Skip to main content

Vitamin A-coupled liposomes carrying TLR4-silencing shRNA induce apoptosis of pancreatic stellate cells and resolution of pancreatic fibrosis

Journal of Molecular Medicine volume 96, pages 445–458 (2018)Cite this article

Abstract

Chronic pancreatitis leads to irreversible damage in pancreatic endocrine and exocrine functions. However, there is no clinically available antifibrotic drug. Pancreatic stellate cells (PSCs) can be activated by Toll-like receptor 4 (TLR4) responses to its ligands and they contribute to the formation of pancreatic fibrosis. Silencing the expression of TLR4 in PSCs by RNAi may be a novel therapeutic strategy for the treatment of pancreatic fibrosis. In addition, PSCs have a remarkable capacity for vitamin A uptake most likely through cellular retinol binding protein (CRBP). In our study, to ensure the efficient delivery of RNAi therapeutic agents to PSCs, VitA-coupled liposomes (VA-lips) were used as drug carriers to deliver plasmids expressing TLR4-specific short hairpin RNA (shRNA) to treat pancreatic fibrosis. Our study demonstrated that silencing the expression of TLR4 could induce mitochondrial apoptosis in aPSCs and might be an effective therapeutic strategy for the treatment of pancreatic fibrosis.

Key messages

  • VA-lip-shRNA-TLR4 recovers pancreatic tissue damage.

  • VA-lip-shRNA-TLR4 resolution of pancreatic fibrosis.

  • VA-lip-shRNA-TLR4 accelerates ECM degradation and inhibits ECM synthesis.

  • Silencing TLR4 induces aPSCs mitochondrial apoptosis.

  • Silencing TLR4 inhibits the activation of NF-κB.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  1. Michalski CW, Gorbachevski A, Erkan M, Reiser C, Deucker S, Bergmann F, Giese T, Weigand M, Giese NA, Friess H, Kleeff J (2007) Mononuclear cells modulate the activity of pancreatic stellate cells which in turn promote fibrosis and inflammation in chronic pancreatitis. J Transl Med 5:63

    Article  PubMed  PubMed Central  Google Scholar 

  2. Erkan M, Adler G, Apte MV, Bachem MG, Buchholz M, Detlefsen S, Esposito I, Friess H, Gress TM, Habisch HJ, Hwang RF, Jaster R, Kleeff J, Klöppel G, Kordes C, Logsdon CD, Masamune A, Michalski CW, Oh J, Phillips PA, Pinzani M, Reiser-Erkan C, Tsukamoto H, Wilson J (2012) StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut 61:172–178

    CAS  Article  PubMed  Google Scholar 

  3. Kollmann TR, Levy O, Montgomery RR, Goriely S (2012) Innate immune function by Toll-like receptors: distinct responses in newborns and the elderly. Immunity 37:771–783

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Masamune A, Kikuta K, Watanabe T, Satoh K, Satoh A, Shimosegawa T (2008) Pancreatic stellate cells express Toll-like receptors. J Gastroenterol 43:352–362

    CAS  Article  PubMed  Google Scholar 

  5. Jagavelu K, Routray C, Shergill U, O’Hara SP, Faubion W, Shah VH (2010) Endothelial cell toll-like receptor 4 regulates fibrosis-associated angiogenesis in the liver. Hepatology 52:590–601

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Kesar V, Odin JA (2014) Toll-like receptors and liver disease. Liver Int 34:184–196

    CAS  Article  PubMed  Google Scholar 

  7. Ford LP, Toloue MM (2010) Delivery of RNAi mediators. Wiley Interdiscip Rev RNA 1:341–350

    CAS  Article  PubMed  Google Scholar 

  8. McCarroll JA, Phillips PA, Santucci N, Pirola RC, Wilson JS, Apte MV (2006) Vitamin A inhibits pancreatic stellate cell activation: implications for treatment of pancreatic fibrosis. Gut 55:79–89

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Xiao W, Jiang W, Shen J, Yin G, Fan Y, Wu D, Qiu L, Yu G, Xing M, Hu G et al (2015) Retinoic acid ameliorates pancreatic fibrosis and inhibits the activation of pancreatic stellate cells in mice with experimental chronic pancreatitis via suppressing the Wnt/beta-catenin signaling pathway. PLoS One 10:e0141462. https://doi.org/10.1371/journal.pone.0141462

    Article  PubMed  PubMed Central  Google Scholar 

  10. Sato Y, Murase K, Kato J, Kobune M, Sato T, Kawano Y, Takimoto R, Takada K, Miyanishi K, Matsunaga T et al (2008) Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nat Biotechnol 26:431–442

    CAS  Article  PubMed  Google Scholar 

  11. Narmada BC, Kang Y, Venkatraman L, Peng Q, Sakban RB, Nugraha B, Jiang X, Bunte RM, So PT, Tucker-Kellogg L et al (2013) Hepatic stellate cell-targeted delivery of hepatocyte growth factor transgene via bile duct infusion enhances its expression at fibrotic foci to regress dimethylnitrosamine-induced liver fibrosis. Hum Gene Ther 24:508–519

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Xue J, Zhao Q, Sharma V, Nguyen LP, Lee YN, Pham KL, Edderkaoui M, Pandol SJ, Park W, Habtezion A (2016) Aryl hydrocarbon receptor ligands in cigarette smoke induce production of Interleukin-22 to promote pancreatic fibrosis in models of chronic pancreatitis. Gastroenterology 151:1206–1217

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Neuschwander-Tetri BA, Bridle KR, Wells LD, Marcu M, Ramm GA (2000) Repetitive acute pancreatic injury in the mouse induces procollagen alpha1(I) expression colocalized to pancreatic stellate cells. Lab Investig 80:143–150

    CAS  Article  PubMed  Google Scholar 

  14. Ishiwatari H, Sato Y, Murase K, Yoneda A, Fujita R, Nishita H, Birukawa NK, Hayashi T, Sato T, Miyanishi K et al (2013) Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes. Gut 62:1328–1339

    CAS  Article  PubMed  Google Scholar 

  15. Rongione AJ, Kusske AM, Kwan K, Ashley SW, Reber HA, McFadden DW (1997) Interleukin 10 reduces the severity of acute pancreatitis in rats. Gastroenterology 112:960–967

    CAS  Article  PubMed  Google Scholar 

  16. Zhang Y, Liu P, Gao X, Qian W, Xu K (2010) rAAV2-TGF-beta(3) decreases collagen synthesis and deposition in the liver of experimental hepatic fibrosis rat. Dig Dis Sci 55:2821–2830

    Article  PubMed  Google Scholar 

  17. van Westerloo DJ, Florquin S, de Boer AM, Daalhuisen J, de Vos AF, Bruno MJ, van der Poll T (2005) Therapeutic effects of troglitazone in experimental chronic pancreatitis in mice. Am J Pathol 166:721–728

    Article  PubMed  PubMed Central  Google Scholar 

  18. Cheng P, Li Y, Yang L, Wen Y, Shi W, Mao Y, Chen P, Lv H, Tang Q, Wei Y (2009) Hepatitis B virus X protein (HBx) induces G2/M arrest and apoptosis through sustained activation of cyclin B1-CDK1 kinase. Oncol Rep 22:1101–1107

    CAS  PubMed  Google Scholar 

  19. Zhang Y, Peng T, Zhu H, Zheng X, Zhang X, Jiang N, Cheng X, Lai X, Shunnar A, Singh M et al (2010) Prevention of hyperglycemia-induced myocardial apoptosis by gene silencing of toll-like receptor-4. J Transl Med 8:133

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Alabsi AM, Lim KL, Paterson IC, Ali-Saeed R, Muharram BA (2016) Cell cycle arrest and apoptosis induction via modulation of mitochondrial integrity by Bcl-2 family members and caspase dependence in dracaena cinnabari-treated H400 human oral squamous cell carcinoma. Biomed Res Int 2016:4904016

    Article  PubMed  PubMed Central  Google Scholar 

  21. Masamune A, Watanabe T, Kikuta K, Shimosegawa T (2009) Roles of pancreatic stellate cells in pancreatic inflammation and fibrosis. Clin Gastroenterol Hepatol 7:S48–S54

    CAS  Article  PubMed  Google Scholar 

  22. Freund C, Gotthardt DN (2017) Vitamin A deficiency in chronic cholestatic liver disease: is vitamin A therapy beneficial? Liver Int 37:1752–1758

    Article  PubMed  Google Scholar 

  23. Parra-Guillen ZP, Gonzalez-Aseguinolaza G, Berraondo P, Troconiz IF (2010) Gene therapy: a pharmacokinetic/pharmacodynamic modelling overview. Pharm Res 27:1487–1497

    CAS  Article  PubMed  Google Scholar 

  24. Wieland CW, van Lieshout MH, Hoogendijk AJ, van der Poll T (2011) Host defence during Klebsiella pneumonia relies on haematopoietic-expressed Toll-like receptors 4 and 2. Eur Respir J 37:848–857

    CAS  Article  PubMed  Google Scholar 

  25. Standiford LR, Standiford TJ, Newstead MJ, Zeng X, Ballinger MN, Kovach MA, Reka AK, Bhan U (2012) TLR4-dependent GM-CSF protects against lung injury in gram-negative bacterial pneumonia. Am J Physiol Lung Cell Mol Physiol 302:L447–L454

    CAS  Article  PubMed  Google Scholar 

  26. Ahmadishoar S, Kariminik A (2017) Toll-like receptor 2 and its roles in immune responses against Legionella pneumophila. Life Sci 188:158–162

    CAS  Article  PubMed  Google Scholar 

  27. Albiger B, Dahlberg S, Sandgren A, Wartha F, Beiter K, Katsuragi H, Akira S, Normark S, Henriques-Normark B (2007) Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection. Cell Microbiol 9:633–644

    CAS  Article  PubMed  Google Scholar 

  28. Neuschwander-Tetri BA, Burton FR, Presti ME, Britton RS, Janney CG, Garvin PR, Brunt EM, Galvin NJ, Poulos JE (2000) Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse. Dig Dis Sci 45:665–674

    CAS  Article  PubMed  Google Scholar 

  29. Sun Z, Gou W, Kim DS, Dong X, Strange C, Tan Y, Adams DB, Wang H (2017) Adipose stem cell therapy mitigates chronic pancreatitis via differentiation into acinar-like cells in mice. Mol Ther 25:2490–2501

    CAS  Article  PubMed  Google Scholar 

  30. Shimizu K (2008) Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis. J Gastroenterol 43:823–832

    CAS  Article  PubMed  Google Scholar 

  31. Yokota T, Denham W, Murayama K, Pelham C, Joehl R, Bell RH Jr (2002) Pancreatic stellate cell activation and MMP production in experimental pancreatic fibrosis. J Surg Res 104:106–111

    CAS  Article  PubMed  Google Scholar 

  32. Phillips PA, McCarroll JA, Park S, Wu MJ, Pirola R, Korsten M, Wilson JS, Apte MV (2003) Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover. Gut 52:275–282

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Zhu X, Zhao H, Graveline AR, Buys ES, Schmidt U, Bloch KD, Rosenzweig A, Chao W (2006) MyD88 and NOS2 are essential for toll-like receptor 4-mediated survival effect in cardiomyocytes. Am J Physiol Heart Circ Physiol 291:H1900–H1909

    CAS  Article  PubMed  Google Scholar 

  34. Lu M, Zhang Q, Chen K, Xu W, Xiang X, Xia S (2017) The regulatory effect of oxymatrine on the TLR4/MyD88/NF-kappaB signaling pathway in lipopolysaccharide-induced MS1 cells. Phytomedicine 36:153–159

    CAS  Article  PubMed  Google Scholar 

  35. Bhardwaj A, Sethi G, Vadhan-Raj S, Bueso-Ramos C, Takada Y, Gaur U, Nair AS, Shishodia S, Aggarwal BB (2007) Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells. Blood 109:2293–2302

    CAS  Article  PubMed  Google Scholar 

  36. Laird MH, Rhee SH, Perkins DJ, Medvedev AE, Piao W, Fenton MJ, Vogel SN (2009) TLR4/MyD88/PI3K interactions regulate TLR4 signaling. J Leukoc Biol 85:966–977

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Park S, Lim W, Bazer FW, Song G (2017) Naringenin induces mitochondria-mediated apoptosis and endoplasmic reticulum stress by regulating MAPK and AKT signal transduction pathways in endometriosis cells. Mol Hum Reprod. https://doi.org/10.1093/molehr/gax057

  38. Manohar M, Verma AK, Venkateshaiah SU, Sanders NL, Mishra A (2017) Pathogenic mechanisms of pancreatitis. World J Gastrointest Pharmacol Ther 8:10–25

    Article  PubMed  PubMed Central  Google Scholar 

  39. Rockey DC, Bell PD, Hill JA (2015) Fibrosis—a common pathway to organ injury and failure. N Engl J Med 372:1138–1149

    CAS  Article  PubMed  Google Scholar 

  40. Wu Y, Lu J, Antony S, Juhasz A, Liu H, Jiang G, Meitzler JL, Hollingshead M, Haines DC, Butcher D et al (2013) Activation of TLR4 is required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by IFN-gamma and lipopolysaccharide in human pancreatic cancer cell lines. J Immunol 190:1859–1872

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81400665).

Author information

Affiliations

  1. Department of Endocrinology and Metabolism, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, No.37 Guo Xue Xiang, Chengdu, 610041, China

    Yuwei Zhang & Nanwei Tong

  2. Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, No.17 Section 3 People’s South Road, Chengdu, 610041, China

    Dan Yue, Liuliu Cheng & Ping Cheng

  3. Department of Pathology, West China Second Hospital, Sichuan University, No.20 Section 3 People’s South Road, Chengdu, 610041, China

    Anliang Huang

Authors
  1. Yuwei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dan Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liuliu Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anliang Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nanwei Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ping Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ping Cheng.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest related to this study.

Electronic supplementary material

Supplementary Fig. 1

Evaluation of the side effects of VA-lip-shRNA-TLR4. a Representative images of H&E staining for liver (top panel) and lung (bottom panel) (× 200). b Alanine aminotransferase and c aspartate aminotransferase levels in the sera of mice. Values are the mean ± SD for each group (n = 10 per group). (JPEG 2933 kb)

ESM 2

(DOCX 16 kb)

ESM 3

(DOCX 17 kb)

ESM 4

(DOCX 17 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, Y., Yue, D., Cheng, L. et al. Vitamin A-coupled liposomes carrying TLR4-silencing shRNA induce apoptosis of pancreatic stellate cells and resolution of pancreatic fibrosis. J Mol Med 96, 445–458 (2018). https://doi.org/10.1007/s00109-018-1629-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00109-018-1629-6

Keywords

  • Pancreatic fibrosis
  • Pancreatic stellate cells
  • Toll-like receptor 4
  • Gene therapy
  • RNAi
  • Liposome