Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

TGR5 agonist inhibits intestinal epithelial cell apoptosis via cAMP/PKA/c-FLIP/JNK signaling pathway and ameliorates dextran sulfate sodium-induced ulcerative colitis

Acta Pharmacologica Sinica volume 44, pages 1649–1664 (2023)Cite this article

Abstract

Excessive apoptosis of intestinal epithelial cell (IEC) is a crucial cause of disrupted epithelium homeostasis, leading to the pathogenesis of ulcerative colitis (UC). The regulation of Takeda G protein-coupled receptor-5 (TGR5) in IEC apoptosis and the underlying molecular mechanisms remained unclear, and the direct evidence from selective TGR5 agonists for the treatment of UC is also lacking. Here, we synthesized a potent and selective TGR5 agonist OM8 with high distribution in intestinal tract and investigated its effect on IEC apoptosis and UC treatment. We showed that OM8 potently activated hTGR5 and mTGR5 with EC50 values of 202 ± 55 nM and 74 ± 17 nM, respectively. After oral administration, a large amount of OM8 was maintained in intestinal tract with very low absorption into the blood. In DSS-induced colitis mice, oral administration of OM8 alleviated colitis symptoms, pathological changes and impaired tight junction proteins expression. In addition to enhancing intestinal stem cell (ISC) proliferation and differentiation, OM8 administration significantly reduced the rate of apoptotic cells in colonic epithelium in colitis mice. The direct inhibition by OM8 on IEC apoptosis was further demonstrated in HT-29 and Caco-2 cells in vitro. In HT-29 cells, we demonstrated that silencing TGR5, inhibition of adenylate cyclase or protein kinase A (PKA) all blocked the suppression of JNK phosphorylation induced by OM8, thus abolished its antagonizing effect against TNF-α induced apoptosis, suggesting that the inhibition by OM8 on IEC apoptosis was mediated via activation of TGR5 and cAMP/PKA signaling pathway. Further studies showed that OM8 upregulated cellular FLICE-inhibitory protein (c-FLIP) expression in a TGR5-dependent manner in HT-29 cells. Knockdown of c-FLIP blocked the inhibition by OM8 on TNF-α induced JNK phosphorylation and apoptosis, suggesting that c-FLIP was indispensable for the suppression of OM8 on IEC apoptosis induced by OM8. In conclusion, our study demonstrated a new mechanism of TGR5 agonist on inhibiting IEC apoptosis via cAMP/PKA/c-FLIP/JNK signaling pathway in vitro, and highlighted the value of TGR5 agonist as a novel therapeutic strategy for the treatment of UC.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: OM8 is a potent and selective TGR5 agonist.
Fig. 2: OM8 could attenuate DSS-induced colitis.
Fig. 3: OM8 restored tight junction proteins expression in DSS-induced colitis mice.
Fig. 4: OM8 drove ISCs proliferation and differentiation in DSS-induced colitis mice.
Fig. 5: OM8 inhibited intestinal epithelial cell apoptosis both in vivo and in vitro.
Fig. 6: OM8 suppressed intestinal epithelial cell apoptosis in a TGR5-dependent manner.
Fig. 7: OM8 inhibited TNF-ɑ induced JNK phosphorylation in intestinal epithelial cells through TGR5/cAMP/PKA signaling pathway.
Fig. 8: Upregulation of c-FLIP expression by TGR5/cAMP/PKA signaling pathway is indispensable for the inhibition by OM8 on TNF-ɑ induced JNK phosphorylation and apoptosis in intestinal epithelial cells.
Fig. 9: Mechanisms of TGR5 agonist on inhibiting intestinal epithelial cell apoptosis and ameliorating ulcerative colitis.

Similar content being viewed by others

References

  1. Pavlidis P, Tsakmaki A, Pantazi E, Li K, Cozzetto D, Digby-Bell J, et al. Interleukin-22 regulates neutrophil recruitment in ulcerative colitis and is associated with resistance to ustekinumab therapy. Nat Commun. 2022;13:5820.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gao C, Zhou Y, Chen Z, Li H, Xiao Y, Hao W, et al. Turmeric-derived nanovesicles as novel nanobiologics for targeted therapy of ulcerative colitis. Theranostics. 2022;12:5596–614.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhao J, Lin Z, Ying P, Zhao Z, Yang H, Qian J, et al. circSMAD4 promotes experimental colitis and impairs intestinal barrier functions by targeting JAK2 through sponging miR-135a-5p. J Crohns Colitis. 2022;jjac154. https://doi.org/10.1093/ecco-jcc/jjac154.

  4. Scalavino V, Piccinno E, Bianco G, Schena N, Armentano R, Giannelli G, et al. The increase of miR-195-5p reduces intestinal permeability in ulcerative colitis, modulating tight junctions’ expression. Int J Mol Sci. 2022;23:5840.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Li X, Li Q, Xiong B, Chen H, Wang X, Zhang D. Discoidin domain receptor 1(DDR1) promote intestinal barrier disruption in ulcerative colitis through tight junction proteins degradation and epithelium apoptosis. Pharmacol Res. 2022;183:106368.

    Article  CAS  PubMed  Google Scholar 

  6. Wan Y, Yang L, Jiang S, Qian D, Duan J. Excessive apoptosis in ulcerative colitis: crosstalk between apoptosis, ROS, ER stress, and intestinal homeostasis. Inflamm Bowel Dis. 2022;28:639–48.

    Article  PubMed  Google Scholar 

  7. Yu TX, Kalakonda S, Liu X, Han N, Chung HK, Xiao L, et al. Long noncoding RNA uc.230/CUG-binding protein 1 axis sustains intestinal epithelial homeostasis and response to tissue injury. JCI Insight. 2022;7:e156612.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Shao M, Yan Y, Zhu F, Yang X, Qi Q, Yang F, et al. Artemisinin analog SM934 alleviates epithelial barrier dysfunction via inhibiting apoptosis and caspase-1-mediated pyroptosis in experimental colitis. Front Pharmacol. 2022;13:849014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Duan C, Xu X, Lu X, Wang L, Lu Z. RIP3 knockdown inhibits necroptosis of human intestinal epithelial cells via TLR4/MyD88/NF-kappaB signaling and ameliorates murine colitis. BMC Gastroenterol. 2022;22:137.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rusu I, Mennillo E, Bain JL, Li Z, Sun X, Ly KM, et al. Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage. J Clin Invest. 2022;132:e154993.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zheng C, Lu T, Fan Z. miR-200b-3p alleviates TNF-alpha-induced apoptosis and inflammation of intestinal epithelial cells and ulcerative colitis progression in rats via negatively regulating KHDRBS1. Cytotechnology. 2021;73:727–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kawamata Y, Fujii R, Hosoya M, Harada M, Yoshida H, Miwa M, et al. A G protein-coupled receptor responsive to bile acids. J Biol Chem. 2003;278:9435–40.

    Article  CAS  PubMed  Google Scholar 

  13. Finn PD, Rodriguez D, Kohler J, Jiang Z, Wan S, Blanco E, et al. Intestinal TGR5 agonism improves hepatic steatosis and insulin sensitivity in Western diet-fed mice. Am J Physiol Gastrointest Liver Physiol. 2019;316:G412–G424.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ma SY, Ning MM, Zou QA, Feng Y, Ye YL, Shen JH, et al. OL3, a novel low-absorbed TGR5 agonist with reduced side effects, lowered blood glucose via dual actions on TGR5 activation and DPP-4 inhibition. Acta Pharmacol Sin. 2016;37:1359–69.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cipriani S, Mencarelli A, Chini MG, Distrutti E, Renga B, Bifulco G, et al. The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis. PLoS One. 2011;6:e25637.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sorrentino G, Perino A, Yildiz E, El Alam G, Bou Sleiman M, Gioiello A, et al. Bile acids signal via TGR5 to activate intestinal stem cells and epithelial regeneration. Gastroenterology. 2020;159:956–68.

    Article  CAS  PubMed  Google Scholar 

  17. Cao H, Chen ZX, Wang K, Ning MM, Zou QA, Feng Y, et al. Intestinally-targeted TGR5 agonists equipped with quaternary ammonium have an improved hypoglycemic effect and reduced gallbladder filling effect. Sci Rep. 2016;6:28676.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Han F, Ning M, Wang K, Gu Y, Qu H, Leng Y, et al. Design and exploration of gut-restricted bifunctional molecule with TGR5 agonistic and DPP4 inhibitory effects for treating ulcerative colitis. Eur J Med Chem. 2022;242:114697.

    Article  CAS  PubMed  Google Scholar 

  19. Tang X, Ning M, Ye Y, Gu Y, Yan H, Leng Y, et al. Discovery of novel ketoxime ether derivatives with potent FXR agonistic activity, oral effectiveness and high liver/blood ratio. Bioorg Med Chem. 2021;43:116280.

    Article  CAS  PubMed  Google Scholar 

  20. Zou Y, Lin J, Li W, Wu Z, He Z, Huang G, et al. Huangqin-tang ameliorates dextran sodium sulphate-induced colitis by regulating intestinal epithelial cell homeostasis, inflammation and immune response. Sci Rep. 2016;6:39299.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ning MM, Yang WJ, Guan WB, Gu YP, Feng Y, Leng Y. Dipeptidyl peptidase 4 inhibitor sitagliptin protected against dextran sulfate sodium-induced experimental colitis by potentiating the action of GLP-2. Acta Pharmacol Sin. 2020;41:1446–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Varfolomeev EE, Ashkenazi A. Tumor necrosis factor: an apoptosis JuNKie? Cell. 2004;116:491–7.

    Article  CAS  PubMed  Google Scholar 

  23. Guicciardi ME, Gores GJ. AIP1: a new player in TNF signaling. J Clin Invest. 2003;111:1813–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jin S, Ray RM, Johnson LR. Rac1 mediates intestinal epithelial cell apoptosis via JNK. Am J Physiol Gastrointest Liver Physiol. 2006;291:G1137–1147.

    Article  CAS  PubMed  Google Scholar 

  25. Ibraheem K, Yhmed AMA, Nasef MM, Georgopoulos NT. TRAF3/p38-JNK signalling crosstalk with intracellular-TRAIL/Caspase-10-Induced apoptosis accelerates ROS-Driven cancer cell-specific death by CD40. Cells. 2022;11:3274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Siggers RH, Hackam DJ. The role of innate immune-stimulated epithelial apoptosis during gastrointestinal inflammatory diseases. Cell Mol Life Sci. 2011;68:3623–34.

    Article  CAS  PubMed  Google Scholar 

  27. Mennillo E, Yang X, Paszek M, Auwerx J, Benner C, Chen S. NCoR1 protects mice from dextran sodium sulfate-induced colitis by guarding colonic crypt cells from luminal insult. Cell Mol Gastroenterol Hepatol. 2020;10:133–47.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Wang K, Ding Y, Xu C, Hao M, Li H, Ding L. Cldn-7 deficiency promotes experimental colitis and associated carcinogenesis by regulating intestinal epithelial integrity. Oncoimmunology. 2021;10:1923910.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol. 2014;14:141–53.

    Article  CAS  PubMed  Google Scholar 

  30. Lin S, Stoll B, Robinson J, Pastor JJ, Marini JC, Ipharraguerre IR, et al. Differential action of TGR5 agonists on GLP-2 secretion and promotion of intestinal adaptation in a piglet short bowel model. Am J Physiol Gastrointest Liver Physiol. 2019;316:G641–G652.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Watanabe S, Chen Z, Fujita K, Nishikawa M, Ueda H, Iguchi Y, et al. Hyodeoxycholic acid (HDCA) prevents development of dextran sulfate sodium (DSS)-induced colitis in mice: possible role of synergism between DSS and HDCA in increasing fecal bile acid levels. Biol Pharm Bull. 2022;45:1503–9.

    Article  CAS  PubMed  Google Scholar 

  32. Chen Y, Le TH, Du Q, Zhao Z, Liu Y, Zou J, et al. Genistein protects against DSS-induced colitis by inhibiting NLRP3 inflammasome via TGR5-cAMP signaling. Int Immunopharmacol. 2019;71:144–54.

    Article  CAS  PubMed  Google Scholar 

  33. Sakanaka T, Inoue T, Yorifuji N, Iguchi M, Fujiwara K, Narabayashi K, et al. The effects of a TGR5 agonist and a dipeptidyl peptidase IV inhibitor on dextran sulfate sodium-induced colitis in mice. J Gastroenterol Hepatol. 2015;30:60–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gu M, Zhao P, Zhang S, Fan S, Yang L, Tong Q, et al. Betulinic acid alleviates endoplasmic reticulum stress-mediated nonalcoholic fatty liver disease through activation of farnesoid X receptors in mice. Br J Pharmacol. 2019;176:847–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ye Z, Zhu Y, Tang N, Zhao X, Jiang J, Ma J, et al. Alpha7 nicotinic acetylcholine receptor agonist GTS-21 attenuates DSS-induced intestinal colitis by improving intestinal mucosal barrier function. Mol Med. 2022;28:59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lin W, Ma C, Su F, Jiang Y, Lai R, Zhang T, et al. Raf kinase inhibitor protein mediates intestinal epithelial cell apoptosis and promotes IBDs in humans and mice. Gut. 2017;66:597–610.

    Article  CAS  PubMed  Google Scholar 

  37. Xu S, Hu G, Wu D, Kan X, Oishi H, Takahashi S, et al. MafK accelerates Salmonella mucosal infection through caspase-3 activation. Aging. 2022;14:2287–303.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zuo G, Zhang T, Huang L, Araujo C, Peng J, Travis Z, et al. Activation of TGR5 with INT-777 attenuates oxidative stress and neuronal apoptosis via cAMP/PKCepsilon/ALDH2 pathway after subarachnoid hemorrhage in rats. Free Radic Biol Med. 2019;143:441–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Reich M, Deutschmann K, Sommerfeld A, Klindt C, Kluge S, Kubitz R, et al. TGR5 is essential for bile acid-dependent cholangiocyte proliferation in vivo and in vitro. Gut. 2016;65:487–501.

    Article  CAS  PubMed  Google Scholar 

  40. Ye X, Wu J, Li J, Wang H. Anterior gradient protein 2 promotes mucosal repair in pediatric ulcerative colitis. Biomed Res Int. 2021;2021:6483860.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Hsu NY, Nayar S, Gettler K, Talware S, Giri M, Alter I, et al. NOX1 is essential for TNFalpha-induced intestinal epithelial ROS secretion and inhibits M cell signatures. Gut. 2023;72:654–62.

    Article  CAS  PubMed  Google Scholar 

  42. Assi K, Pillai R, Gomez-Munoz A, Owen D, Salh B. The specific JNK inhibitor SP600125 targets tumour necrosis factor-alpha production and epithelial cell apoptosis in acute murine colitis. Immunology. 2006;118:112–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang X, Cui X, Zhu C, Li M, Zhao J, Shen Z, et al. FKBP11 protects intestinal epithelial cells against inflammation‑induced apoptosis via the JNK‑caspase pathway in Crohn’s disease. Mol Med Rep. 2018;18:4428–38.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhang J, Wang Q, Zhu N, Yu M, Shen B, Xiang J, et al. Cyclic AMP inhibits JNK activation by CREB-mediated induction of c-FLIP(L) and MKP-1, thereby antagonizing UV-induced apoptosis. Cell Death Differ. 2008;15:1654–62.

    Article  CAS  PubMed  Google Scholar 

  45. Zhang Y, Chen P, Liang XF, Han J, Wu XF, Yang YH, et al. Metabolic disorder induces fatty liver in Japanese seabass, Lateolabrax japonicas fed a full plant protein diet and regulated by cAMP-JNK/NF-kB-caspase signal pathway. Fish Shellfish Immunol. 2019;90:223–34.

    Article  CAS  PubMed  Google Scholar 

  46. Huang S, Ma S, Ning M, Yang W, Ye Y, Zhang L, et al. TGR5 agonist ameliorates insulin resistance in the skeletal muscles and improves glucose homeostasis in diabetic mice. Metabolism. 2019;99:45–56.

    Article  CAS  PubMed  Google Scholar 

  47. Tian F, Hu Y, Sun X, Lu G, Li Y, Yang J, et al. Suppression of cFLIPL promotes JNK activation in malignant melanoma cells. Mol Med Rep. 2016;13:2904–8.

    Article  CAS  PubMed  Google Scholar 

  48. Nakajima A, Kojima Y, Nakayama M, Yagita H, Okumura K, Nakano H. Downregulation of c-FLIP promotes caspase-dependent JNK activation and reactive oxygen species accumulation in tumor cells. Oncogene. 2008;27:76–84.

    Article  CAS  PubMed  Google Scholar 

  49. Piao X, Komazawa-Sakon S, Nishina T, Koike M, Piao JH, Ehlken H, et al. c-FLIP maintains tissue homeostasis by preventing apoptosis and programmed necrosis. Sci Signal. 2012;5:ra93.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Seidelin JB, Coskun M, Vainer B, Riis L, Soendergaard C, Nielsen OH. ERK controls epithelial cell death receptor signalling and cellular FLICE-like inhibitory protein (c-FLIP) in ulcerative colitis. J Mol Med. 2013;91:839–49.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was financially supported by National Natural Science Foundation of China (No. 81872922 and 82073683). The authors thank Dr. Wei Tang from Shanghai Institute of Materia Medica, Chinese Academy of Sciences for giving valuable suggestions.

Author information

Authors and Affiliations

  1. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China

    Wen-ji Yang, Fang-hui Han, Yi-pei Gu, Hui Qu, Jian-hua Shen & Ying Leng

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Wen-ji Yang, Jian-hua Shen & Ying Leng

  3. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China

    Jia Liu

Authors
  1. Wen-ji Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fang-hui Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yi-pei Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hui Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jia Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jian-hua Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ying Leng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YL, JHS and WJY designed the research. WJY, FHH, YPG, HQ and JL performed the research. WJY and YPG analyzed and interpreted the data. WJY and YL wrote the paper. All authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Jian-hua Shen or Ying Leng.

Ethics declarations

Competing interests

The authors declare no competing interests.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, Wj., Han, Fh., Gu, Yp. et al. TGR5 agonist inhibits intestinal epithelial cell apoptosis via cAMP/PKA/c-FLIP/JNK signaling pathway and ameliorates dextran sulfate sodium-induced ulcerative colitis. Acta Pharmacol Sin 44, 1649–1664 (2023). https://doi.org/10.1038/s41401-023-01081-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41401-023-01081-y

Keywords

This article is cited by

Search

Advanced search

Quick links