Inhibition of Fibroblast Activation by Components of Rhizoma Curcumae and Rhizoma Sparganii in A Rat Model of Uterine Leiomyoma

Background: Traditional Chinese medicine (TCM) often uses Rhizoma Curcumae and Rhizoma Sparganii (RCRS) (cid:0) the natural herbs for the treatment of UL. RCRS has been shown to be effective in the treatment of UL in our previous study. This study was to investigate the molecular mechanism by which RCRS inhibits broblast activation protein (FAP) activation and prevents uterine leiomyoma in rats. Methods: The SD rat model of uterine leiomyoma was established by estrogen and progesterone load combined with external stimulation. Subsequently, histological analyses, enzyme-Linked immunosorbent assays, western blotting were performed to evaluate the effect of the drug on uterine leiomyoma and its mechanism. Results: Our data showed that treatment of rats with RCRS signicantly reduced the expression of FAP, TGF-β (the FAP activating factor), and signicantly decreased the phosphorylation of cell proliferation pathway-related signaling factors AKT/MEK/ERK, as well as the expression of the extracellular matrix component collagen. Conclusions: Our results showed that RCRS is very effective in prevention and treatment of uterine leiomyoma in rats, and RCRS may exert its actions by inhibiting the activation of tumor-associated broblasts, inhibiting the cell proliferation, and improving tumor extracellular matrix. that AKT inhibitors can activate the AKT pathway in uterine broids, which increase the phosphorylation level of AKT compared with normal uterine tissue, suggesting that AKT is a potential biomarker for UL. Our results showed that AKT, ERK, and MEK phosphorylation levels were elevated in the model group, whereas phosphorylation levels of AKT, ERK, and MEK were reduced after treatment with RCRS component compatibility. It has been shown in the literature that(Higashino 2019) silencing of the TAF phenotype protein FAP can reduce the phosphorylation levels of AKT, ERK and MEK, indicating that AKT, ERK, and MEK phosphorylation levels are associated with FAP gene expression and TAF activation. This is consistent with our ndings. These results indicate that the therapeutic effects of total avonoids of Rhizoma Sparganii, volatile oil of Rhizoma Curcumae and their compatibility on UL may be related to the inhibition of FAP of TAF. 2009). Therefore, the effects of RCRS component compatibility on collagen I and bronectin in uterine tissue of UL model rats were investigated. The results showed that the expression level of collagen I in the uterus of UL model rats were reduced by the total avonoids of Rhizoma Sparganii and volatile oil of Rhizoma Curcumae after treatment. However, the RCRS component compatibility ability to regulate the expression of collagen I in uterine tissue suggests that its mechanism of treatment of UL may be related to RCRS component compatibility inhibiting TAF activation by inhibiting FAP expression.


Background
Uterine leiomyoma (UL) is the most common benign tumors of reproductive system in women of childbearing age with an incidence of about 70%, of which 25-50% of the patients show clinical symptoms (Stewart et al., 2016).Because the main stromal cells of UL are broblasts, it is also called uterine broids (Bhowmick et al., 2004). The main drugs used clinically to treat UL include mifepristone, letrozole, and vitamin D. Surgical treatment is currently the primary means of clinical treatment of UL, and hysterectomy is the only way to cure UL. However, these treatments have serious side effects, so it is urgent to use an effective and safe way to treat UL.
Rhizoma Curcumae and Rhizoma Sparganii (RCRS) is frequently used in patients with gynecological diseases such as uterine broids, dysmenorrhea and ovarian cysts improving blood circulation and remove stasis.Compatibility of Rhizoma Curcumae(Curcuma phaeocaulis Valeton) and Rhizoma Sparganii (Sparganium stoloniferum Buch. -Ham. ) is a representative medicine for the treatment of UL in TCM (Yu et al., 2012). Chemical analyses show that Rhizoma Sparganii (RS) mainly contains volatile oil, avonoids, saponins, organic acids, and other functional ingredients (Xu et al., 2015).Total avonoids are one of the main active ingredients of RS and have pharmacological effects such as anticoagulation, antithrombotic and anticancer. Zedoary turmeric oil is a volatile oil extracted from Rhizoma Sparganii (RC). As an effective anti-tumor drug (Chang et al., 2019), the main active ingredients of zedoary turmeric oil are curcumin, curcumin diketone and β-elemene (Dosoky and Setzer, 2018). RCRS, a combination of total avonoids and zedoary turmeric oil, has a signi cant therapeutic effect on experimental uterine leiomyoma (Yu et al., 2014). Our previous studies have shown that the therapeutic effects of RCRS on UL include improving pathological status, reducing volume, and inhibiting myoma cell proliferation (Yu et al., 2014). Another study showed that the therapeutic effect of RCRS on UL may be related to the regulation of endocrine levels (Yu et al., 2019). The results of metabolomics prove that RCRS can reverse the abnormal metabolism in rats. But its further mechanism remains to be studied . Due to the multi-channel and multi-target characteristics of TCM, the key targets for its e cacy are not clear and need to be studied. UL are composed of leiomyoma cells, broblasts and a large number of extracellular matrices (ECM) (Islam et al., 2018). Studies have shown that RCRS inhibits the proliferation of leiomyoma cells, but the mechanism is still unclear. Most studies of UL focused on leiomyoma cells, the mechanisms underlying tumor-associated broblasts (TAF) activation and UL development are not clear.
In this study, we used a rat model of qi stagnation and blood stasis UL, in which UL was induced by estrogen and progesterone load combined with external stimulation (Zhao et al., 2018). The model is more suitable for TCM experiments according to the basic theory of TCM combined with western medical theory and experimental zoology. Since broblast activation leads to UL, the study assumes that RCRS inhibits UL through inhibiting TAF activation. Results of this study should provide experimental basis for using traditional Chinese medicine to treat UL.

Preparation of herbal extract
The volatile oil of Rhizoma Curcumae was prepared and validated as described in our previous study (Zhao et al., 2018 Compound was used in four concentrations: original extract (1g crude extract/ml), 66.7% extract, 33.3% extract, and 16.7% extract. All extracts were stored at 4℃ until use.

Preparation of herbal decoction
Herbs (Rhizome Sparganii: Rhizome Curcumae =1:1) were crush and soaked for 2h. Herb fragments were decocted in two steps, 1h for the rst time and half an hour for the second time. After mixing and ltering, the decoction was concentrated to 0.67 crude drug/mg and stored at 4℃.

Animal
Rats care and experimental procedures were performed under a protocol approved by the Animal Ethical Committee at the Chengdu University of Traditional Chinese Medicine (Animal Ethics Approval Number NO. 2017-08). Female Speci c pathogen-free (SPF) SD rats (Animal License No. SYXK (Chuang) 2014-049) were purchased from Chengdu Dashuo Experimental Animal Tech (China). All rats were adaptively fed with ad libitum access to standard rodent diet and water for 4 days. All animals were sacri ced by cervical dislocation under 2% pentobarbital sodium(0.25ml/100g) after5 weeks.

Study design
Seventy-two SD rats were randomly divided into 9 groups control group, model group, RCRS-treated groups (66.7%, 33.3%, and 16.7%), RC-treated group, RS-treated group, decoction-treated group, and a positive control group. Model establishment began after adaptively feeding. The control group rats received no treatment. The remaining 8 groups were administered intragastrically with diethylstilbestrol (1.35 mg/kg) every day, and intramuscularly with 1 mg progesterone three times a week for ve weeks.
At the beginning of the fourth week, the eight experimental groups of rats were injected subcutaneously with epinephrine hydrochloride (0.5 mg/kg/d), and one external stimulus was given 4 hours after the injection: (1) Upside down day and night for 24 hour; (2) 60db noise continuous stimulation for 3 hours; (3) continuous hanging tail upside down for 10min; (4) 5-10℃ water bath for 4 minutes. It lasts for two weeks and guarantees that each stimulus is ≥ 2 times in two weeks.
At the beginning of modeling, drug (10ml/kg) was given to the intervention groups at the same time, and the corresponding groups were given separately ( Fig.1). After 5 weeks, the rat serum was collected for ELISA after anesthesia; the rat uterus was divided into two parts, one for para n embedding and one for western blot analysis.

Histological analyses
For hematoxylin and eosin (H&E) staining, uterus was xed overnight in 4% paraformaldehyde and then embedded in para n, sectioned, and stained.

Enzyme-Linked immunosorbent assays
Rat sera were collected, diluted, and divided in two aliquots. One aliquot was used to measure estradiol (E2) and the other aliquot used to measure progesterone (P). The assays were performed using ELISA kits purchased from Nanjing Jiancheng Bioengineering Institute (#R20181226, and #R20181227 for E2 and P, respectively). and a Multiskan MK3 microplate reader (Thermo Lab systems).

Western blotting
The uterus tissue was cut, homogenized and centrifuged, and the supernatant was aspirated for use. Detection was performed using a BCA protein quanti cation kit (PICPI23223, Thermo). GAPDH used as loading control for error correction.

Statistical analysis
Data were analyzed using one-way analysis of variance. The results are expressed as the mean+SD. A pvalue less than 0.05 was considered signi cant. If the data did not conform to a normal distribution, a nonparametric test was used.

Development of rat model
Rats of the model group showed hair loss, decreased diet, and slow increase of body weight.
The uterus of the model group was dull, the texture was hard, and there were abnormalities, nodules and edema macroscopic (Fig.3A). At the same time, the transverse diameter and vertical diameter of the uterus in the model group increased, and the volume of the uterus also increased signi cantly.
Pathological examination showed that compared with the control group, the myometrial cells of the uterus of the model group were disorganized, the thickness of the muscle layer was different, the outline of the muscle bers was unclear, and the muscle ber cells showed various degrees of deformation or even necrosis (Fig.3B). This is similar to the histopathological changes in uterine broids in clinical people. Meanwhile, the levels of estrogen and progesterone in the serum and uterus of the model group were signi cantly higher (Fig.3C-F).
These results demonstrated that the rat model of UL is successful.
Effects of RCRS on the appearance of uterus in rats Compared with the model group, the uterus of rat received medication was mostly symmetrical, the texture became soft and the thickness was uniform, and the uterine surface was smoother than the model group with no obvious swelling and ecchymoses. A small number of nodules can be seen on some uteri (Fig.3A).

Effects of RCRS on histological changes of uterus
There were 6 (6/8) cases in the positive group, 6 (6/8) cases in the water group, 6 (6/8) cases in the RCtreated group, 5 (5/8) cases in the RS-treated group, 3 (3/8) cases in the RS-treated (16.7%) group, and 5 (5/8) cases in the RS-treated (33.3%) group, and 5 (5/8) cases in the RS-treated (66.7%) group showed different degrees of degeneration (Fig.3B). Myometrium is disorderly arranged and changes in thickness. The muscle bers are unclear and Light-stain. Muscle bers show varying degrees of degeneration and necrosis. The nucleus volume increased and hyalinization can be seen in muscle bers. These results show that RCRS treatment can signi cantly improve the histological conditions of UL.

Effects of RCRS on the expression of FAP in uterus
Immunohistochemistry studies showed that the expression of FAP in the model group (P<0.05) increased signi cantly compared with the control group. The expression of FAP in RCRS-treated group (16.7%) was also increased. The effect of RCRS seemed to be dose-dependent as the low dose of the drug had a weaker inhibitory effect on FAP expression. Compared with the model group, FAP expression was signi cantly decreased after RCRS (66.7%) or RC treatment (P<0.05). Positive control group showed a similar inhibitory effect on FAP expression (p<0.01) (Fig.4).

Effects of RCRS on the expression of TGF-β in uterus
Western blot analysis showed the expression of TGF-β was signi cantly increased in the model group compared with the control group (p<0.01). Compared with the model group, the expression of TGF-β in the uterus in all drug intervention groups was signi cantly decreased (p<0.05). However, no statistical differences were detected in the control group. In the positive group, the expression of TGF-β was similar to that in each drug intervention group (p<0.01) (Fig.5A).

Effects of RCRS on the proliferation signaling pathway in uterus
To investigate the effects of RCRS on the expression of proteins regulating cell proliferation and apoptosis we analyzed the expression of AKT, ERK1/2 and MEK by western blot. Results showed that the drug treatment had no signi cant effect on the expression of these proteins. However, compared with the control group, the phosphorylation levels of AKT, ERK1/2 and MEK in the model group were signi cantly increased (p<0.01; p<0.001; p<0.001). The phosphorylation levels of AKT, ERK/2 and MEK were signi cantly reduced in each drug intervention group compared to the model group. Positive group had similar effects on AKT and ERK1/2 phosphorylation (p<0.01; p<0.001; p<0.05) (Fig.6).

Effects of RCRS on the extracellular matrix in uterus
In order to study the effect of RCRS on the extracellular matrix of rat uterus, the expression of Collagen I and bronectin, an important component of extracellular matrix, was analyzed by Western blot. Compared with the control group, the expression of collagen I and bronectin protein in the model group was signi cantly increased (p<0.001; p<0.05). Compared with the model group, except for the RS-treated group, the drug intervention groups reversed the expression of collagen I to varying degrees (p<0.05). This difference was particularly signi cant in the RCRS-treated (16.7%)/(66.7%) group (p < 0.01). Compared with the model group, the level of Collagen in the positive group was also decreased (p<0.001). there was no signi cant difference in the control group (Fig.5B).
The drug treatment showed no signi cant effect on bronectin expression (Fig.5C).

Discussion
Uterine leiomyomas, also known as broids, affect up to 70% of women before menopause (Stewart et al., 2017) and cause various clinical problems, such as excessive uterine bleeding, algomenorrhea, infertility and miscarriage. Uterine broids are recognized as estrogen-dependent tumors, and their development is closely related to the coordination of estrogen and progesterone. However, no report was found in literature regarding the effects of RCRS component compatibility on TAF of UL. Therefore, in this study, we aimed to address this issue using a well-established rat model of UL. Morphological and pathologic data showed that RSRS treatment signi cantly improved. Based on that, we further examined whether RCRS improves UL by inhibiting TAF activation. As studies have shown that about 80% of broblasts are activated in tumor tissue, and FAP plays a key role in broblast activation ( Räsänen et  TCM has been practicing the concept of drug combination for thousands of years and has been validated in the clinical treatment of diseases. Our experimental results also illustrate the scienti c and effective theory of the compatibility of TCM.

Conclusion
In this study, the UL model in rats was established to evaluate the effect of RCRS on UL. The results show that RCRS can signi cantly improve the macroscopic and pathological state of uterus in rats with UL, reduce the expression of TGF -β and FAP protein in the uterus, decrease the expression of signal factors (AKT, ERK and MEK) in the cell proliferation related pathway and regulate the secretion of collagen I in ECM. It is suggested that RCRS may achieve the effect of treating UL by inhibiting the activation of TAF, the expression of factors of cell proliferation pathway (AKT, ERK and MEK) and reducing collagen I in ECM. The data presented in this study may provide a new idea and therapeutic approach for UL and lays the foundation for further investigation in the treatment of UL.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.