The inhibitory effects of 20(R)-ginsenoside Rg3 on the proliferation, angiogenesis and collagen synthesis of hypertrophic scar derived fibroblasts in vitro

Document Type: Original Article

Authors

Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital Affiliated Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, China

Abstract

Objective(s): Therapeutic effect of many selectable methods applied in clinical practice for treating hypertrophic scar (HS) is not still so satisfactory. Meanwhile, a few medicines may lead to several undesirable complications. The traditional Chinese medicine, Rg3, has been reported for multiple antitumor effects previously. We have conducted series of animal experiments and confirmed the inhibitory effect of Rg3 in HS before. The aim of this study was to further verify the conclusions of previous studies and reveal the specific functional mechanisms of Rg3.
Materials and Methods: The HS specimens were obtained from the patients aged from 15 to 36 years without systemic diseases and the primary cultured cells were isolated from the scar tissue and expanded in vitro. In every experiment, hypertrophic scar fibroblasts (HSFs) were divided into three groups and respectively cultured in medium with or without different Rg3 concentrations (50, 100 μg/ml). Cell viability assay, flow cytometry analysis (FCM), quantitative PCR, cell migration assay, immunofluorescence staining, western blot and ELISA were employed.
Results: The outcomes demonstrated that Rg3 could suppress cell proliferation, vascularization and extracellular matrix (ECM) deposition of HSFs in vitro by TGF-β/SMAD and Erk signaling pathways. Significant statistical differences were between control group and Rg3-treated groups (P<0.05).
Conclusion: This study provides sufficient in vitro evidences for Rg3 as a promising drug in the treatment of human HS.

Keywords

Main Subjects


1. Lorden ER, Miller KJ, Ibrahim MM, Bashirov L, Hammett E, Chakraborty S, et al. Biostable electrospun microfibrous scaffolds mitigate hypertrophic scar contraction in an immune-competent murine model. Acta Biomater 2016; 32: 100-109.

2. Guadanhim LR, Gonçalves RG, Bagatin E. Observational retrospective study evaluating the effects of oral isotretinoin in keloids and hypertrophic scars. Int J Dermatol 2016; 55: 1255-1258.

3. Ud-Din S, Bayat A. New insights on keloids, hypertrophic scars, and striae. Dermatol Clin 2014; 32: 193-209.

4. Van der Veer WM, Bloemen MC, Ulrich MM, Molema G, van Zuijlen PP, Middelkoop E, et al. Potential cellular and molecular causes of hypertrophic scar formation. Burns 2009; 35: 15-29.

5. Hayashi T, Furukawa H, Oyama A, Funayama E, Saito A, Murao N, et al. A new uniform protocol of combined corticosteroid injections and ointment application reduces recurrence rates after surgical keloid/hypertrophic scar excision. Dermatol Surg 2012; 38: 893-897.

6. Yang SY, Yang JY, Hsiao YC. Comparison of combination therapy (steroid, calcium channel blocker, and interferon) with steroid monotherapy for treating human hypertrophic scars in an animal model. Ann Plast Surg 2015; 74 Suppl 2: S162-167.

7. Wang J, Jiao H, Stewart TL, Shankowsky HA, Scott PG, Tredget EE. Improvement in postburn hypertrophic scar after treatment with IFN-alpha2b is associated with decreased fibrocytes. J Interferon Cytokine Res 2007; 27: 921-930.

8. Berman B, Flores F. The treatment of hypertrophic scars and keloids. Eur J Dermatol 1998; 8: 591-595.

9. He B, Chen P, Xie Y, Li S, Zhang X, Yang R, et al. 20(R)-Ginsenoside Rg3 protects SH-SY5Y cells against apoptosis induced by oxygen and glucose deprivation/reperfusion. Bioorg Med Chem Lett 2017; 27: 3867-3871.

10. Keung MH, Chan LS, Kwok HH, Wong RN, Yue PY. Role of microRNA-520h in 20(R)-ginsenoside-Rg3-mediated angiosuppression. J Ginseng Res 2016; 40: 151-159.

11. Cheng L, Sun X, Hu C, Jin R, Sun B, Shi Y, et al. In vivo early intervention and the therapeutic effects of 20(S)-ginsenoside Rg3 on hypertrophic scar formation. PLoS One 2014; 9: e113640.

12. Kim SM, Lee SY, Cho JS, Son SM, Choi SS, Yun YP, et al. Combination of ginsenoside Rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of NF-Kb. Eur J Pharmacol 2010; 10: 1-9.

13. Shin JU, Lee WJ, Tran TN, Jung I, Lee JH. Hsp70 knockdown by siRNA decreased collagen production in keloid fibroblasts. Yonsei Med J 2015; 56: 1619-1626.

14. Chen QJ, Zhang MZ, Wang LX. Gensenoside Rg3 inhibits hypoxia-induced VEGF expression in human cancer cells. Cell Physiol Biochem 2010; 26: 849-858.

15. Shin YM, Jung HJ, Choi WY, Lim CJ. Antioxidative, anti-inflammatory, and matrix metalloproteinase inhibitory activities of 20(S)-ginsenoside Rg3 in cultured mammalian cell lines. Mol Biol Rep 2013; 40: 269-279.

16. Li Y, Yang T, Li J, Hao HL, Wang SY, Yang J, et al. Inhibition of multiple myeloma cell proliferation by ginsenoside Rg3 via reduction in the secretion of IGF-1. Mol Med Rep 2016; 14: 2222-2230.

17. Shan X, Fu YS, Aziz F, Wang XQ, Yan Q, Liu JW. Ginsenoside Rg3 inhibits melanoma cell proliferation through down-regulation of histone deacetylase 3 (HDAC3) and increase of p53 acetylation. PLoS One 2014; 9: e115401.

18. Xia T, Wang YN, Zhou CX, Wu LM, Liu Y, Zeng QH, et al. Ginsenoside Rh2 and Rg3 inhibit cell proliferation and induce apoptosis by increasing mitochondrial reactive oxygen species in human leukemia Jurkat cells. Mol Med Rep 2017; 15: 3591-3598.

19. Berman B, Maderal A, Raphael B. Keloids and Hypertrophic Scars: pathophysiology, classification, and treatment. Dermatol Surg 2017; 43 Suppl 1:S3-S18.

20. Ehrlich HP, Desmoulière A, Diegelmann RF, Cohen IK, Compton CC, Garner WL, et al. Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol 1994; 145:105-113.

21. Mu S, Kang B, Zenfresultg W, Sun Y, Yang F. MicroRNA-143-3p inhibits hyperplastic scar formation by targeting connective tissue growth factor CTGF/CCN2 via the Akt/mTOR pathway. Mol Cell Biochem 2016; 416: 99-108.

22. Yamamoto T, Hartmann K, Eckes B, Krieg T. Role of stem cell factor and monocyte chemoattractant protein-1 in the interaction between fibroblasts and mast cells in fibrosis. J Dermatol Sci 2001; 26: 106-111.

23. Salgado RM, Alcántara L, Mendoza-Rodríguez CA, Cerbón M, Hidalgo-González C, Mercadillo P, et al. Post-burn hypertrophic scars are characterized by high levels of IL-1β mRNA and protein and TNF-α type I receptors. Burns 2012; 38: 668-676.

24. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92: 827-839.

25. Uchida G, Yoshimura K, Kitano Y, Okazaki M, Harii K. Tretinoin reverses upregulation of matrix metalloproteinase-13 in human keloid-derived fibroblasts. Exp Dermatol 2003; 12 Suppl 2: 35-42.

26. Huang D, Liu Y, Huang Y, Xie Y, Shen K, Zhang D, et al. Mechanical compression upregulates MMP9 through SMAD3 but not SMAD2 modulation in hypertrophic scar fibroblasts. Connect Tissue Res 2014; 55: 391-396.

27. Cheng F, Shen Y, Mohanasundaram P, Lindström M3, Ivaska J4, Ny T, et al. Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β-Slug signaling. Proc Natl Acad Sci U S A 2016; 113: E4320-7.

28. Bullard KM, Longaker MT, Lorenz HP. Fetal wound healing: current biology. World J Surg 2003; 27: 54-61.

29. Kose O, Waseem A. Keloids and hypertrophic scars: are they two different sides of the same coin? Dermatol Surg 2003; 34: 336-346.

30. Branton MH, Kopp JB. TGF-β and fibrosis. Microbes Infect 1999; 1: 1349-1365.

31. Chun Q, ZhiYong W, Fei S, XiQiao W. Dynamic biological changes in fibroblasts during hypertrophic scar formation and regression. Int Wound J 2016; 13:257-262.

32. Wu Y, Zhang Q, Ann DK, Akhondzadeh A, Duong HS, Messadi DV, et al. Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts. Am J Physiol Cell Physiol 2004; 286: C905-912.

33. Li J, Zhang M, Ma J. Myricitrin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation and migration through suppressing PDGFRβ/Akt/Erk signaling. Int J Clin Exp Med 2015; 8: 21715-21723.

34. Zeng D, Wang J, Kong P, Chang C, Li J, Li J. Ginsenoside Rg3 inhibits HIF-1α and VEGF expression in patient with acute leukemia via inhibiting the activation of PI3K/Akt and ERK1/2 pathways. Int J Clin Exp Pathol 2014; 7:2172-2178.

35. Chen QJ, Zhang MZ, Wang LX. Gensenoside Rg3 inhibits hypoxia-induced VEGF expression in human cancer cells. Cell Physiol Biochem 2010; 26: 849-858.