Inducible nitric oxide synthase promotes differentiation of satellite cells and prevents stress urinary incontinence via HGF-mediated p38/MAPK signaling



Satellite cells (SCs) are multipotent skeletal muscle precursor cells, which can lead to muscle regeneration, a crucial process for the treatment of stress urinary incontinence (SUI). The activation of SCs is likely to be caused by inducible nitric oxide synthase (iNOS). This study examines the underlying mechanism of iNOS in SC activation, and the implications of a potential treatment of SUI.


SCs were isolated from the levator ani muscles of rats, followed by measurement of iNOS, paired box gene 7 (Pax7) and myosin D (MyoD) protein expression. Flow cytometric and cell counting kit-8 assays were conducted to assess SC proliferation, apoptosis, cell cycle distribution. SC myotube formation was observed under a light microscope. A rat model of SUI was established using vaginal dilation and bilateral ovarian castration, followed by assessment of leaking point pressure (LPP) and a sneezing test. The levels of MyoD, Pax7, hepatocyte growth factor (HGF) and p-38 in the levator ani muscles were detected by immunohistochemical and western blot analyses.


SCs successfully isolated from rat levator ani muscles showed increased MyoD and reduced Pax7 expression. Upregulation of iNOS promoted differentiation and myotube formation of SCs. iNOS elevated the expression of HGF, which in turn activated the p38/MAPK signaling pathway, promoting the differentiation of SCs. SCs overexpressing iNOS increased LPP, thus preventing SUI in an in vivo rat model.


Our results show that iNOS could activate the HGF-dependent p38/MAPK signaling pathway to alleviate SUI, potentially providing a novel therapeutic strategy for SUI.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5



Stress urinary incontinence


Nitric oxide


Inducible nitric oxide synthase


Hepatocyte growth factor


Jun amino-terminal kinase


Mitogen-activated protein kinase




Phosphate-buffered saline


Dulbecco's modified Eagle's medium


Fetal bovine serum


Polymerase chain reaction




Dimethyl sulfoxide


Small interfering RNA


Negative control


Cell counting kit-8


Optical density




Complementary DNA


Reverse transcription quantitative polymerase chain reaction


Glyceraldehyde-3-phosphate dehydrogenase






Paired box gene 7


Myosin D


Tris-buffered saline with Tween-20


Horseradish peroxidase


Enhanced chemiluminescence


Immunoglobulin G


Hematoxylin and eosin




PBS containing 0.1% Tween-20


Fluorescein isothiocyanate


Propidium iodide


Maximum bladder capacity


Leaking point pressure


Analysis of variance


  1. 1.

    Vardar E, Vythilingam G, Pinnagoda K, Engelhardt EM, Zambelli PY, Hubbell JA, Lutolf MP, Frey P, Larsson HM (2019) A bioactive injectable bulking material; a potential therapeutic approach for stress urinary incontinence. Biomaterials 206:41–48

    CAS  PubMed  Google Scholar 

  2. 2.

    El-Hefnawy AS, Wadie BS (2011) Severe stress urinary incontinence: objective analysis of risk factors. Maturitas 68(4):374–377

    PubMed  Google Scholar 

  3. 3.

    Saaby ML (2014) The urethral closure function in continent and stress urinary incontinent women assessed by urethral pressure reflectometry. Dan Med J 61(2):B4795

    PubMed  Google Scholar 

  4. 4.

    Lecoeur C, Swieb S, Zini L, Riviere C, Combrisson H, Gherardi R, Abbou C, Yiou R (2007) Intraurethral transfer of satellite cells by myofiber implants results in the formation of innervated myotubes exerting tonic contractions. J Urol 178(1):332–337

    PubMed  Google Scholar 

  5. 5.

    Wu R, Huang C, Wu Q, Jia X, Liu M, Xue Z, Qiu Y, Niu X, Wang Y (2019) Exosomes secreted by urine-derived stem cells improve stress urinary incontinence by promoting repair of pubococcygeus muscle injury in rats. Stem Cell Res Ther 10(1):80

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Lee YC, Juan YS, Liu CC, Bao BY, Wang CJ, Wu WJ, Huang CN, Huang SP (2016) The association of endothelial nitric oxide synthase (eNOS) G894T gene polymorphism with responsiveness to a selective alpha1 -blocker in men with benign prostatic hyperplasia related lower urinary tract symptoms. BJU Int 118(2):313–319

    PubMed  Google Scholar 

  7. 7.

    Filippin LI, Cuevas MJ, Lima E, Marroni NP, Gonzalez-Gallego J, Xavier RM (2011) Nitric oxide regulates the repair of injured skeletal muscle. Nitric Oxide 24(1):43–49

    CAS  PubMed  Google Scholar 

  8. 8.

    Lee M, Choy JC (2013) Positive feedback regulation of human inducible nitric-oxide synthase expression by Ras protein S-nitrosylation. J Biol Chem 288(22):15677–15686

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Chen M, Cheng C, Yan M, Niu S, Gao S, Shi S, Liu H, Qin Y, Shen A (2008) Involvement of CAPON and nitric oxide synthases in rat muscle regeneration after peripheral nerve injury. J Mol Neurosci 34(1):89–100

    CAS  PubMed  Google Scholar 

  10. 10.

    Filippin LI, Moreira AJ, Marroni NP, Xavier RM (2009) Nitric oxide and repair of skeletal muscle injury. Nitric Oxide 21(3–4):157–163

    CAS  PubMed  Google Scholar 

  11. 11.

    Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102(6):777–786

    CAS  Google Scholar 

  12. 12.

    Sambasivan R, Yao R, Kissenpfennig A, Van Wittenberghe L, Paldi A, Gayraud-Morel B, Guenou H, Malissen B, Tajbakhsh S, Galy A (2011) Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development 138(17):3647–3656

    CAS  PubMed  Google Scholar 

  13. 13.

    Zhang K, Sha J, Harter ML (2010) Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells. J Cell Biol 188(1):39–48

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Fajardo-Puerta AB, Mato Prado M, Frampton AE, Jiao LR (2016) Gene of the month: HGF. J Clin Pathol 69(7):575–579

    CAS  PubMed  Google Scholar 

  15. 15.

    Zhang H, Anderson JE (2014) Satellite cell activation and populations on single muscle-fiber cultures from adult zebrafish (Danio rerio). J Exp Biol 217(Pt 11):1910–1917

    PubMed  Google Scholar 

  16. 16.

    Anderson JE (2016) Hepatocyte growth factor and satellite cell activation. Adv Exp Med Biol 900:1–25

    CAS  PubMed  Google Scholar 

  17. 17.

    Su Y, Xie W, Wang C, Peng L, Zhou X, Ye L (2012) JNK/P38 mitogen-activated protein kinase used for hepatocyte growth factor-induced proliferation, differentiation, and migration in human dental papilla cells. J Endod 38(9):1207–1213

    PubMed  Google Scholar 

  18. 18.

    Liang H, Yan X, Pan Y, Wang Y, Wang N, Li L, Liu Y, Chen X, Zhang CY, Gu H, Zen K (2015) MicroRNA-223 delivered by platelet-derived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3. Mol Cancer 14:58

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Segales J, Perdiguero E, Munoz-Canoves P (2016) Regulation of muscle stem cell functions: a focus on the p38 MAPK signaling pathway. Front Cell Dev Biol 4:91

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Feng A, Zhou G, Yuan X, Huang X, Zhang Z, Zhang T (2013) Inhibitory effect of baicalin on iNOS and NO expression in intestinal mucosa of rats with acute endotoxemia. PLoS one 8(12):e80997

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Liu D, Adams MS, Burdette EC, Diederich CJ (2018) Transurethral high-intensity ultrasound for treatment of stress urinary incontinence (SUI): simulation studies with patient-specific models. Int J Hyperthermia 34(8):1236–1247

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Aicher WK, Hart ML, Stallkamp J, Klunder M, Ederer M, Sawodny O, Vaegler M, Amend B, Sievert KD, Stenzl A (2014) Towards a treatment of stress urinary incontinence: application of mesenchymal stromal cells for regeneration of the sphincter muscle. J Clin Med 3(1):197–215

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Otto A, Collins-Hooper H, Patel A, Dash PR, Patel K (2011) Adult skeletal muscle stem cell migration is mediated by a blebbing/amoeboid mechanism. Rejuvenation Res 14(3):249–260

    CAS  PubMed  Google Scholar 

  24. 24.

    Ding S, Swennen GNM, Messmer T, Gagliardi M, Molin DGM, Li C, Zhou G, Post MJ (2018) Maintaining bovine satellite cells stemness through p38 pathway. Sci Rep 8(1):10808

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Olguin HC, Yang Z, Tapscott SJ, Olwin BB (2007) Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination. J Cell Biol 177(5):769–779

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Tatsumi R (2010) Mechano-biology of skeletal muscle hypertrophy and regeneration: possible mechanism of stretch-induced activation of resident myogenic stem cells. Anim Sci J 81(1):11–20

    CAS  PubMed  Google Scholar 

  27. 27.

    Gill R, Hitchins L, Fletcher F, Dhoot GK (2010) Sulf1A and HGF regulate satellite-cell growth. J Cell Sci 123(Pt 11):1873–1883

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Witt R, Weigand A, Boos AM, Cai A, Dippold D, Boccaccini AR, Schubert DW, Hardt M, Lange C, Arkudas A, Horch RE, Beier JP (2017) Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering. BMC Cell Biol 18(1):15

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Lee SJ, Yoo M, Go GY, Hwang J, Lee HG, Kim YK, Seo DW, Baek NI, Ryu JH, Kang JS, Bae GU (2014) Tetrahydropalmatine promotes myoblast differentiation through activation of p38MAPK and MyoD. Biochem Biophys Res Commun 455(3–4):147–152

    CAS  PubMed  Google Scholar 

  30. 30.

    Lu T, Yang C, Sun H, Lv J, Zhang F, Dong XJ (2014) FGF4 and HGF promote differentiation of mouse bone marrow mesenchymal stem cells into hepatocytes via the MAPK pathway. Genet Mol Res 13(1):415–424

    CAS  PubMed  Google Scholar 

  31. 31.

    Kuang W, Deng Q, Deng C, Li W, Shu S, Zhou M (2017) Hepatocyte growth factor induces breast cancer cell invasion via the PI3K/Akt and p38 MAPK signaling pathways to up-regulate the expression of COX2. Am J Transl Res 9(8):3816–3826

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Aenlle KK, Curtis KM, Roos BA, Howard GA (2014) Hepatocyte growth factor and p38 promote osteogenic differentiation of human mesenchymal stem cells. Mol Endocrinol 28(5):722–730

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Shinohara M, Sumino Y, Sato F, Kiyono T, Hashimoto N, Mimata H (2017) Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter. Int J Urol 24(6):461–467

    CAS  PubMed  Google Scholar 

  34. 34.

    Kim BS, Chun SY, Lee JK, Lim HJ, Bae JS, Chung HY, Atala A, Soker S, Yoo JJ, Kwon TG (2012) Human amniotic fluid stem cell injection therapy for urethral sphincter regeneration in an animal model. BMC Med 10:94

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Lin CS, Lue TF (2012) Stem cell therapy for stress urinary incontinence: a critical review. Stem Cells Dev 21(6):834–843

    CAS  PubMed  Google Scholar 

Download references


The authors would like to acknowledge the helpful comments on this paper received from the reviewers.


This study was supported by the Yunnan Provincial Science and Technology Department (No. 2015FA007); National Natural Science Foundation of China (81860127); Medical Discipline Leaders of Health and Family Planning Commission of Yunnan Province (D-201615); Young and Middle-Aged Academic and Technical Leaders Reserved Scholar of Yunnan Province (No. 2017HB038); the Engineering and Research Center of Yunnan College and University for Female Pelvic Floor Disease Diagnosis and Treatment.

Author information




KF and FG designed the study. JW, JL and TY collated the data, carried out data analyses and produced the initial draft of the manuscript. SL, YW and TC contributed to drafting and polishing the manuscript. All authors have read and approved the final submitted manuscript.

Corresponding author

Correspondence to Kewei Fang.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Guan, F., Wu, J., Li, J. et al. Inducible nitric oxide synthase promotes differentiation of satellite cells and prevents stress urinary incontinence via HGF-mediated p38/MAPK signaling. World J Urol (2020).

Download citation


  • Stress urinary incontinence
  • Inducible nitric oxide synthase
  • Satellite cells
  • Hepatocyte growth factor
  • p38 mitogen-activated protein kinase signaling pathway