Ingestion of Lactobacillus strain reduces anxiety and improves cognitive function in the hyperammonemia rat


Evidence suggests that the hyperammonemia (HA)-induced neuroinflammation and alterations in the serotonin (5-HT) system may contribute to cognitive decline and anxiety disorder during hepatic encephalopathy (HE). Probiotics that maintain immune system homeostasis and regulate the 5-HT system may be potential treatment for HA-mediated neurological disorders in HE. In this study, we tested the efficacy of probiotic Lactobacillus helveticus strain NS8 in preventing cognitive decline and anxiety-like behavior in HA rats. Chronic HA was induced by intraperitoneal injection of ammonium acetate for four weeks in male Sprague-Dawley rats. HA rats were then given Lactobacillus helveticus strain NS8 (109 CFU mL−1) in drinking water as a daily supplementation. The Morris water maze task assessed cognitive function, and the elevated plus maze test evaluated anxiety-like behavior. Neuroinflammation was assessed by measuring the inflammatory markers: inducible nitric oxide synthase, prostaglandin E2, and interleukin-1 β in the brain. 5-HT system activity was evaluated by measuring 5-HT and its metabolite, 5-HIAA, and the 5-HT precursor, tryptophan. Probiotic treatment of HA rats significantly reduced the level of inflammatory markers, decreased 5-HT metabolism, restored cognitive function and improved anxiety-like behavior. These results indicate that probiotic L. helveticus strain NS8 is beneficial for the treatment of cognitive decline and anxiety-like behavior in HA rats.


  1. 1

    Rodrigo R, Cauli O, Gomez-Pinedo U, Agusti A, Hernandez-Rabaza V, Garcia-Verdugo JM, Felipo V. Hyperammonemia induces neuro-inflammation that contributes to cognitive impairment in rats with hepatic encephalopathy. Gastroenterology, 2010, 139: 675–684

    PubMed  CAS  Article  Google Scholar 

  2. 2

    Saul WSWB. Hyperammonemic encephalopathy. Medicine (Abingdon), 2002, 81: 240–249

    Google Scholar 

  3. 3

    Felipo V, Butterworth RF. Neurobiology of ammonia. Prog Neurobiol, 2002, 67: 259–279

    PubMed  CAS  Article  Google Scholar 

  4. 4

    Lajtha A, Reith MEA, eds. Handbook of Neurochemistry and Molecular Neurobiology. 3rd ed. New York: Springer, 2008

    Google Scholar 

  5. 5

    Basile AS, Jones EA. Ammonia and GABA-ergic neurotransmission: Interrelated factors in the pathogenesis of hepatic encephalopathy. Hepatology, 1997, 25: 1303–1305

    PubMed  CAS  Article  Google Scholar 

  6. 6

    Montoliu C, Piedrafita B, Serra MA, del Olmo JA, Urios A, Rodrigo JM, Felipo V. IL-6 and IL-18 in blood may discriminate cirrhotic patients with and without minimal hepatic encephalopathy. J Clin Gastroenterol, 2009, 43: 272–279

    PubMed  CAS  Article  Google Scholar 

  7. 7

    Gibertini M, Newton C, Friedman H, Klein TW. Spatial learning impairment in mice infected with legionella pneumophila or administered exogenous interleukin-1β. Brain Behav Immun, 1995, 9: 113–128

    PubMed  CAS  Article  Google Scholar 

  8. 8

    Wiltfang J, Nolte W, Weißenborn K, Komhuber J, Rüther E. Psychiatric aspects of portal-systemic encephalopathy. Metab Brain Dis, 1998, 13: 379–389

    PubMed  CAS  Article  Google Scholar 

  9. 9

    Erecinska M, Pastuszko A, Wilson DF, Nelson D. Ammonia-induced release of neurotransmitters from rat brain synaptosomes: differences between the effects on amines and amino acids. J Neurochem, 1987, 49: 1258–1265

    PubMed  CAS  Article  Google Scholar 

  10. 10

    Rössle M, Luft M, Herz R, Klein B, Lehmann M, Gerok W. Amino acid, ammonia and neurotransmitter concentrations in hepatic encephalopathy: serial analysis in plasma and cerebrospinal fluid during treatment with an adapted amino acid solution. Klin Wochenschr, 1984, 62: 867–875

    PubMed  Article  Google Scholar 

  11. 11

    Murphy DL, Moya PR, Fox MA, Rubenstein LM, Wendland JR, Timpano KR. Anxiety and affective disorder comorbidity related to serotonin and other neurotransmitter systems: obsessive-compulsive disorder as an example of overlapping clinical and genetic heterogeneity. Philos Trans R Soc Lond B Biol Sci, 2013, 368: 20120435

    PubMed  PubMed Central  Article  Google Scholar 

  12. 12

    Bergqvist PBF, Hjorth S, Audet RM, Apelqvist G, Bengtsson F, Butterworth RF. Ammonium acetate challenge in experimental chronic hepatic encephalopathy induces a transient increase of brain 5-HT release in vivo. Eur Neuropsychopharm, 1996, 6: 317–322

    Article  Google Scholar 

  13. 13

    Miura H, Ozaki N, Sawada M, Isobe K, Ohta T, Nagatsu T. A link between stress and depression: shifts in the balance between the kynurenine and serotonin pathways of tryptophan metabolism and the etiology and pathophysiology of depression. Stress, 2008, 11: 198–209

    PubMed  CAS  Article  Google Scholar 

  14. 14

    Vignau J, Costisella O, Canva V, Imbenotte M, Duhamel A, Lhermitte M. Impact of interferon alpha immunotherapy on tryptophan metabolism in patients with chronic hepatitis C. Results of a pilot studies on ten patients. Encephale, 2009, 35: 477–483

    PubMed  CAS  Article  Google Scholar 

  15. 15

    Laugeray A, Launay JM, Callebert J, Surget A, Belzung C, Barone PR. Peripheral and cerebral metabolic abnormalities of the tryptophan-kynurenine pathway in a murine model of major depression. Behav Brain Res, 2010, 210: 84–91

    PubMed  CAS  Article  Google Scholar 

  16. 16

    Butterworth RF, Norenberg MD, Felipo V, Ferenci P, Albrecht J, Blei AT, Members of the ICoEMoHE. Experimental models of hepatic encephalopathy: ISHEN guidelines. Liver Int, 2009, 29: 783–788

    PubMed  Article  Google Scholar 

  17. 17

    Lena PJ, Subramanian P. Effects of melatonin on the levels of antioxidants and lipid peroxidation products in rats treated with ammonium acetate. Pharmazie, 2004, 59: 636–639

    PubMed  CAS  Google Scholar 

  18. 18

    Moroni F, Lombardi G, Moneti G, Cortesini C. The release and neosynthesis of glutamic acid are increased in experimental models of hepatic encephalopathy. J Neurochem, 1983, 40: 850–854

    PubMed  CAS  Article  Google Scholar 

  19. 19

    Subash S, Subramanian P. Morin a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: a biochemical and histopathological study. Mol Cell Biochem, 2009, 327: 153–161

    PubMed  CAS  Article  Google Scholar 

  20. 20

    Bajaj JS, Sanyal AJ, Bell D, Gilles H, Heuman DM. Predictors of the recurrence of hepatic encephalopathy in lactulose-treated patients. Aliment Pharm Therap, 2010, 31: 1012–1017

    CAS  Article  Google Scholar 

  21. 21

    Flamm SL. Rifaximin treatment for reduction of risk of overt hepatic encephalopathy recurrence. Therap Adv Gastroenterol, 2011, 4: 199–206

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  22. 22

    Nicaise C, Prozzi D, Viaene E, Moreno C, Gustot T, Quertinmont E, Demetter P, Suain V, Goffin P, Devière J, Hols P. Control of acute, chronic, and constitutive hyperammonemia by wild-type and genetically engineered Lactobacillus plantarum in rodents. Hepatology, 2008, 48: 1184–1192

    PubMed  CAS  Article  Google Scholar 

  23. 23

    Isolauri E, Sütas Y, Kankaanp P, Arvilommi H, Salminen S. Probiotics: effects on immunity. Am J Clin Nutr, 2001, 73: 444S–450S

    PubMed  CAS  Google Scholar 

  24. 24

    Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science, 2000, 289: 1352–1355

    PubMed  CAS  Article  Google Scholar 

  25. 25

    Desbonnet L, Garrett L, Clarke G, Bienenstock J, Dinan TG. The probiotic Bifidobacteria infantis: an assessment of potential antidepressant properties in the rat. J Psychiat Res, 2008, 43: 164–174

    PubMed  Article  Google Scholar 

  26. 26

    Gareau MG, Wine E, Rodrigues DM, Cho JH, Whary MT, Philpott DJ, MacQueen G, Sherman PM. Bacterial infection causes stress-induced memory dysfunction in mice. Gut, 2011, 60: 307–317

    PubMed  Article  Google Scholar 

  27. 27

    Benton D, Williams C, Brown A. Impact of consuming a milk drink containing a probiotic on mood and cognition. Eur J Clin Nutr, 2007, 61: 355–361

    PubMed  CAS  Article  Google Scholar 

  28. 28

    Rao AV, Bested AC, Beaulne TM, Katzman MA, Iorio C, Berardi JM, Logan AC. A randomized, double-blind, placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue syndrome. Gut Pathog, 2009, 1: 6

    PubMed  PubMed Central  Article  Google Scholar 

  29. 29

    Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, Bienenstock J, Cryan JF. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA, 2011, 108: 16050–16055

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  30. 30

    Joo HM, Kim KA, Myoung KS, Ahn YT, Lee JH, Huh CS, Han MJ, Kim DH. Lactobacillus helveticus HY7801 ameliorates vulvovaginal candidiasis in mice by inhibiting fungal growth and NF-κB activation. Int Immunopharmacol, 2012, 14: 39–46

    PubMed  CAS  Article  Google Scholar 

  31. 31

    Ohland CL, Kish L, Bell H, Thiesen A, Hotte N, Pankiv E, Madsen KL. Effects of Lactobacillus helveticus on murine behavior are dependent on diet and genotype and correlate with alterations in the gut microbiome. Psychoneuroendocrinology, 2013, 38:1738–1747

    PubMed  CAS  Article  Google Scholar 

  32. 32

    Gokcimen A, Kocak A, Gulle K, Sutcu R, Elmas O, Caliskan S, Ozguner F. The effects of allopurinol on rat liver and spleen tissues in a chronic hyperammonemia animal model. Saudi Med J, 2007, 28: 1648–1653

    PubMed  Google Scholar 

  33. 33

    Sgouras D, Maragkoudakis P, Petraki K, Martinez-Gonzalez B, Eriotou E, Michopoulos S, Kalantzopoulos G, Tsakalidou E, Mentis A. In vitro and in vivo inhibition of helicobacter pylori by lactobacillus casei strain shirota. Appl Environ Microb, 2004, 70: 518–526

    CAS  Article  Google Scholar 

  34. 34

    Walf AA, Frye CA. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protocols, 2007, 2: 322–328

    PubMed  CAS  Article  Google Scholar 

  35. 35

    Wolfer DP, Stagljar-Bozicevic M, Errington ML, Lipp HP. Spatial memory and learning in transgenic mice: fact or artifact? Physiology, 1998, 13: 118–123

    Google Scholar 

  36. 36

    Monfort P, Cauli O, Montoliu C, Rodrigo R, Llansola M, Piedrafita B, el Mlili N, Boix J, Agustí A, Felipo V. Mechanisms of cognitive alterations in hyperammonemia and hepatic encephalopathy: therapeutical implications. Neurochem Int, 2009, 55: 106–112

    PubMed  CAS  Article  Google Scholar 

  37. 37

    Reznikov L, Fadel J, Reagan L. Glutamate-mediated neuroplasticity deficits in mood disorders. Neuroplasticity, 2009, 13-26

  38. 38

    Cauli O, Rodrigo R, Piedrafita B, Boix J, Felipo V. Inflammation and hepatic encephalopathy: Ibuprofen restores learning ability in rats with portacaval shunts. Hepatology, 2007, 46: 514–519

    PubMed  CAS  Article  Google Scholar 

  39. 39

    Khasnavis S, Jana A, Roy A, Wood T, Ghosh S, Watson R, Pahan K. Suppression of nuclear factor-?B activation and inflammation in microglia by a physically-modified saline. J Biol Chem, 2012, 287: 29529–29542

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  40. 40

    Riedel CU, Foata F, Philippe D, Adolfsson O, Eikmanns BJ, Blum S. Anti-inflammatory effects of bifidobacteria by inhibition of LPS-induced NF-κB activation. World J Gastroenterol, 2006, 12: 3729–3735

    PubMed  PubMed Central  Google Scholar 

  41. 41

    Enck P, Klosterhalfen S, Martens U. Probiotic therapy for irritable bowel syndrome. Dtsch Med Wochenschr, 2011, 136: 371–375

    PubMed  CAS  Article  Google Scholar 

  42. 42

    Laugeray A, Launay JM, Callebert J, Surget A, Belzung C, Barone PR. Evidence for a key role of the peripheral kynurenine pathway in the modulation of anxiety- and depression-like behaviours in mice: focus on individual differences. Pharmacol Biochem Be, 2011, 98: 161–168

    CAS  Article  Google Scholar 

  43. 43

    Iversen SD. 5-HT and anxiety. 1984, 23: 1553–1560

    CAS  Google Scholar 

  44. 44

    Jennings KA, Loder MK, Sheward WJ, Pei Q, Deacon RMJ, Benson MA, Olverman HJ, Hastie ND, Harmar AJ, Shen S, Sharp T. Increased expression of the 5-HT transporter confers a low-anxiety phenotype linked to decreased 5-HT transmission. J Neurosci, 2006, 26: 8955–8964

    PubMed  CAS  Article  Google Scholar 

  45. 45

    Dejong CH, van de Poll MC, Soeters PB, Jalan R, Olde Damink SW. Aromatic amino acid metabolism during liver failure. J Nutr, 2007, 137: 1579S–1585S

    PubMed  CAS  Google Scholar 

  46. 46

    O’Connor JC, André C, Wang Y, Lawson MA, Szegedi SS, Lestage J, Castanon N, Kelley KW, Dantzer R. Interferon-γ and tumor necrosis factor-α mediate the upregulation of indoleamine 2,3-dioxygenase and the induction of depressive-like behavior in mice in response to bacillus calmette-guérin. J Neurosci, 2009, 29: 4200–4209

    PubMed  PubMed Central  Article  Google Scholar 

  47. 47

    Däubener W, Schmidt SK, Heseler K, Spekker KH, MacKenzie CR. Antimicrobial and immunoregulatory effector mechanisms in human endothelial cells. Indoleamine 2,3-dioxygenase versus inducible nitric oxide synthase. Thromb Haemost, 2009, 102: 1110–1116

    PubMed  Google Scholar 

  48. 48

    Gurtner GJ, Newberry RD, Schloemann SR, McDonald KG, Stenson WF. Inhibition of indoleamine 2,3-dioxygenase augments trinitrobenzene sulfonic acid colitis in mice. Gastroenterology, 2003, 125: 1762–1773

    PubMed  CAS  Article  Google Scholar 

  49. 49

    Forsythe P, Inman MD, Bienenstock J. Oral treatment with live Lactobacillus reuteri inhibits the allergic airway response in mice. Am J Respir Crit Care Med, 2007, 15: 561–569

    Article  Google Scholar 

  50. 50

    Valladares R, Bojilova L, Potts AH, Cameron E, Gardner C, Lorca G, Gonzalez CF. Lactobacillus johnsonii inhibits indoleamine 2,3-dioxygenase and alters tryptophan metabolite levels in BioBreeding rats. FASEB J, 2013, 27: 1711–1720

    PubMed  CAS  Article  Google Scholar 

  51. 51

    Wu HQ, Pereira ER, Bruno J, Pellicciari R, Albuquerque E, Schwarcz R. The astrocyte-derived α7 nicotinic receptor antagonist kynurenic acid controls extracellular glutamate levels in the prefrontal cortex. J Mol Neurosci, 2010, 40: 204–210

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  52. 52

    Cortese BM, Mitchell TR, Galloway MP, Prevost KE, Fang J, Moore GJ, Uhde TW. Region-specific alteration in brain glutamate: possible relationship to risk-taking behavior. Physiol Behav, 2010, 99: 445–450

    PubMed  CAS  Article  Google Scholar 

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Correspondence to Feng Jin.

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Luo, J., Wang, T., Liang, S. et al. Ingestion of Lactobacillus strain reduces anxiety and improves cognitive function in the hyperammonemia rat. Sci. China Life Sci. 57, 327–335 (2014).

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  • hyperammonemia
  • probiotics
  • cognition
  • anxiety
  • neuroinflammation
  • serotonin