five views were chosen on each slide for statistical analysis in a blinded manner.

Masson staining and sirius red staining

To further detect collagen fibers levels, Masson trichrome staining and sirius red staining were used to stain ovary tissues as previous studies^{10, 11}. After the staining, the slides were photographed under a light microscope. Collagen fibers were stained blue, the nuclei were black, and muscle fibers and cytoplasm were red. Five views were chosen on each slide for statistical analysis.

MCs staining and immunohistochemical analysis

Slides were stained in 0.05 % toluidine blue for ovary sections. A 1 % stock solution in 70 % ethanol was dissolved in 0.5 % NaCl solution (pH 2.2–2.3), and the slides were immersed 30 min in the above concentration. They were then washed twice in distilled water, dehydrated in a series of increasing concentrations of ethanol, followed by placement in butyl acetate ester. Samples were coverslipped using Eukitt® mounting medium and were allowed to dry overnight.

MC counting was always performed in a blinded fashion by an experimenter that was unaware of the sample identity. Criteria for degranulation included loss of purple staining, fuzzy appearance, distorted shape, or multiple granules visible in the vicinity of the cell. The entire surface area of the ovary was scanned manually using a light microscope.

In addition, immunohistochemistry was used to stain MCs with mast cell tryptase (1:50, Proteintech, China) monoclonal antibody as previously described²³.

Primary ovarian theca–stroma cells culture

Ovarian theca–stroma cells were obtained as our previous study¹¹. Briefly, ovaries from 3- 4-week-old C57BL/6 immature mice were isolated from the bursa and poked with insulin needles. After about 100 times, the granulosa cells and oocytes were released. The tissue mixture was digested with pre-equilibrated enzymatic media containing 0.7 mg/ml Collagenase IV (Thermo Fisher Scientific, USA) and 0.2 mg/ml DNAse I (Thermo Fisher Scientific, USA) at 37°C for 1 h. The enzymatic digestion was neutralized with an equal volume of media with 10% FBS and strained by a strainer to remove undigested tissue. Cells were centrifuged (1,000 rpm, 5 min) and suspended in McCoy’s 5A medium with 10% fetal bovine serum and 100 U/ml penicillin. After overnight incubation, the morphological appearance of the theca–stromal cells was observed under a microscope. The attached cells exhibited a long fusiform and fibroblast-like shape. The purity of theca–stromal cells was also identified by immunofluorescence staining of vimentin before further analysis.

Enzyme-linked immunosorbent assay

Follicular fluid and serum sample of each patient and the serum of each mouse were collected and stored at −80°C for analysis. The expression of tryptase in samples from patients and the levels of E2, FSH, LH and AMH in mouse serum were measured using an ELISA kit according to the manufacturer’s instructions (Cloud-Clone Corp, China).

Immunofluorescence staining

After different treatment, stromal cells in the chamber slide were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100. After washing with phosphate-buffered saline (PBS), stromal cells were blocked with bovine serum for 1 hour and incubated with primary antibodies against PAR2 (1:50, Santa Cruz, USA) and vimentin (1:100, Santa Cruz, USA) overnight at 4°C. After washing with PBS, cells were incubated in the dark for 2 h with Alexa Fluor 488 or 598-labeled secondary antibodies (Proteintech, China) at room temperature. The nuclei were stained with DAPI (Servicebio, China). Images were obtained using a microscope with an image analysis system (Zeiss, Germany).

PCR

Total RNA from primary theca–stroma cells was isolated with an RNA extraction kit (Foregene, China), and reverse transcription to cDNA with a PrimeScript RT Master Mix Perfect Real-Time kit (TaKaRa, Japan) according to the manufacturer’s instructions. The target mRNA was quantified with qRT-PCR.

Primers are listed as the following: Bcl-xL forward 5′-GAGCTGGTGGTTGACTTTCTCT-3 ′, reverse 5′ -TCCATCTCCGATTCAGTCCCT-3′, and GAPDH forward 5 ′-AGGCCGGTGCTGAGTATGTC-3 ′, reverse 5 ′-TGCCTGCTTCACCACCTTCT-3′. The relative mRNA values were normalized to the GAPDH gene control values and calculated using the comparative cycle threshold (ΔΔCt) method.

Small interfering RNAs transfection

RNAi-negative controls and specific small-interfering RNAs (siRNAs) against PAR-2, RNF152 and Bcl-xL were purchased from GenePharma (Shanghai, China). To clarify the molecular mechanism ovarian fibrosis, the primary ovarian theca–stroma cells were transfected with the corresponding siRNAs using Lipofectamine 3000 Reagent (Invitrogen, USA) before the different treatments.

Western bloting

Ovary tissues and ovarian theca–stroma cells were homogenized in RIPA lysis buffer (Biyuntian,

Shanghai, China), which contained 50 mM Tris (pH 7.4), 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), sodium orthovanadate, sodium fluoride, EDTA, leupeptin, etc. The homogenates were centrifuged at 12,000g for 20 min (4 °C), and the supernatants were then harvested as cytosolic fractions for immune blot analysis. After incubation for 20 min on ice, lysate was centrifuged and protein concentration was determined by the BCA kit. 40μg of each sample was separated by 10% or 15% SDS-PAGE gels according the molecular weight and then transferred onto PVDF membranes. After being blocked with 5% BSA for 1 hour at room temperature, the blots were incubated with various antibodies against COL1A1 (1:10000, proteintech, China), COL1A2 (1:1000, proteintech, China), Bcl-xL (1:500, proteintech, China), Myc (1:1000, Cell Signaling Technology), Flag (1:1000, Cell Signaling Technology), HA (1:10000, Sigma-Aldrich), Tubulin (1:10000, proteintech, China) and GAPDH (1:20000, proteintech, China). The bands were visualized and captured using a G-Box iChemi Chemiluminescence Image Capture System from Syngene.

Ubiquitination analysis

HA-tagged ubiquitin, Flag-Bcl-xL and Myc-RNF152 plasmid were used to transfect HEK293T cells, and then 10 μM MG-132 were added for 12 h before treated with 100 μM PAR2-AP for 1 h. Equal amouts of proteins were incubated with anti-flag magnetic bead overnight. For negative control, proteins were incubated overnight with IgG bead and anti-IgG antibody. The beads were washed five times with IP lysis buffer and boiled at 95℃ for 5 minutes.

Statistical analysis

The data were presented as mean ± SEM. Independent-sample Student's t-tests or Mann-Whitney U tests were used to analyze differences between two groups for nonparametrically distributed data. For comparisons among more than two groups, a one-way analysis of variance (ANOVA) followed by Turkey's post hoc test was appropriately employed. Spearman’s correlation was applied to evaluate the correlations between tryptase levels in serum or follicular fluid and ovarian reserve indicators. A P value < 0.05 was considered statistically significant.

Construction of the POI mouse model

Plasma E2 and AMH levels were declined, while FSH and LH levels were elevated in the cisplatin-induced POI mice compared to the control group (P<0.01, Figure 1A-D). Cisplatin significantly decreased the ratio of ovary to body weight (P<0.01, Figure 1E). Ovarian morphological examination demonstrated that the number of functional follicles, including primordial, primary, and secondary follicles, was significantly reduced in the POI group (P<0.01, P<0.01and P<0.05, respectively, Figure 1F, 1G). These results indicated that the POI mouse model was successfully established.

Abundance of collagen and mast cells in the ovaries of POI mice

Masson and Sirius red staining showed increased collagen abundance in the ovarian stroma of POI mice(Figure 2A,B). In addition, Western blot was conducted on the collected mice ovarian tissues. The expression of COL1A1 and COL1A2, two subunits of collagen I, were significantly higher in the ovaries from POI mice compared to the control mice (P<0.01, Figure 2C, 2D). To understand whether mast cells are involved in cisplatin-induced ovarian fibrosis, we first quantified mast cells in toluidine blue (TB) and tryptase stained section of ovarian tissue sections from the control group and the POI group. As shown in Fig. 2E-2H, the number of activated mast cells was increased in the ovaries of the POI group than in those of the control group. These results suggested that cisplatin injection might induce the degranulation of mast cells.

Upregulation of COL1A1 and COL1A2 by tryptase in ovarian theca-stroma cells

Our previous study demonstrated that treatment with 1μg/ml LPS for 24h could induce P815 cells degranulation by promoting histamine and tryptase release²⁴.To test whether activated mast cell is an underlying factor in ovarian fibrosis, we established an in vitro co-culture model in which primary mouse ovarian theca-stroma cells were incubated with conditioned medium (CM) from P815 cells given different treatment. Vimentin, a marker for ovarian theca-stroma cells was stained positively(Figure 3A). As shown in Figure 3B-D, the transfer of CM from P815 cells stimulated with LPS induced a significant increase in COL1A1 and COL1A2 protein levels (P<0.05). However, LPS alone or CM from P815 cells without LPS treatment had no effect on Collagen I expression (P>0.05). We next examined whether histamine, a classic component of mast cell granules, is involved in collagen synthesis, we incubated primary mouse ovarian theca-stroma cells with different histamine concentrations ranging from 0 to 1μg/ml for 24 hours. In vitro studies demonstrated that there were no change in the protein levels of COL1A1 and COL1A2 (P>0.05, Figure 3E-G) . To address the role of tryptase in ovarian fibrosis, we conducted Western blotting of primary mouse ovarian theca-stroma cells following various concentrations of tryptase for 24h. A marked increase in COL1A1 and COL1A2 expression was observed in 10nM tryptase-treated cells (P<0.05, Figure 3H-J) .

Involvement of PAR2 in regulation of Collagen I by tryptase

Because proteinase-activated receptor 2 (PAR2) is the receptor of tryptase²⁵, we evaluated the presence of PAR2 in the primary mouse ovarian theca-stroma cells by immunofluorescence staining. As expected, ovarian theca-stroma cells showed positive staining for PAR2 (Figure 4A). The upregulation of COL1A1 and COL1A2 in ovarian theca-stroma cells caused by tryptase was abolished by knockdown of PAR2 (P<0.05, Figure 4B-D), indicating that PAR2 is required for tryptase-mediated induction of collagen I.

Tryptase induced COL1A1 and COL1A2 through Bcl-xL

Silencing Bcl-xL has been reported to suppress collagen expression in hypertrophic scar fibroblasts²⁶. Therefore, we first examined the role of Bcl-xL in tryptase-induced COL1A1 and COL1A2 production. Western blot showed an increase in Bcl-xL expression after 10nM tryptase treatment (P<0.05, Figure 5A-B). We then transfected ovarian theca-stroma cells with Bcl-xL small interfering RNA (siRNA). As shown in Figure 5C-E, the induction of COL1A1 and COL1A2 caused by tryptase was abolished by inhibition of Bcl-xL (P<0.05).

A previous study has identified the role of PAR2 in the stabilization of Bcl-xL in colon cancer cell lines²⁷. Since knockdown of PAR2 attenuated tryptase-induced collagen production(Figure 4B-D), we examined whether Bcl-xL was regulated by PAR2 in mouse ovarian theca-stroma cells. Western blot demonstrated that activation of PAR2 with activating peptide (PAR2-AP) significantly increased Bcl-xL protein level, whereas knockdown of PAR2 by siRNA decreased the protein level of Bcl-xL(P<0.05, Figure 5G-J). Treatment with MG-132, a reversible proteasome inhibitor, completely reversed the downregulation of Bcl-xL by knockdown of PAR2 in HEK293T cells (Figure. S1A). However, activation of PAR2 had no effect on the mRNA level of Bcl-xL(P>0.05, Figure 5F). In addition, The ubiquitination of Bcl-xL was decreased by PAR2 activation(Figure. S1B). The above results suggested that tryptase/PAR2 might regulate collagen expression by Bcl-xL through through ubiquitin-proteasome pathway.

E3 ligase RNF152 mediated Tryptase-induced collagen expression.

Ectopic RNF152 promoted the the ubiquitination of Bcl-xL. In addition, activation of PAR2 reversed the degradation of Bcl-xL(Figure. S1B). Knockdown of RNF152 significantly increased the protein level of Bcl-xL and blocked the inhibition of Bcl-xL caused by si-PAR2(Figure. 6A-B). We therefore hypothesized that RNF152 might be involved in tryptase-induced collagen production. To verify this hypothesis, we transfected si-RNF152 into mouse ovarian theca-stroma cells in the presence of tryptase. Western blot analysis showed that knockdown of RNF152 reversed the downregulation of COL1A1 and COL1A2 protein caused by si-Bcl-xL(Figure. 6C-E). Taken together, our results indicated that RNF152 regulated the collagen production by tryptase through modulating Bcl-xL stabilization.

Cromolyn inhibited cisplatin-induced collagen deposition in vivo

Cromolyn sodium is commonly used to impair mast cell degranulation. We hypothesized that inhibiting the degranulation of mase cells might play a protective role in the ovarian function. We found that there was no difference in plasma E2, FSH, LH and AMH levels between POI mice treated with cromolyn or sterile saline(P>0.05, Figure 7A-D). Additionally, ovarian index and the number of total follicle were not affected by cromolyn treatment(P>0.05, Figure 7E-G). Intriguingly, administration of cromolyn to POI mice resulted in decreased collagen deposition(Figure 7G-J). Masson and Sirius red staining also showed no difference in collagen depostion between control mice and cromolyn-treated mice(Figure 7G). Upon mast cell inhibition, we observed a significantly decrease in the protein levels of COL1A1 and COL1A2 in POI mice(Figure 7H-J). These observations implied that inhibiting mast cell degranulation attenuated ovarian fibrosis, but had no effect on ovarian function in mice.

Higher level of tryptase in serum of POI and bPOI patients

Sixty patients were recruited in our study and classified into three groups: control (n=20), POI(n=20) and POI(n=20) groups. The clinical features are shown in Table 1. There were no differences in age, body mass index (BMI) or basal levels of serum estradiol and progesterone among three groups. Both bPOI and POI patients had significantly higher level of FSH, whereas serum level of AMH and basal antral follicle counts (AFC) were markedly declined compared with the control group(P<0.001). Higher level of LH was observed in POI group than bPOI and control group (P<0.001).

The concentrations of tryptase in serum and follicular fluid from bPOI patients were elevated compared with those from control women (P<0.05, Figure 8A, 8D). The level of tryptase in serum was higher in POI patients than in normal people (P<0.01, Figure 8A). Additionally, tryptase in serum and follicular fluid levels were positively associated with FSH, but negatively with AMH (P<0.01, Figure 8B-C and 8E-F).

The etiologies of POI include genetic defects, iatrogenic factors and autoimmune diseases²⁸. However, a significant number of POI patients are idiopathic and their causes are not defined². Recently, the role of cellular immunity and chronic inflammation in the etiopathogenesis of POI have gained great concern²⁹. Cytokines and immune cells, the critical components of cellular immunity, lead to inflammation³⁰. The development of oocytes rely on a complex ovarian stromal milieu³¹. Excessive amount of ECM, indicative of tissue fibrosis, is responsible for impaired ovarian function³². Increased inflammation and fibrosis occur before the exhaustion of follicles and probably lead to ovarian insufficiency³³. Mast cells are mostly best known for their role in inflammation, and they are mainly located in ovarian stromal tissues³⁴. However, the role of mast cells in ovarian fibrosis and in the pathogenesis of POI remains unknown.

The POI mouse model was successfully established using cisplatin as reported³⁵. In line with a previous study, increased collagen deposition was observed in the ovaries of cisplatin-induced POI mice¹⁰. We found that the POI mice exhibited an increase number of mast cells. In addition to the animal study, the ovarian theca-stroma cells are used for in vitro study to explore the mechanism of ovarian fibrosis³⁶. Therefore, we constructed a co-culture model in which primary mouse ovarian theca-stroma cells were treated with conditioned medium from mouse mast cell line P815 under different conditions. Interestingly, our results demonstrated a pro-fibrotic role of degranulated mast induced by LPS in ovarian theca-stroma cells. Notably, LPS alone had no effect on collagen production. Mast cells are sentinel immune cells that probably respond early to ovary injury induced by cisplatin. Some studies have shown that mast cells are related to inflammation and subsequent fibrosis in some organs³⁷. A previous study showed that degranulated mast cells were noted in the mouse fibrotic colons in the dextran sodium sulfate-induced intestinal fibrosis models^{37, 38}. In the in vitro experiment, mast cells tryptase activated fibroblasts, thus secreting more ECM proteins through PAR-2/Akt/mTOR pathway³⁷. Kim and colleagues claimed that pharmacological inhibition of SIRT2 alleviated allergic inflammation and collagen accumulation by inhibiting mast cells activation via suppression intracellular calcium influx³⁸. We therefore wonder which mediator released by activated mast cells is involved in the development of ovarian fibrosis. We treated ovarian stromal cells with only tryptase to exclude the effects of other factors secreted by mast cells. In the current study, data showed that tryptase promoted the production of both COL1A1 and COL1A2 proteins, major members of ECM family, in ovarian theca-stroma cells. These results were the first evidence of a relationship between mast cells and ovarian fibrosis, suggesting that mast cells and its meditor, tryptase might be involved in the development of POI.

In the present study, administration of cromolyn sodium, a clinically used mast cell stabilizer, in the cisplatin-induced POI model reversed fibrotic phenotype. To back our findings, an early study found that treatment with cromolyn sodium in Mdr2-/- mice, which mimic human primary sclerosing cholangitis, reduced the degree of inflammation and the progression of fibrosis³⁹. However, hormone levels were not affected by stabilization of mast cells. As severe loss of follicles number in the POI mouse model, inhibiting of mast cells degranulation might not be sufficient to improve ovarian function. The effect of cromolyn sodium on ovarian function in a mouse model of diminished ovarian reserve constructed with low-dose cisplatin could be further investigated. A recent study showed that combining cromolyn sodium with anti-IL-17A reduced clinical severity and attenuated bone destruction in a mouse model of arthritis⁴⁰. It is likely that drug combination treatment might be beneficial than single-drug therapy.

Tryptase exerts its effect via binding to the PAR2 of effector cells. Our data indicated that ovarian theca-stroma cells expressed PAR2. Furthermore, knockdown of PAR2 abolished the increased collagen production induced by tryptase. These results suggested that the stimulatory action of tryptase on ovarian theca-stroma cells might be a PAR2-mediated event. Our results were coincidence with Tan’s study. The authors demonstrated that tryptase increased the protein level of Collagen I by activating PAR2 and PPARγ pathway in atrial fibroblasts⁴¹. Another study revealed that in cardiac fibroblasts, tryptase elicited the fibrotic response by binding to the PAR2 receptor⁴². A majority of studies focusing on follicle fluid due to its accessibility⁴³. Low-grade inflammation with increased levels of pro-inflammatory cytokines in follicle fluid might be associated with fibrosis in the ovarian stroma⁴⁴. To clarify whether similar mechanisms might be conserved in humans, we analyzed the tryptase levels in follicular fluid samples from women with diverse ovarian reserves. Tryptase was detectable in follicular fluid. The levels of follicular fluid tryptase in bPOI patients were higher than in women with normal ovarian function. Our study also detected that serum typtase levels were higher in bPOI patients and POI patients than in controls. The increase in human tryptase levels with decreasing ovarian function implied that similar mechanisms might occur in humans. Moreover, a positive correlation between tryptase levels and FSH and a negative correlation between tryptase and AMH were found in our study. We hypothesized that the higher levels of follicular fluid tryptase in bPOI patients might be caused by more mast cells infiltrated into damaged ovaries. It is worth noting that tryptase levels in follicular fluid may not necessarily reflect those in the ovarian theca-stroma cells. More studies are needed to evaluate the role of tryptase on human ovarian theca-stroma cells in the future.

Multiple studies have suggested that Bcl-xL is associated with fibrosis in various cells^{26, 45}. Knockdown of Bcl-xL reduced the expression of fibrotic factors in hypertrophic scar fibroblasts²⁶. UBX1967, a Bcl-xL small molecule inhibitor, depleted a population of COL1A1-positive endothelial cells related to retinopathy by single-cell RNA sequencing analysis⁴⁵. Interestingly, Li et al reported that upon PAR2 activation, the ubiquitination of Bcl-xL was decreased⁴⁶. However, it is unclear whether tryptase activation of PAR2 stabilizes Bcl-xL protein, thus promoting fibrosis of ovarian theca-stroma cells. In the current study, we found that tryptase increased the expression of Bcl-xL, while inhibition of Bcl-xL by siRNA ameliorated the upregulation of COL1A1 and COL1A2 induced by tryptase. Bcl-xL pretein level of ovarian theca-stroma cells was decreased or increased when transfected with PAR2 siRNA or treated with PAR2 activating protein. Collectively, these findings suggested a major role of Bcl-xL in the modulation of collagen production in ovarian theca-stroma cells.

Recently, many studies have pointed out that E3 ubiquitin ligases are involved in organ fibrosis^47-49. In a model of hypertensive renal disease performed by Zhang et al, renal inflammatory and fibrotic response was significantly ameliorated by overexpression of TRIM31, an E3 ubiguitin ligase⁴⁹. In addition, severe fibrosis in kidneys was found in a TRIM31^-/- mice than TRIM3^+/+ mice. Another study observed that hepatocyte-specific RNF5 deficiency mice had exacerbated hepatic fibrosis and inflammation during the progression of nonalcoholic fatty liver disease induced by high fat diet⁴⁸. RNF152 is a well-known Bcl-xL E3 lagase⁴⁶. RNF152 knockdown blocked the downregulation of Bcl-xL by PAR2 inhibition. We observed an increase of collagen production by ablation of RNF152. Further in vivo studies are needed to confirm the role of RNF152 in the ovarian fibrosis by conditional knockout mice or by injecting with overexpression virus.

The present study has a few limitations. Firstly, the number of control, bPOI and POI subjects was relatively small. Secondly, we failed to collect follicle fluid from POI group. Thirdly, further in vivo studies are needed to clarify the effect of mast cells in the development of ovarian fibrosis using mast cell-deficient mice. There are various methods to establish the POI mouse model. It is not clear whether mast cell and its mediator, tryptase, play pathogenic roles in ovarian fibrosis in other types of POI mouse model. Moreover, it is important to assess the therapeutic potential of PAR2 inhibitors or Bcl-xL inhibitors in POI mice models.

To conclude, our results show that tryptase, the main mediator released by activated mast cells, promotes collagen deposition in ovarian theca-stroma cells through its receptor PAR2. Activating PAR2 increased collagen production by stabilization of Bcl-xL protein. Cromolyn sodium, a mast cell stabilizer, attenuated ovarian fibrosis, but has no effect on hormone synthesis and follicle number in the chemotherapy-induced POI mouse model. These findings might provide a new insight into the mechanism and therapeutic potential of mast cells in ovarian fibrosis.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81801408, 81901996). We would like to thank all the participants who were included in this study.

Author Contributions

X.Z.：Investigation，methodology, data acquisition and drafting the article. J.Z.：Conceptualization, statistical analysis and editing the draft. C.W.: Conceptualization, design and supervision.

Conflicts of Interest : All authors declare no conflict of interest in relation to this work.

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Tabel 1 Demographic Features of Recruited Patients

Characteristic	Control (n=20)	bPOI (n=20)	POI(n=20)	P value
Age (y)	31.35±0.65	32.85±0.62	32.95±0.67	0.155
BMI (kg/m2)	21.11±0.29	22.56±0.61	21.73±0.52	0.696
AFC	14.55±0.94	4.80±0.47^a	1.50±0.22^ab	＜0.001
FSH (IU/L)	5.86±0.33	14.52±0.98^a	38.32±4.39^ab	＜0.001
LH (IU/L)	4.74±0.36	6.46±0.88	18.77±3.69^ab	＜0.001
Estradiol (pg/ml)	27.72±2.55	38.80±9.03	23.58±3.88	0.091
P (ng/ml）	0.23±0.03	0.20±0.02	0.22±0.03	0.639
AMH (ng/ml)	3.80±0.38	0.54±0.07^a	0.19±0.03^ab	＜.001

Hormone levels were measured in serum on day 3 of the menstrual cycle. AFC was detected as the number of follicles (3–8 mm in diameter) by transvaginal ultrasound scan during the early follicular phase.

bPOI, biochemical premature ovarian insufficiency; POI, premature ovarian insufficiency; AFC, antral follicle count; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P4, progesterone; AMH, anti-Müllerian hormone.

a P < 0.05 vs. control.

b P < 0.05 vs. bPOI.

The authors declare no competing interests.

supplementaryfigure.tif
Figure S1. Activation of PAR2 blocked RNF-152-mediated Bcl-xL ubiquitination. (A) HEK-293 cells with or without PAR2 knockdown were treated with 20 μM MG-132 for 12 h. HEK293T cells were co-infected with Ub-tagged HA, Flag-tagged Bcl-xL and Myc-tagged RNF152. After 36h, the cells were treated with MG-132 at a dose of 10 μM for 12 h before challenged with 100 μM PAR2-AP for 1 h. Flag antibody was used to immunoprecipitate Bcl-xL protein, and ubiquitinated Bcl-xL was detected with HA antibody via Western blot.

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Mast cell tryptase-PAR2 axis promotes ovarian fibrosis through RNF152-mediated stabilization of Bcl-xL

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