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.
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References
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Varfolomeev EE, Ashkenazi A. Tumor necrosis factor: an apoptosis JuNKie? Cell. 2004;116:491–7.
Guicciardi ME, Gores GJ. AIP1: a new player in TNF signaling. J Clin Invest. 2003;111:1813–5.
Jin S, Ray RM, Johnson LR. Rac1 mediates intestinal epithelial cell apoptosis via JNK. Am J Physiol Gastrointest Liver Physiol. 2006;291:G1137–1147.
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.
Siggers RH, Hackam DJ. The role of innate immune-stimulated epithelial apoptosis during gastrointestinal inflammatory diseases. Cell Mol Life Sci. 2011;68:3623–34.
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.
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.
Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol. 2014;14:141–53.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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.
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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
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DOI: https://doi.org/10.1038/s41401-023-01081-y
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