β-Sitosterol Inhibits The Proliferation of Endometrial Cells via Regulating Smad7-Mediated TGF-β/Smads Signaling Pathway

Document Type : Original Article


Departments of Gynecology, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China


Objective: To investigate the effect of β-sitosterol on endometrial cells to understand the underlying mechanism.
Materials and Methods: This is a laboratory-based experimental study conducted on animals and cells. Histological
assays were performed to determine the effect of β-sitosterol on endometrial cells. The CCK-8 assay was used to
assess the inhibitory effect of β-sitosterol on the proliferation of ectopic endometrial stromal cells (hEM15A). Flow
cytometry was performed to evaluate the induction of apoptosis by β-sitosterol in hEM15A cells. The transwell invasion
assay was conducted to measure the suppression of hEM15A cell migration by β-sitosterol. Western blot analyses
were performed to analyze the effect of β-sitosterol on the expression of Smad family member 7 (Smad7) and the
activity of transforming growth factor-β (TGF-β1), as well as the phosphorylation of Smad2 and Smad3.
Results: Histological assays showed that β-sitosterol regulates histopathology and induces apoptosis of endometrial
cells in vivo. The CCK-8 assay revealed that β-sitosterol could inhibit the proliferation of hEM15A in human endometriosis
patients. Flow cytometry showed that apoptosis was triggered by β-sitosterol in hEM15A. The transwell invasion assay
indicated that the hEM15A migration under the β-sitosterol treatment group was suppressed. Western blot analyses
suggested that β-sitosterol increased the expression of Smad7, decreased the activity of TGF-β1, and reduced the
phosphorylation of Smad2 and Smad3. The effect of β-sitosterol was weakened by the silence of Smad7.
Conclusion: The results suggest that β-sitosterol can inhibit the proliferation of endometrial cells and relieve
endometriosis by inhibiting TGF-β-induced phosphorylation of Smads through regulation of Smad7.


Main Subjects

  1. Amro B, Ramirez Aristondo ME, Alsuwaidi S, Almaamari B, Hakim Z, Tahlak M, et al. New understanding of diagnosis, treatment and prevention of endometriosis. Int J Environ Res Public Health. 2022; 19(11): 6725.
  2. Pirtea P, Vulliemoz N, de Ziegler D, Ayoubi JM. Infertility workup: identifying endometriosis. Fertil Steril. 2022; 118(1): 29-33.
  3. Song Y, Fu J, Zhou M, Xiao L, Feng X, Chen H, et al. Activated Hippo/Yes-associated protein pathway promotes cell proliferation and anti-apoptosis in endometrial stromal cells of endometriosis. J Clin Endocrinol Metab. 2016; 101(4): 1552-1561.
  4. D’Hooghe TM, Debrock S, Hill JA, Meuleman C. Endometriosis and subfertility: is the relationship resolved? Semin Reprod Med. 2003; 21(2): 243-254.
  5. Mechsner S. Endometriosis, an ongoing pain-step-by-step treatment. J Clin Med. 2022; 11(2): 467.
  6. Ni L, Tang C, Wang Y, Wan J, Charles MG, Zhang Z, et al. Construction of a miRNA-Based nomogram model to predict the prognosis of endometrial cancer. J Pers Med. 2022; 12(7): 1154.
  7. Liu Z, Yi L, Du M, Gong G, Zhu Y. Overexpression of TGF-β enhances the migration and invasive ability of ectopic endometrial cells via ERK/MAPK signaling pathway. Exp Ther Med. 2019; 17(6): 4457-4464.
  8. Young VJ, Brown JK, Maybin J, Saunders PT, Duncan WC, Horne AW. Transforming growth factor-β induced Warburg-like metabolic reprogramming may underpin the development of peritoneal endometriosis. J Clin Endocrinol Metab. 2014; 99(9): 3450-3459.
  9. Hu Z, Li B, Wang Z, Hu X, Zhang M, Chen R, et al. The sympathetic transmitter norepinephrine inhibits VSMC proliferation induced byTGFβ by suppressing the expression of the TGFβ receptor ALK5 in aorta remodeling. Mol Med Rep. 2020; 22(1): 387-397.
  10. Li H, Rong P, Ma X, Nie W, Chen Y, Zhang J, et al. Mouse umbilical cord mesenchymal stem cell paracrine alleviates renal fibrosis in diabetic nephropathy by reducing myofibroblast transdifferentiation and cell proliferation and upregulating MMPs in mesangial cells. J Diabetes Res. 2020; 2020: 3847171.
  11. Chung AC, Dong Y, Yang W, Zhong X, Li R, Lan HY. Smad7 suppresses renal fibrosis via altering expression of TGF-β/Smad3- regulated microRNAs. Mol Ther. 2013; 21(2): 388-398.
  12. Li JH, Zhu HJ, Huang XR, Lai KN, Johnson RJ, Lan HY. Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation. J Am Soc Nephrol. 2002; 13(6): 1464-1472.
  13. Kim KS, Yang HJ, Lee JY, Na YC, Kwon SY, Kim YC, et al. Effects of β-sitosterol derived from Artemisia capillaris on the activated human hepatic stellate cells and dimethylnitrosamineinduced mouse liver fibrosis. BMC Complement Altern Med. 2014; 14: 363.
  14. Vijaya, Yadav AK. In vitro anthelmintic assessment of selected phytochemicals against Hymenolepis diminuta, a zoonotic tapeworm. J Parasit Dis. 2016; 40(3): 1082-1086.
  15. Cheng D, Guo Z, Zhang S. Effect of β-sitosterol on the expression of HPV E6 and p53 in cervical carcinoma cells. Contemp Oncol (Pozn). 2015; 19(1): 36-42.
  16. Sharmila R, Sindhu G. Modulation of angiogenesis, proliferative response and apoptosis by β-Sitosterol in rat model of renal carcinogenesis. Indian J Clin Biochem. 2017; 32(2): 142-152.
  17. Park YJ, Bang IJ, Jeong MH, Kim HR, Lee DE, Kwak JH, et al. Effects of β-sitosterol from corn silk on TGf-β1-induced epithelialmesenchymal transition in lung alveolar epithelial cells. J Agric Food Chem. 2019; 67(35): 9789-9795.
  18. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Viganò P. Endometriosis. Nat Rev Dis Primers. 2018; 4(1): 9.
  19. Wang X, Li M, Hu M, Wei P, Zhu W. BAMBI overexpression together with β-sitosterol ameliorates NSCLC via inhibiting autophagy and inactivating TGF-β/Smad2/3 pathway. Oncol Rep. 2017; 37(5): 3046-3054.
  20. Wen Y, Fan L, Pang L, Zhao T, Li R, Zhang Y, et al. NeiyiKangfu tablets control the progression of endometriosis through inhibiting RAF/MEK/ERK signal pathway by targeting RKIP. Gynecol Endocrinol. 2022; 38(12): 1136-1146.
  21. Laitinen K, Gylling H. Dose-dependent LDL-cholesterol lowering effect by plant stanol ester consumption: clinical evidence. Lipids Health Dis. 2012; 11: 140.
  22. Lohrey EE, Hughes IR, Gray IK. Effect of dietary lipid oxidation on measurement of protein efficiency ratios. J Assoc Off Anal Chem. 1978; 61(1): 104-110.
  23. Guo SW. The pathogenesis of adenomyosis vis-à-vis endometriosis. J Clin Med. 2020; 9(2): 485.
  24. Ciarmela P, Critchley H, Christman GM, Reis FM. Pathogenesis of endometriosis and uterine fibroids. Obstet Gynecol Int. 2013; 2013: 656571.
  25. Harada T, Kaponis A, Iwabe T, Taniguchi F, Makrydimas G, Sofikitis N, et al. Apoptosis in human endometrium and endometriosis. Hum Reprod Update. 2004; 10(1): 29-38.
  26. Wang J, Zhang L, Jiang W, Zhang R, Zhang B, Silayiding A, et al. MicroRNA-135a promotes proliferation, migration, invasion and induces chemoresistance of endometrial cancer cells. Eur J Obstet Gynecol Reprod Biol X. 2019; 5: 100103.
  27. Awad AB, Roy R, Fink CS. Beta-sitosterol, a plant sterol, induces apoptosis and activates key caspases in MDA-MB-231 human breast cancer cells. Oncol Rep. 2003; 10(2): 497-500.
  28. Sundstrøm T, Prestegarden L, Azuaje F, Aasen SN, Røsland GV, Varughese JK, et al. Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis. Acta Neuropathol Commun. 2019; 7(1): 55.
  29. Awad AB, Gan Y, Fink CS. Effect of beta-sitosterol, a plant sterol, on growth, protein phosphatase 2A, and phospholipase D in LNCaP cells. Nutr Cancer. 2000; 36(1): 74-78.
  30. Choi YH, Kong KR, Kim YA, Jung KO, Kil JH, Rhee SH, et al. Induction of Bax and activation of caspases during beta-sitosterol- mediated apoptosis in human colon cancer cells. Int J Oncol. 2003; 23(6): 1657-1662.
  31. Yin X, Li X, Feng G, Qu Y, Wang H. LINC00565 enhances proliferative ability in endometrial carcinoma by downregulating KLF9. Onco Targets Ther. 2020; 13: 6181-6189.
  32. Moreau JM, Velegraki M, Bolyard C, Rosenblum MD, Li Z. Transforming growth factor-β1 in regulatory T cell biology. Sci Immunol. 2022; 7(69): eabi4613.
  33. Wang J, Xu Z, Wang Z, Du G, Lun L. TGF-beta signaling in cancer radiotherapy. Cytokine. 2021; 148: 155709.
  34. Shu Y, Liu Y, Li X, Cao L, Yuan X, Li W, et al. Aspirin-triggered resolving D1 inhibits TGF-β1-induced EndMT through increasing the expression of Smad7 and is closely related to oxidative stress. Biomol Ther (Seoul). 2016; 24(2): 132-139.
  35. Huang ZX, Wu RF, Mao XM, Huang SM, Liu TT, Chen QH, et al. Establishment of an immortalized stromal cell line derived from human Endometriotic lesion. Reprod Biol Endocrinol. 2020; 18(1): 119.
  36. Wieser F, Vigne JL, Ryan I, Hornung D, Djalali S, Taylor RN. Sulindac suppresses nuclear factor-kappaB activation and RANTES gene and protein expression in endometrial stromal cells from women with endometriosis. J Clin Endocrinol Metab. 2005; 90(12): 6441-6447.
  37. Becker CM, Gattrell WT, Gude K, Singh SS. Reevaluating response and failure of medical treatment of endometriosis: a systematic review. Fertil Steril. 2017; 108(1): 125-136.
  38. Barra F, Scala C, Mais V, Guerriero S, Ferrero S. Investigational drugs for the treatment of endometriosis, an update on recent developments. Expert Opin Investig Drugs. 2018; 27(5): 445-458.
  39. Yu Y, Cao Y, Huang W, Liu Y, Lu Y, Zhao J. β-Sitosterol Ameliorates Endometrium Receptivity in PCOS-Like Mice: The Mediation of Gut Microbiota. Front Nutr. 2021; 8: 667130.
  40. Young VJ, Ahmad SF, Brown JK, Duncan WC, Horne AW. Peritoneal VEGF-A expression is regulated by TGF-β1 through an ID1 pathway in women with endometriosis. Sci Rep. 2015; 5: 16859.