Korean J Physiol Pharmacol.  2021 Jul;25(4):375-383. 10.4196/kjpp.2021.25.4.375.

Sepsis induces variation of intestinal barrier function in different phase through nuclear factor kappa B signaling

• Cao YY
• Wang ZH
• Xu QC
• Chen Q
• Wang Z
• Lu WH

• KMID: 2516876
• DOI: http://doi.org/10.4196/kjpp.2021.25.4.375

Abstract

The intestinal barrier function disrupted in sepsis, while little is known about the variation in different phases of sepsis. In this study, mouse models of sepsis were established by caecal ligation and puncture (CLP). The H&E staining of sections and serum diamine oxidase concentration were evaluated at different timepoint after CLP. TUNEL assay and EdU staining were performed to evaluate the apoptosis and proliferation of intestinal epithelium. Relative protein expression was assessed by Western blotting and serum concentrations of pro-inflammatory cytokines was measured by ELISA. The disruption of intestinal barrier worsened in the first 24 h after the onset of sepsis and gradually recovered over the next 24 h. The percentage of apoptotic cell increased in the first 24 h and dropped at 48 h, accompanied with the proliferative rate of intestinal epithelium inhibited in the first 6 h and regained in the later period. Furthermore, the activity of nuclear factor kappa B (NF-κB) presented similar trend with the intestinal barrier function, shared positive correction with apoptosis of intestinal epithelium. These findings reveal the conversion  process of intestinal barrier function in sepsis and this process is closely correlated with the activity of NF-κB signaling.

Keyword

Apoptosis; Cell proliferation; Intestinal epithelium; NF-kappa B; Sepsis

Figure

• 

  Fig. 1 Changes of intestinal barrier function in sepsis. (A) Typical H&E-stained sections of intestinal tissues at different timepoint after caecal ligation and puncture (CLP) (bar, 10 μm; magnification, 200×). (B) The Chiu’s score of H&E-stained sections. (C) The serum concentration of diamine oxidase (DAO) at different timepoint after CLP. (D) The level of zona occludens 1 (ZO-1) and occludin were measured at different timepoint after CLP. The graph represented the relative band densities. *p < 0.05 vs. the original timepoint.

• 

  Fig. 2 The variation of apoptosis of intestinal epithelium in sepsis. (A) Representative images of TUNEL staining of the indicated timepoint (bar, 20 μm; magnification, 400×). (B) The percentage of positive TUNEL stained cell at different timepoint after caecal ligation and puncture (CLP). (C) The level of cleaved-caspase3 was measured at different timepoint after CLP. The graph represented the relative band densities. *p < 0.05 vs. the original timepoint.

• 

  Fig. 3 The proliferation rate of intestinal epithelium in sepsis. (A) The representative images of EdU assays of the indicated timepoint (bar, 20 μm; magnification, 400×). The positive cells were marked with red arrows. (B) The percentage of positive EdU stained cell at different timepoint after caecal ligation and puncture (CLP). (C) The level of proliferating cell nuclear antigen (PCNA) was measured at different timepoint after CLP. The graph represented the relative band densities. *p < 0.05 vs. the original timepoint.

• 

  Fig. 4 The activity of nuclear factor kappa B (NF-κB) signaling positively corrected with apoptosis during sepsis. (A, B) The serum concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined by ELISA at different timepoint after caecal ligation and puncture (CLP). (C) The level of nuclear p-NF-κB (p-P65) in intestinal epithelium was measured at different timepoint after CLP. The graph represented the relative band densities. (D) The level of IκB-α in intestinal epithelium was measured at different timepoint after CLP. The graph represented the relative band densities. (E) The serum level of inhibitor kappa B kinase beta (IKKβ) at different timepoint after CLP. (F) Correlation analysis for the percentage of apoptotic cells and IKKβ levels. *p < 0.05 vs. the original timepoint.

Reference

1. Rhodes A, Evans LE, Alhazzani W, Levy MM, Antonelli M, Ferrer R, Kumar A, Sevransky JE, Sprung CL, Nunnally ME, Rochwerg B, Rubenfeld GD, Angus DC, Annane D, Beale RJ, Bellinghan GJ, Bernard GR, Chiche JD, Coopersmith C, De Backer DP, et al. 2017; Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016. Intensive Care Med. 43:304–377. DOI: 10.1007/s00134-017-4683-6. PMID: 28101605.

2. Gotts JE, Matthay MA. 2016; Sepsis: pathophysiology and clinical management. BMJ. 353:i1585. DOI: 10.1136/bmj.i1585. PMID: 27217054.

3. Mayr FB, Yende S, Angus DC. 2014; Epidemiology of severe sepsis. Virulence. 5:4–11. DOI: 10.4161/viru.27372. PMID: 24335434. PMCID: PMC3916382.

4. Mittal R, Coopersmith CM. 2014; Redefining the gut as the motor of critical illness. Trends Mol Med. 20:214–223. DOI: 10.1016/j.molmed.2013.08.004. PMID: 24055446. PMCID: PMC3959633.

5. Li Q, Zhang Q, Wang C, Liu X, Li N, Li J. 2009; Disruption of tight junctions during polymicrobial sepsis in vivo. J Pathol. 218:210–221. DOI: 10.1002/path.2525. PMID: 19235836.

6. Hu Q, Ren H, Li G, Wang D, Zhou Q, Wu J, Zheng J, Huang J, Slade DA, Wu X, Ren J. 2019; STING-mediated intestinal barrier dysfunction contributes to lethal sepsis. EBioMedicine. 41:497–508. DOI: 10.1016/j.ebiom.2019.02.055. PMID: 30878597. PMCID: PMC6443583.

7. Haussner F, Chakraborty S, Halbgebauer R, Huber-Lang M. 2019; Challenge to the intestinal mucosa during sepsis. Front Immunol. 10:891. DOI: 10.3389/fimmu.2019.00891. PMID: 31114571. PMCID: PMC6502990.

8. Eslamian G, Ardehali SH, Vahdat Shariatpanahi Z. 2019; Association of intestinal permeability with a NUTRIC score in critically ill patients. Nutrition. 63-64:1–8. DOI: 10.1016/j.nut.2019.01.010. PMID: 30925269.

9. Assimakopoulos SF, Triantos C, Thomopoulos K, Fligou F, Maroulis I, Marangos M, Gogos CA. 2018; Gut-origin sepsis in the critically ill patient: pathophysiology and treatment. Infection. 46:751–760. DOI: 10.1007/s15010-018-1178-5. PMID: 30003491.

10. Günther C, Buchen B, Neurath MF, Becker C. 2014; Regulation and pathophysiological role of epithelial turnover in the gut. Semin Cell Dev Biol. 35:40–50. DOI: 10.1016/j.semcdb.2014.06.004. PMID: 24973733.

11. Günther C, Neumann H, Neurath MF, Becker C. 2013; Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium. Gut. 62:1062–1071. DOI: 10.1136/gutjnl-2011-301364. PMID: 22689519.

12. Klingensmith NJ, Coopersmith CM. 2016; The gut as the motor of multiple organ dysfunction in critical illness. Crit Care Clin. 32:203–212. DOI: 10.1016/j.ccc.2015.11.004. PMID: 27016162. PMCID: PMC4808565.

13. Fischer A, Gluth M, Pape UF, Wiedenmann B, Theuring F, Baumgart DC. 2013; Adalimumab prevents barrier dysfunction and antagonizes distinct effects of TNF-α on tight junction proteins and signaling pathways in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 304:G970–G979. DOI: 10.1152/ajpgi.00183.2012. PMID: 23538493.

14. Chen LW, Chen PH, Chang WJ, Wang JS, Karin M, Hsu CM. 2007; IKappaB-kinase/nuclear factor-kappaB signaling prevents thermal injury-induced gut damage by inhibiting c-Jun NH2-terminal kinase activation. Crit Care Med. 35:1332–1340. DOI: 10.1097/01.CCM.0000261891.30360.F0. PMID: 17414734.

15. Wullaert A, Bonnet MC, Pasparakis M. 2011; NF-κB in the regulation of epithelial homeostasis and inflammation. Cell Res. 21:146–158. DOI: 10.1038/cr.2010.175. PMID: 21151201. PMCID: PMC3193399.

16. Kondylis V, Kumari S, Vlantis K, Pasparakis M. 2017; The interplay of IKK, NF-κB and RIPK1 signaling in the regulation of cell death, tissue homeostasis and inflammation. Immunol Rev. 277:113–127. DOI: 10.1111/imr.12550. PMID: 28462531.

17. Vlantis K, Wullaert A, Polykratis A, Kondylis V, Dannappel M, Schwarzer R, Welz P, Corona T, Walczak H, Weih F, Klein U, Kelliher M, Pasparakis M. 2016; NEMO prevents RIP kinase 1-mediated epithelial cell death and chronic intestinal inflammation by NF-κB-dependent and -independent functions. Immunity. 44:553–567. DOI: 10.1016/j.immuni.2016.02.020. PMID: 26982364. PMCID: PMC4803910.

18. Cao YY, Wang Z, Wang ZH, Jiang XG, Lu WH. 2021; Inhibition of miR-155 alleviates sepsis-induced inflammation and intestinal barrier dysfunction by inactivating NF-κB signaling. Int Immunopharmacol. 90:107218. DOI: 10.1016/j.intimp.2020.107218. PMID: 33296782.

19. Cao Y, Chen Q, Wang Z, Yu T, Wu J, Jiang X, Jin X, Lu W. 2018; PLK1 protects against sepsis-induced intestinal barrier dysfunction. Sci Rep. 8:1055. DOI: 10.1038/s41598-018-19573-x. PMID: 29348559. PMCID: PMC5773589.

20. Peuker K, Muff S, Wang J, Künzel S, Bosse E, Zeissig Y, Luzzi G, Basic M, Strigli A, Ulbricht A, Kaser A, Arlt A, Chavakis T, van den Brink GR, Schafmayer C, Egberts JH, Becker T, Bianchi ME, Bleich A, Röcken C, et al. 2016; Epithelial calcineurin controls microbiota-dependent intestinal tumor development. Nat Med. 22:506–515. DOI: 10.1038/nm.4072. PMID: 27043494. PMCID: PMC5570457.

21. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, Clevers H. 2011; Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. 469:415–418. DOI: 10.1038/nature09637. PMID: 21113151. PMCID: PMC3547360.

22. Gehart H, Clevers H. 2019; Tales from the crypt: new insights into intestinal stem cells. Nat Rev Gastroenterol Hepatol. 16:19–34. DOI: 10.1038/s41575-018-0081-y. PMID: 30429586.

23. de Sousa E Melo F, de Sauvage FJ. 2019; Cellular plasticity in intestinal homeostasis and disease. Cell Stem Cell. 24:54–64. DOI: 10.1016/j.stem.2018.11.019. PMID: 30595498.

24. Yoseph BP, Klingensmith NJ, Liang Z, Breed ER, Burd EM, Mittal R, Dominguez JA, Petrie B, Ford ML, Coopersmith CM. 2016; Mechanisms of intestinal barrier dysfunction in sepsis. Shock. 46:52–59. DOI: 10.1097/SHK.0000000000000565. PMID: 27299587. PMCID: PMC4910519.

25. Assimakopoulos SF, Tsamandas AC, Tsiaoussis GI, Karatza E, Zisimopoulos D, Maroulis I, Kontogeorgou E, Georgiou CD, Scopa CD, Thomopoulos KC. 2013; Intestinal mucosal proliferation, apoptosis and oxidative stress in patients with liver cirrhosis. Ann Hepatol. 12:301–307. DOI: 10.1016/S1665-2681(19)31369-9. PMID: 23396742.

26. Obermüller B, Frisina N, Meischel M, Singer G, Stanzl-Tschegg S, Lichtenegger H, Kolb D, Klymiuk I, Till H, Castellani C. 2020; Examination of intestinal ultrastructure, bowel wall apoptosis and tight junctions in the early phase of sepsis. Sci Rep. 10:11507. DOI: 10.1038/s41598-020-68109-9. PMID: 32661347. PMCID: PMC7359326.

27. Li GX, Wang XM, Jiang T, Gong JF, Niu LY, Li N. 2015; Berberine prevents intestinal mucosal barrier damage during early phase of sepsis in rat through the toll-like receptors signaling pathway. Korean J Physiol Pharmacol. 19:1–7. DOI: 10.4196/kjpp.2015.19.1.1. PMID: 25605990. PMCID: PMC4297756.

28. Li GX, Wang XM, Jiang T, Gong JF, Niu LY, Li N. 2014; Berberine prevents damage to the intestinal mucosal barrier during early phase of sepsis in rat through mechanisms independent of the NOD-like receptors signaling pathway. Eur J Pharmacol. 730:1–7. DOI: 10.1016/j.ejphar.2014.02.006. PMID: 24530556.

29. Dong LW, Yang J, Tong LJ, Tang C, Liu MS. 1999; Transcriptional regulation of alpha1-adrenoceptor gene in the rat liver during different phases of sepsis. Biochim Biophys Acta. 1453:207–215. DOI: 10.1016/S0925-4439(98)00102-1. PMID: 10036318.

30. Maitra SR, Wojnar MM, Lang CH. 2000; Alterations in tissue glucose uptake during the hyperglycemic and hypoglycemic phases of sepsis. Shock. 13:379–385. DOI: 10.1097/00024382-200005000-00006. PMID: 10807013.

31. Yang S, Zhou M, Fowler DE, Wang P. 2002; Mechanisms of the beneficial effect of adrenomedullin and adrenomedullin-binding protein-1 in sepsis: down-regulation of proinflammatory cytokines. Crit Care Med. 30:2729–2735. DOI: 10.1097/00003246-200212000-00018. PMID: 12483065.

32. Remick DG, Bolgos GR, Siddiqui J, Shin J, Nemzek JA. 2002; Six at six: interleukin-6 measured 6 h after the initiation of sepsis predicts mortality over 3 days. Shock. 17:463–467. DOI: 10.1097/00024382-200206000-00004. PMID: 12069181.

33. Turnbull IR, Javadi P, Buchman TG, Hotchkiss RS, Karl IE, Coopersmith CM. 2004; Antibiotics improve survival in sepsis independent of injury severity but do not change mortality in mice with markedly elevated interleukin 6 levels. Shock. 21:121–125. DOI: 10.1097/01.shk.0000108399.56565.e7. PMID: 14752284.

34. Walley KR, Lukacs NW, Standiford TJ, Strieter RM, Kunkel SL. 1996; Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun. 64:4733–4738. DOI: 10.1128/iai.64.11.4733-4738.1996. PMID: 8890233. PMCID: PMC174439.

35. Ruiz S, Vardon-Bounes F, Merlet-Dupuy V, Conil JM, Buléon M, Fourcade O, Tack I, Minville V. 2016; Sepsis modeling in mice: ligation length is a major severity factor in cecal ligation and puncture. Intensive Care Med Exp. 4:22. DOI: 10.1186/s40635-016-0096-z. PMID: 27430881. PMCID: PMC4949182.

36. Ghosh S, Karin M. 2002; Missing pieces in the NF-kappaB puzzle. Cell. 109 Suppl:S81–S96. DOI: 10.1016/S0092-8674(02)00703-1. PMID: 11983155.

37. Hayden MS, Ghosh S. 2008; Shared principles in NF-kappaB signaling. Cell. 132:344–362. DOI: 10.1016/j.cell.2008.01.020. PMID: 18267068.

38. Gao MY, Chen L, Yang L, Yu X, Kou JP, Yu BY. 2014; Berberine inhibits LPS-induced TF procoagulant activity and expression through NF-κB/p65, Akt and MAPK pathway in THP-1 cells. Pharmacol Rep. 66:480–484. DOI: 10.1016/j.pharep.2013.12.004. PMID: 24905527.

39. Li X, Li X, Zheng Z, Liu Y, Ma X. 2014; Unfractionated heparin suppresses lipopolysaccharide-induced monocyte chemoattractant protein-1 expression in human microvascular endothelial cells by blocking Krüppel-like factor 5 and nuclear factor-κB pathway. Immunobiology. 219:778–785. DOI: 10.1016/j.imbio.2014.06.005. PMID: 25023776.

40. Spehlmann ME, Eckmann L. 2009; Nuclear factor-kappa B in intestinal protection and destruction. Curr Opin Gastroenterol. 25:92–99. DOI: 10.1097/MOG.0b013e328324f857. PMID: 19528876.

41. Lawrence T. 2009; The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 1:a001651. DOI: 10.1101/cshperspect.a001651. PMID: 20457564. PMCID: PMC2882124.

42. Chen F, Castranova V, Shi X. 2001; New insights into the role of nuclear factor-kappaB in cell growth regulation. Am J Pathol. 159:387–397. DOI: 10.1016/S0002-9440(10)61708-7. PMID: 11485895. PMCID: PMC1850555.

43. Bergmann MW, Loser P, Dietz R, von Harsdorf R. 2001; Effect of NF-kappa B inhibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes. J Mol Cell Cardiol. 33:1223–1232. DOI: 10.1006/jmcc.2001.1385. PMID: 11444925.

44. Fujioka S, Schmidt C, Sclabas GM, Li Z, Pelicano H, Peng B, Yao A, Niu J, Zhang W, Evans DB, Abbruzzese JL, Huang P, Chiao PJ. 2004; Stabilization of p53 is a novel mechanism for proapoptotic function of NF-kappaB. J Biol Chem. 279:27549–27559. DOI: 10.1074/jbc.M313435200. PMID: 15102862.

45. Co NN, Tsang WP, Tsang TY, Yeung CL, Yau PL, Kong SK, Kwok TT. 2010; AF1q enhancement of gamma irradiation-induced apoptosis by up-regulation of BAD expression via NF-kappaB in human squamous carcinoma A431 cells. Oncol Rep. 24:547–554. DOI: 10.3892/or_00000891. PMID: 20596645.

46. Li L, Aggarwal BB, Shishodia S, Abbruzzese J, Kurzrock R. 2004; Nuclear factor-kappaB and IkappaB kinase are constitutively active in human pancreatic cells, and their down-regulation by curcumin (diferuloylmethane) is associated with the suppression of proliferation and the induction of apoptosis. Cancer. 101:2351–2362. DOI: 10.1002/cncr.20605. PMID: 15476283.

47. Yang DW, Qian GB, Jiang MJ, Wang P, Wang KZ. 2019; Inhibition of microRNA-495 suppresses chondrocyte apoptosis through activation of the NF-κB signaling pathway by regulating CCL4 in osteoarthritis. Gene Ther. 26:217–229. DOI: 10.1038/s41434-019-0068-5. PMID: 30940879.