Age-Related NAFLD: The Use Of Probiotics As A Supportive Therapeutic Intervention Part 1

: Human aging, a natural process characterized by structural and physiological changes,  leads to alterations of homeostatic mechanisms, decline of biological functions, and subsequently,  the organism becomes vulnerable to external stress or damage. The elderly population is prone to develop diseases due to the deterioration of physiological and biological systems. With aging, the production of reactive oxygen species (ROS) increases, and this causes lipid, protein,  and DNA damage, leading to cellular dysfunction and altered cellular processes. Indeed, oxidative stress plays a key role in the pathogenesis of several chronic disorders, including hepatic diseases,  such as non-alcoholic fatty liver disease (NAFLD). NAFLD, the most common liver disorder in the Western world, is characterized by intrahepatic lipid accumulation; is highly prevalent in the aging population; and is closely associated with obesity, insulin resistance, hypertension, and dyslipidemia. Among the risk factors involved in the pathogenesis of NAFLD, the dysbiotic gut microbiota plays an essential role, leading to low-grade chronic inflammation, oxidative stress, and the production of various toxic metabolites. The intestinal microbiota is a dynamic ecosystem of microbes involved in the maintenance of physiological homeostasis; the alteration of its composition and function, during aging, is implicated in different liver diseases. Therefore, gut microbiota restoration might be a  complementary approach to treating NAFLD. The administration of probiotics, which can relieve oxidative stress and elicit several anti-aging properties, could be a strategy to modify the composition and restore healthy gut microbiota. Indeed, probiotics could represent a valid supplement to prevent and/or help treat some diseases, such as NAFLD, thus improving the already available pharmacological intervention. Moreover, in aging, the intervention of prebiotics and fecal microbiota transplantation, as well as probiotics, will provide novel therapeutic approaches. However, the relevant research is limited, and several scientific research works need to be done shortly to confirm their efficacy.

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1. Introduction

The so-called gut microbiota (GM) is constituted of numerous different populations of microorganisms (bacteria, archaea, fungi, and viruses) that reside in the gastrointestinal tract of mammals. In recent years, a significant interest in intestinal microbiota has spread, as it is considered one of the key factors contributing to the maintenance of physiological intestinal homeostasis, the protection against pathogens, and the modulation of the immune system. All these important functions make the GM a fundamental system able to regulate the host's health [1,2]. Many types of research on GM composition, conducted both in animals and humans, have highlighted its involvement in the onset and progression of several disorders, including neurodegenerative; cardiovascular; gastrointestinal; and metabolic diseases, such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD) [3].

The progressive degeneration of the tissues, with consequent alteration of organs'  structure and function, and the loss of homeostasis, make elderly people more prone to develop diseases [4–6]. During aging, it is widely reported that the increased imbalance between reactive oxygen species (ROS) production and antioxidant enzyme expression leads to the onset of oxidative stress (OS), with consequent damage to proteins, DNA,  and cellular organelles [7]. Specifically, in the gut, OS, together with a sedentary lifestyle,  changes in diet, and administration of drugs, causes GM dysbiosis, which contributes to the increase in intestinal permeability, resulting in the release of bacteria, endotoxins, and pro-oxidants into the systemic circulation. Ultimately, all these factors contribute to the development of hepatic diseases, such as NAFLD [8]. Currently, NAFLD is considered the most common chronic liver disease in the Western world and it is characterized by an excessive intrahepatic fat accumulation and is strongly associated with obesity, hypertension,  and insulin resistance [9]. The pathogenesis of NAFLD is not completely understood, but the most accredited hypothesis is the interaction among environmental factors (such as a  hypercaloric diet), GM changes, a sedentary lifestyle, and genetic predisposition [10]. Over time, NAFLD can become non-alcoholic steatohepatitis (NASH), and eventually progress into fibrosis, cirrhosis, and hepatocellular carcinoma [11]. To block the progression of NAFLD, thus improving the elderly's health, the prevention of the disease is important. The use of probiotics, which are alive microorganisms with numerous health benefits, could be a valid strategy, thanks to their ability to restore the GM and relieve oxidative stress [12].

This review aims to underline the possible factors causing GM dysbiosis and intestinal permeability disruption in elderly people, focusing above all on OS, with particular attention to the association between an altered GM and the development of NAFLD. We also discuss the NAFLD-associated GM signatures and the use of probiotics as a potential therapeutic strategy to restore GM to a healthy condition and counteract NAFLD progression.

2. Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis

Non-alcoholic fatty liver disease is an umbrella term including simple steatosis or nonalcoholic fatty liver (NAFL) and its progression into non-alcoholic steatohepatitis. NAFLD  is the hepatic manifestation of the "metabolic syndrome", which also comprises dyslipidemia, hypertension, insulin resistance, and diabetes [13,14]. Recently, the term NAFDL  has been proposed to be replaced by the more generic definition of metabolic-associated fatty liver disease (MAFLD), even though, in general, the traditional nomenclature is still preferred by the majority of experts, mainly because many clinical trials are currently specifically targeting NASH [15]. About a quarter of the world population suffers from NAFLD [16], with rates exceeding 43 percent in patients with metabolic syndrome [17]. Progression into NASH has been observed in about 10 percent of patients suffering from NAFLD, and more commonly in patients suffering from diabetes (37.7 percent ), who also presents the highest prevalence rate of NAFL (55.5 percent ) [18]. In NASH, the hepatic fat deposition is accompanied by an increased free fatty acid oxidation and mitochondrial dysfunction, leading to a  chronic inflammatory state, which in turn can lead to a high risk of fibrosis, cirrhosis, and hepatocellular carcinoma development [11,19]. Traditionally, two main "hits" were believed to be involved in NAFLD pathogenesis, being the first intrahepatic fat accumulation triggered by a sedentary lifestyle, bad nutritional habits, and insulin resistance [20], and the second a  lipid-induced over-production of ROS [21]. The two-hit hypothesis, by a consensus, is now considered too simplistic and a "multiple-hit hypothesis" has been proposed instead [11]. The multiple-hit hypothesis has been described as an "integrated response"  of the organism to the combination of hypercaloric nutrition and a sedentary lifestyle in a genetically predisposed host, leading to metabolic syndrome and obesity [22]. These events are accompanied by insulin resistance in the muscle in response to the increased levels of circulating free fatty acids, leading to an increase in hepatic de novo lipogenesis (DNL) and an imbalance in adipose tissue lipolysis, resulting in higher levels of circulating fatty acids conveyed to the liver [22]. Insulin resistance also contributes to the release of adipokines and inflammatory cytokines from the adipose tissue [23]. Aging affects the process of de novo lipogenesis (DNL) mostly through changes in systemic mediators such as insulin and leptin; in fact, aging is an insulin- and leptin-resistant state [24]. Many factors contribute to insulin resistance in aging, including an increase in body adiposity and visceral fat, increased adipose tissue inflammation, an increase in circulating cytokines, a sedentary lifestyle, and changes in the growth hormone/insulin-like-growth-factor I (GH-IGF)  axis [25]. Insulin resistance has been shown to induce an increase in the percent contribution of DNL to hepatic lipid accumulation [26]. Transcription factors sterol regulatory element-binding protein (SREBP)-1c and carbohydrate-responsive element-binding protein (ChREBP)-1, driven by insulin and glucose respectively, play a crucial role in stimulating DNL in the hepatocytes through an increase in the transcription of rate-limiting DNL  enzymes such as fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD1), and acyl coA carboxylase (ACC) [27]. Similarly to insulin resistance, in aging also serum leptin levels are increased, along with a paradoxical lack of effects due to multiple causes, including receptor desensitization, mutations in the genes encoding leptin and its receptors as well as proteins involved in self-regulation of leptin synthesis, and changes in blood–brain barrier permeability [28,29]. Leptin serves as the "satiety signal", acting primarily at the level of the hypothalamus to decrease appetite; so, reduced leptin levels or leptin resistance result in a higher food intake [30]. In addition to its central role, leptin may have a direct action on DNL; receptors for leptin have also been found in peripheral tissues including the liver [31], so changes in leptin signaling may also result in direct DNL enzyme positive modulation [24]. In the liver, excessive fat accumulation leads to lipotoxicity, a condition promoting oxidative stress and affecting mitochondrial and endoplasmic reticulum physiological functions [32]. Altogether, these processes lead to hepatic chronic inflammation accompanied by cell death,  hepatic stellate cell (HSC) activation, and fibrosis. However, the original assumption that steatosis always precedes inflammation is not always correct; in fact, NASH can also be the initial hepatic injury: it is the timing and the combination of insults that determine whether steatosis or NASH will occur first [33]. Recently, a deficit of lipophagy has been identified as a further contributor to lipid overaccumulation in NAFLD pathogenesis [34]. Lipophagy is a highly regulated step process that consists of (1) protein-mediated sequestration of lipid droplets within cytosolic vesicles and formation of a phagosome; (2) transport of a  phagosome to a lysosome and formation of the autophagolysosome; and (3) lipid degradation by lysosomal lipases [35]. Many proteins are involved in this process. The cargo adapter p62 is essential as it connects the lipidic cargo with autophagosomes; elevated P62  levels usually are a marker of decreased autophagy. LC3-II, a protein that targets the elongated autophagosome membrane, is degraded by lysosomal proteases; therefore, the increase in LC3-II indicates its impaired turnover [36]. Both P62 and LC3-II proteins were found to accumulate in high-fat diet-fed C57BL/6J male mice and high-fat/high-glucose cultured Huh7 cells [35]. In addition, in NAFLD patients, lipid droplet-loaded lysosomes and P62/sequestosome (SQSTM)1 cluster were associated with NAFLD activity score (NAS) and fibrosis stage, respectively, as well as expression levels of lysosomal genes and autophagy-related genes, showing that impaired autophagy is associated with features of advanced disease [35].

3. Gut Microbiota and Oxidative Stress 

The human microbiota consists of a wide range of microorganisms that reside in different parts of the body, including the skin and the gastrointestinal, genitourinary, and respiratory tracts [37]. In addition to these body districts, the urethra and the mammary glands have their microbes [38]. The GM is a complex and dynamic ecosystem of trillions of commensal microorganisms, including different communities of bacteria and some members of archaea, fungi, and viruses, which live in the gastrointestinal tract and give rise to a mutual relationship with the host [39,40]. The colonization of the gastrointestinal tract by bacteria begins in utero, via the placenta and the mother's amniotic fluid [41], while after birth, the mode of delivery (natural or cesarean), feeding (breastfeeding or artificial),  ingestion of antibiotics or probiotics during the early days of life, genetics, and environmental factors influence the composition of GM [39,42]. The Bifidobacterium mainly dominates the microbiota profile of infants, which can continually change during the first 3 years of life [43] according to a variety of factors, such as nutrition, geographic distribution [44],  genetic background, and immunological stimuli [45]. After 3 years of age, the GM acquires a more complex adult pattern that is relatively stable throughout adulthood [37]. Namely,  the GM of healthy adults is composed of anaerobic bacteria, most of which (more than 90 percent ) belong to the phyla of Bacteroidetes (Bacteroides, Prevotella, and Porphyromonas) and Firmicutes (Clostridia), followed by a small percentage (1–8 percent ) of Actinobacteria (Bifidobacterium), Proteobacteria, and Verrucomicrobia [46,47]. GM is necessary for human health; indeed, it can modulate innate and adaptive immune responses, regulate cellular growth, and preserve epithelial barrier function [48]. Furthermore, GM is also involved in glucose and lipid metabolism, energy balance, detoxification, vitamin K synthesis, and production of short-chain fatty acids (SCFAs; acetate, propionate, and butyrate) [45,47].

On the whole, based on its composition, the GM is described as an organ with a high degree of variability and heterogeneity, able to change and adapt to the needs of the host's human body [37]. As a result, each person's GM has a unique composition that differs from others [49]. Nevertheless, GM composition changes drastically as people age due to various factors, including lifestyle, dietary habits, stress, use of antibiotics and drugs,  and environmental stimuli [39,50] (Figure 1). This event results in damage and loss of intestinal homeostasis [51], and thus, in the elderly, the GM acquires specific features such as fewer beneficial bacteria, changes in dominant gut species (reduction of Firmicutes and increase in facultative aerobic bacteria), and proliferation of pathobionts proliferation (streptococci, staphylococci, enterobacteria, and enterococci), which are responsible for the onset in the gut of an inflammatory state in the gut [37,52,53]. Further, the dysbiotic microbiota is no longer able to perform its primary beneficial functions, thus leading to the production of toxic metabolites and to inflammation, which in turn cause the development of a variety of metabolic diseases, such as hypercholesterolemia, diabetes, obesity, NAFLD,  and its progression into NASH [54,55].

Since elderly people have difficulties in swelling and chewing, in association with decreased digestive motility [56], nutrition plays a key role in changing the GM pro-file [57]. A diet lacking in fibers and proteins, for example, as well as vitamin D and calcium deficiency, can alter the composition of GM, [58,59]. Moreover, the consumption of plant-based proteins, animal-based proteins, inulin, olive oil, and omega-3 polyunsaturated fatty acids (PUFA) can also modulate GM [60]. Besides diet, GM dysbiosis can as well be caused by oxidative stress and treatment with drugs aimed at targeting human cells rather than microorganisms, such as antidiabetics (metformin), proton pump inhibitors (PPIs), nonsteroidal anti-inflammatory drugs (NSAIDs), and atypical antipsychotics (AAPs) [61,62].

The accumulation of ROS produced by cellular metabolic and respiratory processes is recognized as one of the causes that promote aging [63]. Namely, ROS and reactive nitrogen species (RNS) are essential for cellular proliferation and differentiation, cytokines release, metabolism, and immune response. They are naturally produced by the organism's cells at low levels [7]. Under physiological conditions, the organism has several antioxidative defense mechanisms, including enzymes (catalase, glutathione peroxidase,  and superoxide dismutase) and antioxidants (such as vitamin C, vitamin E, uric acid,  carotenoids, and flavonoids), which can protect it against oxidant species; instead, as people get older, there is a cellular imbalance between these defenses and ROS generation, in favor of oxidants, resulting in OS [7,64]. OS causes molecular and cellular damage, particularly to proteins, lipids, DNA, and organelles [7,65], thus contributing to uncontrolled proliferation, inflammation, and cell death through apoptosis [66].

During aging, the alteration of cellular macromolecules, as well as the dysfunction at the level of mitochondria, which represents the primary source of energy, and the subsequent further production of ROS, eventually lead to the onset of age-related disorders [64].

Specifically, at the intestinal level, the continuous exposure of the mucosa to oxidants derived from diet and bacteria results in excessive production of ROS and the consequent onset of oxidative stress [66]. OS may disrupt colonic epithelial tight junctions, subsequently increasing the intestinal mucosa permeability, thus leading to a phenomenon known as "leaky gut" syndrome. This condition is characterized by the translocation of pro-oxidants and antigens, such as lipopolysaccharides (LPS), bacteria, and their endotoxins into the systemic circulation, where they reach various target organs [40,46], resulting in several pathological conditions, including metabolic disorders and infectious and systemic diseases (such as cardiac, neurodegenerative, and neoplastic) [40,67].

As mentioned before, whereas the composition of GM seems to be directly associated with ROS production in the intestine [68], other authors [60], conversely revealed that the abundance and composition of GM may influence the intestinal production of ROS. The consumption of probiotic bacteria and antioxidant nutrients able to change GM may lower ROS production by inhibiting pro-oxidant enzymes and stimulating antioxidant enzymes and related pathways [60]. Furthermore, GM itself can produce antioxidant molecules (glutathione, butyrate, and folate) able to protect the gut from toxins and ROS [69].

Finally, given that an imbalance between oxygen species generation and antioxidant defenses causes intestinal damage, an excessive amount of ROS and RNS leads to an elevated cellular oxidative stress, contributing to GM dysbiosis, which favors several gastrointestinal conditions, such as inflammation and metabolic disorders, like NAFLD [60]. The alteration of the intestinal microbiota is an important factor that contributes to the pathogenesis of NAFLD and its progression into NASH [54]. In particular, dysbiosis and oxidative stress lead to a dysregulation of intestinal permeability, resulting in the release of endotoxins, and microbiota metabolites, derived from saccharolytic and proteolytic fermentation, at the level of the liver, with a consequent increase in the accumulation of hepatic fat and inflammation [8], typical signs of this disease. Over time, if these conditions persist, NAFLD progresses into NASH, the more severe form, characterized by hepatocellular injury, chronic inflammation, and fibrosis [70] (Figure 2).

4. Gut Microbiota and NAFLD Development in Animal Models

In the last decades, fecal transplantation experiments in mice have provided a growing body of evidence about a causal role between GM alterations and NAFLD/NASH development [71,72]. GM was studied in various animal models of NAFLD, and its alteration was found to be associated with NAFLD genesis and progression. Adult germ-free mice fed with a regular diet, when exposed to a microbiota harvested from conventionally raised animals, showed a 60 percent increase in body fat content and the insurgence of insulin resistance,  possibly due to the increase in the absorption of monosaccharides from the gut lumen,  resulting in the induction of hepatic de novo lipogenesis [73]. Similarly, wild-type germ-free mice fed with a Western-style, high-fat, sugar-rich diet, are less prone to develop steatosis when compared to animals raised in conventional conditions [74,75]. On the contrary,  steatosis develops regularly in germ-free knockout (KO) mice lacking fasting-induced adipose factor (FIAF), a circulating lipoprotein lipase inhibitor normally suppressed by GM,  suggesting that FIAF is a mediator of microbial-regulated energy storage [74]. Recognizing the role of GM in the development of NAFLD also implies the concept that NAFLD is potentially a transmissible process [76]. Germ-free mice colonized with the cecal content collected from donors either responders or non-responders to a high-fat diet developed symptoms comparable to the respective donor when fed with the same diet. In other words, germ-free mice receiving intestinal microbiota from responder mice developed macrosteatosis and hyperglycemia; differently, mice receiving intestinal microbiota from non-responder mice do not develop NAFLD when fed with a high-fat diet [77]. A  similar effect was seen in mice colonized with human GM from healthy individuals or NAFLD patients: mice fed with a high-fat diet developed more severe NAFLD symptoms when receiving the microbiota from NAFLD patients and vice-versa [78]. In more recent work, quercetin was administered to donor mice fed with a high-fat diet to modulate the microbiota composition; the transplantation of microbiota from quercetin-treated donors in germ-free mice resulted in a protective phenotype against diet-induced NAFLD [79]. Further, in mice fed with a Western diet, a worsening of NASH symptoms was associated with the depletion of G protein-coupled chemokine receptor CX3CR1; the depletion of GM  using broad-spectrum antibiotics was also found to protect mice from diet-induced NASH,  similarly to what was demonstrated in germ-free mice [80].

The methionine-choline deficient (MCD) diet is another well-established animal model of NAFLD. In this model, choline deficiency affects triglyceride export via very low-density lipoproteins (VLDLs), resulting in hepatic steatosis; in addition, the lack of methionine impairs glutathione synthesis, causing a significant increase in oxidative injury [14]. The MCD diet-induced NAFLD is characterized by hepatic ballooning, marked oxidative stress,  chronic inflammation, and fibrosis, without the development of hyperglycemia, dyslipidemia,  and insulin resistance; therefore, it is more suitable for the study of inflammation and fibrosis [81]. In mice, the administration of the MCD diet induces persistent alterations in the GM and impairment of the intestinal barrier [82]. However, unexpectedly, in MCD mice the treatment with broad-spectrum antibiotics, aimed to deplete the microbiota, does not produce the same effect seen in germ-free or antibiotic-treated mice fed with a Western diet,  resulting in the aggravation of steatosis, inflammation, a higher histopathological NAFLD  activity score (NAS), and a significantly higher liver-to-body weight ratio [83]. In contrast,  the microbiota modulation via probiotics has shown beneficial effects both in the high-fat NAFLD model and in the MCD-induced NASH [84], suggesting that in the MCD model,  the microbiota preserves its protective activity, which is lost in high-fat and Western diet models of NAFLD. These last works suggest that the microbiota should be seen as both a  potential therapeutic agent and a drug target for the treatment of NAFLD.

An alternative NAFLD model consists of the supplementation of high doses of fructose in a regular diet [14]. Interestingly, high fructose supplementation does not necessarily result in body weight gain, but indeed in the increase in liver weight/body weight ratio [75], supporting the newly acquired notion that diet-induced liver steatosis does not necessarily precede body weight gain [85]. Fructose at high doses is associated with microbiota overgrowth and increased intestinal permeability, leading to an endotoxin-dependent activation of hepatic Kupffer cells. The suppression of endotoxin-mediated activation of Kupffer cells in toll-like receptor (TLR)-4 mutant mice resulted in the reduction of hepatic triglyceride accumulation by approximately 40 percent in comparison with fructose-fed wild-type mice [86]. Fructose-induced steatosis is also absent in germ-free mice, confirming that bacterial products such as LPS are required to induce liver steatosis and indicating that the gut microbiota is involved in the pathogenesis of the experimental fatty liver disease [75].

Several hypotheses have been formulated as to how GM may contribute to NAFLD  development and progression into NASH. As previously mentioned, they include increased intestinal permeability, leading to increased absorption by the host of microbially produced toxins and metabolites, such as LPS, trimethylamine N-oxide (TMAO), choline,  and ethanol, which trigger inflammation and affect immunity [71]. Infiltrating immune cells such as monocyte-derived macrophages and neutrophil granulocytes, two mediators of the hepatic inflammation during NASH, seem to have a relevant role in the microbiota-mediated worsening of NAFLD; chemokine receptor antagonists, by inhibiting monocyte recruitment, reduce hepatocyte ballooning, fibrosis, and inflammation in both the Western diet and the MCD diet models [87]. Infiltrating immune cells express high levels of pathogen recognition receptors (PRRs), including the NLR inflammasome family members,  designated to recognize toxins released by the microbiota that reach the liver via portal circulation [88]. Interestingly, in NLRP3 and NLRP6 inflammasome-deficient mice, an unfavorable intestinal microbiota has been linked to a loss of intestinal barrier integrity and increased translocation of toxins of microbial origin into the liver, where they activate hepatic inflammation [89]. These data indicate that the translocation of bacterial products from the gut into the liver is part of a highly regulated series of complex interactions among the gut, its microbiota, and the liver, often referred to as the gut–liver axis, and contributing to liver fat accumulation and inflammation in NASH [90].

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