{"paper_id":"0df57d88-cd91-4b54-ad29-8c79149b4bf3","body_text":"Endometriosis is a gynecological disease caused\nby women’s endometrial tissue metastasis, invasion,\nand growth outside the uterine cavity. It can lead to\nsevere clinical symptoms such as lower abdomen pain,\ninfertility, and dysmenorrhea, which threaten women’s\nreproductive health, and affect their quality of life\nand work efficiency ( 1 ,  2 ). According to studies, the\nproliferation and apoptosis of normal endometrial stromal\ncells (ESC) in patients with endometriosis are important\nreasons for decreased endometrial receptivity, difficult\nembryo implantation, and infertility ( 3 ,  4 ). At present,\nhormone treatment of endometriosis is inefficient, and\nthe annual recurrence rate after surgical treatment is\nhigh ( 5 ). Therefore, exploring alternative therapies for\nendometriosis is expected to alleviate the burden of\npatients with endometriosis.\nThere are many causative factors for endometriosis,\nsuch as miRNAs which may promote the proliferation,\ninvasion, and metastasis of cancer cells by regulating the expression of upstream target genes, thereby affecting the\nprognosis of endometrial cancer patients ( 6 ). Previous\nstudies have reported that the activity of transforming\ngrowth factor-β (TGF-β) in the intimal tissue of\nendometriosis significantly increased, confirming that the\nabnormal expression of TGF-β is related to the abnormal\ngrowth of ectopic intima and enhanced aggressiveness\n( 7 ,  8 ). TGF-β1 is one of the vital cytokines, which has\na regulation effect on cell division and proliferation\n( 9 ). Smad2/3 is the first signal molecule transmitted by\nthe TGF-β1 signal, and they play pivotal roles in the\nbiological effect of TGF-β1 ( 10 ). Smad3 is the main\nsignal transmission protein of the TGF-β signaling\nsystem to promote adhesion, while smad7 can inhibit\nthe phosphorylation of Smad3 and block the TGF-β\nsignaling system ( 11 ). Furthermore, smad7 can inhibit\nthe promoting effect of TGF-β on fibrosis in renal tubular\nepithelial cells by impairing Smad2 activation ( 12 ).\nPhytosterols are a class of steroid compounds with multiple\nbiological activities and high clinical application value ( 13 ). Traditional Chinese herbs rich in β-sitosterol, include\nTrifolium repens, Houttuynia cordata, and Lasia spinosa\n( 14 ). The chemical formula of β-sitosterol is C30H52O and its\nmolecular weight is 414.71. It is a white amorphous powder\nat room temperature and is insoluble in water. Studies have\nshown that β-sitosterol has various biological activities such\nas anti-inflammatory, antiproliferative, and anticancer effects\n( 15 ,  16 ). In human alveolar epithelial cells, β-sitosterol can\ninhibit TGF-β1-induced epithelial-mesenchymal transition\n(EMT) by inhibiting the TGF-1/Smad pathway ( 17 ). In\naddition, the pathogenesis of endometriosis involves EMT,\nwhich is a complex process of epithelial cells transforming\ninto mesenchymal cells ( 18 ). Overexpression of the\nBAMBI gene encoding the type I receptor of TGF-beta and\napplication of β-sitosterol can inhibit autophagy in non-small\ncell lung cancer (NSCLC) cells, induce G0/G1 cell cycle\narrest, and then inhibit cell proliferation by inactivating the\nTGF-β/Smad2/3/c-Myc pathway ( 19 ). However, the effect\nof β-sitosterol on the growth of endometrial grafts and the\nproliferation of hEM15A cells derived from Endometriosis\nthrough the Smad7-mediated TGF-β/Smads signaling\npathway has received little attention.\nThe proliferation and apoptosis of ESC play an important\nrole in the pathogenesis of endometriosis. Considering\nthe relationship between Smad7 and cell proliferation,\nSmad7 could also be a key target for β-sitosterol to inhibit\nthe development of endometriosis. The purpose of this\nresearch is to investigate the protective mechanism of\nβ-sitosterol on endometriosis in vivo and in vitro through\nthe Smad7-mediated TGF-β/Smads signaling pathway.\n\nThis is a laboratory-based experimental study conducted\non animals and cells. 10-week-old sexually mature C57BL/6\nfemale mice (20 ± 2 g) were all purchased from Chengdu\nDashuo Biological Technology Co., Ltd., (Chengdu, China).\nIn this research, all animal experiment operations followed the\nAnimal Experimental Committee and the Ethics Committee\nof the Hospital of Chengdu University of Traditional Chinese\nMedicine requirements (No. is 2021DL-02). The animals\nwere housed in an SPF-grade laboratory, fed, and watered ad\nlibitum. Allogeneic endometrial transplantation was used to\nestablish endometriosis models ( 20 ). Briefly, mice were given\nsubcutaneous injections of estradiol benzoate (E2, 0.1 mg,\nSolarbio, China) and anesthetized by intraperitoneal injection\nof 1% sodium pentobarbital (40 mg/kg). After anesthesia,\nthe donor uterus was removed and isolated under aseptic\nconditions. The membrane was cut into less than 1 mm3\npieces and put into serum-free DMEM/F-12 medium. The\nexperimental mice were separated into 3 groups, a control\ngroup (endometriosis), β-sitosterol (Meilun, China) high-dose, and low-dose treatment groups, 6 mice per group. In the\ncontrol group, the harvested uterine tissue was rinsed twice\nwith sterile saline and then cut into pieces, ensuring that the\nlargest diameter fragments were less than 1 mm3\n. The uterine\nfragments were injected intraperitoneally from one donor mouse into recipient mice, and the mice were gavaged with\nthe same amount of normal saline containing β-sitosterol as\nthe treatment groups. The β-sitosterol high-dose (350 µg/\nkg) and low-dose (35 µg/kg) treatment groups received\nintragastric administration once a day for 21 consecutive\ndays. After finishing the experiment, the implant was taken\nout for the next experiment.\nThe tissues of normal endometrium and ectopic\nendometrium were fixed in 4% paraformaldehyde, and\nethanol was used for gradient dehydration. Then, paraffin\nsections (4 μm) of endometrial tissue were made for\nH&E staining (Solarbio, Beijing, China). An optical\nmicroscope was used to observe the pathomorphological\ncharacteristics of the normal uterus endometrium and\nectopic endometrium. For Tunel assay, the sections were\npermeabilized with proteinase-K (Non-specific serine\nprotease) for 20 min and blocked with 5% goat serum\nfor 30 minutes, respectively. Sections were stained with\nAlexa Fluor 488 (Elabscience, China) for 30 minutes at\n37°C and protected from light. Binding Alexa Fluor 488-\ndUTP to nicked DNA by TdT transferase is one of the\ncommon methods to detect apoptosis. Then, the tissues\nwere washed with phosphate buffer saline (PBS), and\n1×Equilibration Buffer to immerse the sample. Last, the\nsections were fixed on slides with a DAPI sealer (Yeasen,\nChina) and were observed through an Olympus BX51\nfluorescence microscope (Olympus, Japan).\nAfter creating 4 μm paraffin slices, incubating them\nwith primary antibodies for Smad7 (ab216428, Abcam,\nCambridge, MA, USA), TGF-β1 (ab215715, Abcam, USA),\np-smad2 (ab280888, Abcam, USA), and p-smad3 (ab52903,\nAbcam, Cambridge, MA, USA) was performed overnight\nat 4°C. Then, the slices were incubated with an appropriate\ndosage of biotinylated goat anti-rabbit IgG secondary\nantibody (D110065, BBILIFE, China) for 30 minutes at\n37°C. Finally, the sections were stained with DAB (AR1025,\nBOSTER, China), and re-stained with hematoxylin (Solarbio,\nChina). The positive cells that appeared under an optical\nmicroscope for Smad7/TGF-1/Smad2/3 protein expression\nwere identified and colored yellow or brown. Positive cells\nwere counted using image-Pro+60 image analysis software.\nThe percentage of positive cells=number of positive cells/\ntotal number of cells×100.\nThe ectopic endometrial stromal cell line (hEM15A) from\nhuman endometriosis patients was purchased from American\nType Culture Collection (ATCC) (Manassas, VA, USA).\nhEM15A cells were cultured in DMEM/high glucose (4.5\ng/L D-Glucose) medium (Hyclone, South Logan, UT, USA).\nCells were passaged in the logarithmic growth phase. DMEM\nwas supplemented with 10% fetal bovine serum (FBS, Gibco,\nRockville, MD, USA) and 1% glutamine (Sigma, St. Louis,\nMO, USA) and incubated at 37°C.\nDigestion of the logarithmic growth phase hEM15A cells\nwith 0.25% trypsin, hEM15A cells were collected to adjust\nthe cell concentration to 5×10 4 \ncells/mL-1. Then, the cells were\nseeded into a 96-well culture plate with a volume of 200 μL\nper well at 37°C and 5% CO 2 \n. After 24 hours of cell adhesion,\nthe culture media was swapped out for the wells that contained\nvarious concentrations of β-sitosterol (30, 60, 90 μmol/L), and\nthen the plate was incubated at 37°C in 5% CO 2 \nfor 48 hours.\n20 μL of CCK-8 (GLPBIO, USA) solution (5 mg/mL) was\nadded to each well, and incubation was continued for 1 hour\nat 37°C. A microplate reader assessed each well’s absorbance\n(D) value at 450 nm (Thermo Fisher Scientific, Waltham,\nMA, USA).\nhEM15A cells were collected following treatment with\nβ-sitosterol for 48 hours, and then washed twice with\nphosphate buffer. 100 µL of 1x binding buffer was taken to\nresuspend the cells. 1×10 5 \ncells were used to stain for loss\nof apoptosis assay. Sequential additions of 5 μL of Annexin\nV-FITC (Yuanxin, China) and 5 μL of PI (Yuanxin, China)\nwere made, and the mixture was stained after 15 minutes of\ndark incubation. Within an hour, flow cytometry was utilized\nto find the cells that had undergone apoptosis.\nA 24-well Transwell chamber (Corning, NY, USA)\nwith 8.0-μm pore membranes covered with Matrigel (BD\nBiosciences, San Jose, CA, USA) was used to conduct a\ncell invasion experiment. Briefly, after being suspended\nin serum-free DMEM, hEM15A cells (1×10 5 ) were plated\nin the upper chamber of the Transwell (200 µl). 600 µl\nof a full medium comprising 10% fetal bovine serum\n(FBS, Gibco, Rockville, MD, USA) was then added to\nthe lower chamber. Transwells were collected 24 hours\nafter incubation, preserved for 30 minutes in -20°C cold\nmethanol, and then stained with 0.5% crystal violet at\nroom temperature for 20 minutes (Solarbio, China). Under\nan optical microscope (Olympus, Japan), invasive cells\nwere seen.\nRIPA buffer was used to lyse cells and tissues in\norder to extract the total protein. After the BCA protein\ndetection kit (Abcam, USA) had measured the protein\nconcentration, sodium dodecyl sulfate polyacrylamide\ngel electrophoresis (10% SDS-PAGE) and electrotransfer\nwere used to estimate the total protein. The protein was\ntransferred to the PVDF membrane, and the membrane\nwas blocked by skim milk solution. The primary\nantibodies for Smad7 (1:1000, Abcam, UK), TGF-β1\n(1:1000, Abcam, UK), p-Smad2 (1:500, Abcam, UK),\nSmad2 (1:1000, Abcam, UK), p-Smad3 (1:1000, Abcam,\nUK), Smad3 (1:500, Abcam, UK) were then added, the\nmembrane was sealed with 5% skimmed milk solution, and incubated at 4°C for 12 hours. The membrane was then\nrinsed with PBST (every 15 minutes) four times before\nbeing incubated with the diluted secondary antibody\n(1:100000, abcam, UK) for 2 hours at room temperature\nto strengthen the immunological signal detected by the\nchemiluminescence detection equipment. Optical density\nanalysis was carefully performed using Image J software\nusing β-actin as the reference.\nTotal RNA was extracted from hEM15A cells using the\nTRIzol kit (TaKaRa, Japan), and its concentration and\npurity were assessed using UV spectrophotometry. The\nsample is deemed eligible when the A260/A280 ratio is\nbetween 1.8 and 2.0. Next, total RNA was reversed into\ncDNA by Real-time quantitative reverse transcription\npolymeras chain reaction (RT-qPCR) kit steps (TaKaRa,\nJapan). The primer sequence is shown in Table 1. With\nGAPDH as a reference, the 2 -ΔΔCt  method was used to\ncalculate Smad7 relative expression.\nPrimers used in this study\nThe siRNA transfection was carried out according\nto the instructions of lipofectamine 2000TM (Carlsbad,\nCA, USA). 50 µL of serum-free media was used to\ndilute one microliter of the transfection reagent before\nit was incubated at room temperature for 5 min. Then,\nthe cationic vesicles were combined with the diluted\nsiRNA (50 ng) for 20 minutes at room temperature with\nthe diluted transfection reagent. A hundred microliters\nof RNA cationic vesicles were added to the wells of the\ncell culture plate, and the plate was then incubated at\n37°C with 5% CO 2 \nfor 24 to 48 hours. The siRNAs were\nsynthesized from GenePharma (Shanghai, China), and\nthe sequences are as follows:\nsi- SMAD7 ( 1 ) sense:\n5´-AAGGAAAAAGCCUCUUUCCCC-3´\nantisense:\n5´-GGAAAGAGGCUUUUUCCUUCU-3´\nsi- SMAD7 ( 2 ) sense:\n5´-AA AUCCAUCGGGUAUCUGGAG-3´\nantisense:\n5´-CCAGAUACCCGAUGGAUUUUC-3´\nThe research data were statistically analyzed using GraphPad\nPrism8 software (La Jolla, CA, USA). Measurement data are\nexpressed as mean ± standard deviation (x̅ ± SD). Multiple\ngroups underwent one-way analysis, and P<0.05 was regarded\nas statistically significant.\n\nβ-sitosterol is a tetracyclic triterpenoid compound with\ncyclopentane perhydrophenanthrene as the basic skeleton.\nThe endometrial tissue of each group of mice was\nstained by H&E, and the endometrial mucosa injury,\nproliferation and inflammation were observed under a\nmicroscope. In addition, as shown in Figure 1A, in the\ncontrol group, the cyst-like structure of ectopic endometrial\ntissue had a structure similar to that of the endometrium\nunder microscopic observation. Pathological changes were\ndegeneration, necrosis, and hyperplasia of endometrioid epithelial cells, neonatal capillary formation, and neutrophil\ninfiltration in the interstitial layer. In summary, pathological\nchanges such as endometrial epithelial cell hyperplasia and\nneonatal capillary formation in the control group indicated\nthat the cyst-like structure in this group was in the growth\nstage. Compared with the control group, the β-sitosterol\nlow-dose (35 µg/kg) group and the β-sitosterol high-dose\n(350 µg/kg) group had more endometrialoid epithelial cell\ndegeneration and necrosis. And the β-sitosterol high-dose\n(350 µg/kg) group thinned the propria layer and significantly\nreduced cells. These results indicate that the cyst-like structure\ngrowth was inhibited in the β-sitosterol low- (35 µg/kg) and\nhigh-dose (350 µg/kg) groups, among which the cyst-like\nstructure growth of β-sitosterol high-dose (350 µg/kg) group\nwas relatively the weakest, and of the β-sitosterol low-dose\n(35 µg/kg) group was relatively poor. TUNEL staining was\nused to detect the effect of β-sitosterol on endometrial cells.\nAs shown in Figure 1B and C, the control group have no\nobvious green fluorescence, the cells are not stained by Alexa\nFluor 488, and there is no apoptosis. But when β-sitosterol\nwas used in both low (35 µg/kg) and high (350 µg/kg) levels,\nthe green fluorescence is obvious, and the endometrial cells\nappear to have undergone apoptosis. Apoptotic cells were\nincreased in the high-dose (350 µg/kg) β-sitosterol compared\nto the low-dose group.\nEffects of β-sitosterol on histopathology of uterus in endometriosis mice. A. H&E staining images of the endometrium (scale bar: 50 µm). B. Images\nof endometrium tissue following TUNEL staining of Alexa Flour 488 (green fluorescence) (scale bar: 20 µm). C. Statistical analysis of apoptosis cells by\nTUNEL staining. **; P˂0.01, ***; P˂0.001 compared with the control group.\nImmunohistochemistry was used to detect the changes of\nSmad7/TGF-β1/Smad2/3 proteins in endometrial tissues.\nAs for smad7, compared with the control group, smad7\nwas increased in the β-sitosterol low (35 µg/kg) and high-dose (350 µg/kg) treatment groups. However, compared\nwith the control group, the expression of TGF-β1, Smad2,\nand Smad3 was downregulated in the treatment of high and low-dose β-sitosterol groups ( Fig .2A, B ). The above\nresults suggest that changes in Smad7, TGF-β1, and\nsmad2/3 signaling are involved in endometriosis.\nEffects of β-sitosterol on Smad7-mediated TGF-β1/Smad2/3 in endometriosis mice. A. Immunohistochemistry of Smad7/TGF-β1/Smad2/3 signaling\npathway related proteins (scale bar: 40 µm). B. Statistics of Smad7/TGF-β1/Smad2/3 signal pathway related protein. *; P˂0.05 and **; P˂0.01 compared\nwith the control group.\n\nThe biological behavior of β-sitosterol on hEM15A\ncells was detected by CCK-8, flow cytometry, and\ntranswell test. As shown in Figure 3A, cell viability was\ndecreased in a dose-dependent manner by β-sitosterol,\nand it was the lowest at 90 μM. As shown in Figure 3B\nand C, the apoptosis rate was increased by β-sitosterol\nin a dose-dependent manner, and the highest at 90 μM.\nAs shown in Figure 3D and E, cell migration ability\nwas dose-dependently decreased by β-sitosterol. The\nmigration ability of the blank group was the strongest, and the migration ability was the weakest at 90 μM of\nβ-sitosterol.\nTo investigate whether the β-sitosterol regulates Smad7/\nTGF-β1/Smad2/3 expression levels in vivo, total protein\nin hEM15A cells treated with β-sitosterol was detected.\nAs shown in Figure 3F and G, the expression level of\nSmad7 is the highest at 90 μM of β-sitosterol, and it has a\ndose-dependent effect. The expression level of TGF-β1/p Smad2/3 is the highest without treatment of β-sitosterol,\nand the expression level is the lowest at 90 μM and has a\ndose-dependent effect.\nThe effect of β-sitosterol on the biological behavior of hEM15A cells. A. Inhibition effect of β-sitosterol on hEM15A cells detected by CCK8. B.\nApoptotic rate of hEM15A cells induced by β-sitosterol. C. Statistics of apoptotic cells. D. β-sitosterol inhibits hEM15A migration by transwell chamber.\nE. statistics of transwell experimental. F. Western blot of Smad7/TGF-β1/Smad2/3 signaling pathway-related proteins. G. Statistics of Smad7/TGF-β1/\nSmad2/3 related protein expression. *; P˂0.05, **; P˂0.001, and ***; P˂0.0001, compared with the 0 group.\n\nAfter hEM15A cells were treated with si-Smad7 or 60\nμM β-sitosterol, RNA and protein were extracted to detect\nthe relative expression of Smad7/TGF-β1/Smad2/3. Then,\nthe biological behavior of β-sitosterol and Smad7 on\nhEM15A cells were detected by CCK-8, flow cytometry,\nand transwell. As shown in Figure 4A, B, and C, compared\nwith si-Smad7( 2 ), si-Smad7( 1 ) had a better inhibitory\neffect on smad7 at the protein and mRNA levels. Based\non the above results, si-Smad7( 1 ) was chosen for further\ninvestigation. As shown in Figure 4D, compared with the\nblank group, β-sitosterol could cause a significant decrease\nin cell viability in hEM15A. While compared with the\nβ-sitosterol treatment group, si-Smad7( 1 ) could reverse\nthe decrease in cell viability caused by β-sitosterol. As\nshown in Figure 4E and F, the β-sitosterol treatment group had the highest apoptotic rate, while the si-Smad7( 1 )\ntransfection group had the lowest apoptotic rate.\nMoreover, the apoptosis caused by β-sitosterol could be\nreduced by transfected si-Smad7( 1 ). As shown in Figure\n4G and H, the si-Smad7( 1 ) transfection group had the\nstrongest migration ability while the β-sitosterol treatment\ngroup had the weakest migration ability. Moreover, the\ndecline in cell migration caused by β-sitosterol was\nsignificantly reversed under the action of si-Smad7( 1 ).\nAs shown in Figure 4I and J, compared with the blank\ngroup [si-Smad7( 1 ) and β-sitosterol were both free],\nthe expression level of TGF-β1, p-smad2, and p-smad3\nin the β-sitosterol treatment group was low, while the\nexpression level of TGF-β1, p-smad2, and p-smad3 in the\nsi-Smad7( 1 ) transfection group was high. si-Smad7( 1 )\ncan effectively inhibit increased protein levels caused by\nβ-sitosterol.\nThe effect of Smad7 on the biological behavior of hEM15A cells caused by 60μM β-sitosterol. A. The protein expression of Smad7. B. The statistics\nof Smad7. C. The mRNA level of Smad7. D. The effect of β-sitosterol and si-Smad7 on cell survival of hEM15A cells by CCK8. E. Effects of β-sitosterol\nand si-Smad7 on the apoptosis of hEM15A cells. F. Statistics of apoptotic cells. G. Effects of β-sitosterol and si-Smad7 on the migration of hEM15A cells.\nH. statistics of transwell experimental. I. Western blot of TGF-β1/Smad2/3 signaling pathway-related proteins. J. Statistics of TGF-β1/Smad2/3 related\nprotein expression. *; Means compared with the blank group, #; Means compared with the β-sitosterol group, *; P˂0.05, **; P˂0.001, ***; P˂0.0001,\ncompared with the blank group, #; P˂0.05, ##; P˂0.001, and ###; P˂0.0001, compared with the β-sitosterol group.\n\nSterols are important physiologically active molecules\nin various foods and are also important components of\ncell membranes of all eukaryotes (humans, animals,\nplants), involved in important life activities, known as\nthe “key” to life, because of its solid state, also known\nas sterols ( 21 ). The most well-known and extensively\nresearched sterol is animal sterol, or cholesterol, which\nis mostly found in the brain, spinal cord, liver, and blood\nof both humans and animals ( 22 ). β-sitosterol is a natural\nproducts, the development and research of which have\ngrown extensilvely, but there are still only a few drugs\nof natural product origin in the field of gynecological\ndiseases. Therefore, the study of β-sitosterol in\nendometriosis enriches the study of natural products in\ngynecological diseases.\nThere are many reasons for the formation and\ndevelopment of EMS, such as inflammation, abnormal\ngrowth, and immune factors ( 23 ,  24 ). The biological\ncharacteristics of the ectopic endometrium cells of\npatients are different from those of normal endometrial\ncells ( 25 ). The eutopic endometrial cells of patients have a\nstronger ability for migration, invasion, proliferation, and\nblood vessel formation ( 26 ).\nPrevious research proved that miRNAs might promote\nthe proliferation, invasion, and metastasis of cancer\ncells by regulating the expression of upstream target\ngenes, thereby affecting the prognosis of patients with\nendometrial cancer ( 6 ). Studies have shown that the\nexpression of TGF-β1 in the eutopic endometrium tissue\nis higher than that of the normal tissue ( 7 ). In the present\nstudy, β-sitosterol inhibited the expression level of\nTGF-β1 and suppressed cell proliferation and migration,\nthereby inhibiting the formation and progression of\nendometriosis lesions. Smad7 can inhibit the expression\nof TGF-β1, which could reveal that Smad7 is involved in\nthe regulation of β-sitosterol on TGF-β1. Therefore, based\non the smad7-mediated TGF-β/Smads signaling pathway\nfor the treatment of endometriosis β-sitosterol contributes\nto the study of clinical treatment.\nβ-sitosterol is a natural active substance which was\nwidely found in many medicinal plants ( 27 ). Previous\nstudies have shown that β-sitosterol plays a helpful role in\nthe prevention and treatment of tumors ( 28 ). β-sitosterol\nwas previously reported to inhibit the proliferation and\noccurrence of tumor cells, inhibit the differentiation and\nproliferation of tumor or cancer cells, and induce tumor\ncell apoptosis ( 39 ,  30 ). In addition, after nearly ten years\nof basic research, it was proved that ectopic endometrial\ncells were found to be more proliferative than normal\nendometrial cells ( 31 ). Therefore, effectively inhibiting\nthe proliferation of endometrial cells and promoting their\napoptosis is a classic method for the improvement of\nendometriosis.\nTGF-β1 was previously reported to be involved in\nvarious cell functions such as proliferation, differentiation, adhesion, migration, infiltration, and angiogenesis ( 32 ).\nPrevious studies have shown that the level of TGF-β1 is\npositively correlated with the severity of endometriosis\n( 8 ). The Smad protein family which is the substrate of the\nTGF-β receptor, exists in the cytoplasm, and can transmit\nthe signal directly from the cell membrane to the nucleus\n( 32 ). Smad7 was proven an inhibitor of TGF-β-Smads\nsignal transduction. Smad7 can inhibit TGF-β-Smads\nsignal transduction at the three levels of the TGF-β1\nreceptor, the Smad2/3 complex ( 34 ), and the nucleus.\nIn this study, under-treatment of high-dose β-sitosterol,\nSmad7 expression level was the highest, while TGF-β1/\nSmads protein expression level was the lowest. From\nthe above description, TGF-β1 and Smad7 are present\nin endometriosis, and the two factors exist in the same\nsignal transduction process. It is speculated that TGF-β1\nand Smad7 are in normal endometrial tissue. There is a\nbalanced relationship.\nhEM15 is an immortalized cell line of eutopic\nendometrial stromal cells in EMS patients. It retains some\nof the characteristics of endometrial cell morphology and\nmolecular biology. It has high cell homogeneity and a\nlong survival time, which can be used as research work\nfor endometriosis as  in vitro  models ( 35 ). Sulindac has\nbeen demonstrated to control the expression of genes and\nproteins in ESCs from endometriosis-affected women,\nas well as to reduce nuclear factor-B activation ( 36 ).\nConsistent with the above research, the results showed that\nβ-sitosterol shows a proliferation inhibitory effect, proapoptotic effect on hEM15, and cell migration inhibition\nwhich was the weakest at 90 μM. β-sitosterol-induced\nSmad7 inhibited TGF-β1/Smad2/3 signaling, and this\neffect was positively correlated with the concentration of\nβ-sitosterol.\nThe Smad7-mediated change in the activity of the\nTGF-β1/Smad2/3 signaling pathway could be the key\nto β-sitosterol’s influence on the biological behavior of\nhEM15 cells. In this study, si-SMAD7( 1 ) can significantly\nsilence the expression of Smad7 protein. The ability of\ncell proliferation and migration was the strongest under\nthe treatment of si- SMAD7 ( 1 ), and apoptotic cells were\nsignificantly reduced. Moreover, the signal transduction\nof TGF-β1/Smad2/3 becomes stronger after transfection\nwith si- SMAD7 ( 1 ). The effects of β-sitosterol and si- SMAD7 ( 1 ) were opposed, indicating that the action of\nβ-sitosterol on hEM15 cells might be caused through\nSmad7.\nIn addition, there are already many drugs for the\ntreatment of endometriosis, during which the pain is\nmostly relieved, but the symptoms usually recur soon\nafter stopping the drug administration ( 37 ). Tamoxifen,\nmifepristone, and aromatase inhibitors are currently in the\ndevelopment stage ( 38 ). β-sitosterol could regulate the\ngut microbiota to treat endometrial disease ( 39 ). Young\net al. ( 40 ) reported that TGF-β1 regulates intraperitoneal\nVEGF-A expression via the ID1 pathway in patients\nwith endometriosis for the treatment of uterine disease.\nOur study was in agreement with the Young results that β-sitosterol has a significant modulatory effect on TGF-β1\nsignaling and also confirms the therapeutic effect of\nβ-sitosterol on the endometriosis of the animal models.\n\nThis study explored the therapeutic effect of β-sitosterol\non endometriosis in animal models. Experiments have\nfound that β-sitosterol can effectively promote apoptosis\nof endometriotic cells and improve endometrial tissue\nlesions  in vivo. In vitro  experiments show that β-sitosterol\ncan inhibit the proliferation of hEM15A cells and promote\ntheir apoptosis. In addition, the combined application of\nsi-Smad7 and β-sitosterol counteract the positive effects\nof β-sitosterol. Smad7-mediated TGF-β/Smads signaling\npathway could be the key molecular target for β-sitosterol.\nThese results in this research indicate that β-sitosterol\nand Smad7 could be used as potential new drugs for the\ntreatment of endometriosis. Admittedly, the limitation is\nthat the species difference between mice and humans is a\ndifficult problem for clinical translation. There are many\npathogenic factors of endometriosis, Smad7/TGFβ did not\nreflect all signaling pathways and future research should\nbe undertaken to explore the more in-depth mechanism.","source_license":"CC0","license_restricted":false}