Rosiglitazone alleviates LPS-induced endometritis via suppression of TLR4-mediated NF-κB activation

doi:10.21203/rs.3.rs-1856909/v1

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Rosiglitazone alleviates LPS-induced endometritis via suppression of TLR4-mediated NF-κB activation

https://doi.org/10.21203/rs.3.rs-1856909/v1

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Endometritis is a common reproductive system disease that seriously affects the reproductive health of women of childbearing age. Rosiglitazone (RGZ) is known to have anti-inflammatory and immunomodulatory effects. The effect of RGZ on the treatment of endometritis has not been reported. In the present study, we aimed to investigate the anti-inflammatory effect of RGZ on lipopolysaccharide (LPS)-induced endometritis and to explore its possible mechanism of action. Using LPS-induced endometritis mice as a model, the preventive and therapeutic effects of RGZ on endometrial inflammation were explored. Histopathology revealed that RGZ significantly attenuated LPS-induced endometritis in C57BL/6 mice. The anti-inflammatory effects of RGZ and possible related signaling pathways were investigated in vitro using human endometrial stromal cells (HESCs). The results of reverse transcription-polymerase chain reaction and western blot analysis showed that RGZ pretreatment could inhibit the expression of Toll-like receptor 4 (TLR4) and its downstream activation of nuclear factor-κB (NF-κB). Collectively, these results suggest that RGZ may inhibit LPS-induced endometritis through the TLR4-mediated NF-κB pathway.

Rosiglitazone

endometritis

TLR4

NF-κB

Endometritis not only affects embryo implantation to cause infertility but also leads to pregnancy-related diseases, such as miscarriage, premature rupture of membranes, premature birth, and intrauterine infection. Bacterial infection and dysbacteriosis are the causes of endometritis. Although the accepted method is antibiotic treatment, it has been reported that up to 24.6% of patients still have signs of endometritis after antibiotic treatment[1–2]. 91.4% and 70.7% of E. coli isolates isolated from cows suffering from endometritis isolates were resistant to cefotaxime and doxycycline, respectively[3].

These indicated that antibiotic resistance should not be ignored. Therefore, it is of great significance to explore new drugs for the treatment of endometritis[4, 5]. As the main component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS) is a key factor in the induction of endometritis[6, 7]. LPS can induce the cascade release of inflammatory factors, such as interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor -alpha (TNF-α), to exacerbate endometrial tissue damage[8] and inhibit the production of cytokines to achieve therapeutic effects.

Toll-like receptors (TLRs) are a class of intracellular pattern recognition receptors (PRRs) that participate in the body's innate and adaptive immune responses by recognizing molecules such as LPS and membrane lipoproteins. TLR4 is an important member of the TLR family and can recognize the exogenous ligand LPS. The binding of LPS and TLR4 can cause intracellular signal transduction response, activate the NF-κB signaling pathway, and lead to the generation of inflammatory mediators to trigger inflammatory response[9, 10].

Rosiglitazone (RGZ) is a thiazolidinedione insulin sensitizer, mainly used for the treatment of type 2 diabetes. Studies have shown that RGZ has both insulin-sensitizing effects and anti-inflammatory and immune-modulating effects. RGZ plays its anti-inflammatory roles in the acute and chronic inflammatory responses of many organs such as the heart, lung, kidney, and gastrointestinal tract[11–14]. Bo et al. reported that RGZ pretreatment alleviated LPS-induced placental inflammation and reduced embryonic mortality in mice[15]. Since RGZ is known to have anti-inflammatory effects, it is meaningful to study its role and potential mechanism in LPS induced endometritis. Therefore, in this study, we established an LPS-induced mouse endometritis model to determine the pathological changes of the mouse endometrium. Human endometrial stromal cells (HESCs) were stimulated with LPS and the anti-inflammatory mechanism of RGZ was assessed by pre-treatment with RGZ to detect TLR4, p-p65, p-IκBα, IL-1β and IL-6. The results of this study provide new ideas and directions for the treatment of endometritis.

Reagents

Rosiglitazone (BRL-49653) were purchased from MedChemExpress (NJ, USA) and dissolved in dimethyl sulfoxide (DMSO). LPS was purchased from servicebio (Wuhan, China). The NF-κB (p65) (F-6) antibody was purchased from Santa Cruz (TX, USA). IκBα (L35A5) mouse mAb, p-IκBα (Ser32) (14D4) rabbit mAb and p-p65 (Ser536) (93H1) rabbit mAb were purchased from Cell Signaling Technology (Beverly, MA, USA). TLR4 (D220102) and GAPDH (D110016) antibodies were obtained from Sangon Biotech (Shanghai, China).

Animals and treatment

All animal studies were conducted in accordance with the ARRIVE guidelines and the guidelines and approval of Animal Use and Care Committee at YanTai Yuhuangding Hospital(Permit No. Permit No. 2021S31). C57BL/6 mice (40 females aged 8 weeks) were randomly divided into four groups (n = 10). The selection of doses was based on a previous report[16, 17]. The murine model of LPS-induced endometritis was performed as the paper described[18]. RGZ + LPS group: mice were intra-uterus infused with 50uL LPS(1μg/mL) two hours after intraperitoneal injection of RGZ (10 mg/kg). RGZ control group: mice were intra-uterus infused with phosphate buffered saline (PBS) two hours after intraperitoneal injection of RGZ. LPS group: mice were Intra-Uterus infused with LPS two hours after intraperitoneal injection of 200 μL PBS. DMSO control group received equal volumes of normal PBS. (RGZ solvent is DMSO, PBS containing 0.1% DMSO for intraperitoneal injection). After 24 hours later, the mice were sacrificed by cervical dislocation, and the mouse uterus was taken for fixation, embedding, Hematoxylin and eosin (HE) staining and immunohistochemical staining.

Histopathologic evaluation of the uterus tissue

All endometritis tissues isolated from cervical dislocation mice were fixed in 10 % formalin, dehydrated in ethanol, and embedded in paraffin. After that, the tissue was sliced into 5µm and stained with HE. The sections were observed under optical microscope (Leica, Wetzlar, Germany). Histology scoring was carried out according to injury degree such as the integrity of tissue structure and the numbers of infiltrated inflammatory cells^[17].

Immunohistochemistry

Immunostaining for TLR4 was performed on 5 µm sections of formalin-fixed, paraffin-embedded endometrioid biopsies. The slides were deparaffinized using fresh xylene and then rehydrated with gradient-grade ethanol. Antigen retrieval was carried out in a microwave oven at 100°C for 20 min, with subsequent cooling to ambient temperature. The slides were then incubated with 3% H₂O₂ for 10 min and then blocked with 3% bovine serum albumin (BSA). The sections were incubated with anti-TLR4 antibody at a dilution of 1:100 at 4°C overnight and incubated with horseradish peroxidase (HRP) conjugated goat anti-rat secondary antibody for 30 min, at 1:400 dilution and 37˚C to amplify the signal. The sections were incubated with immunoreactivity complexes detected by 3, 3’-diaminobenzidine tetrahydrocholoride. The slides were then dehydrated using a gradient-grade ethanol and xylene series according to routine dehydration steps, and finally fixed with neutral resin. Immunohistochemical images were taken with an optical microscope. The immunohistochemical staining was analysed with ImageJ software to obtain the mean optical density (integrated optical density/total area). Four slides per mouse sample were randomly analysed for immunohistochemical quantification.

Cell culture and treatment

Human endometrial stromal cells (HESCs, ATCC, Manassas, VA, USA) were cultured with phenol red-free DMEM/F12 (GIBCO, Grand Island, NY, USA) supplemented 10% charcoal stripped fetal bovine serum (FBS) and 1% Penicillin-Streptomycin and maintained at 37℃ with 5% CO₂. Once HESCs reached 90% confluence, and then digested with 0.5% Trypsin and passed down from 1 to 2 or 3. The cultured HESCs were stimulated with LPS in a dose range of (0.01,0.05,0.25,1,5μg/mL) for 24 h to detect the secreation of inflammatory factors of IL-1β and IL-6. To study the anti-inflammatory effects of RGZ, the cells were pretreated with various concentrations of RGZ (10 and 20 μM/L) for 30 min thereafter stimulated with LPS (1μg/mL) for 24 h.

CCK8 assay

Cell viability was determined by a Cell Counting Kit-8 (CCK-8) assay kit (sparkjade, China). HESCs cells were seeded at 0.3× 10⁵ cells/well in 96-well plates overnight and then treated with various concentrations of RGZ (0,5,10,20,40 μM/L) for 24 h. Subsequently, the cells were treated with 100 μL mixture containing DMEM/F12 medium and 10% CCK-8 reagent. After 30min culture at 37 °C, HESCs cells was analyzed the optical density (OD) immediately at 450 nm by microplate reader (Thermo scientific, Waltham, MA, USA).

RT-PCR

Total RNA was extracted from obtained HESCs by TRIzol reagent according to routine procedures. Total RNA and complementary DNA were prepared in accordance with the manufacturer’s instructions and performed on a real time PCR machine (FTC-3000P Funglyn Biotech, Toronto,Canada). The relative expression of each target gene compared with the corresponding β-actin threshold cycle (CT) values calculated using the 2^-ΔΔCt method. Table.1 showed the primers of IL-1β, IL-6, TLR4 and β-actin.

Western blot

Total protein was harvested from uterus tissues and cells using RIPA lysis buffer supplemented with a protease inhibitor. The total protein concentration was measured with a BCA protein assay kit (sparkjade, China). Next, equal amounts of protein were fractionated using 10% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF, Millipore, USA) membranes. The membranes were blocked with 5% skim milk for 1 h and then incubated with the indicated primary antibodies (1:1000 dilution) at 4 °C overnight. After incubation with the horseradish peroxidase-conjugated second antibodies, the intensities of the proteins were analyzed with a Vilber Fusion FX5 Spectra (Vilber Lourmat, France). GAPDH served as an internal standard.

Immunofluorescence

To study the anti-inflammatory effects of RGZ, the cells were pretreated with various concentrations of RGZ (10 and 20 μM/L) for 30 min thereafter stimulated with LPS (1μg/mL) for 24 h. HESCs were fixed with paraformaldehyde, permeabilized with PBS containing 0.3% Triton X-100, exposed to 3% BSA, and incubated with indicated antibodies at 1:200 dilution at 4 °C overnight. Thereafter, the cells were incubated with FITC-labelled secondary antibodies at 1:100 dilution for 1 h at 37 °C, and the nuclei were stained for 10 min with DAPI dye and visualized under a fluorescence microscope.

Statistical analysis

All data were analyzed using GraphPad Prism 6. Statistical significance was determined using Student’s t-test for two groups or one-way analysis of variance (ANOVA) for multiple group comparisons. The data was expressed as the means ± SEM. Values of p < 0.05 were considered statistically significant.

The effect of RGZ on uterus tissue pathologic changes during LPS-induced endometritis

The effect of RGZ on LPS-induced uterus tissues histopathologic changes in mice is shown in Fig.1. Both DMSO control and RGZ control groups exhibited a normal structure. In the LPS group, the uterus tissue appeared markedly damaged, including hyperemia, hemorrhage, many neutrophils infiltrating the uterus and the shedding of epithelial cells. Compared with the LPS group, the degree of tissue loss in the RGZ+LPS group was reduced. Student’s t-test was used to statistically analyze the histological scores of the control groups and the experimental test groups. The results showed that there was no difference between the control groups, and there was a significant difference between LPS and LPS+RGZ, suggesting that RGZ could significantly improve the pathological changes of endometritis caused by LPS. To further explore the protective effect of RGZ on LPS-induced endometritis, we determined the expression of TLR4 in uterine tissue in different test groups. The IHC （全称是什么）results showed that the expression of TLR4 between the control groups was very low and there was no difference, and the expression of TLR4 after RGZ treatment was significantly lower than that of the LPS group(Fig.2). Taken together, these results suggest that RGZ may suppress inflammation by inhibiting TLR4.

The effect of RGZ on TLR4 expression and phosphorylated IκBα and p65 during LPS-induced endometritis

As shown in Fig. 3a, b, compared with other groups, the uterine tissue of LPS-stimulated group had the highest expression of TLR4. The expression of TLR4 in uterine tissue decreased after RGZ treatment. Furthermore, overexpression of TLR4 weakened the effect of RGZ on LPS-induced p65 phosphorylation and IκB expression in HESCs (Fig. 3c, d). These results strongly suggest that RGz inhibits LPS- TLR4- pathway to alleviate LPS induced inflammatory response.

Effects of RGZ on cell viability and cytokines production

The cells were grown in experimental media containing various concentrations and the potential cytotoxic effects of RGZ on HESCs were examined using the CCK-8 assay.

The results are provided in Fig. 4 and 5. After 24 hours, there was no significant difference in the optical density values between the groups of cells after exposure to RGZ treatment below 20 μM/L (Fig. 4). The results of the CCK-8 assay suggested that RGZ at the concentrations used (10 μM/L and 20 μM/L) had no cytotoxic effect on HESCs(p =0.15 and p =0.15 ), but RGZ at the concentrations used (40 μM/L) had significant cytotoxic effect on HESCs(p =0.04 ) . To explore whether RGZ has a similar anti-inflammatory effect in vitro. We first treated the cells with different concentrations of LPS (0.01,0.05,0.25,1,5μg/mL) for 24 h to mimic the inflammatory state induced by LPS in vitro. Since both IL-1β and IL-6 expressed the highest at LPS concentration of 1μg/mL, the subsequent experiments used LPS at a concentration of 1μg/mL. Next, we measured the protein levels of IL-1β and IL-6, and the results showed that the levels of IL-1β, IL-6 and TLR4 were decreased in a dose-dependent manner by RGZ treatment (Fig. 5 c-e). This was consistent with the in vivo results.

RGZ blunts LPS-induced activation of TLR4 and NF-κB in vitro

As displayed in Fig. 6, the levels of TLR4, Phospho-p65 (p-p65) and Phospho-IκBα (p-IκBα) were noticeably increased in the LPS group relative to the control group. Their levels were greatly reduced in the RGZ-treated group. And this effect showed some dose-dependent, high concentrations of RGZ abolished these expressions higher than low concentrations. Moreover, immunofluorescence staining demonstrated that RGZ treatment significantly reduced nuclear translocation of NF-κB p65 (Fig. 7), and these observations further confirmed the inhibitory effect of RGZ on NF-κB activation, which is in full agreement with the in vivo findings.

In recent years, animal models of LPS-induced endometritis have been widely used in the study of this disease[18, 20]. In this study, intrauterine infusion of LPS was used in mice to construct an endometritis model[19]. We found that after LPS exposure, the mouse endometrium had pathological changes and obvious inflammatory cell infiltration, indicating that LPS intrauterine injection successfully established the endometritis mouse model. Intraperitoneal injection of RGZ before LPS perfusion significantly relieved endometritis, along with decreased inflammatory cell infiltration and decreased TLR4 expression. These results indicated that RGZ inhibited LPS-induced endometritis.

In this study, the toxic effect of RGZ on HESCs was evaluated by MTT assay, and RGZ treatment did not significantly affect the normal growth activity of HESC until the concentration of RGZ reached 40 µM/L when cell viability decreased significantly. This demonstrates that the drug concentrations selected for treatment in this study did not cause significant cytotoxicity. Under normal conditions, p65 binds to IκBα in the cytoplasm. When stimulated by LPS, IκBα will be phosphorylated and degraded, while p65 will be phosphorylated and enter the nucleus to promote the transcription of nuclear genes, which leads to the release of a large number of inflammatory factors[21, 22]. In this study we found that LPS dose-dependently induced HESC to secrete IL-1β and IL-6, while RGZ dose-dependently reduced LPS-induced IκBα and p65 phosphorylation, inhibited the activation of NF-κB signaling pathway, and inhibited the expression of IL-1β and IL-6.

As a key receptor of LPS, TLR4 mediates immune response, which can initiate a cascade of downstream factors, regulate the synthesis and expression of downstream inflammatory mediators, and further activate the translocation of nuclear factor NF-κB to trigger an inflammatory response. Therefore, inhibiting the activation of TLR4/NF-κB signaling pathway proteins may be a target for the treatment of inflammation[23, 24]. We found in this study that RGZ not only reduced the expression of TLR4 in mouse endometrium but also down-regulated TLR4 in HESC cells, accompanied by a decrease in the expression of inflammatory factors, which were similar to the effects of a TLR4 inhibitor (Fig. 8).Jiang et al. reported that TLR4 knockout significantly reduced the phosphorylation level of p65 and the expression of IL-1β and TNF-α after LPS stimulation[25]. Kadam et al. reported that in an LPS-induced preterm birth model, RGZ reversed the inflammatory effect of LPS to prevent preterm birth by down-regulating TLR4 and up-regulating antioxidant responses[17]. RGZ inhibits LPS-induced inflammation by reducing pro-inflammatory factor production through the TLR4/NF-κB signaling pathway[26, 27]. Although RGZ is an agonist of PPARγ, and the activation of PPARγ is known to have anti-inflammatory effects[28, 29], PPARγ was not detected in HESCs by Western blot in this study due to the limitation of experimental conditions and the low expression of PPARγ in HESCs. Therefore, whether RGZ exerts anti-inflammatory effects through PPARγ receptors in addition to the TLR4/NF-κB pathway in HESCs remains unclear.

In conclusion, LPS can induce HESCs to secrete inflammatory factors and participate in the initiation and development of endometritis. RGZ not only inhibited endometritis in mice but also inhibited the production of inflammatory factors induced by LPS in HESCs in a dose-dependent manner. RGZ was involved in the inhibition of TLR4/NF-κB-mediated inflammatory pathways. These findings suggest that RGZ has the potential to treat endometritis. RGZ may become a new drug for the treatment of endometritis, and the in-depth study of its mechanism of action has important theoretical significance for the prevention and treatment of this disease.

Ethics approval and consent to participate

All animal experiments confirmed in the manuscript were carried in accordance with the ARRIVE guidelines. (https://arriveguidelines.org).

(The Ethics committee of the Yantai Yuhuangding Hospital of Qingdao University, Yantai, China, approved on February 05, 2021; Protocol No. 2021S31).

Consent for publication

Not applicable.

Availability of data and materials

Data and materials available on request from the Corresponding author.

Competing interests

The author declares no competing interests.

Funding

The study was supported by Shandong Medical and Health Science and Technology Development Project (No.202005030944).

Authors’ contributions

Ding Ma, Hongchu Bao, Qinglan Qu and Jianxiang Cong performed the experiments. Ding Ma, Shuyuan Yin, Huishan Zhao, Shunzhi he and Dongmei Zhao designed supervised the study, performed the statistical analyses, and wrote the manuscript. All authors read and approved the manuscript, all data were generated in-house, and that no paper mill was used.

Acknowledgements

Not applicable.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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Rosiglitazone alleviates LPS-induced endometritis via suppression of TLR4-mediated NF-κB activation

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