SCM-198 Prevents Endometriosis By Reversing Low Autophagy Levels Of Endometriotic Stromal Cells Via Inhibiting TNF-Α-Aromatase-Estrogen-Erα Pathway And Promoting PR Expression

Background: Endometriosis (EMS), an estrogen-dependent disease, is characterized by dysregulated in�ammation and increased estrogen in ectopic lesions. However, the crosstalk and pathogenic mechanism of in�ammation and estrogen has not been fully explored. SCM-198 is the synthetic form of leonurine with multiple pharmacological activities. Whether SCM-198 could inhibit the progress of EMS by regulating in�ammation and estrogen signaling remains unknown. Methods: The therapeutic effects and potential mechanisms of SCM-198 on EMS were analyzed by establishing EMS mice models and performing RNA-seq assay. ELISA was performed to detect estrogen and TNF-α concentration in normal endometrial stromal cells (nESCs) and ectopic endometrial stromal cells (eESCs), with or without SCM-198 treatment. Western blotting, RNA silencing and plasmid overexpression were utilized to analyze the relationship among in�ammation, endocrine and autophagy as well as the regulation of SCM-198 on inammation-endocrine-autophagy axis. Results: Increased estrogen-ERα signaling and decreased PR expression co-led to the hypo-autophagy state in eESCs, which further inhibited the apoptosis of eESCs. Highly expressed TNF-α in eESCs enhanced low-autophagy mediated anti-apoptosis effect by activating aromatase-estrogen-ERα signaling. SCM-198 inhibited the growth of ectopic lesions in EMS mouse model and promoted the apoptosis of eESCs both in vivo and in vitro. The apoptosis effect of SCM-198 on eESCs were realized by upregulating the autophagy level via inhibiting TNF-α activated aromatase-estrogen-ERα signaling and increasing PR expression. Conclusion: In�ammation


Background
Endometriosis (EMS) is de ned as the presence of endometrial-like tissue outside the uterine cavity, with periodic bleeding of the ectopic lesions under the in uence of ovarian hormones [1].EMS is an estrogendependent and in ammation disordered disease that affects 10% women in reproductive age [2], and is associated with pelvic pain and infertility [3][4][5][6].None of the proposed pathogenic theories can (retrograde menstruation, coelomic metaplasia and immune disorder theory) fully explain the origin and progression of EMS [7,8].Recent studies have pointed out that the aetiology of endometriosis is more related to the interaction between in ammation and endocrine [9,10].
The treatment of EMS is tricky for its high recurrent rate (~50%) [11].The surgical treatment is traumatic [12,13] and medical therapy (e.g., progesterone, gonadotropin-releasing hormone (GnRH) agonists) is ine cient reducing the locally produced estrogen by ectopic lesions [11].The expenditure of EMS is identi ed with the cost of diabetes, while the curative effect fails to meet the expectations.Therefore, it is urgent to comprehensively understand the pathogenesis of EMS and to nd appropriate treatment.
Enhanced survivability leads to continuous development of EMS [14].Elevated estrogen level and resisted progesterone effect co-enhanced the survival of ectopic endometrium [15,16].Accumulated proin ammatory factors TNF-α enhanced the proliferation of ectopic endometrial stromal cells (eESCs) [17,18].Moreover, meta-analysis showed that TNF-α was associated with susceptibility to EMS and anti-TNFα therapy can relieve the pelvic pain associated with EMS [19,20].However, the speci c crosstalk between endocrine and in ammation and its role in the occurrence and development of EMS needs to be further investigated.
Recently, the role of autophagy has been emphasized on the progress of EMS [21].The autophagy level of eESCs is downregulated, which further promotes the survival and inhibits the apoptosis of eESCs [22][23][24].
Abnormal upregulated estrogen signaling has been found to inhibit autophagy in EMS [10,25].In ammation and autophagy can negatively regulate each other.Autophagy activation ameliorates in ammation [26,27] and enhancing in ammation inhibits autophagy [28,29].Whether in ammation, endocrine and autophagy have regulatory networks to jointly mediate the process of EMS needs to be further explored.SCM-198 is a synthetic form of leonurine [30,31].Studies have shown that SCM-198 has therapeutic effects on cardiovascular [32,33] and cerebrovascular disease [34].Importantly, SCM-198 has been demonstrated to alleviate hyperalgesia in adenomyosis mice [35].However, no study was reported to explore the effect of SCM-198 on EMS.
In the present study, we focused on the therapeutic effects of SCM-198 on EMS and explored the regulatory roles of SCM-198 in in ammation, endocrine and autophagy network of EMS.We found that SCM-198 obviously reversed the downregulated expression of PR and alleviated the higher production of TNF-α in eESCs.The activation of aromatase-estrogen-ERα signaling induced by augmented TNF-α was dramatically inhibited by SCM-198.Further, SCM-198 promoted the apoptosis of eESCs via reversing the inhibited autophagy level facilitated by activated TNF-α-aromatase-estrogen-ERα signaling and weakened PR expression.This study explained the complicated mechanisms involving immuneendocrine dysregulation of EMS and supplied a promising therapy for the refractory disease.

Methods
Reagents and mice EMS model SCM-198 was kindly gifted by Dr Zhu Yizhun's lab.Female C57BL/6 mice (6-8 weeks old) were purchased from Shanghai JieSiJie Laboratory Animal Co. Ltd (shanghai., China).After two weeks of adaptation, mice were randomly selected as the donors of EMS model.Donor mice were intraperitoneally injected with 17β estradiol (E2) (#E2758, Sigma, St. Louis, USA) (0.2ug/g bodyweight) three times for one week.Vaginal smears were utilized to select estrus mice as the recipients of EMS mice model Mixture of the donor mouse uterine fragments and PBS were intraperitoneally injected to recipient mouse.Recipient mice were allowed to rest for one week.
To investigate the effects of SCM-198 on EMS, recipient mice were randomly divided into three groups: EMS group, EMS + low-dose SCM-198 group (EMS+SCM-198 L, 7.5mg/kg) and EMS + high-dose SCM-198 group (EMS+SCM-198 H, 15mg/kg).Intraperitoneally inject 200µl of SCM-198 to the recipient mice according to the corresponding dose (once a day for a total one week).EMS group were given the same dose and frequency of PBS.One week later, the mice were sacri ced.Collect endometriotic tissue, uterus and peritoneal uid for subsequent treatment.

Collection of human samples and isolation of endometrial stromal cells
Human ectopic endometrial tissues were obtained from 46 women with EMS (aged 22-45 years) via laparoscopy and human normal endometrial samples were collected from 10 healthy women (aged 23-46 years) by uterine curettage at the Obstetrics and Gynecology Hospital of Fudan University.The endometrial tissues were minced (2-3-mm pieces) and digested in DMEM/F-12 containing collagenase type IV (0.1%, Sigma, USA) for 30min at 37°C.The dispersed cells were then ltered through a 400-mesh wire sieve to remove the undigested tissue pieces containing the glandular epithelium.After gentle centrifugation, the supernatant was discarded, and the cells were resuspended in DMEM/F-12 containing 10% fetal bovine serum (Gemini, Calabasas, USA), 100 IU/ml penicillin (Sigma, USA), 100 μg/ml streptomycin (Sigma, USA), and 1 μg/ml amphotericin B (Sangon, Shanghai, China) at 37°C in 5% CO 2 .

Immunohistochemical
Placing the immunohistochemical sections at 60°C for 2h.Using xylene and gradient alcohol to Dewax and rehydrate the sections.It was incubated with 3% hydrogen peroxide and 5% bovine serum albumin successively to block endogenous peroxidase.Tissue sections were incubated with anti-mouse ERα (#ab32063, Abcam, Cambridge, UK), PR (#ab101688, Abcam, UK), overnight in a humid chamber at 4°C.Wash the sections 3 times with PBS for 5 minutes each time.Cover the section with peroxidaseconjugated goat anti-rabbit or mouse IgG (#GK500710, Gene Teck, San Francisco, USA) and incubate the secondary antibody for 30 minutes.Then it was reacted with 3,3-diaminobenzidine (DAB) and stained the nucleus with hematoxylin.Finally, the slices are dehydrated in gradient alcohol and xylene, and then the slices are mounted.

Western blotting analysis
The total proteins of endometrial stromal cells (ESCs), mouse uterine and ectopic lesions were extracted using radioimmunoprecipitation (RIPA) assay buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Sigma, USA).The protein concentration was measured using a BCA protein assay kit (Beyotime, China).After denaturation, equal amounts of protein were separated via SDSpolyacrylamide gel electrophoresis (PAGE) before wet transfer onto polyvinylidene di uoride membranes.

RNA-Seq Data Analysis
Differential expression was computed with DESeq2, and the corrected gene or transcriptome expression value, the corrected fold-change, and the p-value and FDR value of the signi cance of the difference can be obtained.Set p-value<0.05and Fold change greater than 1.2 times or less than 0.83333 times as difference, then we get 1616 differential genes.Based on the differentially expressed genes between the different groups, the GO database and the KEGG database were used for functional enrichment and pathway enrichment, respectively.Statistical analysis was performed in the R version 4.0.4 according to p-value<0.05.Bubble charts and volcano plot were performed by using ggplot and cluster pro ler packages.

Quantitative real-time PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, California, USA) and then reverse-transcribed into rst-strand complementary DNA (cDNA) (Takara, Kyoto, Japan) according to the manufacturer's instructions.The synthesized cDNA was ampli ed with speci c primers and SYBR Green (Takara, Japan) using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, California, USA).Triplicate samples were examined for each condition.A comparative threshold cycle value was normalized for each sample using the 2 -ΔΔ Ct method.

Plasmid Overexpression and siRNA transfection
Aromatase overexpression (Aromatase over ) plasmid and negative control plasmid were purchased from Shanghai Genechem Co., LTD.(shanghai, China).Aromatase siRNA (siAromatase) and control siRNA were purchased from Shanghai Genepharma Co., LTD.(shanghai, China).Aromatase overexpressed plasmid and negative control plasmid (Ctrl) were transfected into eESCs cells by liposome transient transfection when the fusion degree reached about 70-80% in 6-well plate.Transfected cells were incubated for 24 h at 37°C and then collected for further study.The transfection process of siAromatase were similar to the overexpressed aromatase transfection.

Statistical analysis
Prism 8 software (GraphPad) was used for data analysis.Statistical signi cance was determined using Student's t-test for 2-group or one-way ANOVA for multiple group comparisons.The data are presented as mean±SD.Statistical signi cance was attained when P < 0.05.

SCM-198 suppresses the endometriotic growth both in vivo and in vitro
We rst explored whether SCM-198 could alleviate the development of EMS by using mice EMS model.Fig. 1A showed the general process of establishing EMS mice model.As shown, SCM-198 not only decreased the weight and size of mice ectopic lesions (Fig. 1B, C), but also reduced the wall thickness of EMS lesions (Fig. 1D).In addition, masson staining showed that SCM-198 treatment signi cantly reduced collagen accumulation in ectopic lesions (Fig. 1E).Western blotting results revealed that SCM-198 inhibited the expression of anti-apoptosis protein Bcl-2 and promoted the expression of pro-apoptosis protein Bax in ectopic lesions at both low and high administration (Fig. 1F).Consistent with in vivo results, in vitro analysis showed that SCM-198 inhibited Bcl-2 and promoted Bax expression in eESCs (Fig. 1G).Meanwhile, brosis related molecules bronectin 1 (FN1) and vimentin were also decreased in SCM-198 dose-dependently treated eESCs (Fig. 1H).These results suggest that SCM-198 has the ability to accelerate the apoptosis and attenuate the growth and brosis of EMS both in vivo and in vitro.

SCM-198 promotes the autophagy level and inhibits estrogen signaling of EMS
To investigate the under mechanism of SCM-198 in restraining EMS, we performed RNA-seq in SCM-198 treated and untreated ectopic lesions of EMS mice model.We observed a total of 1616 differentially expressed genes, with 701 genes being upregulated and 915 genes being downregulated in SCM-198 treated ectopic lesions compared with that in EMS groups (Fig. 2A).GO enrichment and KEGG pathway analysis revealed that SCM-198 downregulated the autophagy inhibitor molecules and estrogen receptor pathway of ectopic lesions (Fig. 2B, C).
To con rm the results from bioinformatics analysis, we rst analyzed the autophagy related gene expression in ectopic lesions.The results in Fig. 2D showed that SCM-198 could extensively promote the mRNA expression of autophagy positively-related proteins in ectopic lesions, such as Map1lc3b, Becn1Ulk1, Atg3, Atg4b, Atg5, Atg7, Gabarap, Atg9a and Atg10.Further, western blotting results con rmed that SCM-198 could promote the autophagy level by upregulating the ratio of autophagy-positively related proteins LC3B-II/ and BECN1 expression (Fig. 2E).Meanwhile, SCM-198 reversed the imbalanced expression of ERα and PR in EMS ectopic lesions by up-regulating PR and down-regulating ERα expressions (Fig. 2F, G).These results suggest that SCM-198 could promote the autophagy level and inhibit estrogen signaling of EMS.
The ERα/PR imbalance contributes to the hypo-autophagy state of eESCs.
We then assessed estrogen, hormone receptor, and autophagy level of eESCs in EMS patients.Higher production of estrogen (Fig. 3A) and upregulated ERα (Fig. 3B) were observed in eESCs.Compared with nESCs, LC3B-II/ and BECN1 were downregulated in eESCs, suggesting a lower autophagy level in eESCs (Fig. 3C).To explore the relationship between estrogen signaling and autophagy, eESCs were treated with E2.The results showed that E2 treatment dose dependently increased ERα expression and inhibited autophagy level by reducing LC3B-II/ and BECN1 expression (Fig. 3D).Previous study has demonstrated that ERα inhibited autophagy in eESCs [10].Thus, locally produced high level of estrogen led to increased ERα, which further inhibited autophagy.Meanwhile, PR expression was decreased in eESCs (Fig. 3E).
Progesterone increased PR expression and promoted autophagy in a dose-dependent manner (Fig. 3F).Further, PR silencing downregulated the autophagy of eESCs by decreasing LC3B-II/ and BECN1 protein expression, implying that decreased PR contributed to the hypo-autophagy state in eESCs (Fig. 3G).Together, these results suggest that local high estrogen level leads to increased ERα level and the imbalanced of ERα/PR expression in ectopic lesions promotes the hypo-autophagy in eESC.
Together, these results imply that SCM-198 could promote the apoptosis of eESCs by enhancing autophagy via inhibiting estrogen pathway and promoting PR expression.

TNF-α positively regulates aromatase-estrogen-ERα pathway in eESCs
Locally disordered in ammatory factors and increased estrogen both promoted the growth and proliferation of EMS ectopic lesions.To investigate the crosstalk between in ammation and estrogen signaling pathway in EMS, we rst detected the level of pro-in ammation cytokine TNF-α and the expression of aromatase (key enzyme of estrogen production) in eESCs.As shown, the mRNA expression and concentration of TNF-α was signi cantly increased (Fig. 5A) and the expression of aromatase was also upregulated in eESCs (Fig. 5B).Next, eESCs were treated with TNF-α or R-7050, a tumor necrosis factor receptor (TNFR) antagonist.TNF-α obviously promoted estrogen signaling pathway by increasing the expressions of aromatase and ERα (Fig. 5C), and elevating estrogen concentration (Fig. 5D).Aromatase silencing decreased estrogen level and ERα expression (Fig. 5E, F).Aromatase overexpression increased estrogen level and ERα expression (Fig. 5G, H), indicating that aromatase-estrogen signaling positively regulate ERα.Further, to demonstrate TNF-α positively regulate estrogen-ERα signaling through aromatase, aromatase was silenced under the treatment of TNF-α.As shown in Fig. 5I and J, aromatase silencing abolished the promotive effect of TNF-α on estrogen production and ERα expression.These data indicate that TNF-α can positively regulate the aromatase-estrogen-ERα pathway and in ammatory disorders can promote estrogen production to accelerate the development of EMS.

Discussion
EMS, a common obstetrics and gynecology diseases, is characterized by the growth of endometrial tissue appears outside the uterine cavity.Owing to the severe complications (pelvic pain and infertility) and high prevalence (~10%), multiple therapies have been proposed to deal with the disease [36,37].However, still up to 50% recurrence rate has been observed in EMS patients, because neither medication nor surgical treatment is effective in eradicating the growth of ectopic lesions [11].Finding and identifying effective medication for the treatment of EMS is bene cial for improving the quality of women life and relieving substantial economic burden.SCM-198, a synthetic form of leonurine, proved to have pain relief pharmacological effects on adenomyosis [35].In the present study, we rst identi ed the therapeutic effects of SCM-198 on EMS, as evidenced by decreased weight and size, reduced collagen accumulation and augmented Bax/Bcl-2 ratio of mice ectopic lesions.
Aberrant autophagy has stepped into the spotlight on the stage of EMS pathogenesis [21,38].Cumulative studies have uncovered that the autophagy level of ectopic endometrium is decreased [39,40].More important, low-autophagy level contributes to the survival of ectopic endometrium, as corroborated by enhanced apoptosis and decreased proliferation of eESCs under the induction of autophagy [24,41].
Then, we inquired whether autophagy is involved in the mechanisms of SCM-198 on treating EMS.RNA-Seq results based on ectopic foci suggested that SCM-198 treatment signi cantly promoted autophagy of eESCs.The upregulated mRNA levels of autophagy related genes in ectopic lesions and increased LC3B and BECN1 expression both in vitro and in vivo under SCM-198 treatment further con rmed the promotive effects of SCM-198 on autophagy level of eESCs.
The imbalanced endocrine microenvironment of ectopic endometrium, manifested with high estrogen signaling and progesterone resistance, have inescapable responsibility for the growth of ectopic endometrium.Increased estrogen production supported by elevated aromatase expression potentiates the proliferation of ectopic lesion [42][43][44], which could be reinforced by resisted progesterone signaling mediated by decreased PR expression [45,46].Multiple studies have emphasized that inhibiting autophagy is an important pathway for estrogen to restrain the apoptosis and facilitate growth of ectopic lesions [10,25].Whether the promotive effects of SCM-198 on autophagy are realized by repairing the damaged hormonal endocrine networks.We veri ed that increased estrogen signaling and impaired progesterone signaling co-drive the decline of autophagy in eESCs.In consistent with the hypothesis, the upregulation effects of SCM-198 on autophagy are mediated by increasing PR expression and decreasing aromatase-estrogen-ERα signaling in eESCs.
The current consensus is that dysregulated pelvic in ammatory process plays a crucial role in the initiation and progression of EMS [47].Abnormally upregulated pro-in ammatory cytokine TNF-α is closely related to the pelvic pain and infertility associated with EMS [20,48].Our data con rmed that there was an obviously upregulation in both mRNA expression and concentration of TNF-α.The upraised production of TNF-α could be suppressed the treatment of SCM-198, which has been identi ed as an antiin ammation drug.More important, we demonstrated that elevated TNF-α augmented the activation of estrogen-ERα signaling by increasing the expression of aromatase.Further, TNF-α inhibited the autophagy of eESCs by promoting estrogen signaling, suggesting in ammation-endocrine-autophagy axis plays an indispensable role in ectopic endometrial survival.Notably, we found that SCM-198 abated TNF-α induced estrogen signaling activation and autophagy inhibition, ultimately restrained the apoptosis of eESCs.

Conclusions
In summary, the disturbed in ammation, endocrine and autophagy made EMS to be complicated and di cult to cure.Elevated levels of in ammation, imbalanced estrogen and progesterone and inactivated autophagy co-contribute to the progression of EMS.SCM-198 focused on rectifying the aberrant in ammation-endocrine-autophagy axis.SCM-198 reversed the low autophagy level of eESCs via inhibiting TNF-α-aromatase-estrogen-ERα pathway and promoting PR expression, and ultimately alleviated the development of EMS.To sum up, this study provided a theoretical basis for the potential application of SCM-198 on the treatment of EMS.Compared with nESCs, the production of TNF-α was higher in eESCs.Elevated TNF-α augmented the activation of aromatase-estrogen-ERα signaling.The upregulated estrogen signaling and downregulated progesterone signaling co-suppressed the autophagy level, which further conduced to the growth of eESCs.SCM-198 inhibited TNF-α-aromatase-estrogen-ERα signaling and increased PR expression.

Abbreviations Figures
Consequently, SCM-198 promoted autophagy-mediated apoptosis of eESCs by reconstructing the balance of estrogen and progesterone signals.

Figure 3 The
Figure 3

Figure 6 The
Figure 6