Probiotics for the treatment of inflammatory changes in the intestinal mucosa, particularly obesity

Abstract

The use of probiotics, in particular of the Enterococcus faecium strain named SF68, for the treatment of diseases characterized by changes in the intestinal mucosa, in particular of inflammations associated with changes in intestinal permeability, is described.

Description

Technical Field

[0001] The present invention relates to disorders characterized by inflammation associated with changes in the intestinal mucosa, particularly changes in intestinal permeability, and more specifically to the use of probiotics for the treatment of obesity, preferably.

Background Art

[0002] In recent years, for example, in several pathological conditions characterized by the presence of intestinal inflammation such as chronic intestinal inflammatory diseases (MICI), irritable bowel syndrome (IBS), obesity, and also in neurodegenerative pathologies such as Parkinson's disease and Alzheimer's disease, changes in the intestinal mucosa have been clinically demonstrated [Genser L. et al., Enhanced jejunal permeability in human obesity is revealed by lipid challenge and is associated with inflammation and type 2 diabetes. J. Pathol. 2018 Oct. 246(2):217 - 230; Michielan A. et al., Intestinal permeability in inflammatory bowel disease: etiology, clinical evaluation, and treatment of intestinal leak. Mediators Inflamm. 2015, 2015:628157; Shulman et al., Association between intestinal permeability, inflammatory markers, and symptoms in patients with irritable bowel syndrome, J. Gastroenterol. 2014 Nov, 49(11):1467 - 76; Sharma S. et al., Changes in gut microbiota and intestinal permeability in Parkinson's disease: from pathological highlights to management. Neurosci. Lett. 2019 Nov. 1, 712:134516; Sochocka M. et al., Critical review on the pathogenesis of Alzheimer's disease due to gut microbiota changes and inflammation, Mol Neurobiol. 2019 Mar, 56(3):1841 - 1851〕]

[0003] In particular, this enhanced intestinal permeability has been observed to be associated with reduced expression of tight junction proteins such as occludin, zonulin, and claudin, which are essential elements for maintaining the integrity of the barrier [Chelakkot C. et al., Mechanisms Controlling Intestinal Barrier Integrity and Their Pathological Significance, Exp Mol Med. 2018 Aug 16, 50(8):1-9]. Among the major systems involved in maintaining epithelial integrity, the importance of butyric acid, a short-chain fatty acid (SCFA), has been emphasized in several preclinical and clinical studies [Ma X. et al., Butyric Acid Promotes Recovery of Intestinal Wound Healing through Positive Effects on Tight Junctions, J Anim Sci. 2012 Dec, 90 Suppl 4:266-8, doi:10.2527 / jas.50965, PMID:23365351; Yang T. et al., Improvement of Nonalcoholic Fatty Liver Disease by Sodium Butyrate Is Associated with Regulation of Intestinal Tight Junctions in db / db Mice, Food Funct. 2020 Dec 1, 11(12):10675-10689; Vargas-Robles H. et al., Beneficial Effects of Supplements on Intestinal Barrier Function in an In Vivo Experimental Colitis Model, World J Gastroenterol. 2019 Aug 14, 25(30):4181-4198; Gao Y. et al., The Breast Milk Metabolite Short-Chain Fatty Acid Butyric Acid Enhances Immature Intestinal Barrier Function Genes in Response to Inflammation In Vitro and In Vivo, Am J Physiol Gastrointest Liver Physiol. 2020 Oct 21; Tabat et al., Acute Effects of Butyric Acid on Inducible Hypersensitivity and Tight Junction Protein Expression in Human Colonic Tissue, Biomolecules. 2020 May 14, 10(5):766; Liu J. et al., Beneficial Effects of Butyric Acid on Intestinal Injury, J Pediatr Surg. 2020 Jun, 55(6):1088-1093].

[0004] This SCFA, mainly produced by the gut microbiota, brings beneficial nutritional effects to the intestinal epithelium by stimulating the expression of tight junctions [Ma X. et al., supra, Yang T. et al., supra; Vargas-Robles H. et al., supra; Gao Y. et al., supra; Tabat et al., supra; Liu J. et al., supra]. On the other hand, it directly acts on some populations of immune cells to exert an anti-inflammatory effect [Bonomo R.R. et al., Fecal microbiota transplantation and butyrate improve neuropathic pain, immune cell profile, and gene expression in the peripheral nervous system of obese mice, Proc Natl Acad Sci USA, 2020 Oct 20, 117(42):26482-26493; Yang W. et al., Regulation of immune cell IL-22 production and gut immunity by gut microbiota-derived short-chain fatty acids, Nat Commun., 2020 Sep 8, 11(1):4457; Takahashi D. et al., Microbiota-derived butyrate limits autoimmune responses by promoting the differentiation of follicular regulatory T cells, EBioMedicine, 2020 Aug, 58:102913; Furusawa Y. et al., Butyrate produced by symbiotic microflora induces the differentiation of colonic regulatory T cells, Nature, 2013 Dec 19, 504(7480):446-50, doi:10.1038 / nature12721; Chen J. et al., Role of butyrate in reducing pathogen-induced inflammation, Immune Netw. 2020 Feb 4, 20(2):e15].

[0005] The existence of intestinal inflammation having an adverse effect on the use of butyrate is shown by several clinical evidences. In fact, patients with MICI, IBS or colorectal neoplastic diseases are characterized by a reduced expression of butyrate transporters, and as a result, the uptake of this SCFA is defective, deficits at the intestinal cell level occur, and an increase in intestinal permeability has been observed [Bonomo R.R. et al., supra; Yang W. et al., supra; Takahashi D. et al., supra; Furusawa Y. et al., supra; Chen J. et al., supra].

[0006] EP2289505 discloses a nutritional composition containing probiotics, prebiotics and butyric acid or its salts for alleviating intestinal disorders, particularly diarrhea and constipation.

[0007] Therefore, improving the utilization of butyric acid would bring beneficial effects to patients suffering from disorders characterized by intestinal inflammation and / or changes in intestinal permeability.

Summary of the Invention

[0008] We have now found that by treating with probiotics, particularly Enterococcus faecium strains, more specifically the Enterococcus faecium strain with deposit number NCIMB 10415 (referred to as SF68 (registered trademark) as a probiotic active ingredient), not only can the activity of citrate synthase (a key enzyme of the Krebs cycle essential for the correct utilization of butyric acid by cells) be improved, but also the expression ability of transporters can be enhanced, thereby increasing the ability of intestinal epithelial cells to utilize butyric acid. By improving the utilization of butyric acid, probiotic SF68 can increase the expression of tight junctions and strengthen the intestinal barrier.

[0009] Accordingly, an object of the present invention is an Enterococcus faecium-based supplement for use in the treatment of diseases and disorders characterized by intestinal inflammation and / or changes in intestinal permeability, particularly obesity, optionally in combination with butyric acid or its salts.

[0010] Supplements based on SF68 are preferably used. Pharmaceuticals based on SF68 are commercially available, in particular under the name Bioflorin® (Cerbios Pharma SA), and their efficacy and safety as probiotics are widely known (Holzapfel W. et al., Enterococcus faecium SF68 as a model for efficacy and safety evaluation of pharmaceutical probiotics, Beneficial Microbes, 2018, 9(3):375-388).

[0011] Butyric acid and its salts are also commercially available in the form of food supplements. Butyric acid is generally used in the form of its sodium or calcium salt, i.e., sodium butyrate or calcium butyrate, which are preferred forms for the combination according to the present invention.

[0012] When administering probiotics in combination with butyric acid, the two components of the combination can be administered separately or in a single dosage form, preferably separately.

[0013] When administered separately, the two components of the combination can be administered simultaneously, sequentially, or with a time lag between one and the other.

[0014] By way of example, but not limited thereto, the dosage of SF68 is generally in the range of 10 7 - 10 10 and preferably does not fall below 10 9 In the case of butyric acid and its salts, the dosage is generally in the range of 200 - 500 mg and preferably does not fall below 250 mg. The indicated dosage is generally administered 1 - 3 times a day for at least one week.

[0015] It is important to know that the reported dosages can vary depending on the disorder being treated, its severity, the patient's condition, etc. A person skilled in the art can readily identify the most appropriate dosage outside the reported range if so determined.

[0016] The use of the probiotic SF68, optionally in combination with butyric acid or its salts, according to the present invention has beneficial effects on several diseases and disorders characterized by intestinal inflammation and / or changes in intestinal permeability. Among such diseases and disorders, obesity, enteropathy caused by non-steroidal anti-inflammatory drugs, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and diseases characterized by neuroinflammation at the central nervous system (CNS) level, resulting in cognitive impairment, are of particular benefit without limiting the scope of the present invention to these diseases / disorders.

[0017] The particular benefit for these diseases and disorders within the scope of the present invention is due to the availability of animal models for efficacy studies, as will be disclosed in detail later in this specification.

[0018] Obesity

[0019] Overweight and obesity are global public health problems. The prevalence of obesity in Western countries has increased significantly, and as a result, further research is needed to better understand this disease and its complications and comorbidities. Animal models of diet-induced obesity (DIO) can reproduce human overweight and obesity. In fact, many protocols are used to accumulate excess fat in mice [de Moura E. et al., Diet-induced obesity in animal models: Considerations and effects on metabolic markers, Diabetol Metab Syndr., 2021 Mar 18, 13(1):32].

[0020] In this regard, it is worth recalling that high-fat diet (HFD)-induced obesity is currently considered an important tool for understanding the impact of Western diets high in fat content on the development of obesity and related disorders [Wang C.Y., et al., Mouse models of diet-induced obesity and insulin resistance, Methods Mol Biol. 2012, 821:421-433]. In fact, when mice consume a high-fat diet, it may lead to an increase in body fat percentage and leptin, and cause the onset of hypertension and glucose intolerance, thus potentially leading to the development of human-type obesity [de Moura E., et al., supra].

[0021] Based on this experience and past reports [Kim K.A., et al., High-fat diet-induced gut microbiota exacerbates inflammation and obesity in mice via the TLR4 signaling pathway, PLoS One. Laurila A., et al., High-fat, high-cholesterol diet increases the development of gastritis in LDL receptor-negative mice, Arterioscler Thromb Vasc Biol. 2001;21(6):991-996], it was confirmed that this experimental model is suitable because mice fed an HFD for 8 weeks showed a significant increase in body weight, followed by significant changes in several metabolic indicators such as increased blood glucose, cholesterol, and triglyceride levels. Recent studies have also demonstrated that HFD animals show increased expression of inflammatory cytokines (TNF, IL-1β, IL-6) in intestinal tissues, followed by spontaneous granulocyte dysfunction, changes in mucin biosynthesis, and damage to the mucosal barrier [Kim K.A., et al., supra; Ding S., et al., Role of intestinal inflammation in the early stages of obesity and insulin resistance, Curr Opin Clin Nutr Metab Care. 2011;14:328-33; Gulhane M., et al., High-fat diet induces stress and inflammation in colonic epithelial cells and is restored by IL-22, Sci Rep. 2016;6:28990].

[0022] Obesity is also known to be associated with early cognitive impairment, caused by an inflammatory process in the central nervous system supported by changes in the blood-brain barrier [Miller AA, Spencer SJ. Obesity and neuroinflammation: pathways to cognitive impairment. Brain Behav Immun. 2014 Nov;42:10-21; Rhea EM, Salameh TS, Logsdon AF, Hanson AJ, Erickson MA, Banks WA. The blood-brain barrier in obesity. AAPS J. 2017 Jul;19(4):921-930].

[0023] Enteropathy caused by non-steroidal anti-inflammatory drugs (NSAIDs)

[0024] The emergence of new imaging diagnostic techniques such as video capsule endoscopy has enabled the acquisition of new information on site-specific NSAID-induced intestinal damage. The types of mucosal damage seen in up to 75% of NSAID users range from damage mainly observed in the proximal small intestine to distal mucosal erosions and ulcers.

[0025] The etiology of small intestine injury has not yet been fully elucidated. The synthesis of mucosal endogenous prostaglandins is inhibited by NSAIDs throughout the gastrointestinal tract. However, other important pathogenic factors contributing to the damage, such as the presence of bacteria and bile, which are the essential inducing factors of mucosal damage, may differ between the distal and proximal intestinal regions.

[0026] NSAIDs enhance intestinal permeability in patients, and significant bacterial infiltration follows the enhanced permeability of the intestinal barrier, triggering low-grade enteritis. The enhancement of intestinal permeability by the local action of NSAIDs is increased by the inflammatory response (to luminal attack factors) and the microvascular effects of COX inhibition [Bjarnason I. et al., Mechanisms of gastrointestinal damage caused by non-steroidal anti-inflammatory drugs, Gastroenterology. 2018 Feb;154(3):500-514].

[0027] This model is mainly based on the results of mouse models, and several aspects of the damage, such as the enhancement of intestinal permeability by NSAIDs and the location of NSAID-induced enteropathy in the mid-distal part of the small intestine, show significant similarity to humans. As seen in clinical trials, in preclinical models as well, NSAID-induced damage to the intestinal mucosal barrier is the driving force behind a series of pathophysiological events that lead to mucosal damage. Indeed, damaged tight junctions facilitate the invasion and action of bacteria and bacterial antigens (as well as other luminal factors), leading to high expression and release of inflammatory cytokines from the intestinal epithelium. [Colucci R. et al: Pathophysiology of NSAID-related intestinal lesions in rats: Luminal bacteria and mucosal inflammation as targets for prevention, Front Pharmacol. 2018 Nov 29;9:1340; Fornai M. et al, Protection against NSAID-induced small intestine injury in rats: Effect of the poorly absorbed antibiotic rifaximin targeting the gastrointestinal tract. Pharmacol Res. 2016 Feb;104:186-96; Bjarnason I. et al, Mechanisms of gastrointestinal damage by non-steroidal anti-inflammatory drugs. Gastroenterology. 2018 Feb;154(3):500-514].

[0028] IBD / IBS model

[0029] Intestinal diseases (IBD) are a group of inflammatory diseases of the large and small intestines caused by abnormal immune responses. Crohn's disease (CD) and ulcerative colitis (UC) are the main types of IBD. Usually, both are accompanied by severe diarrhea, pain, fatigue, and weight loss. IBD can be debilitating and sometimes cause life-threatening complications.

[0030] When the intestinal barrier is damaged, intestinal permeability is enhanced, exposure to luminal contents is promoted, an immune response is induced, and as a result, intestinal inflammation is promoted. In patients with IBD, several defects are seen in the special components of the mucosal barrier, from the composition of the mucus layer to the adhesion factors that control cell permeability. These changes may be the primary dysfunction in Crohn's disease, but they may also perpetuate the chronic inflammation of the mucosa in ulcerative colitis [Michielan A. et al., Intestinal permeability in inflammatory bowel disease: etiology, clinical evaluation, and treatment of intestinal leakage. Mediators Inflamm. 2015;2015:628157].

[0031] The basic approach to studying the etiology and complexity of human IBD has been the development of various animal models. These animal models have provided important and indispensable detailed analyses of the histopathological and morphological changes in the intestine related to the etiology of human IBD. These models have become essential tools for explaining the histopathological, immunological, and morphological changes in the intestine and potential therapeutic targets [Eichele D.D. et al., The dextran sulfate sodium colitis mouse model: an essential tool for deepening the understanding of the etiology of inflammatory bowel disease. World J Gastroenterol. 2017 Sep 7;23(33):6016-6029].

[0032] In several animal experiments conducted over the past 25 years, a model of dextran sulfate (DSS)-induced colitis has been used as a method of chemically inducing an inflammatory model that is morphologically and symptomatically similar to the epithelial damage observed in human ulcerative colitis. In mice, colitis induced by DSS causes a decrease in the expression of tight junctions, which then leads to an increase in permeability and clinical events of colonic inflammation. In particular, the pattern of tight junction proteins shows rapid changes, such as an increase in the expression of claudin-2 and a decrease in the expression of several claudins and occludin-1. Therefore, impairment of the mucosal barrier is considered to be a secondary event of increased colonic mucosal permeability and the accompanying efflux of inflammatory cells in the intestinal mucosa [Eichele D.D. et al., supra].

[0033] Irritable bowel syndrome (IBS) is a type of functional gastrointestinal disorder (FGID), characterized by symptoms such as abdominal pain, discomfort, and bowel irregularities, without any relation to metabolic or organic abnormalities. Several factors are involved in the pathophysiology of IBS, including visceral hypersensitivity, gastrointestinal (GI) motility, brain-gut interaction, and psychosocial stress. Interestingly, IBS often develops in patients who have recovered from infectious colitis, and symptoms similar to those of IBS are often observed in patients with intestinal inflammatory diseases (IBD) even after the intestinal inflammation has been removed.

[0034] IBS is classified into constipation-predominant (IBS-C), diarrhea-predominant (IBS-D), and mixed (IBS-M) types. There is no cure for IBS, and current treatment strategies often involve taking multiple medications to control symptoms. In particular, the main clinical features of IBS are changes in intestinal motility and excretion and visceral pain [Kodani M. et al., Association between gastrointestinal motility and macrophage / fat cell distribution in the healing stage after DSS-induced colitis in mice. Mol Med Rep. 2018 Jun;17(6):8167-8172].

[0035] Experimental models of IBS can be divided into three types: animal models by central stimulation, animal models by peripheral stimulation, and complex animal models by combined central and peripheral stimulation. Among these models, enteritis induced by chemicals such as DSS may induce IBS symptoms. In fact, in mice that have recovered from DSS-induced colitis, intestinal transit speed is slowed, barrier function is decreased, and EC cell density is decreased [Sharman S.K. et al., Sildenafil normalizes intestinal transit in a preclinical model of constipation. PLoS One.2017 Apr 27;12(4):e0176673].

[0036] Neuropathy

[0037] The microbiota-gut-brain axis not only supports the pathophysiological events underlying several neurological disorders, such as mild cognitive impairment (MCI), dementia, multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), autism, etc., but also emerges as an important communication system involved in maintaining the homeostasis of the central nervous system [Pellegrini C, Antonioli L, Colucci R, Blandizzi C, Fornai M. Interactions between gut microbiota, intestinal mucosal barrier, and enteric neuroimmune system: a common pathway to neurodegenerative diseases? Acta neuropathologica. Sep 2018;136(3):345-361. doi:10.1007 / s00401-018-1856-5; Pellegrini C, Antonioli L, Calderone V, Colucci R, Fornai M, Blandizzi C. The microbiota-gut-brain axis in health and disease: is the NLRP3 inflammasome at the crossroads of microbiota-gut-brain communication? Progress in neurobiology. Aug 2020;191:101806. doi:10.1016 / j.pneurobio.2020.101806]. The mechanisms underlying the microbiota-gut-brain axis mainly rely on the interactions between gut bacteria and their metabolites, the immune system, and ascending nerve fibers, which are accompanied by the intestinal epithelial barrier. In fact, pathogenic bacterial strains and their products can ride on the bloodstream and move to the brain, potentially damaging the integrity of the blood-brain barrier and affecting central nervous circuits. Furthermore, the products of pathogenic bacteria can directly activate circulating immune / inflammatory cells, which can then move to the central nervous system and change the blood-brain barrier [Fung TC, Olson CA, Hsiao EY. Interactions between microbiota, immune system, and nervous system in health and disease. Nature neuroscience. Feb 2017;20(2):145-155. doi:10.1038 / nn.4476; Rothhammer V, Mascanfroni ID, Bunse L, et al. Type I interferons and microbial metabolites of tryptophan regulate astrocyte activity and central nervous system inflammation via the aryl hydrocarbon receptor.Nature medicine. Jun 2016; 22(6):586-97.doi:10.1038 / nm.4106; Rooks MG, Garrett WS. Gut microbiota, metabolites, and host immunity. Nature reviews Immunology. May 27 2016;16(6):341-52. doi:10.1038 / nri.2016.42].

[0038] Therefore, gut immune / inflammatory responses such as changes in the microbiota and intestinal epithelial barrier, and activation of inflammatory enzymes may be early events in neuropathy that promote neuroinflammation and central neuroinflammation via the gut-brain axis [Pellegrini C, Antonioli L, Colucci R, Blandizzi C, Fornai M., supra; Ortiz GG, Pacheco-Moises FP, Macias-Islas MA, et al. Role of the blood-brain barrier in multiple sclerosis. Archives of medical research. Nov 2014;45(8):687-97. doi:10.1016 / j.arcmed.2014.11.013; Zenaro E, Piacentino G, Constantin G. The blood-brain barrier in Alzheimer's disease. Neurobiology of disease. Nov 2017;107:41-56. doi:10.1016 / j.nbd.2016.07.007; Al-Bachari S, Naish JH, Parker GJM, Emsley HCA, Parkes LM. Blood-brain barrier leakage is increased in Parkinson's disease. Frontiers in physiology.2020;11:593026. doi:10.3389 / fphys.2020.593026]. Against this background, manipulation of the gut microbiota by prebiotics and probiotics has been proposed as a useful therapeutic approach for the prevention of central nervous system diseases [Joseph J, Depp C, Shih PB, Cadenhead KS, Schmid-Schonbein G. Modified Mediterranean diet rich in short-chain fatty acids: a potential adjuvant therapy targeting immune and metabolic dysfunctions in schizophrenia? Frontiers in neuroscience.2017;11:155. doi:10.3389 / fnins.2017.00155; Sharma S, Taliyan R, Singh S. Beneficial effects of sodium butyrate against 6-OHDA-induced neurotoxicity and behavioral abnormalities: modulation of histone deacetylase activity.Behavioural brain research. Sep 15 2015;291:306-314. doi:10.1016 / j.bbr.2015.05.052; van de Wouw M, Boehme M, Lyte JM, et al. Short-chain fatty acids: microbial metabolites that mitigate stress-induced changes in the brain-gut correlation. The Journal of physiology. Oct 2018;596(20):4923-4944. doi:10.1113 / JP276431].

Brief description of the drawings

[0039]

Figure 1

Figure 1-2

Figure 2

Figure 2-2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Mode for Carrying Out the Invention

[0040] The present invention is based on the effectiveness of SF68 against several aspects of intestinal inflammation / alterations and its ability to re-uptake butyric acid. Based on these experimental observations, administration of SF68 is particularly effective, but not limited thereto, in the treatment of disorders characterized by changes in intestinal permeability, including obesity, when administered selectively in combination with butyric acid or its salts.

[0041] In particular, by combining them, the beneficial effects of both SF68 and butyric acid on the intestinal mucosa can be combined.

[0042] The effectiveness of SF68 has been demonstrated by experiments on mice treated with a high-fat diet (see Experimental Section).

[0043] In fact, from the results obtained, it seems that: - It was shown that by administering SF68 supplementation for 4 weeks, 4 + 4 weeks, and 8 weeks, the weight gain of animals fed a high-fat diet (HFD) could be significantly suppressed; - Regarding metabolic parameters, HFD significantly increased total cholesterol, LDL, and triglycerides and decreased HDL levels. When SF68 was administered for 8 weeks, such changes were significantly suppressed; - In animals fed HFD, an increase in the circulating levels of IL-1β and LPS-binding protein (LBP, an indicator of increased intestinal permeability) was observed. When SF68 supplementation was ingested for 8 weeks, such changes were significantly suppressed; - As a result of analyzing the feces of animals fed HFD, the calprotectin concentration increased, and no significant change was observed after administration of SF68; - Regarding SCFA levels, there was no change in the level of propionic acid in the feces in HFD. However, in HFD animals, the level of butyric acid in the feces increased significantly. This increase in butyric acid was not observed in HFD animals given SF68 for 8 weeks. - Analysis of the levels of tissue inflammatory parameters (IL-1β and MPO) and tissue oxidative stress (MDA) showed that these parameters were increased in animals fed with HFD. When SF68 was supplemented for 8 weeks, these parameters were significantly decreased, and anti-inflammatory and antioxidant effects were observed; - Experiments conducted in vitro showed that in animals fed with a high-fat diet, the neuronal coding of the enteric nervous system of the large intestine changed, the cholinergic component decreased, and tachycardic tone increased. Such changes are widely known to underlie the motility disorders observed in several pathologies characterized by the presence of enteritis. Most interestingly, SF68 has the effect of counteracting such changes by preserving the cholinergic component and significantly antagonizing excessive tachycardic neurotransmission. Such beneficial effects are thought to be due to a decrease in the levels of inflammation and oxidative stress in the large intestine tissue, or a direct effect on neuronal coding, or both; - In animals fed with HFD, it was demonstrated that the expression of tight junctions in the large intestine tissue was significantly decreased and the intestinal barrier was relaxed. Administration of SF68 increased the expression of tight junctions and strengthened the barrier; - In animals fed with a high-fat diet for 8 weeks, the level of TLR-4 in the large intestine tissue was significantly increased. Such an increase was antagonized by supplementing SF68 for 8 weeks. At the same time, by exploring the intracellular pathway related to TLR-4, an increase in NFK-b was observed after 8 weeks of high-fat diet intake. In the 4+4w and 8w schemes, SF68 antagonized such an increase; - From experiments evaluating the effect of probiotics on the system of butyrate transport and utilization, it was observed that in obese animals, the uptake capacity of this SCFA was decreased due to a decrease in the expression of the butyrate transporter and a decrease in citrate synthase activity. SF68 resulted in improved utilization of this SCFA by the intestinal mucosa by restoring the level of the SMCT1 transporter and normalizing citrate synthase activity in the intestinal tissue; - SF68 has a beneficial effect on intestinal homeostasis, leading to better utilization of butyric acid as demonstrated by the increase in butyrate transporters, improving citrate synthase activity, resulting in improved trophism of the intestinal epithelium, improved expression of tight junctions, accompanied by a decrease in the permeability of luminal antigens, and experimentally proven to reduce the inflammatory burden as inferred from the decrease in inflammatory cytokines. - Furthermore, administration of SF68 improved the expression of tight junctions in the central nervous system, and as a result, the presence of central inflammatory states (decrease in TNF levels) decreased.

[0044] In conclusion, it became clear that administration of SF68 suppresses changes in several systemic and tissue parameters due to high-fat diet intake. The beneficial effect of SF68 on intestinal barrier trophism is demonstrated by an increase in tight junctions, which results from the ability of SF68 to make the intestinal mucosa more sensitive to the uptake and utilization of butyric acid, a SCFA known for its beneficial capabilities on intestinal mucosa trophism and the intestinal immune system, indicating its enhancement. This all seems to occur due to an anti-inflammatory effect that mediates the beneficial effect on the integrity of the intestinal barrier. As demonstrated by histochemical studies of inflammatory infiltration, reduction of circulating LPS, IL-1β, TLR4, and reduction of NF-kB and MPO (signals of reduction of inflammatory cell infiltration), a positive effect on tight junctions has been observed to be associated with improved utilization / re-uptake of butyric acid by intestinal cells.

[0045] This effect is particularly relevant in the context of the present invention, demonstrating the effectiveness of probiotics, selectively in combination with butyric acid (or its salts), for the treatment of diseases and disorders characterized by intestinal inflammation and / or changes in intestinal permeability, including obesity.

[0046] Without being bound by any theory, based on the results in mice, SF68 is thought to normalize the expression of apical transporters of butyric acid altered by a high-fat diet (HFD).

[0047] Next, the usefulness, effectiveness, and advantages of the present invention will be further described in detail by the following examples, which are not intended to limit the scope of the present invention in any way.

[0048] Experimental section

[0049] Five-week-old C57BL / 6 mice (body weight 20 - 22 g) were provided by Envigo srl (Udine, Italy, San Pietro al Natisone). The mice were housed six per cage in a temperature-controlled room with a 12-hour light cycle at 22 - 24 °C and 50 - 60% humidity and acclimated for at least one week. The handling of the animals was in accordance with Directive 2010 / 63 / UE.

[0050] During the adaptation period, all mice were administered a standard diet (SD, 18% fat-derived calories; TD.2018). Subsequently, the animals were randomly divided into six groups, each consisting of 10 mice as follows:

[0051] Protocol A - Group A1: Vehicle with SD + lactose (L) for 4 weeks (4w) - Group A2: Vehicle with HFD + lactose for 4 weeks - Group A3: SD + vehicle + SF68 (P) from the 4th week for 4 weeks - Group A4: SD + vehicle + vehicle with lactose from the 4th week - Group A5: HFD + vehicle + SF68 from the 4th week for 4 weeks - Group A6: HFD + vehicle + vehicle with lactose from the 4th week

[0052] Protocol B - Group B1: SD + SF68 for 4 weeks - Group B2: HFD + SF68 for 4 weeks - Group B3: SD + SF68 for 8 weeks (8w) - Group B4: HFD + SF68 for 8 weeks

[0053] The vehicle was 150 μL of 3% methocel. Bioflorin® used SF68. The high-fat diet (HFD) was 60% calories from fat (TD.06414).

[0054] A comparison of the calories of the two diets is shown in Table 1 below: [Table 1]

[0055] The body weight of the animals was measured once a week starting from the first day of the test, and the rate of change in body weight with the value of the animals in the SD + L group set to 100 is shown in Table 2 below. [Table 2]

[0056] Mice fed a high-fat diet (HFD) showed significant weight gain at 4 and 8 weeks compared to mice fed a normal-calorie diet (SD). Administration of SF68 significantly suppressed weight gain due to HFD at 4 and 8 weeks. Administration of SF68 did not result in a change in weight gain of animals fed SD in all treatment schemes tested.

[0057] Finally, the animals were anesthetized and euthanized. Blood samples and tissue samples were collected and stored at -80 °C for further analysis.

[0058] Body weight and organ changes

[0059] Mice fed a high-fat diet (HFD) showed significant weight gain at 4 and 8 weeks compared to mice fed a normal-calorie diet (SD) (Figure 1A). Administration of SF68 significantly counteracted the weight gain induced by HFD at 4 and 8 weeks (Figure 1A). Administration of SF68 did not result in a change in weight gain of animals fed SD in all treatment schemes tested (Figure 1A).

[0060] Furthermore, Figures 1B - D report the changes in epididymal fat weight (B), spleen weight (C), and liver weight (D) in mice given SD + lactose, SD + SF68, HFD + lactose, and HFD + SF68 in treatment schemes of 4 weeks (4w), 4 + 4 weeks, or 8 weeks (8w).

[0061] No significant effect was seen on the fat accumulated in the epididymis. This fact is not abnormal since in the HFD model, epididymal fat finally increased and it was considered that this parameter could not be changed in the 8 - week test.

[0062] On the contrary, a decrease in the weight of the spleen, a lymphoid organ, was also observed. The spleen is an organ whose weight increases in the presence of systemic inflammation. The decrease in spleen weight indicates an anti - inflammatory effect.

[0063] Normally, in HFD, adipose tissue hypertrophies the liver (non - alcoholic liver syndrome - NASH), causing a decline in function. In HFD animals administered SF68, a decrease in liver weight was observed compared to non - administered animals, and since the appearance of the HFD liver was mainly good in morphological examinations, a protective effect of SF68 (with a smaller area of fat infiltration) was suggested.

[0064] Metabolic parameters

[0065] Animals given HFD had a significantly increased total cholesterol value (Figure 2A) and LDL value (Figure 2C) and a decreased HDL value (Figure 2B) in plasma compared to mice given SD (Figures 2A - D). When SF68 was administered to high - calorie - fed mice, the levels of total cholesterol (Figure 2A) and LDL (Figure 2C) did not change, and the HDL level was significantly increased compared to HFD mice (Figure 2B). And the HDL / LDL ratio changed favorably.

[0066] Animals fed an HFD showed a significant increase in plasma triglyceride concentrations compared to mice fed an SD (Figure 3A), although hemoglobin (Hb) levels did not appear to change (Figure 3B). Administration of SF68 to mice on a high-calorie diet resulted in a significant increase in triglyceride levels compared to HFD mice (Figure 3A), but glycated Hb levels did not change (Figure 3B).

[0067] In the HFD model, the increase in glycated Hb was only observed at weeks 10 - 12, a period longer than the duration of this study, so it could be predicted that changes in glycated Hb would be scarce.

[0068] Conversely, the effect of SF68 on reducing triglycerides is important and counteracts the effect of triglyceride increase by HFD. The increase in triglycerides is associated with liver damage along with an increase in transaminases, so the observed decrease is favorable for a healthier (and less morphologically infiltrated) liver.

[0069] Plasma levels of IL-1β and LBP (LPS-binding protein)

[0070] Plasma levels of IL-1β and LBP were measured by ELISA (Prodotti Gianni, Milan, Italy) as described by Antonioli L. et al. [Int. J. Obes. (2019), doi:10.1038 / s41366-018-0166-2]. The procedure involved centrifuging blood samples at 4000 rpm for 5 minutes at 2 - 8°C, and after centrifugation, the supernatant was collected. An aliquot (100 μl) was used for analysis. IL-1β levels were expressed as pg / ml in plasma, and LBP levels were expressed as ng / ml in plasma.

[0071] Animals fed with HFD showed a significant increase in plasma concentrations of IL-1β (Figure 4A) and LBP (Figure 4B) 8 weeks after HFD feeding, compared to mice fed with SD (Figure 4A and B). Administration of SF68 to mice on a high-calorie diet resulted in a significant increase in IL-1β levels in the 4 + 4-week and 8-week regimens in HFD mice (Figure 4A), but a decrease in circulating LBP levels was observed only 8 weeks after SF68 administration (Figure 4B).

[0072] IL-1β is pyrogenic, inflammatory, and induces macrophage activation. The decrease in plasma concentration of IL-1β is directly correlated with the anti-inflammatory effect of SF68 administration.

[0073] When the intestinal epithelial barrier is damaged, intestinal microorganisms are more likely to migrate into the blood, and the same microorganisms (in the case of Gram) and / or lipopolysaccharide (LPS) produced by their degradation bind to the LBP protein, and the production by hepatocytes increases significantly when microorganisms are present in the blood. The significant increase in LBP in HFD-fed mice indirectly suggests a decline in the function of the intestinal epithelial barrier. Such an increase was significantly counteracted by SF68 at the 8th week of administration, indicating a decrease in circulating LPS.

[0074] Evaluation of SCFA content in feces

[0075] For the analysis of short-chain fatty acids (SCFAs), fecal samples were freeze-dried and subjected to gas chromatography. The freeze-dried material was dissolved in 100 ml of 5 M formic acid and 400 ml of acetone and centrifuged (4000 rpm for 5 minutes). The SCFA concentration in the supernatant was measured using a GC2010 Plus gas chromatograph (Shimadzu Deutschland GmbH, Duisburg, Germany) equipped with a flame ionization detector and a thin-film capillary column Stabilwax (registered trademark) (Restek, Bad Homburg, Germany). The samples were injected in split mode using an autosampler AOC-20s / I (Shimadzu Deutschland GmbH). GC Solution Chromatography Data System (Shimadzu Deutschland GmbH) was used for data processing. For the quantification of SCFAs, an external standard substance (Supelco TM WSFA-1 Mix, Supelco Sigma-Aldrich Co. Bellefonte PA) was employed.

[0076] In mice fed a high-fat diet (HFD), the butyric acid concentration in feces significantly increased 8 weeks after HFD intake (Figure 5A). Such an increase was antagonized by supplementing SF68 with 4+4 and 8w treatment regimens. No change was observed in the propionic acid level (Figure 5B).

[0077] Statistically significant information regarding SF68 has been deeply explored in relation to the mechanism of action through studies on intestinal butyrate transporters, and it has been found that SF68 normalizes the expression of the butyrate apical transporter SMCT1, which was altered by HFD. What can be considered as the mechanism of action of SF68 is, for example, the improvement of the health state of the intestinal epithelium by improving tight junctions, mainly promoting the reuptake of butyrate, and increasing the expression level of the SFCA transporter that promotes the effective utilization of butyrate by organisms as a nutrient factor and anti-inflammatory factor in intestinal cells. Further studies described later observed that SF68 administration was also associated with an increase in citrate synthase activity (in both the intestine and adipose tissue). This enzyme catalyzes the first reaction of the Krebs cycle, which is an important process of cellular respiration, regulates the overall flux of the cycle, and enables the utilization of butyrate in intestinal cells. A high-calorie diet decreases the enzyme activity of citrate synthase, which is counteracted by SF68. This effect, in addition to the improvement in the expression of SCFA transporters (and the strengthening of tight junctions), may explain the association between SF68 administration and a healthier intestine.

[0078] Fecal concentration of calprotectin

[0079] In mice fed a high-fat diet (HFD), the fecal calprotectin value, a protein belonging to the S100 family that is abundant in neutrophils, was significantly increased. Even when SF68 was administered, no significant change was observed in such parameters (Figure 6).

[0080] That is to say, the anti-inflammatory mechanism of SF68 cannot be explained by a direct action on neutrophils. Myeloperoxidase (MPO), malondialdehyde (MDA), and IL-1β levels in intestinal tissues

[0081] According to the description of Antonioli L. et al. [FASEB J. (2020) 34, 5512-5524], MPO levels in colon tissue were measured by ELISA (Prodotti Gianni, Milan, Italy). Colon samples pre-stored at -80 °C were homogenized on ice using a Polytron Homogenizer (Qiagen, Milan, Italy). The homogenate was centrifuged at 12,000 rpm for 15 minutes at 4 °C. An aliquot (100 μL) of the supernatant was used for analysis. MPO levels were expressed as ng / mg tissue.

[0082] To quantitatively estimate the mucosal infiltration of polymorphonuclear cells, the MDA concentration in intestinal samples was measured. The analysis was performed by the previously described method of Antonioli et al. [Int. J. Obes. (2019), supra]. Intestinal tissue was weighed, pulverized, homogenized in 2 ml of cold buffer (QIAGEN, Milan, Italy) using a Polytron homogenizer, and centrifuged at 1200 rpm for 10 minutes at 4 °C. The MDA concentration was measured using a colorimetric assay kit (Calbiochem, San Diego, CA, USA), and the results were expressed as the number of nanomoles of MDA per milligram of colon tissue (nmoles).

[0083] In colon tissue samples isolated from mice fed a high-fat diet, significant increases in MPO, MDA, and IL-1β levels were observed compared to those observed in tissues collected from animals fed a standard diet (Figures 7A, B, and C) (Figures 7A, B, and C). In HFD mice, administration of SF68 significantly decreased MPO, MDA, and IL-1β levels compared to those observed in HFD mice not receiving SF68 supplementation (Figure 7).

[0084] In the tissues of mice fed an HFD, significant increases in MPO, which is derived from the infiltration of immune inflammatory cells, MDA, a signal of oxidative stress, and IL-1β, a pyrogenic and inflammatory cytokine, were observed. When SF68 was administered for 8 weeks, these three components in the tissues decreased significantly. The decrease in MDA implies a decrease in oxidative stress and is thought to be due to the effect of SF68 in supporting endogenous scavengers (such as glutathione). The decrease in MPO means that there is less infiltration of inflammatory cells. The decrease in IL-1β supports the anti-inflammatory effect of SF68, and indeed, a decrease in this interleukin was also observed in the blood.

[0085] In vitro evaluation of colonic contractile activity

[0086] The contractile activity of the colonic muscle specimens was performed with some modifications to the description by Antonioli et al. [FASEB J., supra]. After disposal, the colon was incised above the anal end and immediately removed and placed in Krebs solution. The colon portion was incised along the mesenteric insertion and the mucosa / mucosa-submucosa was removed. The colon samples were cut lengthwise into elongated strips approximately 4 mm wide and 10 mm long along the longitudinal axis.

[0087] The specimen was set in an organ bath containing Krebs solution at 37°C bubbled with 5% CO2 + 95% O2 and connected to an isometric transducer (constant load = 0.5 g). Mechanical activity was measured with a BIOPAC MP150 (Biomedica Mangoni, Pisa, Italy). The composition of the Krebs solution was as follows: KCl 4.7 nM, NaCl 113 nM, KH2PO4 1.2 nM, CaCl2 2.5 nM, MgSO4 1.2 nM, NaHCO3 25 nM, glucose 11.5 nM (pH 7.4 ± 0.1). Each specimen was equilibrated for at least 30 minutes with washes at 10-minute intervals. A pair of coaxial platinum electrodes was placed at a distance of 10 mm from the longitudinal axis of each specimen to apply electrical stimulation with a BM-ST6 stimulator (Biomedica Mangoni, Pisa, Italy). After the equilibration period, each specimen was given repeated electrical stimulation, and the experiment was started when a reproducible response was obtained (usually after 2 - 3 stimulations).

[0088] In the first experiment, all electrical stimulations were recorded in colonic samples maintained in standard Krebs solution.

[0089] In the second and third experimental sets, cholinergic contractions were recorded. Colonic samples were maintained in Krebs solution containing N-ω-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor, 100 μM), N-acetyl-1-tryptophan 3,5-bis(trifluoromethyl)benzyl ester (L-732,138, a neurokinin NK1 receptor antagonist, 10 μM), 5-fluoro-3-[2-[4-methoxy-4-[[(R)-phenylsulfinyl]methyl]-1-piperidinyl]ethyl]-1H-indole (GR159897, an NK2 receptor antagonist, 1 μM), (R)-[[(2-phenyl-4-quinolinyl)carbonyl]amino]-methyl ester benzeneacetic acid (SB218795, an NK3 receptor antagonist, 1 μM), and guanethidine (an adrenergic blocker 10 μM) to evaluate neurogenic contractions, and carbachol (CCh, 10 μM) was given in Krebs solution containing tetrodotoxin (TTX, 1 μM) to examine myogenic responses.

[0090] The fourth and fifth series of experiments were set up to evaluate tachykinergic NK1 stimulation. Neurogenic NK1 contractions were recorded in colonic specimens maintained in Krebs solution containing L-NAME (100 μM), guanethidine (10 μM), GR159897 (1 μM), SB218795 (1 μM) and atropine sulfate (muscarinic receptor antagonist, 1 μM), while myogenic activity was detected by maintaining the specimens in TTX-supplemented Krebs solution and stimulating with exogenous substance P (SP, 1 μM).

[0091] During the stabilization period of colonic tissue maintained in standard Krebs solution, the specimens showed rapid spontaneous motility, which remained stable throughout the experiment and, in most cases, minimally interfered with the motor responses induced by electrical stimulation (ES). The responses induced by electrical stimulation were characterized by successive contractions, which were sometimes followed by after-contractions of different widths. In colonic specimens from SD or HFD animals maintained in standard Krebs solution, application of electrical stimulation induced comparable contractile responses 4 weeks after SD or HFD administration (Figure 8A). Analysis of the main ENS excitatory systems (acetylcholine and substance P) was performed to evaluate the endogenous and exogenous, cholinergic and tachykinergic contractile responses, and no changes due to HFD or SF68 were observed after 4 weeks (Figure 8B).

[0092] In colonic specimens from SD or HFD animals maintained in standard Krebs solution, application of electrical stimulation induced comparable contractile responses after 8 weeks of SD or HFD administration (Figure 9). Analysis of the main ENS excitatory systems (acetylcholine and substance P) revealed a decrease in cholinergic activity and an increase in tachykinergic activity in animals given HFD after 8 weeks (Figure 9). Administration of SF68 for 8 weeks normalized the functional changes induced by the high-fat diet (Figure 9).

[0093] A high-fat diet causes changes in enteric nerve components. Obese individuals present constipation-type "IBS-like" symptoms due to changes in peristaltic movements in which inhibitory and excitatory stimuli alternate repeatedly.

[0094] The contraction stimuli are acetylcholine, which lasts for several milliseconds, and substance P (tachykinin), which acts longer. The relaxation stimulus is nitric oxide. Physiologically, the contraction is mainly cholinergic, but in inflammatory pathological conditions and HFD, the contraction is mainly tachykininergic.

[0095] HFD (and general tissue inflammation) is associated with 1) a decrease in cholinergic responses, 2) an increase in substance P, and an effect (constipation) that prolongs the contraction of longitudinal smooth muscle compared to the gastrointestinal tract.

[0096] SF68 can statistically significantly cancel both the increase in the tachycardic component due to a high-calorie diet and the disappearance of cholinergic responses. It is noteworthy that acetylcholine not only functions as a neurotransmitter but also exhibits immunomodulatory activity and reduces the activity of immune-inflammatory cells.

[0097] The cholinergic contractions that "ruin themselves" during inflammation are stronger in samples treated with SF68 after 8 weeks than in the HFD control. On the other hand, substance P seems to have decreased. Since these effects are canceled by the addition of tetrodotoxin, which blocks the nerve component, the effect of SF68 appears to be at the systemic level that affects the neuroenteric component rather than at the muscle level.

[0098] Western blot analysis of tight junctions in colon tissue

[0099] The colonic tissues were weighed and homogenized in lysis buffer (50 mg in 400 μl) using a Polytron homogenizer (QIAGEN, Milan, Italy). The homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C, and the resulting supernatant was separated from the pellet and stored at -80 °C. To quantify total protein, the Bradford assay was performed. Subsequently, the proteins were separated on a precast 4%-20% polyacrylamide gel (Mini-PROTEAN TGX Gel, Biorad) and transferred to a PVDF membrane (Trans-Blot TurboTM PVDF Transfer Packs, Biorad). The membrane was blocked with 3% BSA diluted in Tris-buffered saline with 0.1% Tween 20 (TBS, 20 mM Tris-HCl, pH 7.5, 150 nM NaCl). Primary antibodies against β-actin (ab8227, Abcam), occludin (ab167161, Abcam), zonulin-1 (ab96587, Abcam), claudin (ab15098, Abcam), toll-like receptor (TLR) 4 (ab22048, Abcam), TLR2 (ab213676, Abcam), NF-κB-p65 (sc-8008, Santa Cruz), MyD88 (sc-136970, Santa Cruz), sodium-coupled monocarboxylate transporter 1 (SMCT1, BS-6106R, Bioss), monocarboxylate transporter 1 (MCT1, PA5-76687, Thermo Fisher), and monocarboxylate transporter 4 (MCT4, PA5-106683, Thermo Fisher) were used. Secondary antibodies were purchased from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected using an ECL reagent (Clarity Western ECL Blotting Substrate, Biorad). Densitometry analysis was performed using iBright analysis software.

[0100] As a result of administering HFD for 4 and 8 weeks, the tight junction levels in the colon tissue were significantly decreased (Figures 10A, B, and C). When SF68 was supplemented for 8 weeks, the expressions of zonulin 1, occludin, and claudin 1 were normalized (Figures 10A, B, C). On the other hand, with administration for 4 + 4 weeks, the expression of claudin 1 was improved, but the expressions of zonulin 1 and occludin were not improved (Figure 10C).

[0101] Evaluation of toll-like receptor expression in intestinal tissue

[0102] In animals administered HFD for 4 and 8 weeks, no significant changes were observed in the colonic expression of Toll-like receptors. Even when SF68 was administered, no significant changes were seen in the TLR2 level (Figure 11A). Supplementation with probiotics for 8 weeks resulted in a significant decrease in the TLR4 colonic level compared to the group administered HFD (Figure 11B).

[0103] TLR is a type of transmembrane receptor protein mainly present in macrophages and dendritic cells and plays an important role in innate immunity. TLR activates the immune response of sentinel cells by binding to ligands belonging to pathogens.

[0104] Western blot of the molecular signaling pathways of nuclear factor kB (NF-kB) and myeloid differentiation primary response gene 88 (MyD88)

[0105] When HFD was administered for 8 weeks, the NF-kB level in the colon tissue was significantly increased (Figure 12A). When SF68 was supplemented in the diet, this parameter was significantly decreased in both the 4 + 4-week and 8-week treatment schemes (Figure 12A). Conversely, when analyzing the MyD88 level, no significant changes were observed among different treatment groups (Figure 12B).

[0106] NF-kB plays an important role in the inflammatory response to infection, and its increase is associated with inflammation, cancer, and autoimmune diseases.

[0107] Immunohistochemistry and immunofluorescence

[0108] A portion of the formalin-fixed whole colon samples was processed for immunoperoxidase staining according to the description of Ippolito et al. [J. Cell. Mol. Med. (2015), doi:10.1111 / jcmm.12428]. After incubating the sections with the primary antibodies anti-claudin-1 and inflammatory infiltrate (Santa Cruz Biotech, California, USA) overnight at 4°C, they were then exposed to a complex of appropriate biotinylated immunoglobulins, streptavidin labeled with peroxidase, and 3,3'-diaminobenzidine (DakoCytomation, Glostrup, Denmark). Subsequently, the immunostained sections were observed with a Leica DMRB microscope, and representative micrographs were taken with a DFC480 digital camera (Leica Microsystems, Cambridge, UK) for quantitative evaluation.

[0109] As a result of administering HFD for 4 weeks and 8 weeks, the claudin-1 levels (Figures 13A and B) and immune-inflammatory infiltrates (Figure 14) in the colon tissue were significantly decreased. When SF68 was supplemented according to the 4-week, 4+4-week, and 8-week regimens, the claudin-1 expression was normalized (Figures 13A and B), and the intestinal inflammation levels were significantly reduced (Figure 14).

[0110] Evaluation of the protein expression of butyrate transporters

[0111] In mice administered HFD for 8 weeks, compared with SD control mice, the apical transporter SMCT1 was significantly decreased (Figure 15A), while the levels of MCT1 (apical) and MCT4 (basolateral) transporters were not affected (Figures 15B and C). When SF68 was administered for 4 weeks and 8 weeks, the expression of the SMCT1 transporter was normalized in HFD mice (Figure 15A), but no changes were observed in the expression of MCT1 and MCT4 proteins (Figures 15B and C).

[0112] Evaluation of citrate synthase activity in the intestinal mucosa

[0113] Frozen samples obtained from different mouse groups were homogenized on ice in cold buffer (250 mM sucrose, 5 mM Tris, 1 mM EGTA, 0.02% Triton X-100; pH 7.4) using a GentleMACS dissociator (Miltenyi Biotec, Bologna, Italy). The homogenate was centrifuged at 12,000 x g for 15 minutes at 4 °C (EuroClone, Speed Master 14 R centrifuge, Milan, Italy). The pellet was discarded and the supernatant was used for protein quantification by Bradford assay and subsequently for determination of citrate synthase activity.

[0114] Samples were diluted with Tris buffer (100 mM; pH 8.2) containing 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, 100 μM) and acetyl-CoA (100 μM). Analysis was performed in a 96-well multi-plate (1 μg of protein per well) and the reaction was initiated by adding a solution of oxaloacetic acid (500 μM). The reaction was followed spectrophotometrically at a wavelength of 412 nm every 30 seconds for 15 minutes at 37 °C (EnSpire, PerkinElmer, Waltham, MA, USA). Citrate synthase activity was determined by comparing the sample activity with that of a known concentration of the isolated enzyme (Sigma-Aldrich, St. Louis, MO, USA). Citrate synthase activity was expressed as mU / ml.

[0115] In mice fed an HFD for 4 or 8 weeks, citrate synthase activity in the intestinal mucosa and adipose tissue was significantly decreased compared to SD control mice (Figures 16A and B). Administration of SF68 for 8 weeks resulted in a significant increase in citrate synthase activity in the intestine and adipose tissue of HFD mice (Figures 16A and B).

[0116] Evaluation of plasma homocysteine levels

[0117] In animals fed a high-calorie diet, plasma homocysteine levels increased (Figure 17). When SF68 was administered to mice fed a high-calorie diet, the levels of this parameter were significantly decreased in the 4+4-week and 8-week regimens (Figure 17).

[0118] In mice fed a high-calorie diet, plasma homocysteine levels increased, which may also occur in obese people consuming a high-calorie diet. Administration of SF68 significantly decreased plasma homocysteine levels.

[0119] Evaluation of TNF levels

[0120] In animals fed a high-calorie diet, TNF levels in brain tissue increased (Figure 18). When SF68 was administered to mice fed a high-calorie diet, the levels of this parameter were significantly decreased in the 4+4-week and 8-week regimens (Figure 18).

[0121] Statistical analysis

[0122] Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc, USA). Statistical significance was set at a P-value of 0.05. For all comparisons (SD vs HFD, SD vs SD*SF68, HFD vs HFD+SF68), statistical significance was evaluated using the t-Student test for unpaired data.

Claims

1. A supplement based on Enterococcus faecium for use in the treatment of a disease characterized by changes in the intestinal mucosa.

2. The supplement according to claim 1, wherein Enterococcus faecium is the strain with deposit number NCIMB 10415, a probiotic active ingredient called SF68 (registered trademark).

3. The supplement according to claim 1, wherein the disease is an inflammatory change in the intestinal mucosa.

4. The supplement according to claim 3, wherein the disease is obesity.

5. The supplement according to any one of claims 1 to 4, wherein Enterococcus faecium is used in combination with butyric acid or a salt thereof.

6. The supplement according to claim 5, wherein sodium butyrate or calcium butyrate is used.

7. Use of a supplement based on Enterococcus faecium in the treatment of a disease characterized by changes in the intestinal mucosa.

Images