Valuable effects of lactobacillus and citicoline on steatohepatitis: role of Nrf2/HO-1 and gut microbiota
AMB Express volume 13, Article number: 57 (2023)
Non-alcoholic steatohepatitis (NASH) is a more dangerous form of chronic non-alcoholic fatty liver disease (NAFLD). In the current investigation, the influence of citicoline on high-fat diet (HFD)-induced NASH was examined, both alone and in combination with Lactobacillus (probiotic). NASH was induced by feeding HFD (10% sugar, 10% lard stearin, 2% cholesterol, and 0.5% cholic acid) to rats for 13 weeks and received single i.p. injection of streptozotocin (STZ, 30 mg/kg) after 4 weeks. Citicoline was given at two dose levels (250 mg and 500 mg, i.p.) at the beginning of the sixth week, and in combination with an oral suspension of Lactobacillus every day for eight weeks until the study’s conclusion. HFD/STZ induced steatohepatitis as shown by histopathological changes, elevated serum liver enzymes, serum hyperlipidemia and hepatic fat accumulation. Moreover, HFD convinced oxidative stress by increased lipid peroxidation marker (MDA) and decreased antioxidant enzymes (GSH and TAC). Upregulation of TLR4/NF-kB and the downstream inflammatory cascade (TNF-α, and IL-6) as well as Pentaraxin, fetuin-B and apoptotic markers (caspase-3 and Bax) were observed. NASH rats also had massive increase in Bacteroides spp., Fusobacterium spp., E. coli, Clostridium spp., Providencia spp., Prevotella interrmedia, and P. gingivalis while remarkable drop in Bifidobacteria spp. and Lactobacillus spp. Co-treatment with citicoline alone and with Lactobacillus improve histopathological NASH outcomes and reversed all of these molecular pathological alterations linked to NASH via upregulating the expression of Nrf2/HO-1 and downregulating TLR4/NF-kB signaling pathways. These results suggest that citicoline and lactobacillus may represent new hepatoprotective strategies against NASH progression.
Non-alcoholic fatty liver disease (NAFLD) is defined as accumulation of fats in hepatocytes that exceeds 5% of liver weight in a patient who is not alcoholic (Vernon et al. 2011). NAFLD is the most common cause of chronic liver disease in developed countries (Younossi et al. 2016). It can affect children, adolescents, and the elderly (Jiang et al. 2020). NAFLD incidence is assessed to be around 25% worldwide, with higher rate (31.79%) was reported in the Middle East (Younossi et al. 2016). It covers a wide range of histopathological conditions, from simple steatosis to non-alcoholic steatohepatitis (NASH) which can lead to cirrhosis and hepatocellular carcinoma (HCC). HCC is one of the foremost causes of cancer-related deaths worldwide (Dhamija et al. 2019).
Obesity, metabolic syndromes, and diabetes mellitus are common risk factors of emerging NAFLD (Yilmaz and Eren 2017). While only a small percentage of patients with simple steatosis (earliest form of NAFLD) progress to NASH (2–5%). A number of those patients develop NASH-related complications (cardiovascular disease, advanced fibrosis, cirrhosis, and HCC) which place a significant load on the healthcare system (Yilmaz and Eren 2017).
The development of steatosis to NASH can be attributed to the effect of oxidative stress which cause hepatocytes injury. Furthermore, changes in the gut microbiota have been related with NASH pathogenesis and progression (Carter et al. 2021). The release of endotoxins (LPS) of Gram negative bacteria stimulate the immune system-to produce a pro-inflammatory cytokines principal to mitochondrial dysfunction (Braunersreuther et al. 2012).
NASH can be diagnosed by liver biopsy and histopathology (Vernon et al. 2011). Several non-invasive biomarkers can also be measured, for example, oxidative stress and elevated mitochondrial permeability lead to caspase activation. Oxidative stress causes lipid peroxidation, producing malondialdehyde (MDA). The effects of ROS were mitigated by antioxidants, which can be classified as enzymatic or non-enzymatic. Enzymatic antioxidants include superoxide dismutase (SOD), which converts superoxide anion to hydrogen peroxide, whereas catalase (CAT) neutralizes hydrogen peroxide in water. In contrast, non-enzymatic forms comprise a wide range of molecules, such as glutathione. These products serve as helpful in vivo indicators for oxidative stress (Phaniendra et al. 2015). In addition, the diagnosis of NASH is aided by raised liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as higher cytokines like tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. (Neuman et al. 2014).
Biologically, the liver and gut are anatomically linked via a bidirectional functional link called the gut-liver axis (GLA). Because of this strong relationship, changes in gut microbiota might alter the liver’s state (Mazzotti et al. 2016). Dysbiosis of gut microbiota is defined as an “imbalance” in the gut microbial community due to gain or loss of a member or a change in relative abundance of microbes (Hrncir 2022). Gut microbiota dysbiosis is linked to numerous intestinal and extra-intestinal diseases including NAFLD (Rinninella et al. 2019). According to a recent study, NAFLD have less variety in gut microbiota than healthy people (Saltzman et al. 2018).
During the past few years, new technologies have facilitated identification and quantification of gut microbiota components by analyzing nucleic acids (DNA and RNA). In the gut of healthy people, the most prevalent bacterial phyla are Bacteroidetes (65.2%), Firmicutes (29.6%), Proteobacteria (2.9%), and Actinobacteria (0.5%) (Jennison and Byrne 2021). Prevotella and Bacteroides are two of the most common genera in the family Bacteroidetes. Numerous genera, including Lactobacillus, Bacillus, Clostridium, and Enterococcus, are members of the Firmicutes phylum. The Bifidobacterium genus is primarily responsible for representing the Actinobacteria phylum (Arumugam et al. 2011). Because the gut microbiota is thought to have a role in NAFLD, researchers have recently been excited in finding drugs that can regulate NAFLD by modifying the gut dysbiosis (Jiang et al. 2020).
Probiotics are living microorganisms that help the host’s health by restoring normal microbiota and relieving digestive disorders (Jiang et al. 2020). Probiotics have the potential to boost human immunity, especially when used in the right doses. Lactic acid bacilli have been shown to have immune-modulating effects on chronic inflammation due to changes in the gut microbiome (El-Baz et al. 2021). Many Lactobacillus species have been shown to alter gut microbiota dysbiosis by modifying the gut microbiota environment (El-Baz et al. 2020). NAFLD patients treated with Lactobacillus bulgaricus and Streptococcus thermophiles had lower levels of liver aminotransferases, according to a human clinical trial (Aller et al. 2011). Furthermore, another study suggested that Lactobacillus and Bifidobacterium probiotics can be effective in improving pediatric NAFLD (Famouri et al. 2017).
The consequence of choline deficiency on the progress of NAFLD is well known (Corbin and Zeisel 2012). Citicoline is a food supplement that is considered as a source of choline. Choline helps support healthy lipid metabolism and keep the liver functioning normally (Synoradzki and Grieb 2019). It can improve hepatic encephalopathy through reduction of oxidative stress (Iulia et al. 2017). As the prevalence of NAFLD has increased to more than 30% of adults in developed countries and its incidence is still on rise.
The goal of the current study was to evaluate Nrf2/HO-1 and TLR4/NF-kB signaling pathways, as well as identify the dysbiosis in the gut microbiome that was associated with experimentally induced NASH, in order to investigate the potential therapeutic effects of citicoline either alone or in combination with Lactobacillus in experimentally induced NASH.
Materials and methods
Chemicals and reagents
Citicoline was supplied as sterile ampoules containing 500 mg/4 mL (Somazina, Ferrer International S.A., Spain). Lactobacillus was obtained as packets of Lacteol forte (Rameda Pharma Co, Giza, Egypt). STZ, carboxymethyl cellulose, cholesterol and cholic acid were acquired from Sigma Chemical Co. (St. Louis, Missouri, USA).
Male adult Sprague Dawley rats (180–220 g, n = 6–8) supplied by delta university, Mansoura (Egypt) were utilized. Prior to the start of the experiment, rats were acclimated for a week. Throughout the trial, rats were kept under conventional dietary and environmental circumstances. Research Ethics Committee at Faculty of Pharmacy, Delta University approved the experimental protocol (ethical approval number: FPDU6/2022).
Induction of fatty liver in rats
Rats were given a high-fat diet (HFD) for 13 weeks that contained 10% sugar, 10% lard stearin, 2% cholesterol, and 0.5% cholic acid. After four weeks, rats received an IP injection of STZ (30 mg/kg in citrate buffer, 1 mL/kg). Seven days following STZ injection, fasting blood glucose (FBG) levels were measured. Only rats with FBG levels above 200 mg/dL were classified as diabetic and permitted to finish the experiment. After STZ treatment, the HFD remained unchanged, but sugar was removed to prevent severe hyperglycemia (El-Derany and El-Demerdash, 2020). The study’s eight-week treatment period started at the start of the sixth week and continued until it was through (Fig. 1S).
The animals were divided into seven groups that include: (1) Control group (Neg. Control): rats were fed normal chow diet, (2) Citicoline group (Neg. + citi 500 mg): rats were fed normal chow diet and administered citicoline (500 mg/kg, IP). (3) Lactobacillus group (Neg.+ Lacto): rats were fed normal chow diet and administered lactobacillus, the Lacteol forte packet’s contents were dissolved in water to create the probiotic suspension, which contained Lactobacillus fermentum and Lactobacillus delbrueckii. (Garcia-Castillo et al. 2019). In order to offer the probiotic-treated groups 0.5 mL of microbial suspension containing 2.7 × 108 CFU/mL, the concentration of the solution was diluted from the starting concentration of 1 × 1010 microbial cells per package. This was ingested by rats using a gastric tube (Fooladi et al. 2015). (4) HFD group (Posit. Control): rats were fed HFD as mentioned above. (5) Citicoline 250 mg (Citi 250) + HFD: rats were fed HFD and received citicoline (250 mg/kg) beginning with the start of the sixth week and lasting for 8 weeks until the study’s conclusion. (6) Citicoline 500 mg (Citi 500) + HFD: rats were fed HFD and received citicoline (500 mg/kg) beginning with the start of the sixth week and lasting for 8 weeks until the study’s conclusion. (7) Citicoline + Lactobacillus (Citi 500 + Lacto) group + HFD: rats were fed HFD and received citicoline (500 mg/kg) and lactobacillus beginning with the start of the sixth week and lasting for 8 weeks until the study’s conclusion.
Animal sacrifice and sample collection
Rats were deprived overnight after the experiment, and the next day they were weighed and given thiopental to make them unconscious. Serum samples were separated and stored at -80 °C for analysis after blood samples were taken through cardiac puncture and centrifuged at 3000 rpm for 20 min. All animals’ livers were also removed, cleaned, weighed, and cleansed with ice-cold phosphate buffered saline (PBS, pH 7.4). Parts of liver tissues were homogenized (10% w/v) in 20 mM Tris–HCl (containing 1 mM EDTA, pH 7.4) and centrifuged at 3000 g for 20 min at 4 °C. The supernatants were collected and stored at -80 °C for subsequent biochemical analyses. Portions of livers were fixed in 10% neutral buffered formalin solutions to be used for histopathological examination. Additionally, stool samples were obtained by weighing 300 mg cecal feces samples shortly after the animals were euthanized. The QIAamp DNA Stool Mini Kit (Qiagen Inc., cat. # 51,504 Hilden, Germany) was used to extract DNA according to the manufacturer’s instructions. To measure the DNA concentration, the obtained DNA was examined spectrophotometrically using a NanoDrop device (OPTIZEN NanoQ, Mecasys) and stored at -20 °C. The rest of the stool samples were stored at -80 °C until they were required again.
Liver injury blood biochemical parameters analysis and liver index
Using commercially available kits from Spectrum Diagnostics (Cairo, Egypt), serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels were measured spectrophotometrically. Additionally, the following equation was used to determine liver index: (Hepatic Mass/Total Body Mass) x 100.
Measurement of serum lipid profile
Using an enzymatic colorimetric method (Biodiagnostics, Cairo, Egypt), serum levels of total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein cholesterol (HDL-C) were measured in accordance with the procedures outlined by Allain et al. (1974), Fossati and Prencipe (1982) and Lopes-Virella et al. (1977), respectively.
Pentraxin and fetuin-B assays
Fetuin-B and pentraxin 3 (PTX3) plasma concentrations were measured using the rat ELISA Kit (USCN Life Science Inc., USA).
Hepatic oxidative stress parameters
In the liver homogenates’ supernatants, these parameters were estimated. A measure of lipid peroxidation is malondialdehyde (MDA) level, while measures of the liver’s antioxidant capacity are total antioxidant capacity (TAC) and superoxide dismutase (SOD) activity. The previously stated technique was used to determine MDA content (Ohkawa et al. 1979). In a nutshell, MDA was determined by thiobarbituric acid (TBA) reaction and spectrophotometric measurement of absorbance at 532 nm. Monitoring the previously mentioned SOD-inhabitable auto-oxidation of pyrogallol was used to measure the SOD activity (Marklund 1985). At 420 nm, the change in absorbance was seen. By reacting a known amount of exogenously supplied hydrogen peroxide (H2O2) with TAC, the amount of TAC was determined. A portion of the supplied hydrogen peroxide is removed by the antioxidants in the sample. Through an enzymatic reaction that turns 3,5, dichloro-2-hydroxy benzensulphonate into a colored product, the residual H2O2 is measured colorimetrically. At 505 nm, the absorbance was determined using spectrophotometry (Koracevic et al. 2001).
Levels of TNF-α and IL-6 in the supernatants of liver tissues were determined using ELISA kits according to manufacturer’s protocol (RayBiotech Inc., Norcross, GA, USA; and eBioscience Inc., San Diego, CA, USA) respectively.
Estimation of hepatic Nrf2 and HO-1 levels
Nuclear factor erythroid 2-related factor (Nrf2) and Heme oxygenase-1 (HO-1) activity were assessed by ELISA assay kits (Cloud-Clone Co., Houston, USA) according to manufacturer’s instructions.
Estimation of apoptosis markers
The pro-apoptotic markers, Caspase-3 and Bax proteins expression, were evaluated in liver tissue homogenates according to the manufacturers ’instruction, using ELISA kits purchased from (BioVisionInc, Catalog #E4592 Milpitas, USA; and Cusabio, Cat. # CSB-EL002573RA, Houston, USA), respectively.
Hepatic histopathological and immunohistochemical assessments
Dissected liver tissue samples were fixed in neutral buffered formalin at a 10% concentration. The tissue was processed, and paraffin blocks were created using conventional histopathological methods. Using the Ishak-modified HAI system, fibrosis and necroinflammation alterations were assessed in Sect. (5 μm thick) stained with Mallory’s trichrome and hematoxylin-eosin, respectively (Ishak et al. 1995). Using the following NAFLD activity scores (NAS), the degree of hepatic steatosis, inflammation, and ballooning was evaluated. The specimens were categorized into grades 0–3 for hepatocellular steatosis (grade 0, where less than 5% of the hepatic parenchyma were occupied by steatosis; grade 1, where 6–33% of the hepatic parenchyma were occupied by steatosis; grade 2, where 34–66% of the hepatic parenchyma were occupied by steatosis; and grade 3, where more than 66% of the hepatic parenchyma were occupied by steatosis). The specimens were divided into classes 0–3 according to the degree of inflammatory cell infiltration (grade 0, no infiltration; grade 1, one to two foci per 400X field; grade 2, three to four foci per 400X field; and grade 3, more than four foci per 400X field). The specimens were categorized into grades 0–2 for hepatocellular ballooning (grade 0, no ballooning; grade 1, few balloon cells; and grade 2, many cells/prominent ballooning). Stages 0–4 of hepatic fibrosis were determined (stage 0, no fibrosis; stages 1 and 2, mild and moderate perisinusoidal and periportal fibrosis respectively; stage 3, bridging fibrosis; and stage 4, cirrhosis). Anti-NF-kB and anti-TLR4 (Santa Cruz, CA, USA) were utilized as primary antibodies for immunohistochemical investigation in order to identify their intended proteins using conventional immunohistochemistry techniques.
Real-time PCR test for measuring relative abundance of selected gut microbiota
The relative abundance of Bifidobacteria, Lactobacillus, Bacteroides Escherichia coli (E. coli), Clostridium, Fusobacteria, P. gingivalis, Providencia and Prevotella intermedia were determined using the quantitative real-time polymerase chain reaction (qRT-PCR). Total bacterial abundance was estimated using suitable primers for 16 S rRNA housekeeping gene amplification (Table 1). For quantitative DNA measurements, 40–80 ng of extracted faecal DNA was mixed with 12.5 µL (2x) SYBR Green PCR master mix (Willowfort Co., Birmingham, UK.) and 1.5 µL of each forward and reverse primer (10 µmol each). To make a final volume of 25 µL, 7.5 µL of nuclease-free H2O was added. On a MyGo real-time PCR system, all real-time reactions were done at 95 °C for 5 min, followed by 45 cycles of 95 °C for 20 s, annealing for 20 s (Table 1), then 72 °C for 40 s and final extension at 72 °C for 5 min (Wong et al. 2017).
One-way analysis of variance (ANOVA) and the Tukey’s Kramer Multiple Comparison Test were used to assess the data. The non-parametric Kruskal-Wallis test and Dunn’s multiple comparison post-test were used to analyse the histopathological scores. The data are represented as means and standard error of the mean (SEM). Significant was defined as p < 0.05.
Effect of citicoline and its combination with Lactobacillus on HFD induced oxidative stress
MDA levels rose following HFD in comparison to control rats, a marker of oxidative stress. Additionally, reduced levels of SOD and TAC indicated that the liver’s capacity to function as an antioxidant was compromised. However, either alone or in combination with Lactobacillus, citicoline significantly increased SOD and TAC levels in comparison to the NASH group. Levels of MDA were also reduced. Additionally, the effect of Citi 500 on MDA is noticeably larger as compared to Citi 250. (p ˂0.05). Except for its impact on TAC, there is no noticeable difference between Lactobacillus and the aforementioned criteria. (p˂0.01) (Fig. 1).
Effect of citicoline and its combination with Lactobacillus on biochemical markers of liver injury and liver index
When compared to the control group, the NASH group had severe liver damage after 13 weeks as evidenced by a marked increase in blood ALT, AST, and ALP levels. (Fig. 2). Comparing the citicoline-treated groups to the NASH group, it was interesting to see that the levels of ALT, AST, and ALP decreased in a dose-dependent manner. In comparison to the citi 500 group, the modification brought forth by the addition of Lactobacillus is not noteworthy. Contrarily, liver index greatly decreased after treatment with the combination of citicoline and Lactobacillus compared to the HFD group, while liver index dramatically rose in the HFD group compared to the control group (Fig. 2).
Effect of citicoline and its combination with Lactobacillus on IL-6 and TNF-α
In comparison to the rats of a negative control, HFD increased the levels of IL-6 and TNF-α in the liver tissue. As seen in Fig. 3a, all drugs under consideration significantly inhibit IL-6 (p˂0.0001, p˂0.0001, and p˂0.0001). Citi 500 and its combination with Lactobacillus substantially reduce TNF- (p˂ 0.0001, p˂0.0001). When compared to Citi 500 alone, the inclusion of Lactobacillus exhibited a more significant inhibitory effect on TNF-α (p ˂0.0001). Additionally, there is a substantial difference in TNFα-inhibition between Citi 500 and Citi 250 (p ˂0.001) (Fig. 3b).
Effect of citicoline and its combination with lactobacillus on Nrf2 and HO-1
An essential upstream regulator of the expression of antioxidant enzymes, including HO-1, is Nrf2. The levels of Nrf2 and HO-1 in the livers of the NASH group significantly decreased as shown in Fig. 3c, d. As opposed to the NASH group, citicoline treatment either alone or in combination with Lactobacillus significantly increased Nrf2 and HO-1 expression in a dose-dependent manner. Additionally, when Lactobacillus was added compared to Citi 500 alone, HO-1 activation significantly increased (p 0.001) (Fig. 3c, d).
Effect of citicoline and its combination with Lactobacillus on Caspase-3 and Bax
Citi 500 significantly inhibits caspase 3 and Bax when used alone or in conjunction with Lactobacillus (p ˂0.001, p ˂0.0001, respectively). Additionally, the levels of caspase 3 change significantly between Citi 250 and Citi 500 (p ˂0.001) (Fig. 4a, b).
Effect of citicoline and its combination with Lactobacillus on serum pentraxin-3 (pg/mL) and Fetuin (ng/mL)
Citicolline and its combination with Lactobacillus exhibit significant ameliorative effects on pentraxin (p ˂0.01, p ˂0.01, p ˂0.0001) and fetuin (p ˂0.01, p ˂0.01, p ˂0.001) serum levels, as shown in Fig. 4c, d, respectively. Citicoline has a dose-dependent impact.
Effect of citicoline and Lactobacillus on lipid profile
When the NASH group is compared to the negative control group, the level of cholesterol significantly increases. (P˂ 0.0001). All of the treatment groups’ cholesterol levels dramatically dropped, including Citi 250 (p ˂0.001), Citi 500 (p ˂0.001), and Citi plus Lacto (p ˂0.0001). Comparing Citi 500 to combined Citi 500 and Lactobacillus group shows a substantial improvement (p ˂0.001) (Fig. 5).
Only the combination of Citi 500 and Lacto had a significant inhibitory effect on TG when compared to the positive group (p ˂0.001). Adding Lacto to Citi 500 improved TG significantly when compared to Citi 500 alone (p ˂0.001). In terms of high-density lipoprotein (HDL), all treated groups have a significant increase (p ˂0.001, p ˂0.001, p ˂0.0001) of HDL-c compared to positive control. However, increasing citicoline dose or addition of Lactobacillus didn’t confer significant increase of HDL-c when compared to citi 250 group (Fig. 5).
Effect of citicoline and its combination with Lactobacillus on hepatic histopathology
Microscopic pictures of H&E stained liver sections showed normal hepatocytes arranged in radiating plates around central vein (CV) with normal portal areas (PA) and sinusoids in negative control group. Meanwhile, liver sections from NASH group showed congested blood vessels, occluded sinusoids, diffuse hydropic degeneration with macro-vesicular steatosis. Portal fibrosis appears in some sections with foci of hemorrhage and few leukocytic cells infiltration. Milder pathological changes were observed in the treated groups including few micro-vesicular steatosis intermingled with few macro-vesicular steatosis in C250 group, few macro-vesicular steatosis in C500 group and very few micro-vesicular steatosis in C500 + Lacto group (Fig. 6). According to the observations from Mallory’s trichrome stained sections, negative control group showed no excess collagen deposition. However, liver sections from NASH group showed excess collagen deposition around central vein or portal areas. Liver sections from citicoline treated groups showed decreased collagen deposition in a dose dependent manner with lower collagen deposition on concomitant administration of Lactobacillus (Fig. 7).
Effect of citicoline and its combination with Lactobacillus on hepatic expression of TLR-4 and NF-kB
Regarding TLR-4 immunoreactivity, very few positively stained hepatocytes were found in normal rat livers. On the contrary, rats in NASH group showed a significant increase in the number of brownish stained hepatocytes as compared to the normal group. Treatment with citi 250, Citi 500 and further addition of Lactobacillus ameliorated TLR-4 expression as demonstrated by decreased positive brown cytoplasmic staining that appears in fewer hepatocytes respectively (Fig. 8).
On the other hand, immunostaining of NF-kB showed prominent positive brown nuclear staining of many von kupffer cells in liver sections from NASH group. As expected, a dose-dependent reduction in NF-kB was observed in the groups treated with citicoline, and to a greater extent on addition of Lactobacillus, compared to the NASH group (Fig. 9).
Effect of citicoline and its combination with Lactobacillus on gut microbiota abundance
In the current study, an alteration in gut microbiota was observed across the tested groups. A substantial difference in the relative abundance of microbiota, with NAFLD being linked to a considerable drop in Bifidobacteria spp. and Lactobacillus spp. compared to healthy controls. In comparison to a normal healthy control group, there was a massive rise in Bacteroides spp., Fusobacterium spp., E. coli, Clostridium spp., Providencia spp., Prevotella interrmedia, and P. gingivalis. In this study, administration of combined citicoline and Lactobacillus probiotics dramatically increased gut microbiota symbiosis when compared to the positive control group (Fig. 10).
NAFLD is the most common chronic liver disease worldwide. NAFLD is generally benign and non-progressive, whereas NASH can lead to cirrhosis and HCC. In terms of pathology, NASH includes not only liver steatosis but also hepatic inflammation and/or fibrosis (Clapper et al. 2013). The first-line treatment for NAFLD is a weight-loss lifestyle strategy (Promrat et al. 2010). Although numerous pharmaceutical alternatives for NASH therapy are employed (insulin sensitizers, lipid-lowering medications, and antioxidants), they either have modest effects or have long-term negative impact that limit their usage (M et al. 2009). Till now, there is no effective medicine for NASH; therefore, it is vital to search for an effective therapy. During the past few years, several researchers suggested that dysbiosis of intestinal microbiota could contribute to NAFLD occurrence and progression (Aragonès et al. 2019; Mokhtari et al. 2017). Hence, management of gut microbiota may be a promising strategy in NAFLD therapy. In this work, the effects of citicoline and its combination with lactobacillus in HFD and STZ-induced NASH, as well as the underlying mechanisms were investigated in a murine animal model.
Several studies reported that administration of a mixture of probiotics including Lactobacillus spp. has been proven to alleviate NAFLD symptoms in animal models (Esposito et al. 2009; Li et al. 2003; Ma et al. 2008; Nardone et al. 2010; Velayudham et al. 2009). In this study, citicoline administration alone or in combination with lactobacillus significantly reduced body and liver mass as well as hepatic index compared to untreated NASH group. Reduced net calorie intake during citicoline medication may be responsible for weight reduction, most likely due to appetite suppression (Killgore et al. 2010). Additionally, the gut flora plays a significant role in controlling body weight by either influencing energy metabolism or preventing the absorption of lipids (Ley et al. 2006). As a result, the Lactobacillus/Citicoline combined regimen had an advantage in reducing liver mass and liver index, which may be connected to a decrease in the deposition of fat in the liver and a corresponding decline in liver index.
The current findings demonstrated dyslipidemia in NASH group, as evidenced by elevated cholesterol and TG levels in the serum, as well as reduced HDL-C serum levels. These negative consequences have been demonstrated in previous studies (Afrin et al. 2017; Gerges et al. 2020). Notably, citicoline treatment improved lipid profile. The addition of lactobacillus in concomitant with citicoline induced an additional decrease in cholesterol and TG levels as well as increase in HDL-C. Similarly, a previous study reported that probiotics significantly improved serum TG and LDL levels in animal model (Al-Muzafar and Amin 2017). Furthermore, it was reported that liver steatosis can be ameliorated with IV-choline-supplement (Buchman et al. 1995).
Furthermore, the possible molecular pathways involved in the therapeutic effects of citicoline and lactobacillus were investigated. Oxidative stress is a major cause of hepatic injury in NASH according to animal models and individuals with the disease. Cytochrome P450, peroxisomal β-oxidation, mitochondrial electron leak, and recruited inflammatory cells have all been identified as contributors of oxidative stress in fatty liver (Machado et al. 2008; Mitsuyoshi et al. 2006). In mitochondria, ROS are produced during the metabolism of free fatty acids, reactive lipid peroxidation products such as MDA can further aggravate oxidative stress, leading to the development of new sources of oxidants. The current findings confirmed the accumulation of MDA (oxidative stress marker), in rats with NASH, along with low SOD and TAC levels. The increase in oxidative stress provoked liver injury, as shown in this study by elevation of serum ALT, AST and ALP levels. These results were also supported by the histopathological findings in which diffuse hydropic degeneration of hepatocytes was observed along with macro-vesicular steatosis, central vein congestion and leukocytic cells infiltration in NASH group. However, the use of citicoline alone or in combination with lactobacillus improved the antioxidant system and restored SOD and TAC levels, resulting in reduced hepatocyte lipid peroxidation with modest improvement of liver function markers and histopathological abnormalities. These findings are in accordance with a recent study reporting that administration of probiotics improved oxidative stress markers in NAFLD animal model (Azarang et al. 2020). It was also reported that probiotic therapy significantly reduce liver specific markers such as ALT, AST and total cholesterol (Ma et al. 2013; Sharpton et al. 2019). Many investigations have shown that citicoline has a strong inhibitory effect on the production of reactive oxygen species (ROS) (Al-Kuraishy and Al-Gareeb 2020; Aminzadeh and Salarinejad 2019; Qian et al. 2014).
Nrf2 is a redox-sensitive transcription factor that regulates detoxification genes and cellular responses to oxidative stress. Nrf2 is required for protection against oxidative liver damage in NASH mouse models (Chowdhry et al. 2010; Okada et al. 2013). Nrf2 is released and translocated into the nucleus in response to oxidative stress, where it stimulates the expression of many antioxidant genes such as HO-1 (Ma 2013). Overexpression of HO-1 can protect cells against oxidative stress-induced cell death (Min et al. 2011). Nrf2 activators are being developed for the treatment of NASH (Musso et al. 2016). In line with this, our findings revealed that NASH rats showed decreased expression and activity of both Nrf2 and its target genes. On the other hand, therapy with citicoline either alone or in combination with lactobacillus increased Nrf2 and its target gene expression. These findings lead us to believe that citicoline protective effects may be mediated by activation of the Nrf2 pathway, which boosts the liver’s antioxidant capacity.
The GLA mediates NASH progression; Altered gut microbiota is associated with increased production of microbial metabolites such as LPS which is transported to the liver via portal vein (Jiang et al. 2020). It was reported that LPS/toll-like receptor 4 (TLR4)/NF-kB signaling is critical for the activation of inflammatory pathways associated with NASH (Machado and Cortez-Pinto 2012). TLR4 binding with LPS cause NF-kB activation and TNF-α production (Neuman et al. 2012). NF-kB signaling is also activated in response to oxidative stress and regulates inflammation and immunological responses (El-Agamy et al. 2019). NF-kB promotes the release of inflammatory cytokines such IL-6 and TNF-α causing liver tissue damage and increasing NAFLD aggravation (Henkel et al. 2018; Zhang et al. 2016). In line with the aforementioned studies, the results of our investigation indicated an increase in TLR4 expression, NF-kB activation and subsequent release of inflammatory cytokines (TNF-α, IL-6) in the NASH group with marked inter-parenchymal lymphocytes in the histological examination. Citicoline, on the other hand, ameliorated the activated inflammatory pathway, LPS/TLR4/NF-κB/TNF-α and IL-6, demonstrating that citicoline had a significant anti-inflammatory impact. Concomitant administration of lactobacillus caused further improvement of inflammatory markers with only mild lobular and portal inflammation. These findings revealed that suppression of TLR4/NF-kB was involved in citicoline and lactobacillus hepato-protection in NASH. Our findings supported by prior research that indicated citicoline suppressed the release of inflammatory cytokines (Bogdanov et al. 2018; Ek et al. 2014; Masoud et al. 2021). In addition, several studies reported that probiotics including lactobacillus inhibited inflammatory signaling such as NF-κB, reduced serum LPS and liver TLR4 expression and delay NAFLD progression (Li et al. 2003; Nagashimada and Honda 2021; Xue et al. 2017).
Patients with NASH have high plasma levels of TNF-α and PTX-3 (Pearce et al. 2013). When inflammatory cytokines are stimulated, PTX3 is rapidly produced in diverse cells (Bottazzi et al. 2009). A greater fibrosis grade was linked to high plasma levels of PTX3 (Boga et al. 2015). PTX-3 expression contributes to increased synthesis of extracellular protein matrix, suggesting a possible role in liver fibrosis (Feder et al. 2020). In the current study, increased levels of PTX3 and TNF-α were observed in liver tissues, which coincided with histological analysis of stained liver tissues showing excess collagen deposition around central vein and portal areas, validated these prior findings. Citicoline, interestingly, reduced the levels of PTX3 and TNF-α and improved the fibrosis score. Combined regimen with lactobacillus caused further improvement. This matches a prior study reporting that citicoline therapy lowers fibrosis and fibroblast cell density in a rat model of laminotomy and discectomy (Savran et al. 2012). Furthermore, it was reported that probiotic consumption significantly reduced the levels of serum PTX3 (Pourrajab et al. 2020). In addition, TNF-α and IL-6 levels reduced significantly after using probiotics in NAFLD patients (Kobyliak et al. 2018).
High serum level of fetuin was linked to NAFLD (Zhu et al. 2017). Fetuin B is a hepatokine that is upregulated by hepatic steatosis in obese persons and increased in type 2 diabetes (Meex et al. 2015). Furthermore, fetuin B had a positive relationship with serum ALT, TG and LDL-C, and a negative relationship with serum HDL-C (Zhu et al. 2017). This is consistent with our findings, which indicated high serum fetuin B levels in NASH rats. Treatment with citicoline alone or in combination with lactobacillus reduced this rise, indicating their therapeutic impact in NASH.
Apoptosis is a programed cell death that is induced by several factors including oxidative stress. Bcl2, Bax, and caspase-3 are the most common and reliable apoptosis markers (Elmore 2007). Hepatocyte apoptosis plays a significant role in liver injury (Wieckowska et al. 2006). Previous research has connected NASH- liver apoptosis to increased expression of caspases-3 and Bax (Wang et al. 2008). Furthermore, Nrf2 and NF-kB signaling pathways, which contribute to hepatocyte death, have been related to changes in pro-apoptotic proteins in NASH mice (Sharma et al. 2018). In the current study, apoptosis is elevated in the liver of NASH rats, which is indicated by increased levels of Bax and caspase-3. Citicoline, interestingly, reduced HFD-induced changes in these apoptotic markers. These findings coincides with a previous study stated that citicoline alone and in combination with hypothermia is effective in ameliorating cerebral damage after transient focal ischemia by suppressing apoptotic processes as indicated by increased Bax and Caspase-3 (Sahin et al. 2010). On the other hand, addition of lactobacillus further depressed apoptotic markers. Consistent with this, pretreatment with Lactobacillus diminished the levels of Bax and caspase 3 in hepatic cell lines (Kanmani and Kim 2022). These findings support the hypothesis that citicoline alters the Nrf2/NF-kB pathways, suppress hepatocyte apoptosis and improves hepatic damage.
In the current study high level of Lactobacillus and Bifidobacterium were observed in normal group compared to positive (fatty liver) group. It was reported that HFD increased intestinal endotoxin, and reduced enteric concentration of Bifidobacteria (Xu et al. 2011). After treatment with Lactobacillus and citicoline the levels of both Bifidobacterium and Lactobacillus increased to be above the normal group. Similar to this, a prior study showed that giving rats fed a high-cholesterol diet Lactobacillus decreases both cholesterol and TG while increasing the amount of faecal Lactobacilli and Bifidobacteria. (Wang et al. 2009).
This study reported overgrowth of Bacteroides, E. coli, Fusobacterium, Providencia, Clostridium and Prevotella during NAFLD that restored to normal level after treatment with Lactobacillus and citicoline. Consistent with our results, it was reported that Proteobacteria including Escherichia coli were significantly elevated in NASH (Zhu et al. 2013). In addition, Bacteroides overgrowth was reported recently in NASH rat model (Carter et al. 2021), and in a study involving human subjects (Boursier et al. 2016). Stool microbiome of children with NAFLD showed high abundance of Prevotella (Michail et al. 2015). Furthermore, Fusobacteria phyla were more abundant in NAFLD patients than healthy individuals (Shen et al. 2017). In addition, a retrospective study reported that NAFLD is a risk factor of Clostridium difficile GIT infection (Nseir et al. 2020).
Although P. gingivalis is a periodontal pathogen, it was reported that periodontal pathogens can cause changes in gut microbiota. P. gingivalis is acid resistant, so it can pass through the stomach and reach the intestinal tract (Nakajima et al. 2015). Variation in the gut microbiota was seen as periodontal infections moved to the intestine, for example, Firmicutes decreased and Bacteroides increased (Liu et al. 2021). All of this is consistent with the elevation of P. gingivalis observed in fatty liver group in the current study. P. gingivalis infection was found to be an important risk factor for progression of liver fibrosis in NAFLD (Nakahara et al. 2018). Bacteroides and E. coli reach high abundance during liver fibrosis (Ji et al. 2020).
Overall, our data show that citicoline either alone and in combination with a mixture of L. fermentum and L. delbruceki have strong anti-inflammatory and antioxidant properties that are effective against NASH. These properties may be related to Nrf2/HO-1 pathway activation and TLR4/NF-kB mediated inflammatory pathway inhibition, which may attenuate NASH associated altered lipid profile, liver damage, and apoptosis. They are therefore potential new hepatoprotective options for NASH.
Enquiries about data availability should be directed to the authors.
Non-alcoholic fatty liver disease
Total antioxidant capacity
Tumor necrosis factor alpha
Nuclear factor erythroid 2
Toll-like receptor 4
Nuclear factor kappa B
High-density lipoprotein cholesterol.
Afrin MR, Arumugam S, Rahman MA, Karuppagounder V, Harima M, Suzuki H, Miyashita S, Suzuki K, Ueno K, Yoneyama H, Watanabe K (2017) Curcumin reduces the risk of chronic kidney damage in mice with nonalcoholic steatohepatitis by modulating endoplasmic reticulum stress and MAPK signaling. Int Immunopharmacol 49:161–167. https://doi.org/10.1016/j.intimp.2017.05.035
Al-Kuraishy HM, Al-Gareeb AI (2020) Citicoline improves human vigilance and visual Working memory: the role of neuronal activation and oxidative stress. Basic Clin Neurosci 11(4):423–432. https://doi.org/10.32598/bcn.11.4.1097.1
Allain CC, Poon LS, Chan CS, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20(4):470–475
Aller R, De Luis D, Izaola O, Conde R, Gonzalez Sagrado M, Primo D, De La Fuente B, Gonzalez J (2011) Effect of a probiotic on liver aminotransferases in nonalcoholic fatty liver disease patients: a double blind randomized clinical trial. Eur Rev Med Pharmacol Sci 15(9):1090–1095
Al-Muzafar HM, Amin KA (2017) Probiotic mixture improves fatty liver disease by virtue of its action on lipid profiles, leptin, and inflammatory biomarkers. BMC Complement Altern Med 17(1):1–9
Aminzadeh A, Salarinejad A (2019) Citicoline protects against lead-induced oxidative injury in neuronal PC12 cells. Biochem Cell Biol 97(6):715–721. https://doi.org/10.1139/bcb-2018-0218
Aragonès G, González-García S, Aguilar C, Richart C, Auguet T (2019) Gut microbiota-derived mediators as potential markers in nonalcoholic fatty liver disease. Biomed Res Int 2019
Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto J-M (2011) Enterotypes of the human gut microbiome. Nature 473(7346):174–180
Azarang A, Farshad O, Ommati MM, Jamshidzadeh A, Heidari R, Abootalebi SN, Gholami A (2020) Protective role of probiotic supplements in hepatic steatosis: a rat model study. Biomed Res Int 2020
Bartosch S, Fite A, Macfarlane GT, McMurdo ME (2004) Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl Environ Microbiol 70(6):3575–3581
Benkirane RM, Guillot E, Mouton C (1995) Immunomagnetic PCR and DNA probe for detection and identification of Porphyromonas gingivalis. J Clin Microbiol 33(11):2908–2912
Boga S, Koksal AR, Alkim H, Yilmaz Ozguven MB, Bayram M, Ergun M, Sisman G, Tekin Neijmann S, Alkim C (2015) Plasma pentraxin 3 differentiates nonalcoholic steatohepatitis (NASH) from Non-NASH. Metab Syndr Relat Disord 13(9):393–399. https://doi.org/10.1089/met.2015.0046
Bogdanov P, Sampedro J, Solà-Adell C, Simó-Servat O, Russo C, Varela-Sende L, Simó R, Hernández C (2018) Effects of Liposomal Formulation of Citicoline in Experimental Diabetes-Induced Retinal Neurodegeneration. Int J Mol Sci 19(8):2458
Bottazzi B, Garlanda C, Cotena A, Moalli F, Jaillon S, Deban L, Mantovani A (2009) The long pentraxin PTX3 as a prototypic humoral pattern recognition receptor: interplay with cellular innate immunity. Immunol Rev 227(1):9–18. https://doi.org/10.1111/j.1600-065X.2008.00719.x
Boursier J, Mueller O, Barret M, Machado M, Fizanne L, Araujo-Perez F, Guy CD, Seed PC, Rawls JF, David LA (2016) The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota. Hepatology (Baltimore MD) 63(3):764–775
Braunersreuther V, Viviani GL, Mach F, Montecucco F (2012) Role of cytokines and chemokines in non-alcoholic fatty liver disease. World J Gastroenterol 18(8):727
Buchman AL, Dubin MD, Moukarzel AA, Jenden DJ, Roch M, Rice KM, Gornbein J, Ament ME (1995) Choline deficiency: a cause of hepatic steatosis during parenteral nutrition that can be reversed with intravenous choline supplementation. Hepatology (Baltimore MD) 22(5):1399–1403
Burns MB, Lynch J, Starr TK, Knights D, Blekhman R (2015) Virulence genes are a signature of the microbiome in the colorectal tumor microenvironment. Genome Med 7(1):1–12
Carter JK, Bhattacharya D, Borgerding JN, Fiel MI, Faith JJ, Friedman SL (2021) Modeling dysbiosis of human NASH in mice: loss of gut microbiome diversity and overgrowth of Erysipelotrichales. PLoS ONE 16(1):e0244763
Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, Barnes R, Watson P, Allen-Vercoe E, Moore RA (2012) Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res 22(2):299–306
Chowdhry S, Nazmy MH, Meakin PJ, Dinkova-Kostova AT, Walsh SV, Tsujita T, Dillon JF, Ashford ML, Hayes JD (2010) Loss of Nrf2 markedly exacerbates nonalcoholic steatohepatitis. Free Radic Biol Med 48(2):357–371. https://doi.org/10.1016/j.freeradbiomed.2009.11.007
Clapper JR, Hendricks MD, Gu G, Wittmer C, Dolman CS, Herich J, Athanacio J, Villescaz C, Ghosh SS, Heilig JS, Lowe C, Roth JD (2013) Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment. Am J Physiol Gastrointest Liver Physiol 305(7):G483–G495. https://doi.org/10.1152/ajpgi.00079.2013
Collado MC, Isolauri E, Laitinen K, Salminen S (2008) Distinct composition of gut microbiota during pregnancy in overweight and normal-weight women. Am J Clin Nutr 88(4):894–899
Corbin KD, Zeisel SH (2012) Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression. Curr Opin Gastroenterol 28(2):159–165. https://doi.org/10.1097/MOG.0b013e32834e7b4b
Dhamija E, Paul SB, Kedia S (2019) Non-alcoholic fatty liver disease associated with hepatocellular carcinoma: an increasing concern. Indian J Med Res 149(1):9
Ek RO, Serter M, Ergin K, Cecen S, Unsal C, Yildiz Y, Bilgin MD (2014) Protective effects of citicoline on TNBS-induced experimental colitis in rats. Int J Clin Exp Med 7(4):989–997
El-Agamy DS, El-Harbi KM, Khoshhal S, Ahmed N, Elkablawy MA, Shaaban AA, Abo-Haded HM (2019) Pristimerin protects against doxorubicin-induced cardiotoxicity and fibrosis through modulation of Nrf2 and MAPK/NF-kB signaling pathways. Cancer Manag Res 11:47–61. https://doi.org/10.2147/cmar.s186696
El-Baz AM, Khodir AE, Adel El-Sokkary MM, Shata A (2020) The protective effect of Lactobacillus versus 5-aminosalicylic acid in ulcerative colitis model by modulation of gut microbiota and Nrf2/Ho-1 pathway. Life Sci 256:117927. https://doi.org/10.1016/j.lfs.2020.117927
El-Baz AM, Shata A, Hassan HM, El-Sokkary MMA, Khodir AE (2021) The therapeutic role of lactobacillus and montelukast in combination with metformin in diabetes mellitus complications through modulation of gut microbiota and suppression of oxidative stress. Int Immunopharmacol 96:107757. https://doi.org/10.1016/j.intimp.2021.107757
El-Derany MO, El-Demerdash E. (2020 Jul) Pyrvinium pamoate attenuates non-alcoholic steatohepatitis: Insight on hedgehog/Gli and Wnt/?-catenin signaling crosstalk. Biochem Pharmacol. 177:113942. https://doi.org/10.1016/j.bcp.2020.113942
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516
Esposito E, Iacono A, Bianco G, Autore G, Cuzzocrea S, Vajro P, Canani RB, Calignano A, Raso GM, Meli R (2009) Probiotics reduce the inflammatory response induced by a high-fat diet in the liver of young rats. J Nutr 139(5):905–911
Famouri F, Shariat Z, Hashemipour M, Keikha M, Kelishadi R (2017) Effects of Probiotics on nonalcoholic fatty liver disease in obese children and adolescents. J Pediatr Gastroenterol Nutr 64(3):413–417. https://doi.org/10.1097/mpg.0000000000001422
Feder S, Haberl EM, Spirk M, Weiss TS, Wiest R, Buechler C (2020) Pentraxin-3 is not related to disease severity in cirrhosis and hepatocellular carcinoma patients. Clin Exp Med 20(2):289–297. https://doi.org/10.1007/s10238-020-00617-4
Fooladi AAI, Yazdi MH, Pourmand MR, Mirshafiey A, Hassan ZM, Azizi T, Mahdavi M, Dallal MMS (2015) Th1 cytokine production induced by Lactobacillus acidophilus in BALB/c mice bearing transplanted breast tumor. Jundishapur JMicrobiol 8(4)
Fossati P, Prencipe L (1982) Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem 28(10):2077–2080
Garcia-Castillo V, Komatsu R, Clua P, Indo Y, Takagi M, Salva S, Islam MA, Alvarez S, Takahashi H, Garcia-Cancino A (2019) Evaluation of the immunomodulatory activities of the probiotic strain Lactobacillus fermentum UCO-979 C. Front Immunol 10
Gerges SH, Wahdan SA, Elsherbiny DA, El-Demerdash E (2020) Diosmin ameliorates inflammation, insulin resistance, and fibrosis in an experimental model of non-alcoholic steatohepatitis in rats. Toxicol Appl Pharmacol 401:115101. https://doi.org/10.1016/j.taap.2020.115101
Ginige MP, Kaksonen AH, Morris C, Shackelton M, Patterson BM (2013) Bacterial community and groundwater quality changes in an anaerobic aquifer during groundwater recharge with aerobic recycled water. FEMS Microbiol Ecol 85(3):553–567
Henkel J, Coleman CD, Schraplau A, Jöhrens K, Weiss TS, Jonas W, Schürmann A, Püschel GP (2018) Augmented liver inflammation in a microsomal prostaglandin E synthase 1 (mPGES-1)-deficient diet-induced mouse NASH model. Sci Rep 8(1):16127. https://doi.org/10.1038/s41598-018-34633-y
Hrncir T (2022) Gut microbiota dysbiosis: triggers, Consequences, Diagnostic and Therapeutic Options, vol 10. MDPI, Microorganisms, p 578
Huijsdens XW, Linskens RK, Mak M, Meuwissen SG, Vandenbroucke-Grauls CM, Savelkoul PH (2002) Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR. J Clin Microbiol 40(12):4423–4427
Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, Denk H, Desmet V, Korb G, MacSween RN, Phillips MJ, Portman BG, Poulsen H, Schever PJ, Schmid M, a HT (1995) Histological grading and staging of chronic hepatitis. J Hepatol 22(6):696–699. https://doi.org/10.1016/0168-8278(95)80226-6
Iulia C, Ruxandra T, Costin L-B, Liliana-Mary V (2017) Citicoline–a neuroprotector with proven effects on glaucomatous disease. Rom J Ophthalmol 61(3):152
Jennison E, Byrne CD (2021) The role of the gut microbiome and diet in the pathogenesis of non-alcoholic fatty liver disease. Clin Mol Hepatol 27(1):22
Jiang X, Zheng J, Zhang S, Wang B, Wu C, Guo X (2020) Advances in the involvement of Gut Microbiota in Pathophysiology of NAFLD. Front Med 7:361
Ji Y, Yin Y, Sun L, Zhang W (2020) The molecular and mechanistic insights based on gut–liver axis: nutritional target for non-alcoholic fatty liver disease (NAFLD) improvement. Int J Mol Sci 21(9):3066
Kanmani P, Kim H (2022) Probiotics counteract the expression of hepatic profibrotic genes via the attenuation of TGF-β/SMAD signaling and autophagy in hepatic stellate cells. PLoS ONE 17(1):e0262767
Killgore WDS, Ross AJ, Kamiya T, Kawada Y, Renshaw PF, Yurgelun-Todd DA (2010) Citicoline affects appetite and cortico-limbic responses to images of high-calorie foods. Int J Eat Disord 43(1):6–13. https://doi.org/10.1002/eat.20658
Kim MJ, Hwang KH, Lee Y-S, Park J-Y, Kook J-K (2011) Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27. J Microbiol Methods 84(3):394–397
Kobyliak N, Abenavoli L, Mykhalchyshyn G, Kononenko L, Boccuto L, Kyriienko D, Dynnyk O (2018) A multi-strain probiotic reduces the fatty liver index, cytokines and aminotransferase levels in NAFLD patients: evidence from a randomized clinical trial. J Gastrointestin Liver Dis
Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V (2001) Method for the measurement of antioxidant activity in human fluids. J Clin Pathol 54(5):356–361. https://doi.org/10.1136/jcp.54.5.356
Ley RE, Turnbaugh PJ, Klein S, Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444(7122):1022–1023. https://doi.org/10.1038/4441022a
Liu Y, Huang W, Wang J, Ma J, Zhang M, Lu X, Liu J, Kou Y (2021) Multifaceted impacts of Periodontal Pathogens in Disorders of the intestinal barrier. Front Immunol:3010
Li Z, Yang S, Lin H, Huang J, Watkins PA, Moser AB, DeSimone C, Song Xy, Diehl AM (2003) Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease. Hepatology (Baltimore MD) 37(2):343–350
Lopes-Virella MF, Stone P, Ellis S, Colwell JA (1977) Cholesterol determination in high-density lipoproteins separated by three different methods. Clin Chem 23(5):882–884
Machado MV, Cortez-Pinto H (2012) Gut microbiota and nonalcoholic fatty liver disease. Ann Hepatol 11(4):440–449
Machado MV, Ravasco P, Jesus L, Marques-Vidal P, Oliveira CR, Proença T, Baldeiras I, Camilo ME, Cortez-Pinto H (2008) Blood oxidative stress markers in non-alcoholic steatohepatitis and how it correlates with diet. Scand J Gastroenterol 43(1):95–102. https://doi.org/10.1080/00365520701559003
Maheux AF, Picard FJ, Boissinot M, Bissonnette L, Paradis S, Bergeron MG (2009) Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples. Water Res 43(12):3019–3028
Ma Q (2013) Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol 53:401–426. https://doi.org/10.1146/annurev-pharmtox-011112-140320
Marklund SL (1985) Superoxide dismutase isoenzymes in tissues and plasma from New Zealand black mice, nude mice and normal BALB/c mice. Mutat Res 148(1–2):129–134. https://doi.org/10.1016/0027-5107(85)90216-7
Masoud F, Habibi-Asl B, Charkhpour M, Asadpour Y, Mahmoudi J (2021) Dexamethasone and citicoline mitigate cisplatin-induced peripheral neuropathy: a novel experimental study in mice. Turk J Pharm Sci. https://doi.org/10.4274/tjps.galenos.2021.51882
Matsuki T, Watanabe K, Fujimoto J, Miyamoto Y, Takada T, Matsumoto K, Oyaizu H, Tanaka R (2002) Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl Environ Microbiol 68(11):5445–5451
Ma X, Hua J, Li Z (2008) Probiotics improve high fat diet-induced hepatic steatosis and insulin resistance by increasing hepatic NKT cells. J Hepatol 49(5):821–830
Ma Y-Y, Li L, Yu C-H, Shen Z, Chen L-H, Li Y-M (2013) Effects of probiotics on nonalcoholic fatty liver disease: a meta-analysis. World J Gastroenterol 19(40):6911
Mazzotti A, Caletti MT, Sasdelli AS, Brodosi L, Marchesini GJDD (2016) Pathophysiology of nonalcoholic fatty liver disease: lifestyle-gut-gene interaction. Dig Dis 34(Suppl 1):3–10
Meex Ruth C, Hoy Andrew J, Morris A, Brown Russell D, Lo Jennifer CY, Burke M, Goode Robert JA, Kingwell Bronwyn A, Kraakman Michael J, Febbraio Mark A, Greve Jan W, Rensen Sander S, Molloy Mark P, Lancaster Graeme I, Bruce Clinton R, Watt Matthew J (2015) Fetuin B is a secreted hepatocyte factor linking steatosis to impaired glucose metabolism. Cell Metab 22(6):1078–1089. https://doi.org/10.1016/j.cmet.2015.09.023
Michail S, Lin M, Frey MR, Fanter R, Paliy O, Hilbush B, Reo NVJFme (2015) Altered gut microbial energy and metabolism in children with non-alcoholic fatty liver disease. FEMS Microbiol Ecol 91(2):1
Min KJ, Lee JT, Joe EH, Kwon TK (2011) An IκBα phosphorylation inhibitor induces heme oxygenase-1(HO-1) expression through the activation of reactive oxygen species (ROS)-Nrf2-ARE signaling and ROS-PI3K/Akt signaling in an NF-κB-independent mechanism. Cell Signal 23(9):1505–1513. https://doi.org/10.1016/j.cellsig.2011.05.013
Mitsuyoshi H, Itoh Y, Okanoue T (2006) [Role of oxidative stress in non-alcoholic steatohepatitis]. Nihon Rinsho 64(6):1077–1082
Mokhtari Z, Gibson DL, Hekmatdoost A (2017) Nonalcoholic fatty liver disease, the gut microbiome, and diet. Adv Nutr 8(2):240–252
Musso G, Cassader M, Gambino R (2016) Non-alcoholic steatohepatitis: emerging molecular targets and therapeutic strategies. Nat Rev Drug Discov 15(4):249–274. https://doi.org/10.1038/nrd.2015.3
Nagashimada M, Honda M (2021) Effect of Microbiome on non-alcoholic fatty liver Disease and the role of Probiotics, Prebiotics, and Biogenics. Int J Mol Sci 22(15):8008
Nakahara T, Hyogo H, Ono A, Nagaoki Y, Kawaoka T, Miki D, Tsuge M, Hiraga N, Hayes CN, Hiramatsu A (2018) Involvement of Porphyromonas gingivalis in the progression of non-alcoholic fatty liver disease. J Gastroenterol 53(2):269–280
Nakajima M, Arimatsu K, Kato T, Matsuda Y, Minagawa T, Takahashi N, Ohno H, Yamazaki K (2015) Oral administration of P. gingivalis induces dysbiosis of gut microbiota and impaired barrier function leading to dissemination of enterobacteria to the liver. PLoS ONE 10(7):e0134234
Nardone G, Compare D, Liguori E, Di Mauro V, Rocco A, Barone M, Napoli A, Lapi D, Iovene MR, Colantuoni A (2010) Protective effects of Lactobacillus paracasei F19 in a rat model of oxidative and metabolic hepatic injury. Am J Physiol Gastrointest Liver Physiol 299(3):G669–G676
Neuman MG, Cohen LB, Nanau RM (2014) Biomarkers in nonalcoholic fatty liver disease. Can J Gastroenterol Hepatol 28(11):607–618. https://doi.org/10.1155/2014/757929
Neuman M, Hilzenrat N, Cohen L, Winkler RE, Nanau R (2012) Multiple factors involved in nonalcoholic hepatitis pathogenesis. Int J Hepatol 2012:429805. https://doi.org/10.1155/2012/429805
Nseir WB, Hussein SHH, Farah R, Mahamid MN, Khatib HH, Mograbi JM, Peretz A, Amara AE (2020) Nonalcoholic fatty liver disease as a risk factor for Clostridium difficile-associated diarrhea. QJM: monthly journal of the Association of Physicians 113(5):320–323. https://doi.org/10.1093/qjmed/hcz283
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–358. https://doi.org/10.1016/0003-2697(79)90738-3
Okada K, Warabi E, Sugimoto H, Horie M, Gotoh N, Tokushige K, Hashimoto E, Utsunomiya H, Takahashi H, Ishii T, Yamamoto M, Shoda J (2013) Deletion of Nrf2 leads to rapid progression of steatohepatitis in mice fed atherogenic plus high-fat diet. J Gastroenterol 48(5):620–632. https://doi.org/10.1007/s00535-012-0659-z
Pearce SG, Thosani NC, Pan JJ (2013) Noninvasive biomarkers for the diagnosis of steatohepatitis and advanced fibrosis in NAFLD. Biomark Res 1(1):7. https://doi.org/10.1186/2050-7771-1-7
Phaniendra A, Jestadi DB, Periyasamy L (2015) Free radicals: properties, sources, targets, and their implication in various diseases. Indian J Clin biochemistry: IJCB 30(1):11–26. https://doi.org/10.1007/s12291-014-0446-0
Pourrajab B, Fatahi S, Dehnad A, Varkaneh HK, Shidfar F (2020) The impact of probiotic yogurt consumption on lipid profiles in subjects with mild to moderate hypercholesterolemia: a systematic review and meta-analysis of randomized controlled trials. Nutr Metab Cardiovasc Dis 30(1):11–22
Promrat K, Kleiner DE, Niemeier HM, Jackvony E, Kearns M, Wands JR, Fava JL, Wing RR (2010) Randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. Hepatology (Baltimore MD) 51(1):121–129. https://doi.org/10.1002/hep.23276
Qian K, Gu Y, Zhao Y, Li Z, Sun M (2014) Citicoline protects brain against closed Head Injury in rats through suppressing oxidative stress and calpain over-activation. Neurochem Res 39(7):1206–1218. https://doi.org/10.1007/s11064-014-1299-x
Rinninella E, Raoul P, Cintoni M, Franceschi F, Miggiano GAD, Gasbarrini A, Mele MC (2019) What is the healthy gut microbiota composition? A changing ecosystem across age, environment, diet, and diseases. Microorganisms 7(1):14
Sahin S, Alkan T, Temel SG, Tureyen K, Tolunay S, Korfali E (2010) Effects of citicoline used alone and in combination with mild hypothermia on apoptosis induced by focal cerebral ischemia in rats. J Clin Neurosci 17(2):227–231. https://doi.org/10.1016/j.jocn.2009.05.016
Saltzman ET, Palacios T, Thomsen M, Vitetta L (2018) Intestinal microbiome shifts, dysbiosis, inflammation, and non-alcoholic fatty liver disease. Front Microbiol 9:61
Savran M, Bekar A, Cansev M, Tolunay S, Ulus I, Taşkapılıoğlu M (2012) Prevention of Epidural Fibrosis in rats by local or systemic administration of Citicoline. Turk Neurosurg 22:634–640. https://doi.org/10.5137/1019-5149.JTN.6008-12.0
Sharma RS, Harrison DJ, Kisielewski D, Cassidy DM, McNeilly AD, Gallagher JR, Walsh SV, Honda T, McCrimmon RJ, Dinkova-Kostova AT, Ashford MLJ, Dillon JF, Hayes JD (2018) Experimental nonalcoholic steatohepatitis and liver fibrosis are ameliorated by pharmacologic activation of Nrf2 (NF-E2 p45-Related factor 2). Cell Mol Gastroenterol Hepatol 5(3):367–398. https://doi.org/10.1016/j.jcmgh.2017.11.016
Sharpton SR, Maraj B, Harding-Theobald E, Vittinghoff E, Terrault NA (2019) Gut microbiome–targeted therapies in nonalcoholic fatty liver disease: a systematic review, meta-analysis, and meta-regression. Am J Clin Nutr 110(1):139–149
Shen F, Zheng R-D, Sun X-Q, Ding W-J, Wang X-Y, Fan J-G (2017) Gut microbiota dysbiosis in patients with non-alcoholic fatty liver disease. Hepatobiliary Pancreat Dis Int 16(4):375–381
Synoradzki K, Grieb P (2019) Citicoline: a Superior form of Choline? Nutrients 11(7). https://doi.org/10.3390/nu11071569
Velayudham A, Dolganiuc A, Ellis M, Petrasek J, Kodys K, Mandrekar P, Szabo G (2009) VSL# 3 probiotic treatment attenuates fibrosis without changes in steatohepatitis in a diet-induced nonalcoholic steatohepatitis model in mice. Hepatology (Baltimore MD) 49(3):989–997
Vernon G, Baranova A, Younossi Z (2011) Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non‐alcoholic steatohepatitis in adults. Aliment Pharmacol Ther 34(3):274–285
Wang Y, Ausman LM, Russell RM, Greenberg AS, Wang XD (2008) Increased apoptosis in high-fat diet-induced nonalcoholic steatohepatitis in rats is associated with c-Jun NH2-terminal kinase activation and elevated proapoptotic bax. J Nutr 138(10):1866–1871. https://doi.org/10.1093/jn/138.10.1866
Wang Y, Xu N, Xi A, Ahmed Z, Zhang B, Bai X (2009) Effects of Lactobacillus plantarum MA2 isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol diet. Appl Microbiol Biotechnol 84(2):341–347
Wieckowska A, Zein NN, Yerian LM, Lopez AR, McCullough AJ, Feldstein AE (2006) In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology (Baltimore MD) 44(1):27–33. https://doi.org/10.1002/hep.21223
Wong SH, Kwong TN, Chow T-C, Luk AK, Dai RZ, Nakatsu G, Lam TY, Zhang L, Wu JC, Chan FK (2017) Quantitation of faecal Fusobacterium improves faecal immunochemical test in detecting advanced colorectal neoplasia. Gut 66(8):1441–1448
Xue L, He J, Gao N, Lu X, Li M, Wu X, Liu Z, Jin Y, Liu J, Xu J (2017) Probiotics may delay the progression of nonalcoholic fatty liver disease by restoring the gut microbiota structure and improving intestinal endotoxemia. Sci Rep 7(1):1–13
Xu R-y, Wan Y-p, Fang Q-y, Lu W, Cai W (2011) Supplementation with probiotics modifies gut flora and attenuates liver fat accumulation in rat nonalcoholic fatty liver disease model. J Clin Biochem Nutr: 1108190104–1108190104
Yilmaz Y, Eren F (2017) Nonalcoholic steatohepatitis and gut microbiota: future perspectives on probiotics in metabolic liver diseases. Turk J Gastroenterol 28:327–328
Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M (2016) Global epidemiology of nonalcoholic fatty liver disease—meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology (Baltimore MD) 64(1):73–84
Zhang X, Han J, Man K, Li X, Du J, Chu ES, Go MY, Sung JJ, Yu J (2016) CXC chemokine receptor 3 promotes steatohepatitis in mice through mediating inflammatory cytokines, macrophages and autophagy. J Hepatol 64(1):160–170. https://doi.org/10.1016/j.jhep.2015.09.005
Zhu J, Wan X, Wang Y, Zhu K, Li C, Yu C, Li Y (2017) Serum fetuin B level increased in subjects of nonalcoholic fatty liver disease: a case-control study. Endocrine 56(1):208–211. https://doi.org/10.1007/s12020-016-1112-5
Zhu L, Baker SS, Gill C, Liu W, Alkhouri R, Baker RD, Gill SR (2013) Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH. Hepatology (Baltimore MD) 57(2):601–609
Author A.F. E-K express his appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia, for funding this work through research group program under grant number (RGP. 2/232/44).
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
The experimental protocol was permitted by Research Ethics Committee, Faculty of Pharmacy, Delta University for Science and Technology (ethical approval number: FPDU6/2022).
Consent for publication
Conflict of interest
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
El-Baz, A.M., El-Ganiny, A.M., Hellal, D. et al. Valuable effects of lactobacillus and citicoline on steatohepatitis: role of Nrf2/HO-1 and gut microbiota. AMB Expr 13, 57 (2023). https://doi.org/10.1186/s13568-023-01561-8
- Gut microbiota