Inhibition of Jiawei Foshou San on invasion and metastasis through MMP/TIMP signaling in endometriosis


 Background: The new formula Jiawei Foshou San (JFS) is consisted of ligustrazine, ferulic acid and tetrahydropalmatine designed from Foshou San . Previously JFS inhibited the growth of rat autograft endometriosis with unclear mechanism. To uncover the effect of JFS on invasion and metastasis in endometrial cells and xenograft endometriosis. Methods: In vitro , cell viability assay was performed for IC50 measurement in hEM15A and HEC1-B cells after treating JFS. Effects of JFS on invasion and metastasis were analyzed in scratch wound and transwell assay. In vivo , effect of JFS was evaluated in xenogeneic transplantation of endometriosis model. The gene and protein expression of MMP/TIMP signaling were inspected in vitro and in vivo . Results: Inhibitory effects of JFS were investigated with dose-dependent manner in hEM15A and HEC1-B cells. JFS significantly inhibited the invasion and metastasis in dose- and time-dependent manner. In xenograft endometriosis, JFS reduced the volume of ectopic endometrium. In-depth study, inactive MMP/TIMP signaling expressed the lower MMP-2/9, higher TIMP-1 by JFS in vitro and in vivo . Conclusions: JFS prevent invasion and metastasis via inactivation of MMP/TIMP signaling in endometrial cells and xenograft endometriosis. It reveals the potential mechanism of JFS on endometriosis and the benefit for further application.


Background
Endometriosis (EMS) is a disease caused by the active endometrial cells growing outside the endometrium. Although the unclear pathogenesis of EMS, the known menstrual re ux hypothesis regards that implantation of viable endometrial cells is the essential process in the menstrual e uent of menstruation. This pathological step mainly contains invasion and metastasis [1,2]. The imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are the vital mechanism of invasion and metastasis. MMP-2 and MMP-9 promote the invasion and metastasis through degradation of extracellular matrix. On the contrary, TIMP-1 has the antagonistic effects on MMPs [3].
EMS belongs to blood stasis syndromes with treatment of activating blood and dissolving stasis in traditional Chinese medicine [4,5]. Famous gynecological prescription Foshou san is performed in blood stasis syndromes, including endomtriosis [6]. JFS, a new Chinese compound, is designed from Foshou san, which is composed of ferulic acid, ligustrazine and tetrahydropalmatine. In previous studies, the potential therapeutic effect of JFS have been detected in rat autograft EMS model [5,7,8], but with unclear effect on endometrial cells and xenograft EMS model.
In this paper, IC50 of JFS on hEM15A and HEC1-B endometrial cells were measured in vitro. Then the antimetastasis of JFS were detected by scratch wound and transwell assays. Therapeutic effect of JFS on xenograft EMS was observed in vivo. The regulations of MMP/TIMP signaling were investigated by JFS in vitro and in vivo.  Scratch wound assay hEM15A and HEC1-B cells were put in a 24-well plate at a density of 6-8×10 4 , 8-10×10 4 cells/well respectively. When the cell con uence reached approximately 80%, wounded traces were produced by 1 ml pipette tip vertically in the well plate. Added medium with JFS, endometrial cells were observed and photographed under 50´ magni cation at 0, 12, 24 and 48 h. The area of the scratched area was calculated using Image pro plus 6.0 software (Media Cybernetics, Silver Spring, 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
The transwell inserts without matrigel (Corning, New York, USA) were put into a 24-well plate. 3×10 4 hEM15A or HEC1-B cells/well were placed into upper chamber with serum-free JFS medium. 5% FBS medium without JFS were added to the lower chamber. After incubation at 37℃ for 12, 24 and 48 hours, non-migrating cells on the surface of upper chamber were swiped by a cotton swab. The migrated cells in bottom were xed with Paraformaldehyde, and then stained with crystal violet. The migratory cell numbers were counted in 5 random elds at 100×magni cation.

Xenograft EMS model and treatment
As attested by the previous study [9,10], bilateral uterus of estrus C3H mice were cut into 4 mm 2 tissue blocks and transplanted into the subcutaneous abdomen of 71 nude mice. After operation, nude mice were administrated with 2 mg·kg −1 estradiol benzoate by intramuscular injection once every 5 days.
Volume of endometrial allografts was calculated by vernier caliper after 28 days with a formula (0.52×length×width×height). Endometrial allograft explants, larger than 4.5 mm 3 with surface blood, were regarded as the successful EMS models in the second laparotomy. Then 4 groups were randomly established with EMS mice, including were EMS group with 0.5% CMC-Na, 90, 180, 360 mg·kg −1 Neiyixiao groups, and 0.05 mg·kg −1 gestrinone group. Another 6 female C3H mice without transplantation were administered 0.5% CMC-Na as control group. After administration for 28 days by gavage, the volumes of the ectopic tissues were measured again.

RNA isolation and qPCR
Total RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA) and then reversely transcribed into cDNA using the PrimeScript™ RT Reagent Kit (Takara, China). Real-time PCR was conducted in CFX96 Real-Time System (Bio-Rad, USA) and fluorescent quantification was detected with SYBR™ Green Master Mix (Thermo Fisher Scientific, USA). The relative mRNA expression was calculated by the 2 -∆∆CT method using primer sequences (Table 1), which were synthesized by Dingguo Changsheng Biotechnology (Beijing, China). GAPDH gene as the control reference. Table 1  Chemiluminescent signals were captured analyzed with the Tanon 5200 imaging system (Tanon, China). β-actin was used as an internal control.

Statistical analysis
All data were presented as the mean ± SD and likened by one-way ANOVA test applying SPSS 21.0 software. P<0.05 was considered statistically significant.

IC50 of JFS on endometrial cells
To reveal the IC50 of JFS on hEM15A and HEC1-B cells, these two cells viability were measured by MTT method after treated with different concentrations of JFS respectively for 24 or 48 h. In hEM15A cells, IC50 of JFS were 839.30±121.11 or 483.53 ±156.91 μg.ml -1 for 24 or 48 h respectively (Fig. 1A). In HEC1-B cells, the IC50 values were 625.20±59.52 or 250.30±68.12 μg.ml -1 for 24 or 48 h JFS treatment respectively (Fig 1 B). Moreover, signi cant inhibitory effects of JFS were investigated with dosedependent manner in hEM15A and HEC1-B cells. It was also notable that IC50 of JFS on HEC1-B cells were obviously lower than that on hEM15A cells. It was indicated that HEC1-B cells might be more sensitive to JFS.

Constraint of endometrial cell migration and invasion by JFS
Scratch wound assay is commonly used to detect the cell migration. IC50 concentration of JFS was performed as middle dose. The low or high dose was half or double of IC50 concentration. The endometrial cells were observed for 12 and 24h with IC50 24h JFS, or for 24 and 48h with IC50 48h JFS.
After treated with JFS for 12 and 24h, wound closure of JFS were obviously decreased compared with control in hEM15A and HEC1-B cells (P<0.01) ( Fig. 2A-D). Meanwhile, JFS signi cantly resisted the spread of both cell lines along the edge of wound scratch, with no effect of 240 μg.ml -1 JFS in hEM15A cells (Fig. 2E-H). This indicated that JFS markedly inhibited cell migration with dose-and time-dependent manner in scratch wound assay.
Transwell assay was performed to measure the effects of JFS on cell migration and invasion. Maintain identical time and concentration of JFS were applied as previous scratch wound experiment. Results showed that JFS signi cantly reduced the number of migratory cells passing through the chamber membrane vs control group in hEM15A (Fig. 3A, C, E, G) and HEC1-B cells (Fig. 3B, D, F, H). This suggested that JFS obviously suppressed cell migration and invasion with dose-and time-dependent manner in transwell assay.
Inhibition of ectopic endometrium volume using JFS 28 days after transplantation, 56 of 71 nude mice were found xenograft EMS in the second laparotomy.
The successful rate of model was 79%. There was no remarkable difference in the volume of the ectopic endometrium between all groups before administration. After treatment for 28 days, the volume of ectopic issue was detected in different groups and compared with pretreatment. In the EMS group, the volume of ectopic endometrium had no difference between posttreatment with pretreatment. There were significantly depression observed in 90, 180 and 360 mg·kg -1 JFS groups, respectively (P<0.05). The volume of ectopic endometrium tissue was minimized in gestrinone group (P<0.05) (Fig. 6A). This suggests that JFS restrained the growth of transplant in a dose-independent manner.

Adjustment of MMP/TIMP signaling by JFS in vivo
After treatment with JFS, genes and proteins of MMP-2, MMP-9, and TIMP-1 were detected in ectopic endometrial tissues. As evidenced by qRT-PCR, the mRNA level of MMP-2 and MMP-9 were signi cantly higher while TIMP-1 was lower in EMS group than those in control group (P<0.01). The mRNA level of MMP-2 and MMP-9 were obviously downregulated and TIMP-1 was upregulated in JFS groups than those in EMS group (P<0.05) (Fig. 6B-D). The protein levels of MMP-2 and MMP-9 were remarkably raised with reducing TIMP-1 in EMS group compared with control group (P<0.05). JFS obviously decreased the protein level of MMP-2 and MMP-9, and increased the protein level of TIMP-1 (P<0.05) (Fig. 6E-H).

Discussion
JFS has been proved to display the potential anti-endometriosis effects in rat autograft EMS model [5,7,8]. The underlying mechanism of JFS embrace diminishing the growth of EMS, suppression of E2, in ammation and epithelial mesenchymal transformation. Therefore, we detected the effects of JFS on endometrial cells and xenograft EMS model. In this study, JFS markedly inhibited invasion and metastasis both in vitro and in vivo. It related to the decrease of MMP-2, MMP-9, and increase of TIMP-1.
In cell viability assay, IC50 of JFS were different in hEM15A and HEC1-B cells, which were human endometriosis-derived eutopic endometrium stromal cells and endometrial adenocarcinoma cells. It was also notable that IC50 in HEC1-B cells were obviously lower than that in hEM15A cells. It was indicated that HEC1-B cells might be more sensitive to JFS. There are no researches of JFS or JFS ingredients, ferulic acid, ligustrazine, or tetrahydropalmatine on endometrial cancer, except Angelica sinensis showed weak binding with ER in endometrial cancer cells [11]. Therefore, our results are suggested that JFS or JFS ingredients might have the potential effects on endometrial cancer. It needs further research.
In traditional Chinese medicine, blood stasis syndromes should be treated with Huoxue Huayu recipes to activate blood and dissolve stasis [12,13]. Whether activating blood and dissolving stasis mean inhibit invasion and metastasis or not will depend on individual situations. On one hand, the suppression of metastatic potential is found in ligustrazine, ferulic acid, or tetrahydropalmatine in various kinds of cancer, for example osteosarcoma, brosacroma, and breast cancer [14][15][16]. Especially, ligustrazine shows downregulation of MMP-2/9, and upregulation of TIMP-1. In neuropathic pain or blood-brain barrier injury, ligustrazine or tetrahydropalmatine decreases the expression of MMP-2/9 [17,18]. On the other hand, using ligustrazine, bone marrow mesenchymal stem cells are promoted to migrate by raising MMP-2/9 [19]. In our experiment, JFS inhibited the growth of endometrial cells and ectopic endometrium in xenograft EMS model. Invasion and metastasis were restrained by JFS, thrgouth suppressing MMP-2/9 and accumulating TIMP-1 in vivo and in vitro. Considering the con icting reports, it is worthwhile to explore role of JFS on different blood stasis syndrome diseases.

Availability of data and materials
The datasets in this study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate The animal care and experimental procedures in this study were approved by the Institutional Animal Care and Use Committee of Southwest University.

Consent for publication
Not applicable.