Probiotics for treatment of intestinal mucosal inflammatory alterations, in particular obesity

Abstract

Described herein is the use of a probiotic, in particular an Enterococcus faecium strain named SF68, in the treatment of a disease characterized by alteration of intestinal mucosa, in particular inflammation associated with alteration of intestinal permeability.

Description

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

[0002] In the past few years, some clinical evidence has shown that changes in the intestinal mucosa occur in several pathological conditions characterized by intestinal inflammation, such as chronic inflammatory intestinal disease (MICI), irritable bowel syndrome (IBS), obesity, and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease [Genser L. et al., Increased jejunal permeability in human obesity is revealed by a lipid challenge and is linked to inflammation and type 2 diabetes. J. Pathol. 2018 Oct. 246 (2): 217-230; Michielan A. et al., Intestinal Permeability in Inflammatory Bowel Disease: Pathogenesis, Clinical Evaluation, and Therapy of Leaky Gut. Mediators Inflamm. 2015, 2015: 628-157; Shulman et al., Associations among gut permeability, inflammatory markers, and symptoms in patients with irritable bowel syndrome,J.Gastroenterol.2014Nov,49(11):1467-76;Sharma S.et al.,Altered gut microbiotaand intestinal permeability in Parkinson's disease:Pathological highlight tomanagement.Neurosci.Lett.2019 Nov.1,712:134516;Sochocka M.et al.,The GutMicrobiome Alterations and Inflammation-Driven Pathogenesis of Alzheimer'sDisease–a Critical Review, Mol Neurobiol. 2019 Mar, 56(3):1841-1851].

[0003] In particular, it has been observed that this increase in intestinal permeability is associated with a decrease in the expression of tight junction proteins (such as occludin, zonulin and claudin), which are essential elements for maintaining barrier integrity [Chelakkot C. et al., Mechanisms regulating intestinal barrier integrity and its pathological implications, Exp Mol Med. 2018 Aug 16, 50(8): 1-9]. Among the major systems involved in maintaining epithelial integrity, several preclinical and clinical studies have highlighted the importance of the short-chain fatty acid (SCFA) butyrate [Ma X. et al., Butyrate promotes the recovering of intestinal wound healing through its positive effect on the tight junctions, J Anim Sci. 2012 Dec, 90 Suppl 4: 266-8, doi: 10.2527 / jas.50965, PMID: 23365351; Yang T. et al., Amelioration of non-alcoholic fatty liver disease by sodium butyrate is linked to the modulation 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 nutritional supplements on intestinal barrier functions in experimental colitis models invivo, World J Gastroenterol. 2019 Aug 14,25(30):4181-4198; Gao Y.et al.,Shortchain fatty acid butyrate,a breast milk metabolite,enhances immatureintestinal barrier function genes in response to inflammation in vitro and invivo,Am J Physiol Gastrointest Liver Physiol.2020 Oct 21;Tabat et al.,AcuteEffects of Butyrate on Induced Hyperpermeability and Tight Junction ProteinExpression in Human Colonic Tissues,Biomolecules.2020 May 14,10(5):766;LiuJ.et al.,Beneficial effects of butyrate in intestinal injury,J PediatrSurg.2020 Jun,55(6):1088-1093]。.

[0004] This SCFA is mainly produced by the intestinal bacterial flora, which has beneficial nutritional effects on 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], and also exerts anti-inflammatory effects by directly acting on several immune cell populations [Bonomo RR et al., Fecal transplantation and butyrate improve neuropathic pain, modify immune cell profile, and gene expression in the PNS of obese mice, Proc Natl Acad Sci USA, 2020 Oct 20, 117(42): 26482-26493; Yang W. et al., Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity, Nat Commun., 2020 Sep 8,11(1):4457; Takahashi D.et al., Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells, EBioMedicine, 2020Aug, 58:102913; Furusawa Y. et al., Commensalmicrobe-derived butyrate induces the differentiation of colonic regulatory T cells, Nature, 2013 Dec 19,504(7480):446-50, doi:10.1038 / nature12721; Chen J.etal., The Role of Butyrate in Attenuating Pathobiont-Induced Hyperinflammation, Immune Netw.2020 Feb 4,20(2):e15].

[0005] Several clinical lines of evidence suggest that the presence of intestinal inflammation can negatively affect butyrate use. Indeed, it has been observed that patients with MICI, IBS, or colorectal neoplastic disease are characterized by a reduced expression of butyrate transporters, which results in an insufficient uptake of this SCFA, leading to insufficient levels in enterocytes and, in turn, to increased intestinal permeability [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 diseases, especially diarrhea and constipation.

[0007] Therefore, improving butyrate use would have a beneficial impact on patients with diseases characterized by intestinal inflammation and / or altered intestinal permeability.

[0008] We have now found that the use of probiotics, in particular strains of Enterococcus faecium, more specifically strain Enterococcus faecium deposited as NCIMB 10415 (known as ) as a probiotic active ingredient can improve the ability of intestinal epithelial cells to use butyrate, which is achieved by increasing their ability to express transporters and increasing citrate synthase activity (a key enzyme in the tricarboxylic acid cycle, which is essential for the correct use of butyrate by cells). By improving the use of butyrate, the probiotic SF68 is able to strengthen the intestinal barrier by increasing the expression of tight junctions.

[0009] The object of the present invention is therefore a supplement based on Enterococcus faecium, optionally in combination with butyric acid or its salts, for the treatment of diseases and disorders characterized by intestinal inflammation and / or altered intestinal permeability, in particular obesity.

[0010] It is best to use SF68-based supplements. SF68-based medicines are available on the market, especially It is sold under the name of Cerbios Pharma SA, and its efficacy and safety as a probiotic are well 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 as food supplements. It is more commonly used in the form of sodium or calcium salts, i.e. sodium butyrate or calcium butyrate, which are the preferred forms for the combination of the present invention.

[0012] When probiotics are used in combination with butyrate, the two components of the combination may be administered separately or in a single dosage form, preferably separately.

[0013] When they are administered separately, the two components of the combination may be administered simultaneously or sequentially or with a time difference with respect to each other.

[0014] Indicatively, but without limitation, SF68 doses are usually in the range of 10 7 -10 10 Range, preferably not less than 10 9 For butyric acid and its salts, the dosage is usually in the range of 200-500 mg, preferably not less than 250 mg. The indicated dosage is usually administered 1-3 times a day for at least one week.

[0015] It is noted that the above reported dosages may vary depending on the disease to be treated, its severity, the patient's condition, etc. If a person skilled in the art deems it necessary, he / she can easily determine the most appropriate dosage outside the reported range.

[0016] According to the present invention, the use of the probiotic SF68 (optionally in combination with butyric acid or its salts) has a beneficial effect on several diseases and conditions characterized by intestinal inflammation and / or altered intestinal permeability. Among these diseases and conditions, obesity, enteropathy caused by NSAIDs, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and diseases characterized by neuroinflammation at the level of the central nervous system and leading to cognitive impairment are of particular interest, without limiting the scope of the present invention to these diseases / conditions.

[0017] These diseases and disorders are of particular interest within the scope of the present invention since efficacy studies can be performed using animal models, as disclosed in more detail below.

[0018] Obesity

[0019] Overweight and obesity are a global public health problem. The incidence of obesity has increased significantly in Western countries, so further research is needed to better understand the disease and its complications or comorbidities. Animal models of diet-induced obesity (DIO) can reproduce human overweight and obesity. In fact, there are many protocols for causing excessive fat accumulation in mice [de Moura E.et al., Diet-induced obesity in animal models: points to considerand influence on metabolic markers, Diabetol Metab Syndr., 2021Mar 18, 13(1): 32].

[0020] In this regard, it is worth recalling that HFD (high fat diet)-induced obesity is currently considered an important tool for understanding the effects of a Western high-fat diet on the development of obesity and related diseases [Wang CY, et al., A mouse model of diet-induced obesity and insulin resistance, Methods Mol Biol. 2012, 821: 421-433]. In fact, mice fed a fat-rich diet may contribute to the development of human obesity because of increased body fat and leptin and the development of hypertension and glucose intolerance [de Moura E. et al., supra].

[0021] According to previous experience and reports [Kim KA et al. High fat diet-induced gutmicrobiota exacerbates inflammation and obesity in mice via the TLR4signaling pathway, PLoS One. 2012; 7(10): e47713; Laurila A. et al. High-fat, high-cholesterol diet increases the incidence of gastritis in LDL receptor-negative mice, Arterioscler Thromb Vasc Biol. 2001; 21(6): 991–996], mice fed with HFD for 8 weeks showed a significant increase in body weight, and metabolic indicators such as blood glucose, cholesterol, and triglycerides also changed significantly, confirming the feasibility of this experimental model. Recent studies have also demonstrated that the expression of proinflammatory cytokines (TNF, IL-1β, IL-6) in the intestinal tissue of HFD animals is increased, followed by spontaneous dysfunction of goblet cells, altered mucin biosynthesis, and impaired mucosal barrier [Kim KA et al., supra; Ding S. et al., Role of intestinal inflammation as an early event in obesity and insulin resistance, Curr Opin Clin Nutr Metab Care. 2011; 14: 328–33; Gulhane M. et al., High fat diets induce colonic epithelial cell stress and inflammation that is reversed by IL-22, Sci Rep. 2016; 6: 28990].

[0022] It is well known that obesity is also associated with early cognitive impairment, which is triggered by inflammatory processes in the central nervous system, and changes in the blood-brain barrier support this process [Miller AA, Spencer SJ. Obesity and neuroinflammation: a pathway to cognitive impairment. Brain Behav Immun. 2014 Nov; 42: 10-21; Rhea EM, Salameh TS, Logsdon AF, Hanson AJ, Erickson MA, Banks WA. Blood-Brain Barriers in Obesity. AAPS J. 2017 Jul; 19(4): 921-930].

[0023] NSAID-induced enteropathy

[0024] The advent of new imaging techniques such as video capsule endoscopy has allowed us to obtain new information on NSAID-induced intestinal injury, which appears to be site-specific. The type of mucosal injury is seen in up to 75% of NSAID consumers and ranges from damaged segments observed primarily in the proximal small intestine to mucosal erosions and ulcers in distal segments.

[0025] The pathogenesis of small intestinal injury is not fully understood. NSAIDs inhibit the synthesis of endogenous prostaglandins in the mucosa throughout the gastrointestinal tract. However, other important pathogenic factors that contribute to injury, such as the presence of bacteria and bile, may differ between distal and proximal intestinal regions as important triggers of mucosal injury.

[0026] NSAIDs increase intestinal permeability in patients, leading to low-grade intestinal inflammation caused by increased intestinal barrier permeability, followed by significant bacterial infiltration. The increase in intestinal permeability caused by local NSAID action is due to an increase in inflammatory response (to luminal attackers) and microvascular effects of COX inhibition [Bjarnason I. et al., Mechanisms of Damage to the Gastrointestinal Tract From Nonsteroidal Anti-Inflammatory Drugs, Gastroenterology. 2018 Feb; 154 (3): 500-514].

[0027] This model is primarily based on results from mouse models, where some aspects of injury show significant similarities to humans, such as increased intestinal permeability observed with NSAID use, and localization of NSAID-induced enteropathy to the mid-distal small intestine. As seen in clinical studies, in preclinical models, NSAID-induced dysbiosis and impairment of the intestinal mucosal barrier are primary to a series of physiological and pathological events that lead to mucosal injury. In fact, damaged tight junctions make it easier for bacteria and bacterial antigens (and other luminal factors) to enter and exert their effects, thereby inducing the intestinal epithelium to express and release more proinflammatory cytokines [Colucci R.et al.,Pathophysiology of NSAID-Associated Intestinal Lesions in the Rat:Luminal Bacteria and Mucosal Inflammation as Targets for Prevention,Front Pharmacol.2018 Nov 29;9:1340;Fornai M.et al.,Small bowel protection against NSAID-injury in rats:Effect of rifaximin,a poorly absorbed,GItargeted,antibiotic.Pharmacol Res.2016 Feb;104:186-96;Bjarnason I.et al.,Mechanisms of Damage to the Gastrointestinal Tract From Nonsteroidal Anti-Inflammatory Drugs.Gastroenterology.2018 Feb;154(3):500-514].

[0028] IBD / IBS Model

[0029] Intestinal bowel disease (IBD) is a group of inflammatory diseases of the colon and small intestine caused by a dysregulated immune response. Chronic colitis (CD) and ulcerative colitis (UC) are the main types of IBD. Both are usually accompanied by severe diarrhea, pain, fatigue and weight loss. IBD can be debilitating and sometimes lead to complications that can be life-threatening.

[0030] Damage to the intestinal barrier may lead to increased intestinal permeability, which promotes exposure of intestinal contents and triggers immune responses, thereby promoting intestinal inflammation. IBD patients show multiple defects in specific components of the mucosal barrier, from the composition of the mucus layer to the adhesins that regulate cell permeability. These changes may represent the main functional disorder of the chronic disease, but may also perpetuate the chronic inflammation of the mucosa in ulcerative colitis [Michielan A. et al., Intestinal Permeability in Inflammatory Bowel Disease: Pathogenesis, Clinical Evaluation, and Therapy of Leaky Gut. Mediators Inflamm. 2015; 2015: 628157].

[0031] The fundamental approach to studying the pathogenesis and complexity of human IBD is to develop various animal models. These animal models provide important and necessary in-depth analysis of intestinal histopathological and morphological changes associated with the pathogenesis of human IBD. These models have become important tools for explaining intestinal histopathological, immunological and morphological changes as well as potential therapeutic targets [Eichele DDet al., Dextran sodium sulfate colitis murine model: An indispensable tool for advancing our understanding of inflammatory boweldiseases pathogenesis. World J Gastroenterol. 2017 Sep 7; 23(33): 6016-6029].

[0032] Over the past 25 years, several animal studies have used the DSS (dextran sulfate)-induced colitis model as a model of chemically induced inflammation, with morphology and symptoms similar to the epithelial damage observed in human ulcerative colitis. In mice, DSS-induced colitis leads to a decrease in the expression of tight junctions, followed by an increase in permeability, and clinical events of colonic inflammation. In particular, there are rapid changes in the tight junction pattern, such as an increase in claudin-2 expression and a decrease in several claudins and occludin-1. Therefore, mucosal barrier impairment is considered a secondary event to the increase in colonic mucosal permeability, followed by an influx of inflammatory cells into the intestinal mucosa [Eichele DD et al., supra].

[0033] Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder (FGID) characterized by symptoms such as abdominal pain or discomfort and stool irregularity, which are not associated with metabolic or organic abnormalities. The physiopathology of IBS involves multiple factors, such as visceral sensitivity, gastrointestinal (GI) motility, brain-gut interactions, and psychosocial stress. Notably, IBS often occurs in patients recovering from infectious colitis, and patients with inflammatory bowel disease (IBD) often experience IBS-like symptoms even after resolution of intestinal inflammation.

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

[0035] IBS experimental models can be divided into three categories: animal models induced by central stimulation, animal models induced by peripheral stimulation, and complex animal models induced by a combination of central and peripheral stimulation. In these models, intestinal inflammation induced by chemicals such as DSS may induce IBS symptoms. In fact, mice that recovered from DSS-induced colitis showed slower intestinal transit, barrier dysfunction, and reduced EC cell density [Sharman SK et al., Sildenafil normalizes bowel transit in preclinical models of constipation. PLoS One. 2017Apr 27; 12(4): e0176673].

[0036] Neurological disorders

[0037] The microbiome-gut-brain axis is emerging as a key communication system involved in maintaining central nervous system homeostasis and supporting pathophysiological events in a variety of neurological diseases, such as mild cognitive impairment (MCI), dementia, multiple sclerosis (SM), Alzheimer's disease (AD) and Parkinson's disease (PD) and autism [Pellegrini C, Antonioli L, Colucci R, Blandizzi C, Fornai M. Interplay among gut microbiota, intestinal mucosal barrier and enteric neuro-immune system: a common path 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. Microbiota-gut-brain axis in health and disease: Is NLRP3inflammasome at the crossroads of microbiota-gut-brain communications? Progress in neurobiology. Aug 2020; 191: 101806. doi: 10.1016 / j.pneurobio.2020.101806]. The mechanism of action of the microbiome-gut-brain axis mainly depends on the interaction between intestinal bacteria and their metabolites and the intestinal epithelial barrier, immune system and ascending nerve fibers. In fact, it has been observed that pathogenic strains and their products can migrate in the bloodstream and spread upward to the brain, where they may compromise the integrity of the blood-brain barrier and affect central neural circuits.In addition, pathogenic bacterial products can directly activate circulating immune / inflammatory cells, which may then migrate to the central nervous system and alter the blood-brain barrier [Fung TC, Olson CA, Hsiao EY. Interactions between the microbiota, immune and nervous systems 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 modulate 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, metadata and host immunity. Nature reviews Immunology. May 27 2016;16(6):341-52.doi:10.1038 / nri.2016.42].

[0038] Therefore, changes in the microbiota and intestinal epithelial barrier, intestinal immune / inflammatory responses (such as inflammasome activation) may be early events in neurological diseases that promote neuroinflammation and central nervous system inflammation through the gut-brain [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'sDisease.Frontiers in physiology.2020;11:593026.doi:10.3389 / fphys.2020.593026].In this context, modulation of intestinal flora using prebiotics or probiotics has been proposed as an effective treatment for preventing central nervous system diseases [Joseph J, Depp C, Shih PB, Cadenhead KS, Schmid-Schonbein G. Modified Mediterranean Diet for Enrichment of Short Chain FattyAcids: Potential Adjunctive Therapeutic to Target Immune and MetabolicDysfunction 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 in 6-OHDA induced neurotoxicity and behavioral abnormalities: Modulation of histone deacetylase activity.Behavioral brain research.Sep 152015;291:306-314.doi:10.1016 / j.bbr.2015.05.052;van de Wouw M,Boehme M,LyteJM,et al.Short-chain fatty acids:microbial metabolites that alleviate stress-induced brain-gut axis alterations.The Journal of physiology.Oct 2018;596(20):4923-4944.doi:10.1113 / JP276431].

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1: Changes in (A) body weight (%), (B) epididymal fat weight, (C) spleen weight, and (D) liver weight of mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the same 4-week, 4+4-week, and 8-week treatment regimens (**P<0.0001vs SD, *P<0.05vs SD, aa P<0.0001vs HFD,a P < 0.05 vs HFD).

[0041] Figure 2 : Total cholesterol (A), HDL (B), LDL (C) levels and (D) HDL / LDL ratio in mice fed SD+lactose, SD+SF68, HFD+lactose and HFD+SF68 during the 4-week, 4+4-week or 8-week treatment regimen (****P<0.0001vs SD, ***P<0.0005vs SD, *P<0.05vs SD, aa P < 0.0001 vs HFD).

[0042] Figure 3 : Triglyceride (A) and glycohemoglobin (Hb) (B) levels in mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, or 8-week treatment regimens (****P<0.0001vs SD, ***P<0.0005vsSD, *P<0.01vs SD, aa P < 0.0001 vs HFD).

[0043] Figure 4 : IL-1β (A) and LBP (B) levels in mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 in the 4-week, 4+4-week, or 8-week treatment regimens (**P<0.001 vs SD, *P<0.05 vs SD, a P < 0.05 vs HFD).

[0044] Figure 5 : Fecal butyrate (A) and propionate (B) levels in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during 4-week, 4+4-week, or 8-week treatment regimens (*P<0.05 vs SD)).

[0045] Figure 6 : Fecal calprotectin levels in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, or 8-week treatment regimens (***P<0.0001vs SD).

[0046] Figure 7 : Tissue MPO (A), MDA (B), and IL-1β (C) levels in mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, or 8-week treatment regimens (**P<0.001vs SD, *P<0.05vs SD, a P<0.05vsHFD,aa P < 0.0001 vs HFD).

[0047] Figure 8: Colonic contraction responses evoked in vitro by electrical stimulation or stimulation with methacholine or substance P in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 for 4 weeks.

[0048] Fig. 9 : In vitro colonic contraction responses induced by electrical stimulation or stimulation with carbachol or substance P in mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 for 8 weeks (**P<0.001vs SD, *P<0.05vs SD, a P < 0.05 vs HFD).

[0049] Fig.10 Densitometric analysis of (A) zonulin, (B) occludin, and (C) claudin-1 expression in mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, and 8-week treatment regimens (*P<0.05 vs SD, a P<0.05 vs SD).

[0050] Fig.11 : Densitometric analysis of (A) TLR2 and (B) TLR4 expression in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, and 8-week treatment regimens (*P<0.05 vs HFD).

[0051] Fig.12 Densitometric analysis of (A) NF-kB and (B) MyD88 expression in mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, and 8-week treatment regimens (*P<0.05 vs SD, a P < 0.05 vs HFD).

[0052] Fig.13 : Densitometric analysis of claudin-1 expression in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, and 8-week treatment regimens (***P<0.0001 vs SD, **P<0.001 vs SD, aaa P < 0.002 vs HFD, a P < 0.05 vs HFD).

[0053] Fig.14: Densitometric analysis of inflammatory infiltrates in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, and 8-week treatment regimens (**P<0.001 vs SD, aaa P < 0.002 vs HFD, aa P < 0.001 vs HFD).

[0054] Fig.15 : Representative blots and densitometry analysis of (A) SMCT1, (B) MCT1, and (C) MCT4 expression in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4+4 and 8-week treatment regimens (**P<0.01 vs SD, a P < 0.0001 vs HFD).

[0055] Fig.16 : Analysis of citrate synthase activity in intestinal tissue (A) and adipose tissue (B) in mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4+4 and 8-week treatment regimens (*P<0.05vs SD, § P<0.05vsHFD).

[0056] Fig.17 : Plasma homocysteine ​​levels in mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week or 8-week treatment regimen (*P<0.05vs HFD).

[0057] Fig.18 : Tissue levels of TNF in the brain of mice fed SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 8-week treatment regimen.

[0058] The present invention is based on the efficacy of SF68 against several aspects of intestinal inflammation / alterations and its ability to reabsorb butyrate. Based on these experimental observations, administration of SF68 (optionally in combination with butyrate or its salts) is particularly effective for treating diseases characterized by altered intestinal permeability, including but not limited to obesity.

[0059] In particular, combined use could combine the beneficial effects of SF68 and butyrate on the intestinal mucosa.

[0060] The efficacy of SF68 has been confirmed by experiments performed on mice treated with a high-fat diet (see Experimental section).

[0061] In fact, from the results obtained:

[0062] - SF68 supplementation at 4, 4+4 and 8 week dosing schedules was shown to significantly counteract weight gain in animals fed a high fat diet (HFD);

[0063] - Regarding metabolic parameters, HFD resulted in a significant increase in total cholesterol, LDL and triglycerides, and a decrease in HDL levels. Treatment with SF68 for 8 weeks significantly counteracted these changes;

[0064] - Animals fed with HFD showed increased circulating levels of IL-1β and LPS-binding protein (LBP, an index of increased intestinal permeability). Supplementation with SF68 at 8 weeks significantly reduced this change;

[0065] - From analysis of feces of animals fed a HFD, calprotectin levels appeared to increase and did not change significantly after treatment with SF68;

[0066] - As for SCFA levels, HFD did not alter fecal propionate levels. However, in HFD animals we noticed a significant increase in fecal butyrate levels. No increase in butyrate was seen in HFD animals treated with SF68 for 8 weeks.

[0067] - Analyzing the levels of tissue inflammatory parameters (IL-1β and MPO) and tissue oxidative stress (MDA), we noted that the above parameters were increased in animals fed a HFD. After 8 weeks of SF68 supplementation, the above parameters decreased significantly, indicating that the treatment has anti-inflammatory and antioxidant activity;

[0068] - From in vitro experiments we could note that the neuronal coding of the enteric nervous system in the colon of animals fed a high-fat diet is altered, with a reduced cholinergic component and an increased tachykinetic tone. It is known that this alteration underlies the motility disorders observed in several pathologies characterized by intestinal inflammation. Of great interest is the effect of SF68 in counteracting this alteration, by preserving the cholinergic component and significantly antagonizing the excessive tachykinetic transmission. This beneficial effect could be attributed to a reduction in the level of inflammation and oxidative stress in colonic tissue or to a direct effect on the neuronal coding, or both;

[0069] - The expression of tight junctions in colon tissue of animals fed HFD was significantly reduced, indicating a relaxation of the intestinal barrier. SF68 treatment was shown to enhance the barrier capacity by increasing the expression of tight junctions;

[0070] - TLR-4 levels in colon tissues of animals fed a high-fat diet for 8 weeks increased significantly. Supplementation with SF68 for 8 weeks suppressed this increase. Meanwhile, by exploring the intracellular pathways associated with TLR-4, we observed an increase in NFK-b after 8 weeks of high-fat diet. This increase was suppressed by SF68 in both the 4+4 and 8-week regimens;

[0071] - Experiments evaluating the effects of probiotics on the butyrate transport and utilization system resulted in a reduced absorption capacity of this SCFA in obese animals, reduced expression of butyrate transporters, and decreased citrate synthase activity. SF68 improved the use of this SCFA by the intestinal mucosa by restoring the levels of the SMCT1 transporter and normalizing citrate synthase activity in intestinal tissue;

[0072] - SF68 exerts a positive effect on intestinal homeostasis leading to a better utilization of butyrate, as experimentally demonstrated by an increase in butyrate transporters and improved intestinal epithelial nutrition through increased citrate synthase activity, evidence also confirmed by a better expression of tight junctions and therefore a reduced permeability to luminal antigens and then a reduced inflammatory load, inferred by a reduction in pro-inflammatory cytokines.

[0073] - In addition, SF68 treatment improved the expression of tight junctions in the CNS, thereby reducing central inflammatory conditions (reduced TNF levels).

[0074] In conclusion, SF68 treatment highlights the ability to counteract the changes in multiple systemic and tissue parameters caused by a high-fat diet. Beneficial effects of SF68 on gut barrier nutrition have been observed, indicating its enhancement, with an increase in tight junctions attributed to the ability of SF68 to render the intestinal mucosa more sensitive to the absorption and utilization of butyrate, a SCFA known for its beneficial capacity on intestinal mucosal nutrition and the intestinal immune system. All of this appears to occur due to an anti-inflammatory effect mediated by a positive effect on intestinal barrier integrity. It has been observed that the positive effect on tight junctions is associated with an improved utilization / reabsorption of butyrate by enterocytes, which was confirmed by histochemical studies of inflammatory infiltrates, i.e., a decrease in circulating LPS, IL-1β and TLR4, as well as a decrease in NF-kB and MPO (signs of reduced inflammatory cell infiltration).

[0075] This effect is particularly important in the context of the present invention, demonstrating the efficacy of probiotics, optionally in combination with butyrate (or its salts), for the treatment of diseases and disorders characterized by intestinal inflammation and / or altered intestinal permeability, including obesity.

[0076] Without being bound by any theory, based on the results in mice we can hypothesize that SF68 leads to normalization of butyrate apical membrane transporter expression, whereas a high fat diet (HFD) alters this expression.

[0077] The utility, efficacy and advantages of the present invention will be described in more detail by the following examples, but these examples are not intended to limit the scope of the present invention in any way.

[0078] Experimental Section

[0079] Five-week-old C57BL / 6 mice (weight 20-22 g) were provided by Envigo srl (San Pietro al Natisone, Udine, Italy). Six mice were housed in cages in a thermostatically controlled room with a 12-h light cycle, a temperature of 22-24°C, and a humidity of 50-60%, and allowed to acclimate for at least one week. Animals were handled in accordance with Directive 2010 / 63 / UE.

[0080] During the adaptation period, all mice were fed a standard diet (SD, 18% of calories from fat; TD. 2018). The animals were then randomly divided into six groups, each consisting of 10 mice, as follows:

[0081] Plan A

[0082] - Group A1: SD + vehicle containing lactose (L), for 4 weeks

[0083] - Group A2: HFD + vehicle containing lactose for 4 weeks

[0084] - Group A3: SD + vehicle for 4 weeks + SF68 (P) starting from the fourth week

[0085] - Group A4: SD + vehicle for 4 weeks + vehicle with lactose starting from the fourth week

[0086] - Group A5: HFD + vehicle for 4 weeks + SF68 starting from the fourth week

[0087] - Group A6: HFD + vehicle for 4 weeks + lactose vehicle added from the fourth week

[0088] Plan B

[0089] Group B1: SD+SF684 weeks

[0090] Group B2: HFD+SF684 weeks

[0091] Group B3: SD+SF688 weeks

[0092] Group B4: HFD+SF688 weeks

[0093] The carrier was 150 μL 3% methylcellulose.

[0094] SF68 is used as

[0095] A high-fat diet (HFD) provides 60% of calories from fat (TD.06414).

[0096] A comparison of the calorie counts for the two diets is reported in Table 1 below:

[0097] Table 1: Comparison of SD and HFD calorie counts

[0098] diet Carbohydrate intake Protein intake Fat intake SD 58％ 24％ 18%(3.1kcal / g) HFD 21.4％ 18.3％ 60.8%(5.1kcal / g)

[0099] Beginning on the first day of the study, the body weights of the animals were measured weekly and the percent change in body weight (with the value for animals in the SD+L group being 100) is reported in Table 2 below.

[0100] Table 2: Percentage changes in body weight of the test animals

[0101]

[0102] Mice fed a high-fat diet (HFD) gained significantly more weight after 4 and 8 weeks compared to animals fed a normal caloric diet (SD). SF68 treatment significantly counteracted the weight gain induced by HFD after 4 and 8 weeks. SF68 treatment did not result in changes in weight gain in animals fed a SD across all treatment regimens tested.

[0103] Finally, the animals were anesthetized and sacrificed. Blood samples and tissue samples were collected and stored at -80 °C for further analysis.

[0104] Changes in weight and organs

[0105] Mice fed a high-fat diet (HFD) gained significantly more weight after 4 and 8 weeks compared with animals fed a normal calorie diet (SD) ( Figure 1A SF68 treatment significantly counteracted the weight gain induced by HFD after 4 and 8 weeks ( Figure 1A ). Treatment with SF68 did not result in changes in body weight gain in animals fed SD in all treatment regimens tested ( Figure 1A ).

[0106] In addition, Figure 1B-D reports the changes in epididymal fat weight (B), spleen weight (C), and liver weight (D) of mice fed with SD+lactose, SD+SF68, HFD+lactose, and HFD+SF68 during the 4-week, 4+4-week, or 8-week treatment regimen.

[0107] The fact that there was no significant effect on fat accumulated in the epididymis is not unusual, since in the HFD model, epididymal fat is the last to increase and the 8 weeks studied might not have allowed changes in this parameter.

[0108] In contrast, a decrease in spleen weight has been observed, which is a lymphoid organ that increases in weight in the presence of systemic inflammation. A decrease in spleen weight suggests an anti-inflammatory effect.

[0109] Normally, on HFD, adipose tissue makes the liver fatter (non-alcoholic liver syndrome - NASH) and then functionally impaired. A reduction in liver weight has been observed, and HFD animals treated with SF68 showed mainly a better appearance of the HFD liver in morphological examination compared to untreated animals, indicating a protective effect of SF68 (less area of ​​fatty infiltration).

[0110] Metabolic parameters

[0111] Compared with mice fed with SD Figure 2 Plasma total cholesterol levels were significantly increased in animals fed a HFD compared with those in animals fed a HFD (A-D). Figure 2 A) LDL ( Figure 2 C), while HDL( Figure 2 Compared with HFD mice, HDL levels in mice treated with SF68 on a high-calorie diet were significantly increased ( Figure 2 B), while total cholesterol level ( Figure 2 A) and LDL ( Figure 2 C) No change. Then, the HDL / LDL ratio changes favorably.

[0112] Compared with mice fed SD Figure 3 A) compared with HFD-fed animals, plasma triglyceride levels were significantly increased ( Figure 3 A), while the hemoglobin (Hb) level does not seem to change ( Figure 3 B). Mice fed a high-calorie diet with SF68 had significantly increased triglyceride levels compared to mice fed a HFD ( Figure 3 A), while the sugar acid Hb level did not change ( Figure 3 B).

[0113] The lack of changes in glycosylated Hb was to be expected, as in the HFD model, increases were only observed during weeks 10–12, a period longer than the duration of the present study.

[0114] Instead, what is important is the triglyceride-lowering effect of SF68, which counteracts the triglyceride-increasing effect of the HFD. Increased triglycerides, along with increased transaminases, are associated with liver problems, so the observed reduction favors a healthier (morphologically less infiltrated) liver.

[0115] Plasma levels of IL-1β and LBP (LPS binding protein)

[0116] The levels of IL-1β and LBP in plasma have been 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]. In this procedure, blood samples were centrifuged at 4000 rpm for 5 minutes at 2-8°C; after centrifugation, the supernatant was collected. Aliquots (100 μl) were used for analysis. IL-1β levels are expressed in pg / ml plasma, while LBP levels are expressed in ng / ml plasma.

[0117] Compared with SD-fed mice, after 8 weeks of HFD feeding, HFD-fed animals had significantly lower plasma IL-1β ( Figure 4 A) and LBP( Figure 4 B) levels increased significantly ( Figure 4 A and Figure 4 B). In mice fed a high-calorie diet, treatment with SF68 resulted in a significant increase in IL-1β levels in HFD mice in both the 4+4 and 8-week regimens ( Figure 4 A), whereas a decrease in circulating LBP levels was observed after only 8 weeks of treatment with SF68 ( Figure 4 B).

[0118] IL-1β is pyrogenic, proinflammatory, and triggers macrophage activation. The decrease in IL-1β plasma levels is directly related to the anti-inflammatory effect of SF68 treatment.

[0119] When the intestinal epithelial barrier is compromised, intestinal microbes migrate more easily into the blood, and the same microbes (if Gram-negative) and / or the liposaccharides (LPS) produced by their degradation bind to the LBP protein, and when microbes are present in the blood, the production of LBP by hepatocytes increases significantly. The significant increase in LBP in mice fed a HFD indirectly suggests a decrease in intestinal epithelial barrier function. This increase was significantly counteracted by SF68 after 8 weeks of treatment, indicating a decrease in circulating LPS.

[0120] Assessment of fecal SCFA content

[0121] To analyze short-chain fatty acids (SCFA), stool samples were lyophilized and subjected to gas chromatography. The lyophilized material was dissolved in 100 ml of 5 M formic acid and 400 ml of acetone and then centrifuged (5 minutes at 4000 rpm). A capillary column equipped with a thin film was used. The SCFA concentration in the supernatant was determined by a GC2010 Plus gas chromatograph (Shimadzu Deutschland GmbH, Duisburg, Germany) with a flame ionization detector (Restek, Bad Homburg, Germany). The samples were dispersed by split injection using an automatic sampler AOC-20s / I (Shimadzu Deutschland GmbH). Data processing was performed using a GC solution chromatography data system (Shimadzu Deutschland GmbH). For the quantification of SCFA, external standards (Supelco TM WSFA-1Mix, Supelco Sigma-Aldrich Co., Bellefonte PA).

[0122] Mice fed a high-fat diet (HFD) showed a significant increase in fecal butyrate levels after 8 weeks of HFD feeding ( Figure 5 A). SF68 supplementation during the 4+4 and 8 week treatment regimens inhibited this increase. No changes in propionate levels were observed ( Figure 5 B).

[0123] Delving deeper into the statistically significant information on the mechanism of action of SF68, we found that SF68 normalizes the expression of the butyrate apical membrane transporter SMCT1, whereas HFD alters this expression. The mechanism of action of SF68 could be the improvement of intestinal epithelial health, for example by improving tight junctions, mainly by increasing the expression of the SFCA transporter, thus facilitating the reabsorption of butyrate, which is then better utilized by the organism, both as a nutritional factor for intestinal cells and as an anti-inflammatory factor. Further studies described below allowed the observation that SF68 treatment was also associated with an increase in the activity of the citrate synthase enzyme (intestine and adipose tissue). This enzyme catalyzes the first reaction of the tricarboxylic acid cycle, a key process in cellular respiration, able to regulate the flow rate of the entire cycle and allow butyrate to be utilized in intestinal cells. A high-calorie diet leads to a decrease in the activity of the citrate synthase enzyme, which can be counteracted by SF68. This effect, in addition to improving the expression of SCFA transporters (and strengthening tight junctions), could explain how SF68 administration is associated with a healthier gut.

[0124] Fecal calprotectin level

[0125] Mice fed a high-fat diet (HFD) showed a significant increase in fecal calprotectin levels, a protein of the S100 family that is abundant in neutrophils. Treatment with SF68 did not significantly alter this parameter ( Figure 6 ).

[0126] This means that the anti-inflammatory mechanism of SF68 cannot be explained by its direct effect on neutrophils.

[0127] Myeloperoxidase (MPO), malondialdehyde (MDA), and IL-1β levels in intestinal tissue

[0128] MPO levels in colon tissue were measured by ELISA (Prodotti Gianni, Milan, Italy) as described by Antonioli L. et al. [FASEB J. (2020) 34, 5512-5524]. Colon samples previously stored at -80°C were homogenized on ice using a Polytron homogenizer (Qiagen, Milan, Italy). The homogenate was centrifuged at 12000 rpm for 15 minutes at 4°C. An aliquot (100 μL) of the supernatant was then used for analysis. MPO levels are expressed in ng / mg tissue.

[0129] MDA concentrations were measured in intestinal samples to quantitatively estimate mucosal infiltration of polymorphonuclear cells. The analysis was performed as previously described by Antonioli et al. [Int. J. Obes. (2019), supra]. Intestinal tissue was weighed, ground and homogenized with a Polytron homogenizer in 2 ml of cold buffer (QIAGEN, Milan, Italy) and centrifuged at 1200 rpm for 10 min at 4°C. MDA concentrations were measured using a colorimetric assay kit (Calbiochem, San Diego, CA, USA) and the results were expressed as nmoles MDA per mg of colon tissue.

[0130] Compared with tissues from animals fed SD ( Figure 7 A. Figure 7 B and Figure 7 C), MPO, MDA, and IL-1β levels were significantly increased in colon tissue samples from mice fed a high-fat diet ( Figure 7 A. Figure 7 B and Figure 7 C). In HFD mice, treatment with SF68 resulted in a significant decrease in MPO, MDA, and IL-1β levels compared with HFD mice not receiving SF68 dietary supplementation ( Figure 7 ).

[0131] In the tissues of mice fed a HFD, a significant increase in MPO (infiltration from immune inflammatory cells), MDA (signal of oxidative stress), and IL-1β (pyrogenic and pro-inflammatory cytokine) was observed. After 8 weeks of treatment with SF68, these three components were significantly reduced in the tissues. The reduction in MDA means a reduction in oxidative stress, which may be due to the support of SF68 as an endogenous scavenger (such as glutathione). The reduction in MPO means a reduction in the infiltration of inflammatory cells. The anti-inflammatory effect of SF68 was confirmed by the reduction in IL-1β, and indeed, the reduction of this interleukin was also observed in the blood.

[0132] In vitro assessment of colonic contractile activity

[0133] Contractile activity of colonic muscle preparations was performed as described by Antonioli et al. [FASEB J., supra] with slight variations. Immediately after sacrifice, the colon was removed through an incision above the anal terminal and placed in Krebs solution. The colon segments were opened along the mesenteric insertion and the mucosa / submucosa removed. The colon samples were cut along the longitudinal axis into strips approximately 4 mm wide and 10 mm long.

[0134] The preparations were placed in an organ bath containing Krebs solution at 37°C, 5% CO2 + 95% O2, and connected to an isometric sensor (constant load = 0.5 g). Mechanical activity was recorded by BIOPAC MP150 (Biomedica Mangoni, Pisa, Italy). The composition of Krebs solution is as follows: KCl 4.7nM, NaCl 113nM, KH2PO4 1.2nM, CaCl2 2.5nM, MgSO4 1.2nM, NaHCO3 25nM, glucose 11.5nM (pH 7.4±0.1). Each preparation was equilibrated for at least 30 minutes and cleaned every 10 minutes. A pair of coaxial platinum electrodes were placed 10 mm from the longitudinal axis of each preparation to bring electrical stimulation through a BM-ST6 stimulator (Biomedica Mangoni, Pisa, Italy). At the end of the equilibration period, each preparation was repeatedly challenged with electrical stimulation and the experiment was started after a reproducible response was obtained (usually after two to three stimulations).

[0135] In the first set of experiments, total electrical stimulation was recorded in colon samples maintained in standard Krebs solution.

[0136] Cholinergic contractions were recorded in the second and third experiments. Colonic samples were preserved in Krebs solution containing N-ω-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor, 100 μM), N-acetyl-1-tryptophan 3,5-bis(trifluoromethyl)benzyl ester (L-732,138, neurokinin NK1 receptor antagonist, 10 μM), 5-fluoro-3-[2-[4-methoxy-4-[[(R)-phenylsulfinyl]methyl]-1-piperidinyl]ethyl]-1H-indole (GR159897, NK2 receptor antagonist, 1 μM), (R)-[[(2-phenyl-4-quinolyl)carbonyl]amino]-methylphenylacetic acid (SB218795, NK3 receptor antagonist, 1 μM), and guanethidine (adrenergic blocker 10 μM). To evaluate neurogenic contraction, stimulation with methacholine (CCh, 10 μM) was performed in Krebs solution containing tetrodotoxin (TTX, 1 μM) to study myogenic responses.

[0137] The fourth and fifth series of experiments were designed to evaluate tachykinase NK1 stimulation. Neurogenic NK1 contractions were recorded in colon specimens preserved 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), whereas myogenic activity was detected when specimens were preserved in Krebs solution supplemented with TTX and stimulated with exogenous substance P (SP, 1 μM).

[0138] During the stabilization period of colonic tissue stored in standard Krebs solution, preparations exhibited rapid spontaneous motor activity that remained stable throughout the experiment and, in most cases, was small and did not interfere with motor responses evoked by electrical stimulation (ES). Electrically induced responses were characterized by phasic contractions, which in some cases were followed by aftercontractions of variable width. In colonic preparations from SD or HFD animals stored in standard Krebs solution, the use of electrical stimulation evoked similar contractile responses after 4 weeks of treatment with SD or HFD ( Fig. 8A In the analysis of the major ENS excitatory systems (acetylcholine and substance P), endogenous and exogenous, cholinergic and tachykinetic contractile responses were assessed and found to be unaltered by HFD after 4 weeks, nor by SF68 at 4 weeks ( Figure 8B ).

[0139] In colon preparations from SD or HFD animals maintained in standard Krebs solution, electrical stimulation induced similar contractile responses after 8 weeks of treatment with SD or HFD ( Fig. 9In an analysis of the major ENS excitatory systems (acetylcholine and substance P), we observed a decrease in cholinergic activity and an increase in tachykinin activity after 8 weeks of HFD feeding ( Fig. 9 Treatment with SF68 for 8 weeks normalized the functional changes induced by a high-fat diet ( Fig. 9 ).

[0140] A high-fat diet causes changes in the neural components of the gut. Obese individuals have "IBS-like" symptoms of the constipation type due to altered peristalsis involving alternating inhibitory and stimulatory stimulation.

[0141] The contractile stimulus is acetylcholine, which lasts a few milliseconds, and substance P (tachykinins), which act for a longer time. The relaxant stimulus is nitric oxide. Physiologically, the contraction is mainly cholinergic, while in inflammatory pathologies or HFD, the contraction is mainly tachykinin.

[0142] HFD (and tissue inflammation in general) is associated with 1) a decrease in cholinergic responses and 2) an increase in substance P, which acts to prolong the contraction of longitudinal smooth muscle compared to the digestive tract (constipation).

[0143] SF68 was able to significantly offset the increase in tachykinase components and loss of cholinergic responses caused by a high-calorie diet. It is worth noting that in addition to being a neurotransmitter, acetylcholine also has immunomodulatory activity and can reduce the activity of immune inflammatory cells.

[0144] Cholinergic contractions are the first to "self-destruct" during inflammation, and after 8 weeks, they were stronger in samples treated with SF68 than in the HFD control group. At the same time, substance P seemed to be reduced. These effects were abolished by the addition of tetrodotoxin, which blocks the neural component, so the effects of SF68 do not seem to be at the muscle level, but rather at a systemic level affecting its neuroenteric component.

[0145] Western blot analysis of tight junctions in colonic tissue

[0146] Colon tissue was weighed and then homogenized in lysis buffer (50 mg in 400 μl) using a Polytron homogenizer (QIAGEN, Milan, Italy). The homogenate was centrifuged at 12000 rpm for 15 minutes at 4 ° C, and the resulting supernatant was separated from the precipitate and stored at -80 ° C. Bradford test was performed to quantify total protein. Subsequently, proteins were separated on a prefabricated 4%-20% polyacrylamide gel (Mini-PROTEAN TGX gel, Biorad) and transferred to a PVDF membrane (Trans-Blot TurboTM PVDF transfer bag, Biorad). The membrane was closed with 3% BSA diluted in Tris buffered saline (TBS, 20mMTris-HCl, pH 7.5, 150nM NaCl) containing 0.1% Tween 20. The antibodies were directed 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); the secondary antibodies were Abcam products (anti-mouse ab97040 and anti-rabbit ab6721); ECL reagent (Clarity Protein bands were detected using Western ECL chemiluminescent substrate (Biorad), and density analysis was performed using iBright analysis software.

[0147] Feeding with HFD for 4 and 8 weeks resulted in a significant decrease in the level of tight junctions in colonic tissue ( Fig.10 A. Fig.10 B and Fig.10 C). Supplementation with SF68 for 8 weeks resulted in normalization of zonulin 1, occludin, and claudin-1 ( Fig.10 A. Fig.10 B and Fig.10 C), while 4+4 weeks of treatment resulted in improved expression of claudin-1, but not zonulin 1 and occludin ( Fig.10 C).

[0148] Assessment of Toll-like receptor expression in intestinal tissue

[0149] Animals fed a HFD for 4 and 8 weeks did not show significant changes in colonic expression of toll-like receptors. Treatment with SF68 did not significantly alter TLR2 levels ( Fig.11 A). Probiotic supplementation for 8 weeks resulted in a significant decrease in TLR4 colonic levels compared to the group fed a HFD ( Fig.11 B).

[0150] TLR is a class of transmembrane receptor proteins that are mainly found in macrophages and dendritic cells and play a key role in innate immunity. TLR connects to ligands belonging to pathogens, thereby activating the immune response of juxtaglomerular cells.

[0151] Western blot of the molecular signaling pathways of nuclear factor kB (NF-kB) and myeloid differentiation primary response gene 88 (MyD88)

[0152] Eight weeks of HFD administration resulted in a significant increase in NF-kB levels in colon tissue ( Fig.12 A). Dietary supplementation with SF68 resulted in a significant decrease in this parameter during both the 4+4 and 8-week treatment regimens ( Fig.12 A). In contrast, by analyzing MyD88 levels, no significant changes were observed between the different treatment groups ( Fig.12 B).

[0153] NF-kB plays a key role in the inflammatory response to infection, and its increase has been linked to inflammation, cancer, and autoimmune diseases.

[0154] Immunohistochemistry and immunofluorescence

[0155] Sections of formalin-fixed full-thickness colon samples were processed for immunoperoxidase staining as described by Ippolito et al. [J. Cell. Mol. Med. (2015), doi: 10.1111 / jcmm.12428]. Sections were incubated overnight at 4°C with primary antibodies against claudin-1 and inflammatory infiltrates (DakoCytomation, Glostrup, Denmark) and then exposed to a complex of appropriate biotinylated immunoglobulins, streptavidin labeled with peroxidase, and 3,3'-diaminobenzidine (DakoCytomation, Glostrup, Denmark). Immunostained sections were then examined with a Leica DMRB microscope, and representative micrographs were taken with a DFC480 digital camera (Leica Microsystems, Cambridge, UK) for quantitative evaluation.

[0156] Feeding HFD for 4 and 8 weeks significantly reduced claudin-1 levels in colon tissue ( Fig.13 A and Fig.13 B) and immune inflammatory infiltration ( Fig.14 The addition of SF68 to the 4-week, 4+4-week, and 8-week regimens normalized claudin-1 expression ( Fig.13 A and Fig.13 B), and significantly reduced intestinal inflammation levels ( Fig.14 ).

[0157] Assessment of butyrate transporter expression

[0158] Compared with SD control mice, mice fed HFD for 8 weeks had significantly decreased expression of the apical membrane transporter SMCT1 ( Fig.15 A), whereas the levels of MCT1 (apical membrane) and MCT4 (basolateral membrane) transporters were not affected ( Fig.15 B and Fig.15 C). Treatment with SF68 for 4 and 8 weeks resulted in normalization of SMCT1 transporter expression in HFD mice ( Fig.15 A), while no changes were observed in the expression of MCT1 or MCT4 proteins ( Fig.15 B and Fig.15 C).

[0159] Evaluation of citrate synthase activity on intestinal mucosa

[0160] Frozen samples from different groups of mice were homogenized on ice with cold buffer (sucrose 250 mM, Tris 5 mM, EGTA 1 mM, Triton X-100 0.02%; pH 7.4) and GentleMACS dissociation agent (Miltenyi Biotec, Bologna, Italy). The homogenate was centrifuged at 12.000 x g for 15 minutes at 4 ° C (EuroClone, Speed ​​Master 14R centrifuge, Milan, Italy). The precipitate was discarded and the supernatant was used to quantify protein by Bradford assay, followed by determination of citrate synthase activity.

[0161] Samples were diluted in Tris buffer (100 mM; pH 8.2) containing 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, 100 μM) and acetyl-CoA (100 μM). The assay was performed in 96 multiwell plates (1 μg protein per well) and the reaction was started by adding oxaloacetate solution (500 μM). The reaction was followed spectrophotometrically at 412 nm every 30 seconds for 15 minutes at 37°C (EnSpire, PerkinElmer, Waltham, MA, USA). Citrate synthase activity was determined by comparing sample activity with known isolated enzyme concentrations (Sigma-Aldrich, St. Louis, MO, USA). Citrate synthase activity is expressed in mU / ml.

[0162] Compared with SD control mice, mice fed HFD for 4 or 8 weeks had significantly reduced citrate synthase activity in the intestinal mucosa and adipose tissue ( Fig.16 A and Fig.16 B). Treatment with SF68 for 8 weeks resulted in a significant increase in citrate synthase activity in the intestine and adipose tissue of HFD mice ( Fig.16 A and Fig.16 B).

[0163] Plasma homocysteine ​​level assessment

[0164] Animals fed a HFD had elevated plasma homocysteine ​​levels ( Fig.17 Treatment with SF68 in mice fed a high-calorie diet resulted in a significant decrease in the levels of this parameter in both the 4+4 and 8-week regimens ( Fig.17 ).

[0165] Mice fed a HFD had elevated plasma homocysteine ​​levels, which may also occur in obese humans on a high-calorie diet. Treatment with SF68 significantly reduced plasma homocysteine ​​levels.

[0166] TNF level assessment

[0167] TNF levels were increased in brain tissue of animals fed a HFD ( Fig.18 Treatment of mice with SF68 under a high-calorie diet resulted in a significant decrease in the levels of this parameter in both the 4+4 and 8-week regimens ( Fig.18 ).

[0168] Statistical analysis

[0169] Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc, USA). Statistical significance was set at a P value of 0.05.

[0170] For all comparisons (SD vs HFD, SD vs SD*SF68, HFD vs HFD+SF68), statistical significance was assessed using the Student's T-test method on uncoupled data.

Claims

1. Enterococcus faecium-based supplements for the treatment of conditions characterized by changes in the intestinal mucosa.

2. The supplement according to claim 1, wherein Enterococcus faecium is a strain with the deposit number NCIMB10415, which is known as of probiotic active ingredients.

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 the preceding claims, wherein Enterococcus faecium is used in combination with butyric acid or a salt thereof.

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

7. Use of an Enterococcus faecium-based supplement for treating a disease characterized by changes in the intestinal mucosa.

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