Combination of ferulic acid, ligustrazine and tetrahydropalmatine inhibits invasion and metastasis through MMP/TIMP signaling in endometriosis

Animals and chemicals

Female C3H mice, aged 8 ± 1 weeks, weighing 19 ± 1 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certification no. SCXK [Jing]2016-0006) (Beijing, China). Female nude mice, aged 8 ± 1 weeks, weighing 19 ± 1 g were purchased from Hunan Silaike Jingda Laboratory Animal Co., Ltd. (Certification no. SCXK [Xiang]2016-0002) (Hunan, China). The mice were sheltered at a Ta of 20 ± 2 °C and 12 h light/dark cycle. They were supplied with enough food and water in Experimental Center of Southwest University. This study was executed in rigorous accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of Southwest University. This study was approved by the Ethics Committee of the School of Pharmacy, Southwest University (Approval no. 201702). To minimize suffering, anaesthesia and other necessary efforts were performed. A total of 350 or 1,750 mg·kg⁻¹ chloral hydrate was administered intraperitoneally in experimental or sacrificed procedure separately. All animals were sacrificed at human endpoint.

Ferulic acid, ligustrazine and tetrahydropalmatine were purchased from Nanjing Zelang Medical Technology Co., Ltd (Nanjing, China), with the purity of 99.8, 99.3, 98.1% respectively. In vitro, the compounds dissolved in DMSO were mixed at the ratio of 1:0.5:0.3 for cells treatment. In vivo, three compounds mixed with the same ratio in 0.5% CMC-Na for mice treatment. Gestrinone was used as a positive drug provided by Zizhu Pharmaceutical Co., Ltd. Beijing, China.

Allograft EMS model and treatment

Allograft EMS mice model was established following previous protocols (Attaman et al., 2014; Cheng et al., 2011). C3H and nude mice were anaesthetized before operation. Bilateral uterus of estrus C3H mice were cut into 4 mm² blocks and transplanted into subcutaneous abdomen of 40 nude mice. After operation, 2 mg·kg⁻¹ estradiol benzoate was intramuscularly administrated in nude mice once every 5 days. Ectopic volume was calculated by vernier caliper after 28 days with a formula (0.52 × length × width × height). Endometrial ectopic explants were larger than 4.5 mm³ with surface blood, indicating that EMS models were successful established. Next, 30 EMS mice randomly were divided into five groups based on random number list, 6 mice in each group. Then the mice were named after the EMS, FLT, and gestrinone groups and treated with 0.5% CMC-Na, 90, 180, 360 mg·kg⁻¹ FLT and 0.05 mg·kg⁻¹ gestrinone, respectively. Another 6 female C3H mice without transplantation were administered with 0.5% CMC-Na as control group. During 28 days’ treatment, weight of nude mice was measured every 7 days. After administration for 28 days, ectopic volumes were measured again.

Cell culture

Human EMS-derived eutopic endometrium stromal cells hEM15A presented by Professor Xiaohong Chang of Peking University People’s Hospital (Beijing, China). The endometrial adenocarcinoma cells HEC1-B and human kidney 293T cells were purchased from the Chinese Centre for Type Cultures Collections (CCTCC, Wuhan, China). hEM15A, HEC1-B or 293T cells were cultured in DMEM/F12 or MEM medium (Gibco, Grand Island, NY, USA) with 10% FBS (Hyclone, Shanghai, China) at 37 °C with 5% CO₂.

Cell viability assay

hEM15A, HEC1-B and 293T cells were seeded at 6-8 × 10³, 8–10 × 10³, 8–10 × 10³ cells/well in 96-well plates respectively. hEM15A cells were treated with FLT (0, 156, 313, 625, 1,250 and 2,500 μg·ml⁻¹), and HEC1-B cells were cultured with FLT (0, 15, 46, 139, 417 and 1,250 μg·ml⁻¹) for 24 or 48 h. Meanwhile, 0, 840, 1,680 and 3,360 μg·ml⁻¹ FLT were used in 293T cells for 24 or 48 h. After treatment, cells were incubated with MTT solution for 4 h. Then cells were measured in a microplate reader after dissolved in DMSO. IC50 values of hEM15A, HEC1-B cells were calculated by GraphPad Prism5 Software (5.01 version, La Jolla, CA, USA).

Scratch wound assay

hEM15A and HEC1-B cells were put in 24-well plate at a density of 6–8 × 10⁴, 8–10 × 10⁴ cells/well. When cell confluence reached approximately 80%, the middle of well was vertically scratched by 1 ml pipette tip. Using FLT, endometrial cells were observed and photographed under 50× magnification at 0, 6, 12, 24 and 48 h. Scratched area was calculated using Image pro plus 6.0 software (Media Cybernetics, Silver Spring, southeastern Montgomery County, MD, USA). Scratch closure rate = (average scratch area at 0 h − average scratch area at each time point)/(average scratch area at 0 h) × 100%.

Transwell assay

Transwell inserts (Corning, New York, NY, USA) without matrigel were put into 24-well plate. A total of 3 × 10⁴ hEM15A or HEC1-B cells/well were placed into upper chamber with serum-free FLT medium. A total of 5% FBS medium without FLT were added to lower chamber. After incubation at 37 °C for 6, 12, 24 and 48 h, surface cells on upper chamber were swiped by cotton swab. Migrated cells in bottom were fixed with paraformaldehyde, and then stained with crystal violet. Migratory cell numbers were counted in 5 random fields at 100× magnification.

RNA isolation and qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then reversely transcribed into cDNA with PrimeScript™ Reagent Kit (Takara, China). Real-time PCR was conducted in CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) and fluorescent quantification was detected with SYBR™ Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Primer of mice and human were synthesized by Dingguo Changsheng Biotechnology (Beijing, China). Relative mRNA expression was calculated by 2−∆∆CT method using primer sequences (Table 1), which. GAPDH gene as the control reference.

Western blot analysis

Total Proteins of tissues and cells were extracted by RIPA lysis buffer with protease inhibitors on ice. Equal amounts of protein were separated by SDS-PAGE and transferred onto poplvinylidene fluoride membrane (Millipore, USA). The membranes were incubated overnight at 4 °C with primary rabbit anti-MMP-2 and rabbit anti-MMP-9 (1:100 dilution; Boster Biological Technology, Wuhan, China), rabbit anti-TIMP-1 (1:300 dilution; Proteintech Biotechnology, Wuhan, China), rabbit anti-β-actin (1:5,000 dilution; Proteintech Biotechnology, Wuhan, China). After washing with TBST, membranes were incubated with HRP-labeled goat anti-rabbit secondary antibody (1:1,000 dilution; Multi Sciences, Hangzhou, China). Chemiluminescent signals were captured analyzed with the Tanon 5200 imaging system (Tanon, Shanghai, China). β-actin was used as an internal control.

Statistical analysis

All data were presented as the mean ± SD. They were analyzed by one-way ANOVA test in SPSS 21.0 software. P < 0.05 was considered statistically significant. Yi Chen was aware of the group allocation at the different stages of the experiment.

Inhibition of ectopic endometrium volume using FLT

A total of 28 days after transplantation, 30 of 40 nude mice were found allograft EMS in second laparotomy. The successful rate of model was 75%. Meantime, no remarkable difference of ectopic volume was found in all groups before administration. After treatment with FLT for 28 days, ectopic volume was detected and compared with pretreatment. In the EMS group, ectopic volume had no difference between posttreatment and pretreatment. There were significantly reduction in ectopic volume in 90, 180 and 360 mg·kg⁻¹ FLT groups, respectively (P < 0.05). Gestrinone is currently applied in clinic as anti-progestagens steroid (Ferrero, Evangelisti & Barra, 2018), which was used as positive control in this study. Ectopic volume of endometrium tissue was also decreased in the gestrinone group (P < 0.05) (Fig. 1A). Moreover, weight of nude mice had no different between EMS, FLT and gestrinone groups (Fig. 1B). This suggests that FLT restrained EMS growth at all tested concentrations without obvious severe side effects.

Adjustment of MMP/TIMP signaling by FLT in vivo

MMP/TIMP signaling is one of important pathway in metastasis and invasion (Herszenyi et al., 2012). While, interruption of metastasis and invasion lead to EMS amelioration (Chui, Wang & Shih, 2017). In this study, MMP/TIMP signaling was detected in ectopic tissues from EMS, FLT groups, and in eutopic tissues from control group of C3H mice without surgery. As evidenced by qRT-PCR, mRNA level of MMP-2 and MMP-9 were significantly higher while TIMP-1 was lower in the EMS group than those in the control group (P < 0.01). mRNA level of MMP-2 and MMP-9 were obviously downregulated by FLT. By contrast, TIMP-1 was upregulated in 360 mg·kg⁻¹ FLT group than the EMS group (P < 0.05) (Figs. 1C–1E). The protein level was analyzed by Western blotting. MMP-2 and MMP-9 protein levels were remarkably raised while TIMP-1 protein level were decreased in the EMS group compared with those in the control group (P < 0.05). FLT obviously decreased the MMP-2 and MMP-9 protein expression. In contrast, FLT increased TIMP-1 protein level (P < 0.05) (Figs. 1F–1I).

Effect of FLT on endometrial and 293T cells

As noted above, FLT suppressed ectopic growth in vivo. But whether there was similar observation in vitro needs to be examined. Thus we measured the IC50s for the treatment of FLT on hEM15A and HEC1-B cells. 293T cells were performed for toxicity detection. In hEM15A cells, IC50 of FLT was 839.30 ± 121.11 or 483.53 ± 156.91 μg·ml⁻¹ at 24 or 48 h respectively (Fig. 2A). In HEC1-B cells, IC50 value was 625.20 ± 59.52 or 250.30 ± 68.12 μg·ml⁻¹ at 24 or 48 h respectively (Fig. 2B). Moreover, significant inhibitory effects of FLT were investigated with dose-dependent manner in both two cell lines. Notablely, HEC1-B cells was sensitive to FLT. The inhibitory effect of FLT on 293T cells was less than on hEM15A and HEC1-B cells with same concentration indicating a low toxicity in 293T (Fig. 2C).

Constraint of migration and invasion by FLT in endometrial cell

Scratch wound assay is commonly used to detect the cell migration. IC50 concentration of FLT was performed as middle dose. Low or high dose was half or double of IC50 concentration. The endometrial cells were observed for 6, 12 and 24 h with IC50 24h FLT, or for 12, 24 and 48 h with IC50 48 h FLT. Wound closure of FLT were obviously decreased compared with control in hEM15A and HEC1-B cells (P < 0.01), except 420 μg·ml⁻¹ FLT at 6h (Figs. 3A–3D). Meanwhile, FLT significantly resisted cells spread along the edge of wound scratch, with no effect in low dose of FLT at some time point (Figs. 3E–3H). This indicated that FLT markedly inhibited cell migration in scratch wound assay.

Transwell assay was performed to measure FLT effects on cell migration and invasion. Maintain identical time and concentration of FLT were applied as previous scratch wound experiment. FLT significantly reduced migratory cells numbers vs control group in hEM15A (Figs. 4A, 4C, 4E, 4G) and HEC1-B cells (Figs. 4B, 4D, 4F, 4H). This suggested that FLT obviously suppressed cell migration and invasion in transwell assay.

Regulation of FLT on MMP/TIMP signaling in vitro

After treated with FLT for 24 h, mRNA level of MMP-2 and MMP-9 obviously attenuated compared to the control group, while TIMP-1 expanded especially in 960 μg·ml⁻¹ FLT group of hEM15A cells (P < 0.05) (Figs. 5A–5C). Moreover, mRNA level of MMP-2 and MMP-9 were significantly downregulated in HEC1-B cells. By contraries, TIMP-1 was upregulated in 1,250 μg·ml⁻¹ FLT group (P < 0.05) (Figs. 5D–5F).

Furthermore, protein level was consistent with gene expression of MMP/TIMP signaling. MMP-2 and MMP-9 protein decreased in FLT groups, accompanied with TIMP-1 collection vs control group in hEM15A cells (P < 0.01) (Figs. 6A–6D). At the same time, MMP-2 protein in 1,250 μg·ml⁻¹ FLT group, and MMP-9 in 625 μg·ml⁻¹ FLT group were significantly downregulated. Though TIMP-1 were upregulated in 625 and 1,250 μg·ml⁻¹ FLT groups compared with the control group in HEC1-B cells (P < 0.05) (Figs. 6E–6H).