2.1 Cell culture

We brought hEECs from the Procell Life Science & Technology Co., Ltd). The cells were cultured in Ham's F-12 medium (Biological Industries, Kibbutz Beit Haemek) contained with 1% penicillin and streptomycin (Beyotime) and 12% fetal bovine serum (Gibco) in an atmosphere of 5% CO₂, at 37°C.

The hEECs were treated with 1 μg/mL LPS (L5293, Merck) for 3 h to establish an endometritis model in vitro.¹⁹

2.2 Cell treatment

To over-express TNIP2 in hEECs, control-plasmid or TNIP2-plasmid was transfected into hEECs using Lipofectamine 2000 (Invitrogen) for 24 h referring to the manufacturer's protocol.

The hEECs were transfected with control-plasmid or TNIP2-plasmid in the presence or absence of 50 μM CU-T12-9 (NF-κB agonist, HY-110353, purity: 98.94%, MedChemExpress) for 24 h, followed by treatment with 1 μg/mL LPS for 3 h. Then following experiments were performed. The concentration selection of the CU-T12-9 was based on the previously published study.²⁰

2.3 Cell counting kit-8 (CCK-8) assay²¹

Cell growth was tested using a CCK-8 kit (Fcmacs). After LPS stimulation, hEECs were resuspended and seeded into 96-well plates with 2.5 × 10³ cells and treated with 10 μL detection solution for 1.5 h at 37°C with 5% CO₂ in the dark. The optical density (OD) values were measured at 450 nm using an ultraviolet spectrophotometer (Infinite Pro, Tecan).

2.4 Cell apoptosis analysis²²

A total of 2 × 10⁵ LPS-induced cells were harvested with 0.5 × 10³ μL solution containing 5 μL Annexin V-FITC and 5 μL propidium iodide (Beyotime) at room temperature in the dark for 35 min. The apoptotic rate was analyzed using flow cytometry (C6; Thermo Fisher Scientific) and the data were analyzed by Kaluza Analysis (version 2.1.1.20653; Beckman Coulter, Inc.).

2.5 Caspase3 activity assay²³

Activity in caspase3 of cells was calculated using a caspase3 colorimetric kit (Beyotime). The culture medium and cells were then covered with trypsin (BI). Cell samples were incubated with lysis buffer (Proteintech) for 20 min. The samples were stored by centrifugation at 10,000 rpm for 10 min. Finally, the samples were analyzed using a microplate reader (Tecan).

2.6 Enzyme-linked-immunosorbent analysis (ELIZA)²⁴

The cell culture supernatant was harvested and used for detecting the level of interleukin (IL)−1β, IL-6, and tumor necrosis factor-alpha (TNF-α). The ELIZA kits were obtained from Beyotime Biotechnology. All procedures were performed per the manufacturer's instructions.

2.7 Western blot analysis²⁵

hEECs lysis was performed using the RIPA buffer (Beyotime, Shanghai, China). Proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Whatman). Phosphate buffered saline-tween-20 (PBST, Beyotime, Shanghai, China) and 5% nonfat milk powder (CST) were used to block the polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was then incubated for 12 h with primary antibodies (Proteintech) against TNIP2 (15459-1-AP; Proteintech), Caspase3 (19677-1-AP; Proteintech), p65 NF-κB (66535-1-Ig; Proteintech), and phospho-p65 NF-κB (ARG51518; Arigo). The next day, after blocking with a second antibody (Arigo), the pattern was visualized using an ECL luminescent solution (Sangon Biotech), and the grayscale value of the target was counted using ImageJ.

2.8 Lactate dehydrogenase (LDH) assay

The LDH levels of the cells were measured following co-culture with LPS using an LDH activity kit (ARG81306; Arigo), following the manufacturer's instructions. The OD value of each well was quantified at 490 nm to analyze LDH activity.

2.9 Reactive oxygen species (ROS) assay

A ROS kit (ARG81192; Arigo) was used to measure the concentration of ROS after treatment with 1 g/mL LPS per the manufacturer's protocol. Absorbance was measured at 525 nm using a spectrophotometer (Thermo Fisher Scientific).

2.10 Measurement of the activities of catalase (CAT) and superoxide dismutase (SOD)

The CAT and SOD activities of cells were measured following co-culture with LPS using CAT and SOD activity kits (Jiancheng) according to the manufacturer's protocol. The OD of each well was determined at 525 nm.

2.11 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay

Following the supplier's protocol, total RNA was recovered from cells using ISOLATION TRIzol buffer® (Multi Sciences), and cDNA was obtained from reverse-transcribed RNA using an RT-PCR kit (Yeasen). qPCR was performed using the PerfectStart® Sybr qPCR Mix (Vazyme). The expression levels of genes were calculated using the $${2}^{ \mbox{-} \unicode{x02206}\unicode{x02206}{C}_{{\rm{t}}}}$$ method.²⁶ The primer sequences for TNIP2 and-actin were as follows: β-actin:5′-CCATCGCCAGTTGCCGATCC-3′ (F) and 5′-GCGAGAGGAGCACAGATACCACCAA-3′ (R); TNIP2:5′-AAGTCCTGACCAGTCGGAACA-3′ (F) and 5′-CCAGCAGGGACGAATACGTG-3′ (R).

2.12 Statistical analysis

All experiments were repeated for three times. Statistical analyses were performed using SPSS 19.0 statistical software (IBM Corp.). Data are presented as mean ± standard deviation from triplicate experiments. Differences between the two groups were assessed using Student's t test, whereas one-way analysis of variance followed by Tukey's test was performed to analyze differences among multiple groups. Statistical significance was set at p < .05 means statistical difference.

3.1 TNIP2 expression was decreased in LPS-induced endometritis cell model

RT-qPCR and western blot analysis were performed to determine TNIP2 expression in LPS-induced endometritis cell model. Compared to the control group, the TNIP2 protein level was dramatically downregulated in the LPS-stimulated group, and the results of RT-qPCR were the same as those of western blot analysis (Figure 1A and B).

3.2 Overexpression of TNIP2 inhibited the apoptosis of hEECs

To observe the effect of TNIP2 on the endometritis model, hEECs were pretransfected with control plasmid or TNIP2-plasmid; 24 h later, the cells were treated with 1 μg/mL LPS for 3 h, and subsequent experiments were conducted. As shown in Figure 2A and B, the RT-qPCR and western blot analysis results indicated that, compared to the control plasmid group, the TNIP2-plasmid significantly increased the TNIP2 expression in hEECs. Compared to the control group, LPS treatment significantly inhibited cell viability (Figure 2C), enhanced the activity of LDH (Figure 2D), increased the rate of apoptosis (Figure 2E and F), increased the caspase3 activity (Figure 2G), and increased the cleaved-caspase3 protein expression level in hEECs (Figure 2H and I). All of the above results were reversed by TNIP2-plasmid transfection.

3.3 High TNIP2 expression inhibited the inflammation and oxidative stress in LPS-induced hEECs

Endometritis is accompanied by the production of large quantities of inflammatory cytokines. Compared with the control group, the secretion of TNF-α, IL-1β, and IL-6 in hEECs in the LPS group was significantly increased (Figure 3A–C). Similarly, released ROS levels were elevated in the LPS-treated hEECs (Figure 3D). The activities of SOD and CAT were also disturbed (Figure 3E and F) in LPS-induced hEECs. All of the above results were reversed by TNIP2-plasmid transfection.

3.4 NF-κB was activated in LPS-induced hEECs

To explore whether NF-κB was involved in the endometritis pathogenesis, hEECs were pretransfected with control-plasmid or TNIP2-plasmid, after 24 h, cells were induced with 1 μg/mL LPS for 3 h. p-p65 and p65 protein expression was detected by western blot analysis and the p-p65/p65 ratio was calculated. Compared to the control group, the p-p65 protein expression and p-p65/p65 ratio in hEECs in the LPS group were increased, suggesting the activation of NF-κB pathway by LPS in hEECs. And co-transfection with the TNIP2-plasmid reversed this phenomenon (Figure 4A and B).

3.5 Protective effect of TNIP2 in endometritis was destroyed by NF-κB activation

To explore the specific mechanism of TNIP2 in the course of endometritis in this study, hEECs were pretransfected with a control-plasmid/TNIP2-plasmid or treated with NF-κB agonist CU-T12-9 for 24 h and then treated with 1 μg/mL LPS for 3 h. In LPS-induced hEECs, TNIP2-reduced p-p65 protein expression and the p-p65/p65 ratio decline were significantly reversed by CU-T12-9 (Figure 5A and B). In addition, the findings indicated that compared with the LPS + control-plasmid group, TNIP2-plasmid transfection significantly increased cell viability (Figure 6A), downregulated the LDH activity (Figure 6B), reduced the ratio of apoptosis (Figure 6C and D), and decreased caspase3 activity and the protein pattern of cleaved-caspase3 (Figure 6E–G) in LPS-induced hEECs. All of the results described above were reversed by the CU-T12-9 treatment.

Moreover, the TNIP2-plasmid-induced TNF-α, IL-1β, and IL-6 secretion downregulation in LPS-induced hEECs were significantly inhibited by CU-T12-9 co-treatment (Figure 7A–C). Meanwhile, the TNIP2-plasmid induced reduction in ROS production and the increase in SOD and CAT activity was reversed by CU-T12-9 (Figure 7D–F).