Tanshinol inhibits trophoblast cell migration and invasion by regulating Gadd45a in preeclampsia

Abstract Objective Tanshinol is an active constituent of Salvia miltiorrhiza that possesses anti-inflammatory, antioxidant, and antibacterial activities. Therefore, this study attempted to detect whether it has a role in the treatment of preeclampsia (PE). Methods In this study, we explored the effect of tanshinol on the development of PE at the cellular level. The effect of tanshinol on cell proliferation was measured by colony formation and EdU assays. The migration, invasion, and in vitro angiogenesis of HTR-8/SVneo cells were detected by wound-healing, transwell, and tube formation assays, respectively. In addition, a PE cell model was established by overexpression of Gadd45a, and this cell model was assessed with the optimal concentration of tanshinol. Results The results show that tanshinol enhanced proliferation, migration, invasion, and tube formation of HTR-8/SVneo cells in vitro. Furthermore, the reduction in proliferation, migration, invasion, and tube formation of cells by Gadd45a overexpression was partially reversed by tanshinol treatment. Tanshinol also inhibited the apoptosis of HTR-8/SVneo cells transfected with Gadd45a. Conclusions In summary, tanshinol promoted proliferation, migration, invasion, and tube formation and inhibited the apoptosis of HTR-8/SVneo cells. It may be a novel therapeutic compound to attenuate the development of PE. PLAIN LANGUAGE SUMMARY Traditional Chinese medicine has maintained the health of people in Asia for thousands of years and is increasingly used worldwide. Tanshinol has been found to be useful in the treatment and prevention of many diseases. Through experiments, we found that tanshinol is a novel therapeutic compound that promotes the proliferation, migration, invasion and tubular formation of HTR-8/SVneo cells. In addition, tanshinol also inhibited the apoptosis rate of preeclampsia cell models. Follow-up experiments will further validate the results of this study.


Introduction
Preeclampsia (PE) is a pregnancy-related disorder that can develop in pregnant women after 20 weeks of gestation, and it is clinically characterised by hypertension and proteinuria (Burton et al. 2019).PE is a cause of neonatal and maternal morbidity and high mortality, and it occurs in approximately 2%-8% of all pregnancies worldwide (Redman and Sargent 2005).PE is a serious complication of pregnancy that can eventually lead to maternal multisystem dysfunction (Mayrink et al. 2018).It is also a multisystem syndrome, which has both genetic and environmental factors involved in its pathogenesis (Lyall et al. 2013).It is widely thought that the pathogenesis of PE is caused by the invasion of damaged trophoblast and excessive induction of trophoblast cell apoptosis, leading to spiral artery remodelling and placenta-uterus hypoperfusion (Gingras-Charland et al. 2019).During normal pregnancy, trophoblast cells differentiate into the highly infiltrating extravillous trophoblast, which further migrates into the decidua matrix, destroying the uterine spiral blood wall and replacing the vascular endothelial cells (Phipps et al. 2019).Any defects in this process, including reduced migration and invasion or enhanced apoptosis, will result in impaired placental formation and implantation, which in turn will lead to placental diseases, such as PE (El-Sayed 2017, Ramos et al. 2017).Therefore, maintaining the normal function of trophoblasts is necessary to prevent the occurrence of PE.
Gadd45, a ubiquitously expressed and DNA damageresponsive protein, acts as an important sensor of environmental and physiological stress, which can protect the body from genotoxic damage, such as ultraviolet light, X-rays, hypoxia, oxidative stress, and pro-inflammatory cytokines (Xiong et al. 2009, Yang et al. 2013a, Yang and Shang 2021).It has been reported that Gadd45 is useful as a pressure sensor for PE diagnosis (Li et al. 2018).In addition, it has been reported that Gadd45 protein was readily detected only in the placenta of individuals with preeclampsia and was localised in PE placentas, especially in endothelial cells and trophoblast cells, and the expression of Gadd45 downstream effector protein p38 was increased (Geifman-Holtzman et al. 2013, 2022, Li et al. 2018).The mRNA levels of Gadd455 family genes (a, b, and g) have been reported in placental tissues of patients with PE and patients with normal blood pressure (Geifman-Holtzman et al. 2013).Although the expression of these three genes was elevated in PE placentas, only the difference in Gadd45a mRNA was statistically significant.Gadd45a was significantly expressed in syncytiotrophoblast cells, the human villus trophoblast column, and extravillous trophoblast in the maternal decidua.
With thousands of years of clinical practice, traditional Chinese medicine has played an important role in maintaining the health of people in Asia and is increasingly being used worldwide (Ma et al. 2017).Salvia miltiorrhiza has a long history of medicinal use in China owing to its anti-inflammatory activity, anti-arthritis properties, wound and burn healing ability, and antibacterial/anticancer properties (Guo et al. 2014, Wang et al. 2020, Yin et al. 2021).Salvia miltiorrhiza contains various bioactive ingredients, including tanshinol.It has been reported that tanshinol can significantly inhibit lung metastasis, decrease serum TGF-b1 levels, and increase effector molecule levels in mice with liver cancer (Zhu et al. 2019).In addition, studies have shown that tanshinol can exert an antitumor effect on breast cancer by restoring the anticancer activity of NK cells (Yang et al. 2021).Tanshinol has also been reported to inhibit activation of the FoxO3a transcription factor and the expression of target genes Gadd45a and catalase while counteracting the inhibition of the Wnt signalling pathway and the expression of target genes Axin2, alkaline phosphatase, and osteoprotectin (Yang et al. 2013b).To date, tanshinol has been used for the treatment and prevention of various diseases.However, whether it plays a role in the treatment of preeclampsia is still unclear and its molecular mechanisms remain to be elucidated.
In previous studies, we found that Gadd45a was highly expressed in PE patients, and the overexpression of Gadd45a reduced the survival rate of HTR-8/SVneo cells; inhibited migration, invasion, and tube formation; and promoted cell apoptosis (Yang and Shang 2021).Based on the above findings, we speculated that tanshinol may have a potential therapeutic effect on preeclampsia by regulating the expression of Gadd45a.To test our hypothesis, we investigated whether tanshinol could attenuate the development of PE at the cellular level.

Clinical samples, cell culture and transfection
Paired placental tissue samples were derived from PE patients (n ¼ 30) and people with normal pregnancies (n ¼ 30) from Shanxi Bethune Hospital between January 2021 and December 2022.The diagnosis of PE was based on the criteria of the International Society for the Study of Hypertension in Pregnancy.Patient placental tissues were immediately frozen using liquid nitrogen and stored at −80 � C until use.This study was performed in accordance with the ethical standards of the responsible committee on human experimentation (Ethics Committee of Shanxi Bethune Hospital; approval code, SBQKL-2022-015).All patients or their families were informed of the study, agreed to participate, and signed informed consent.
The human extravasated trophoblast cell line HTR-8/SVneo was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).The cells were cultured in RPMI-1640 (Gibco) medium supplemented with 10% foetal bovine serum (FBS, Hyclone).The cells were inoculated in six-well cell culture plates at 37 � C and 5% CO 2 for 48 h.Full-length Gadd45a cDNA was cloned into a pcDNA3.1 vector, and a blank pcDNA3.1 vector was used as a control.Cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.This study was performed in accordance with the ethical standards of the responsible committee on human experimentation (Ethics Committee of Shanxi Bethune Hospital; approval code, SBQKL-2022-015), and followed the Guidelines for Stem Cell Research and Clinical Translation and the National Ethical Guidelines for Human Embryonic Stem Cell Research.

EdU assay
The indicated treated cells were seeded at a density of 5 � 10 4 in a 96-well plate and were cultured with an EdU medium diluent for 2 h following the manufacturer's directions (Ribobio).After fixation and permeabilization, the cells were incubated in a DAPI solution and observed by fluorescence microscopy (Olympus, Tokyo, Japan).

Wound-healing assay
Cell migration was quantified using a wound-healing assay.The cells were inoculated in six-well plates (1 � 10 6 cells), and the cell monolayer was scratched with a 200 lL pipette tip.The cells were washed three times with PBS to remove any cellular debris.RPMI-1640 medium without foetal bovine serum was added to each well and incubated at 37 � C and 5% CO 2 for 24 h.Wound closure was observed using a microscope at the same scratch location at 0 and 24 h.

Transwell invasion
Cells were transfected with the indicated plasmids or treated with tanshinol.The medium in the lower chamber contained 10% FBS, and cells (5 � 10 5 ) suspended in MatrigelV R were added to the upper chambers at the same time.The cells were incubated at 37 � C for 72 h.The cells that passed through the membrane were fixed and stained with methanol and 0.1% crystal violet and photographed.Cell invasion was quantified by direct microscopic visualisation and counting.

Tube formation assay
The tube forming ability of cells was assayed by co-culture with the supernatant of pre-treated cells.The cells were layered on the matrix ECM component gel according to the instructions of the kit (Cell Biolabs, USA), and tube formation was assayed, including the determination of the length and node number of the tube, using an optical microscope after 4-18 h of culture.

Flow cytometry
The collected cells were used to assay for apoptosis.The cells were harvested and stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide, according to the instructions of the FITC Annexin V Apoptosis Assay Kit (BD Biosciences, USA).The cells were fixed in 75% cold ethanol at 24 h intervals for detection.

Western blot
The proteins extracted from Placental tissues were isolated on the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels.Protein quantification was performed in the supernatant using a Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA).Equal protein concentrations were isolated from each sample and transferred onto polyvinylidene fluoride (PVDF) membranes.The membranes were blocked with 5% skim milk in Tris-buffered saline Tween-20 (TBST) for 1 h at room temperature (RT) and then incubated with the primary antibodies: Gadd5a (ab180768, 1:1000, Abcam, Cambridge, MA, USA), VEGF (ab52917, 1:1000, Abcam, USA) and GAPDH (ab8245, 1:10,000, Abcam, USA)) overnight at 4 � C. Secondary antibodies conjugated with HRP were used to detect the primary antibodies.The attached antibodies were visualised using the EZ-ECL chemiluminescence detection kit (Pierce, Rockford, IL, USA).

Immunohistochemistry
The collected tissues were embedded in paraffin, and sectioned, and 5 mm retinal sections were dewaxed and rehydrated as previously described (Redman and Sargent 2005).The sections were washed with PBS and incubated overnight at 4 � C with an anti-VEGF antibody (1:100; ab52917; Abcam; USA).Subsequently, the sections were washed three times in PBS for 5 min and incubated with biotinylated goat anti-rabbit IgG (1:1000; ab150077; Abcam, China) at room temperature for 20 min.Sections were washed with PBS, and the avidin-horseradish peroxidase complex was added (SABC Kit; Bost, Wuhan, China).The 3,3-diaminobenzidine (DAB) was used as a chromogen.The reaction was terminated by adding water.Finally, the sections were washed, and dehydrated, and the cells were visualised under a light microscope (LM).

Statistical analysis
The data are expressed as the mean ± SD.Student's t-test was used for between-group comparisons.Differences among groups were analysed using a one-way analysis of variance (ANOVA).Differences were considered statistically significant at P < 0.05.All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).

Upexpression of Gadd5a in PE tissues
The expression of Gadd5a in the placental tissues from PE patients and from women with normal pregnancies in the control group was analysed by western blot, qRT-PCR and immunohistochemistry.The results showed that the expression levels of Gadd5a in PE placental tissues were significantly increased compared to the control group (Figure 1, P < 0.01).

Tanshinol promotes the proliferation, migration, invasion, and tube formation of HTR-8/SVneo cells
To evaluate the effects of tanshinol on HTR-8/SVneo cells, the cells were treated with different concentrations of tanshinol (0, 1, 5, 10, 20, and 30 mM) for 48 h.As shown in Figure 2(A  and B), the results of the colony formation and EdU assays indicate that tanshinol promoted cell proliferation, reaching the highest proliferation level at 10 mM.The wound-healing assay showed that the cell migration rate in the tanshinol treatment group was higher than that in the untreated group, indicating that tanshinol could enhance the migration of HTR-8/SVneo cells (Figure 2C).Additionally, in the Transwell assay, the number of invasive HTR-8/SVneo cells markedly increased in the tanshinol-treated group (Figure 2D).The tube formation capacity of HTR-8/SVneo cells was also determined.As shown in Figure 2E, tanshinol noticeably augmented HTR-8/SVneo tube formation, resulting in a marked increase in tube length.Western blotting results showed that VEGF protein levels were upregulated in cells and downregulated after tanshinol treatment (Figure 2F).These results indicated that the migration, invasion, and tube forming ability of HTR-8/SVneo cells reached the highest level at the tanshinol concentration of 10 mM, which was consistent with the results of proliferation experiments.Taken together, these findings demonstrate that tanshinol increases HTR-8/SVneo cell proliferation, migration, invasion, and tube formation especially at concentrations of 10 mM.Thus, we used 10 mM tanshinol for all of the subsequent experiments.

Effect of tanshinol on the proliferation, migration, invasion, tube formation and apoptosis of HTR-8/SVneo cells overexpressing Gadd45a
Rescue experiments were performed in HTR-8/SVneo cells by transfection with pcDNA-Gadd45a, or pcDNA-NC as the control.qRT-PCR showed that Gadd45a was upregulated in HTR-8/SVneo cells after transfection with the Gadd45a recombinant expression vector, compared to the control group (Figure 3A).The EdU assay showed that overexpression of Gadd45a slowed the growth of HTR-8/SVneo cells, whereas tanshinol partially reversed this inhibitory effect on cell growth (Figure 3B).Consistent with this, Gadd45a overexpression decreased the colony formation capacity of the cells, whereas tanshinol partially reversed the decrease in cell colony formation (Figure 3C).Moreover, the migration and invasion of HTR-8/ SVneo cells were suppressed after Gadd45a overexpression, and treatment with tanshinol partially reversed this suppressive effect (Figure 3(D and E)).Furthermore, the tube formation assay demonstrated that overexpression of Gadd45a significantly decreased tube formation of HTR-8/SVneo cells compared with that of the control.Conversely, tanshinol treatment partially reversed the decrease in tube formation (Figure 3F).Western blotting results demonstrated that VEGF expression was significantly decreased in the cells that overexpress Gadd45a.However, it partially increased after tanshinol treatment (Figure 3G).In addition, flow cytometry showed that overexpression of Gadd45a promoted apoptosis of the HTR-8/SVneo cells compared with control, but treatment with tanshinol partially inhibited this enhanced rate of apoptosis (Figure 3H).Taken together, these findings indicate that tanshinol affects key features of PE in vitro, and that tanshinol inhibits apoptosis in PE.

Discussion
The pathogenesis of PE may be related to impaired placental development, which leads to endothelial dysfunction, systemic inflammation, oxidative stress, and a series of pathological processes (Anton et al. 2015).Tanshinol is the active ingredient of Salviorrhiza and has anti-inflammatory, anti- fibrosis, and antibacterial activities.It has been reported that tanshinol can be used as a new treatment for osteoporosis (Chen et al. 2017).In addition, tanshinol plays a role in the inhibition of cancer progression (Zhang et al. 2010, Zhu et al. 2019).Thus, tanshinol has potential applications in the treatment and prevention of many diseases.However, whether it plays a role in the treatment of preeclampsia is not clear and its therapeutic mechanism remains to be elucidated.
Trophoblast epithelial-mesenchymal transformation is the process by which trophoblast cells transform from an epithelial phenotype to a mesenchymal phenotype, which is characterised by enhanced migration and invasion.Thus, trophoblast cells can fix the placenta in the tissue of the mother and reshape the spiral artery of the mother (Kakkar et al. 2013).Therefore, a reduction in the migration and invasion ability of trophoblast cells is considered to be the main cause of PE.In this study, we found that tanshinol affected the proliferation, migration, and invasion of HTR-8/ SVneo cells, indicating that tanshinol can inhibit the occurrence and progression of PE.In addition, at the optimal concentration of tanshinol of 10 mM, it had an obvious effect on HTR-8/SVneo cell viability.However, this optimal concentration has only been detected at the cellular level and will need to be validated by more detailed analysis and in vivo experiments.
Our previous study showed that Gadd45a is highly expressed in patients with PE, and overexpression of Gadd45a reduced HTR-8/SVneo cell viability; inhibited cell migration, invasion, and tube formation; and promoted cell apoptosis.Therefore, this study simulated the pathogenesis of PE by overexpressing Gadd45a in HTR-8/SVneo cells and confirmed that upregulation of Gadd45a led to a decline in proliferation, invasion, and migration of HTR-8/SVneo cells, while treatment with tanshinol significantly restored the proliferation, invasion, and migration of trophoblast cells.In addition, tanshinol inhibits key features of PE pathogenesis.Lack of placental blood vessels, dysfunction of vascular endothelial cells, and vascular formation disorders are considered the main causes of PE and are related to hypertension and spasm in PE (Liu et al. 2015, Pratt et al. 2015).In this study, it was found that tanshinol increased the in vitro angiogenic ability of HTR-8/SVneo cells, and in a PE cell model established by overexpression of Gadd45a, tanshinol restored the inhibited tubulogenic ability, suggesting that tanshinol can inhibit the development of PE by promoting the tubulogenic ability of cells.In addition, increased apoptosis of placental trophoblast cells can cause placental malformation and is a factor in PE.In this study, the apoptosis rate of HTR-8/SVneo cells was found to be significantly increased in the PE cell model, but the apoptosis rate was decreased after tanshinol treatment, which is a further indicator of the inhibitory effect of tanshinol on the development of PE.
In summary, the present study identified tanshinol as a novel therapeutic compound that promotes the proliferation, migration, invasion, and tube formation of HTR-8/SVneo cells.In addition, tanshinol inhibited the rate of apoptosis in the PE cell model.This study only explored the role of tanshinol in inhibiting the development of PE at the cellular level, and subsequent in vivo experiments will have to be conducted to further validate the findings of this study.

Figure 1 .
Figure 1.Upexpression of Gadd5a in the placental tissues from PE patients.(A) The expression of Gadd45a in placental tissues from PE patients was assessed by Western blot, with GAPDH serving as a reference gene.(B) The expression of Gadd45a in placental tissues from PE patients was assessed by qRT-PCR.(C) The expression of Gadd45a in placental tissues from PE patients was assessed by immunohistochemistry. � P < 0.05, �� P < 0.01 compared with control.

Figure 3 .
Figure 3.Effect of tanshinol on the proliferation, migration, invasion, and tube formation of HTR-8/SVneo cells overexpressing Gadd45a.(A) The results of qRT-PCR showed that pcDNA-Gadd45a transfection increased the expression of Gadd45a.(B) and (C) The proliferative capacity of cells transfected with Gadd45a was restored by tanshinol.(D) and (E) Migration and invasion of cells transfected with Gadd45a were restored by tanshinol.(F) The tube formation of cells transfected with Gadd45a was restored by tanshinol.(G) The effect of tanshinol on apoptosis was assayed using flow cytometry.Gadd45a promoted apoptosis of the HTR-8/SVneo cells, but treatment with tanshinol partially reduced this enhanced rate of apoptosis.� P < 0.05, �� P < 0.01 compared with control.(magnification, 200�).