Early intervention with probiotics and metformin alleviates liver injury in NAFLD rats via targeting gut microbiota dysbiosis and p-AKT/mTOR/LC-3II pathways

Abstract

Non-alcoholic fatty liver disease (NAFLD) constitutes a major health problem worldwide and intimately links with obesity and diabetes. This study aimed to explore the therapeutic impact of early treatment with metformin (MTF) alone or in combination with Lactobacillus reuteri DSM 17938 (L. reuteri) + metronidazole (MTZ) in male Sprague Dawley rats with high-fat diet (HFD)-induced NAFLD. Hepatic steatosis was induced by feeding rats HFD for 6 weeks. MTF (150 mg/kg/day) or L. reuteri (2 × 10⁹ colony forming unit/day) were given orally for 4 weeks; meanwhile, MTZ (15 mg/kg/day, p.o.) was administered for 1 week. Administration of L. reuteri + MTZ in combination with MTF produced a superior effect concerning insulin resistance (IR), lipid profile, liver function, oxidative stress, inflammatory and autophagic markers than using each treatment alone. Besides, this combination resulted in disappearance of steatosis, inflammation and vacuolation within hepatic architecture. Moreover, it normalized short chain fatty acids (SCFAs) as well as Firmicutes and Bacteroidetes faecal contents. In conclusion, early treatment with L. reuteri + MTZ in combination with MTF could prevent NAFLD progression and liver injury through targeting gut dysbiosis, inflammation and autophagic pathways.

Keywords

Autophagy dysbiosis inflammation NAFLD

Introduction

Non-alcoholic fatty liver disease (NAFLD) is considered one of the major troubling diseases worldwide as we are moving toward a gradual decline of viral hepatitis and a progressive rise in obesity and type-2 diabetes mellitus (T2DM).¹ Globally, the prevalence of NAFLD is estimated at ∼25% and is rising to 75% in obese people.² The reported rates are most noteworthy within the Middle East (32%) and South America (31%) and least in Africa (13.5%).³

NAFLD encompasses a broad spectrum of diseases extended from simple steatosis (SS) at early stages to non-alcoholic steatohepatitis (NASH) which can lead to fibrosis.⁴ While hepatic steatosis itself is frequently clinically silent, this lesion predisposes the liver to serious damage by additional insults, such as exposure to lipopolysaccharide (LPS), certain hepatotoxins or infectious agents, driving to the eventual development of cirrhosis.⁵

The pathogenesis of NAFLD is highly complicated and multifactorial; with two hit hypothesis, the first of which is predominantly caused by accumulation of lipids in hepatocytes, contributing to apoptosis and excessive oxidative stress. The second hit is driven by invading immune cells, which release inflammatory mediators such as cytokines.⁶ Lately, it became rapidly evident that the two hit hypothesis is too simple to recapitulate the complexity of human NAFLD where multiple parallel factors, including insulin resistance (IR), dysbiosis and autophagy acting synergistically are implicated in NAFLD development and progression. IR possesses a crucial role in the development of NAFLD by causing increased lipolysis, promoting hepatocyte injury and inflammation as well as restraining of autophagy, which in turn increases lipid stores and can worse NAFLD.^7,8

Actually, dysbiosis, a compositional change in gut microbiota, has been associated with metabolic risk factors such as IR, T2DM, cardiovascular disease and obesity as well as increased gut permeability to dietary factors and bacterial immunogens, thereby enhancing hepatic exposure to injurious stimuli triggering the development of NAFLD and promoting hepatic inflammation and fibrogenesis.^9,10 The phylum Bacteroidetes is the predominant species of intestinal microbiota, found in 68% of the individuals followed by phylum Firmicutes, which accounts for 40% in many individuals.¹¹ So, the use of probiotic or symbiotic may have a role in attenuating the development and progression of NAFLD.

The current gold standard for the management of NAFLD is weight loss and minimizing metabolic risk factors.¹² However, many patients battled to follow the recommended life style modifications.⁴ Moreover, insulin-sensitizing agents, lipid-lowering drugs and antioxidants have been utilized as treatment alternatives for the management of NAFLD. Nevertheless, the majority of these treatments did not provide complete improvement.⁴ Previous studies indicated that metformin (MTF), as an anti-diabetic drug, could modulate gut microbiota composition in experimental animals with high fat diet (HFD) induced obesity beside its effect on reducing IR which is considered as a critical factor in accelerating NAFLD progression.^13,14 Currently, there are no completely successful medications for the prevention of NAFLD, but our previous research has demonstrated an important role for Lactobacillus reuteri (L. reuteri) (as Firmicutes source) and metronidazole (MTZ) (antibiotic against Bacteroidetes) alone, or in combination with MTF in the treatment of HFD-induced NASH.¹⁵ Whereas, this combined treatment showed superior benefits through modulation of IR and hepatic steatosis as well as engulfing lipids accumulation in the liver through autophagy induction and LPS/TLR4 signaling inhibition. However, the present study investigated the impact of early intervention using this combined regimen on the prevention of NAFLD progression to NASH in NAFLD-induced rats through assessment of liver function, oxidative stress, IR, inflammatory and autophagic markers as well as histopathological and immunohistochemistry studies.

Materials and methods

Animals

Male Sprague Dawley rats (120–180 g) were maintained at the Schistosome Biological Supply Center (SBSC), Theodor Bilharz Research Institute (TBRI), Giza, Egypt. kept under controlled temperature (25 ± 2°C), humidity and lighting (12 h light/dark) with free access to standard rat chow and tap water ad libitum. Animal experiments were conducted in accordance with the guide for care and use of laboratory animals of the National Institutes of Health (NIH) after approval by the Ethics Committee at Faculty of Pharmacy, Cairo University (Permit Number: PT 2072) and the Institutional Review Board of TBRI (FWA 0010609). In this study, all sources of contamination were decreased; where rats were housed in a sterilized room, food pellets were sterilized in an oven (100ºC) for 30 min, additionally, water, drinking bottles, bedding and cages were autoclaved to ensure the presence of only the normal commensals.

Drugs and chemicals

L. reuteri (Ewopharma AG, Romania, batch No: 7TSA054), MTZ (ARENA GROUP SA, Romania, batch No: 0300317) and MTF (Chemical Industries Development, Giza Egypt, under licence of: Madaus GmbH. Germany, batch No: 03180385) were used in the present study. Fine chemicals and reagents, unless otherwise specified, were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Experimental groups and samples collection

Rats were randomly divided into five groups (n = 8); group I served as normal group. Groups II–V fed with HFD consisted of 25% fats and 1% cholesterol (Win lab Laboratory chemicals) in addition to 0.25% bile salts (Alpha chemika, India).¹⁶ After 6 weeks, group II was left without treatment, meanwhile, groups III–V were given orally MTF (150 mg/kg/day),¹⁷ L. reuteri DSM 17938 (2 × 10⁹ colony forming unit/day)¹⁸ and MTZ (15 mg/kg/day)¹⁹ or L. reuteri DSM 17938 + MTZ in combination with MTF, respectively. All the treatments were administered for four successive weeks, except for MTZ that was given for 1 week where long lasting impacts on gut microbiota composition have been recorded with the advantage of reducing its side effects.²⁰

At the end of treatment, faeces were collected aseptically over 24 h for determination of acetate: propionate: butyrate ratios and Firmicutes and Bacteroidetes faecal contents. After overnight fasting, rats were euthanized by cervical dislocation under anesthesia and blood was collected from the retro-orbital sinus using non-heparinized capillary tubes for serum separation. Furthermore, the whole livers were immediately removed, washed with ice-cold saline and frozen at −80 °C pending assay.

Assessed parameters

Homeostasis model assessment of insulin resistance (HOMA-IR)

Serum fasting glucose (enzymatic colorimetric kit, Biodiagnostic, Egypt) and insulin (rat ELISA kit, Cloud-Clone Corp, USA) levels were determined. HOMA-IR was calculated according to the following equation: HOMA-IR = fasting glucose (mg/dl) × fasting insulin (μIU/ml)/405.²¹

Liver function and lipid profile markers

Serum alanine and aspartate aminotransferases (ALT & AST) as well as lipid profile including total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL) were determined according to manufacturer’s instructions using the available commercial kits (Spectrum, MDSS, Hannover, Germany). Low-density lipoprotein (LDL) was calculated using Friedewald formula: [LDL = TC − HDL − TG/5].

Oxidative stress, inflammatory and autophagic markers

One gram of liver tissues was homogenized (Pro Scientific Inc., U.S.A. homogenizer) in ice-cold 100 mM KH₂PO₄ buffer (1:4 w/v; pH 7.4) and centrifuged at 10,000 g for 1 h at 4°C. In liver homogenates, the protein content,²² reduced glutathione (GSH) content²³ and lipid peroxidation products, expressed by malondialdehyde (MDA) formation²⁴ were determined. Moreover, LPS, nuclear factor kappa-B (NF-kB), tumor necrosis factor-alpha (TNF-α) as inflammatory markers as well as mechanistic target of rapamycin (mTOR) and phosphorylated AKT (p-AKT) as autophagic markers were estimated using rat ELISA kits (Cloud-Clone Corp, USA).

Determination of short chain fatty acids (SCFAs) fecal ratios

SCFAs were extracted and estimated as described by Lee et al.²⁵ using HPLC method. Briefly, the extracted samples (20 µl) containing SCFAs (acetate, propionate and butyrate) were separated on Coregel 87H1, 300 × 7.8 mm (Concise separations, San Jose, USA) at 65 ºC. Filtered sulphuric acid (0.01 N H₂SO₄, HPLC grade) (Fisher Chemical, UK) was used as a mobile phase at a flow rate of 0.6 ml/min. The corresponding peak areas of acetate, propionate and butyrate were calculated from the corresponding calibration curves (0.02 M to 0.32 M for acetic acid or propionic acid and 0.04 M to 0.64 M for butyric acid) with correlation coefficient = 0.999 and results were expressed as μmol/g wet faeces.

Determination of Firmicutes and Bacteroidetes faecal contents

Bacterial DNA was isolated from the faecal samples using the QIAamp DNA Stool Mini Kit (Qiagen Hidden, Germany) according to the manufacturer’s instruction. Five µl of isolated DNA was used in a total volume of 20 µl, containing 10 µl 2x SYBR Green PCR Master Mix (Power up™ SYBR™ Green Master Mix, Thermo Scientific, USA) and 5 pmole of each primer. The sequence of the primers used was listed in Table 1.^26–28 Firmicutes and Bacteroidetes faecal contents were calculated based on relative comparative quantitation method from the 2^–ΔΔCT formula using the internal standard gene (universal bacteria).²⁹

Table 1.

Primer sequences for real-time PCR analysis.

Target organism(s)	Amplicon length (bp)	Primer sequence
Universal bacteria primer	67	Forward primer: 5-AAACTCAAAKGAATTGACGG -3
Universal bacteria primer	67	Reverse primer: 5-CTCACRRCACGAGCTGAC-3
Firmicutes	126	Forward primer: 5-GAGYATGTGGTTTAATTCGAAGCA-3
Firmicutes	126	Reverse primer: 5-AGCTGACGACAACCATGCAC-3
Bacteroidetes	136	Forward primer: 5-GAGAGGAAGGTCCCCCAC-3
Bacteroidetes	136	Reverse primer: 5-CGCTACTTGGCTGGTTCAG-3

Histological examination

Five µm thick sections of liver specimens were stained with hematoxylin & eosin (H&E) and examined morphologically by semi-quantitative analysis, at high power microscopic fields of ×200/section, using the computerized image analysis system (Axiovision version 4.8, Zeiss Germany). Liver sections were graded according to the following criteria for steatosis: mild = 5%−30%, moderate = 30%−60%, and severe > 60% of hepatocytes affected and for inflammation and vacuolation: mild = 0%−25%, moderate = 26%−50%, and severe > 51% of hepatocytes affected.³⁰

Immunohistochemical examination

Paraffin sections were deparaffinised, rehydrated and endogenous peroxidase was inactivated, then incubated overnight at 4ºC with anti-rat toll like receptor-4 (TLR4) and anti-class II microtubule-associated protein light chain 3 (LC-3II) primary antibodies (Santa Cruz Biotechnology, USA) at dilutions of 1:100 and 1:150, respectively. Then, sections were washed with PBS and incubated at room temperature for 30 min with secondary antibody (Abcam, Cambridge, USA). The percentages of TLR4 and LC-3II positively stained brown hepatocytes in 10 successive fields at magnification of x400/section were calculated.

Statistical analysis

All data are presented as Mean ± standard error of the mean (SEM). Results were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test (SPSS, software package version 16.0, Chicago, IL, USA). P values < 0.05 were considered statistically significant.

Results

Effect of treatments on glucose and insulin levels and HOMA-IR

Table 2 shows significant elevations (p < 0.05) in fasting glucose and insulin levels as well as HOMA-IR about ∼1.5, 1 and 2 fold, respectively in NAFLD-induced rats when compared to their corresponding values in normal group. Compared to HFD group, administration of all treatments caused normalization of fasting glucose and insulin levels as well as HOMA-IR.

Table 2.

Effect of MTF or co-administration of L. reuteri and MTZ alone or their combination on serum fasting glucose, insulin and HOMA-IR in rats with induced NAFLD.

Groups	Fasting glucose (mg/dL)	Insulin (pg/ml)	HOMA-IR
Normal	74.33 ± 1.96	24.53 ± 0.60	0.65 ± 0.02
HFD	110.00 ± 3.69^a	32.53 ± 1.55^a	1.27 ± 0.88^a
HFD + MTF	78.33 ± 2.1^b	27.14 ± 0.47^b	0.75 ± 0.02^b
HFD + MTF	(–28.79%)	(–15.17%)	(–40.95%)
HFD + (L. reuteri + MTZ)	81.70 ± 2.34^b	28.21 ± 1.05^b	0.82 ± 0.04^b
HFD + (L. reuteri + MTZ)	(–25.73%)	(–13.28%)	(–35.43%)
HFD + MTF + (L. reuteri + MTZ)	76.21 ± 2.93^b	26.89 ± 0.76^b	0.73 ± 0.04^b
HFD + MTF + (L. reuteri + MTZ)	(–30.72%)	(–17.34%)	(–42.52%)

Values presented are means of 8 rats ± SEM. Values in parenthesis represent percentage decrease (–) relative to HFD group. Statistical analysis was carried out using one-way ANOVA followed by Tukey-post hoc test. ^{a, b} Significantly different from normal or HFD groups at p < 0.05, respectively. HOMA-IR: Homeostasis model assessment of insulin resistance, HFD: high-fat diet; MTF: metformin; L. reuteri: Lactobacillus reuteri DSM 17938; MTZ: metronidazole; NAFLD: non-alcoholic fatty liver disease.

Effect of treatments on liver function, lipid profile and oxidative stress markers

Administration of MTF alone caused a significant (p < 0.05) decrease in ALT and AST serum levels by ∼15% and 14%, respectively, and normalization of TC level with a significant (p < 0.05) improvement of TG, HDL and LDL by ∼15%, 51% and 24%, respectively when compared to their corresponding values in HFD group (Table 3). Meanwhile, the co-administration of L. reuteri + MTZ alone or their combination with MTF is able to normalize ALT and AST serum levels, as well as all the lipid profile markers except for HDL, with a substantial difference (p < 0.05) in the ALT level relative to the MTF-treated group. Treatment of HFD with MTF or L. reuteri + MTZ diminished (p < 0.05) MDA levels to 11.36 ± 0.33 and 8.78 ± 0.41 respectively, as well as increased (p < 0.05) GSH contents to 0.92 ± 0.03 and 1.08 ± 0.08 respectively, relative to their corresponding HFD groups (13.34 ± 0.04 and 0.59 ± 0.07 respectively). Meanwhile, administration of the combined regimen succeeded to normalize both GSH and MDA contents, with significant (p < 0.05) difference when compared to the MTF group.

Table 3.

Effect of MTF or co-administration of L. reuteri and MTZ alone or their combination on liver functions and lipid profile markers in rats with induced NAFLD.

Groups	ALT	AST	TC	TG	HDL	LDL
Groups	(IU/ml)	(IU/ml)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)
Normal	63.59 ± 1.4	132.77 ± 3.36	104.60 ± 4.03	130.20 ± 7.22	43.21 ± 1.29	35.35 ± 3.66
HFD	94.75 ± 4.07^a	182.90 ± 8.09^a	131.59 ± 5.09^a	182.59 ± 6.48^a	18.39 ± 0.75^a	76.68 ± 4.91^a
HFD + MTF	80.09 ± 1.62^ab	157.53 ± 2.27^ab	117.35 ± 0.86^b	154.35 ± 2.40^ab	27.74 ± 0.79^ab	58.54 ± 1.29^ab
HFD + MTF	(–15.47%)	(–13.87%)	(–10.82%)	(–15.47%)	(+50.84%)	(–23.66%)
HFD + (L. reuteri + MTZ)	70.99 ± 0.89^bc	146.13 ± 5.63^b	110.44 ± 2.03^b	150.75 ± 5.93^b	31.99 ± 1.58^ab	45.80 ± 3.33^b
HFD + (L. reuteri + MTZ)	(–25.08%)	(–20.10%)	(–16.07%)	(–17.44%)	(+73.95%)	(–40.27%)
HFD + MTF + (L. reuteri + MTZ)	65.24 ± 0.88^bc	143.84 ± 4.97^b	108.97 ± 3.15^b	143.59 ± 4.00^b	34.85 ± 3.60^ab	47.41 ± 5.85^b
HFD + MTF + (L. reuteri + MTZ)	(–31.15%)	(–21.36%)	(–17.19%)	(–21.36%)	(+89.51%)	(–38.17%)

Values presented are means of 8 rats ± SEM. Values in parenthesis represent percentage decrease (–) relative to HFD group. Statistical analysis was carried out using one-way ANOVA followed by Tukey-post hoc test. ^{a, b, c} Significantly different from normal, HFD or MTF groups at p < 0.05, respectively. ALT: alanine aminotransferase; AST: aspartate aminotransferase; TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein; LDL: low-density lipoprotein; HFD: high-fat diet; MTF: metformin; L. reuteri: Lactobacillus reuteri DSM 17938; MTZ: metronidazole; NAFLD: non-alcoholic fatty liver disease.

Effect of treatments on inflammatory and autophagy markers

Administration of MTF or L. reuteri + MTZ decreased the levels of inflammatory markers as LPS (∼13%, 17%), NF-kB (∼14%, 21%) and TNF-α (∼21%, 37%) as well as mitigated mTOR and p-AKT levels as autophagy markers by ∼16%, 21% and 31%, 34% respectively, when compared to HFD group (Figure 1). Conversely, treatment with the combined regimen has been effective in normalizing their hepatic levels.

Figure 1.

Effect of MTF alone or in combination with L. reuteri + MTZ on LPS, NF-kB and TNF-α as well as mTOR and p-AKT in HFD-induced hepatic steatosis rats. Values presented are means of 6−8 rats ± SEM. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey-post hoc test. ^{a, b, c, d} Significantly different from normal, HFD, MTF or L. reuteri + MTZ groups at p < 0.05, respectively.

Effect of treatments on SCFs ratios and Firmicutes/Bacteroidetes faecal contents

The ratio of acetate: propionate: butyrate was 58:26:16 in normal rats. Rats fed with HFD revealed a significant (p < 0.05) increase in faecal acetate by 8% as well as a decline in faecal propionate and butyrate contents by ∼15% and 63%, respectively, with ratio of acetate: propionate: butyrate (71:23:6). In addition, a substantial decrease in faecal Firmicutes content by 33% along with an increase in faecal Bacteriodetes content by 22% was recorded when compared to their corresponding normal values (Figure 2). Treatment with MTF alone failed to cause a significant decline (p < 0.05) in faecal propionate and acetate contents, but significantly increased (p < 0.05) faecal butyrate content by ∼72% when compared to corresponding HFD groups. This was associated with improved faecal Firmicutes content, but with an insignificant change of faecal Bacteriodetes content. Meanwhile, treatment with L. reuteri + MTZ alone or their combination with MTF restored the normal faecal acetate, propionate and butyrate as well as faecal Firmicutes and Bacteroidetes contents with significant (p < 0.05) difference from the MTF group (Figure 2).

Figure 2.

Effect of MTF alone or in combination with L. reuteri + MTZ on acetate, propionate and butyrate as well as Firmicutes and Bacteroidetes faecal contents in HFD-induced hepatic steatosis rats. Values presented are means of 6−8 rats ± SEM. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey-post hoc test. ^{a, b, c} Significantly different from normal, HFD or MTF groups at p < 0.05, respectively.

Histopathological examination

Liver sections of normal rats (Figure 3A) revealed normal hepatic architecture with centrally located nuclei. Meanwhile, liver sections of HFD-induced NAFLD (Figure 3B) showed microvesicular steatosis (∼37%), mild inflammation (∼23%) and vacuolation (∼15%). Liver sections of MTF-treated rats (Figure 3C) revealed hepatocytes with early microvesicular steatosis (20%) as well as mild inflammation (∼13%) and vacuolation (∼6%). Administration of L. reuteri + MTZ (Figure 3D) resulted in healing of steatosis, mild inflammation (∼7%) and vacuolation (∼4%) with almost normal hepatic architecture. Co-administration of L. reuteri + MTZ in combination with MTF (Figure 3E) resulted in healing of steatosis (Figure 3F), inflammation (Figure 3G) and vacuolation (Figure 3H) with normal hepatic architecture.

Figure 3.

(A−H). Effect of MTF alone or in combination with L. reuteri + MTZ on hepatic histopathological changes (H&E) of normal control (A) showing normal histological appearance of the liver, hepatocytes arranged in thin plates and centrally located nuclei (black arrow), central vein (yellow arrow), blood sinusoids (red arrow), no fat accumulation is seen, HFD (B) showing macrovesicular steatosis hepatocytes with predominantly a single large-sized fat droplet (red arrow) multiple small to medium-sized fat droplets (yellow arrow), dilated congested sinusoids (black arrow) and intra-portal lymphocytes (green arrow), rats treated with MTF (C) showing hepatocytes with hepatic architecture and preserved arranged in thin plates, congested central vein and sinusoids with early microvesicular steatosis affecting mild-moderate number of hepatocytes (black arrow) with lymphocytes aggregates intra-parenchymal (red arrow), rats treated with L. reuteri + MTZ (D) showing almost normal hepatic architecture and hepatocytes arranged in thin plates, rats treated with the combination L. reuteri + MTZ + MTF (E) showing hepatocytes preserved hepatic architecture and arranged in thin plates, % of steatosis (F), % of inflammation (G) and % of vacuolation (H). Values presented are means of 6−8 rats ± SEM. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey-post hoc test. ^{a, b, c, d} Significantly different from normal, HFD, MTF and L. reuteri + MTZ groups at p < 0.05, respectively.

Immunohistochemical examination

Liver sections of normal rats showed very few (5%) positively stained hepatocytes of TLR4 as an inflammatory marker and LC-3II as an autophagy marker (Figures 4 & 5A) that were moderately and slightly increased to ∼48% and 18.83%, respectively in the HFD group (Figure 4 & 5B). Administration of MTF dramatically decreased TLR4 positively stained hepatocytes to about 23% (Figure 4C) and, conversely, it moderately increased LC-3II positively stained hepatocytes to ∼38% (Figure 5C). Administration of L. reuteri + MTZ alone or in combination with MTF showed few (∼20% and 11%, respectively) positively stained hepatocytes of TLR4 (Figure 4D & E). However, marked elevations in the numbers of LC-3II positively stained hepatocytes (∼48% and 56%, respectively) were observed after administration of L. reuteri + MTZ alone (Figure 5D) or when combined with MTF (Figure 5E).

Figure 4.

(A-F). Effect of MTF alone or in combination with L. reuteri + MTZ on hepatic TLR4 of normal control (A), HFD (B), rats treated with MTF (C), rats treated with L. reuteri + MTZ (D), rats treated with the combination L. reuteri + MTZ + MTF (E), the expression is nuclear/nucleocytoplasmic, black arrow indicate positive browen expression of TLR4 and percentage of positive stained cells with TLR4 (F). Values presented are means of 6−8 rats ± SEM. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey-post hoc test. ^{a, b, c, d} Significantly different from normal, HFD, MTF and L. reuteri + MTZ groups at p < 0.05, respectively.

Figure 5.

(A-F). Effect of MTF alone or in combination with L. reuteri + MTZ on hepatic LC-3II of normal control (A), HFD (B), rats treated with MTF (C), rats treated with L. reuteri + MTZ (D), rats treated with the combination L. reuteri + MTZ + MTF (E), the expression is nuclear/nucleocytoplasmic, black arrow indicate positive browen expression of LC-3II and percentage of positive stained cells with LC-3II (F). Values presented are means of 6−8 rats ± SEM. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey-post hoc test. ^{a, b, c, d} Significantly different from normal, HFD, MTF and L. reuteri + MTZ groups at p < 0.05, respectively.

Discussion

Gut dysbiosis plays a crucial factor in the pathogenesis of NAFLD, where the alteration in gut microbiota is contributing to NAFLD development by abnormal regulation of the liver gut axis, mainly by changing gut microbial components and microbial metabolites such as SCFAs,³¹ which trigger inflammation and subsequently the progression of NAFLD to NASH.³² Growing evidence proposes that at the early onset of a high-fat diet, not only bacterial products but also complete living bacteria can be translocated from the intestinal lumen toward tissues, such as the adipose tissue, or into the portal circulation, inducing liver damage.³³ Accordingly, we assumed that early intervention with probiotic and antibiotic to modulate gut microbiota could be a high priority to prevent NAFLD development and progression.

In the current study, hepatic steatosis was accompanied with increased ALT, AST, glucose, insulin, HOMA-IR, TC, TG and LDL levels along with decreased HDL. Moreover, increased lipid peroxidation expressed by MDA formation and decreased of GSH hepatic stores were also observed. The accumulated TGs trigger mitochondrial dysfunction and oxidative stress³⁴ and provoke the inflammatory processes in which noteworthy elevations of LPS, NF-kB and TNF-α hepatic contents as well as immunohistochemical increment in the number of positive TLR4 cells were observed in the current study. These results are also confirmed histopathologically where microvesicular steatosis, mild inflammation and vacuolation were observed. Intestinal inflammatory response caused by HFD impedes intestinal tight junctions leading to increased gut permeability, affecting both insulin sensitivity and energy balance.³⁵ Additionally, HFD resulted in inhibition of hepatic autophagy, as demonstrated in this study by increasing p-AKT and mTOR hepatic content along with decreasing the number of positive LC-3II cells immunohistochemically. These results are in harmony with Xiao et al.³⁶ who reported inhibition of autophagy in adult female rats nourished with HFD for 8 weeks to create NAFLD. Aside from the inhibition of autophagy, HFD induced gut dysbiosis, where the faecal content of Firmicutes decreased in addition to the rise in Bacteroidetes. Furthermore, HFD changed the composition of gut metabolites, as shown by the relative elevation of both acetate and propionate faecal contents with relative diminution of the butyrate faecal content, and this may be attributed to an increase in gram-negative bacteria, which in turn would increase LPS levels.³⁷ Acetate is the main SCFA in the colon and acts as a substrate for hepatic cholesterol synthesis and de novo lipogenesis.³⁸ Increased gluconeogenesis and lipogenesis due to increased acetate and propionate production may lead to NAFLD, obesity and subsequently IR and T2DM.³⁹ On the other hand, butyrate has a critical role in modulation of energy harvest, hepatic lipogenesis and gluconeogenesis, adipokine signaling in adipocytes, and intestinal permeability.⁴⁰ Moreover, a correlation between the increased levels of Bacteroidetes and NAFLD development/progression as well as escalation of the Bacteroidetes/Firmicutes ratio in NAFLD patients was previously reported.^41,42

In the current study, treatment with MTF improved ALT, AST, TG, LDL and HDL levels in addition to normalization of serum glucose, insulin, HOMA-IR and TC. This could be attributable to the activation of AMP-activated protein kinase, bringing down of blood glucose levels by decreasing hepatic gluconeogenesis, stimulating glucose uptake into muscles and increasing fatty acid oxidation in adipose tissue leading to reduction of irreversibly glycated LDL-C.⁴³ Furthermore, MTF improved GSH and MDA hepatic contents, which might be ascribed to improvement of dyslipidemia and reduction of TG accumulation, as well as diminished steatosis, inflammation and vacuolation histopathologically. This was associated with improved hepatic contents of LPS, NF-kB and TNF-α as well as a decline in the number of positive TLR4 cells immnuohistochemically. Indeed, Lin et al.⁴⁴ reported that MTF was successful at reversing fatty liver, probably through reduced hepatic expression of TNF-α, which promotes hepatic lipid accumulation and ATP depletion in obese and leptin-deficient mice. Treatment with MTF also moderately induced autophagy, which was observed in this study by diminishing p-AKT and mTOR hepatic contents along with elevating the number of positive LC-3II cells immunohistochemically. Although our findings demonstrated that MTF failed to improve Bacteroidetes, propionate and acetate faecal contents, yet it improved Firmicutes as well as butyrate faecal contents. The possible mechanism by which MTF affect gut microbiota is likely due to its action on increasing the abundance of Akkermansia muciniphila,¹³ which protects the mucus layer and decreases the permeability of the gut barrier, preventing the LPS from entering the blood circulation.⁴⁵

Probiotics have been appeared to have many beneficial health effects,⁴⁶ and its use may have significant modulatory impacts on gut microbiota by improvement of the abnormal lipid metabolism and gut microbiota dysbiosis.⁴⁷ Probiotic, which produces butyrate, is particularly critical where butyrate supplementation in obese mice helps to improve the integrity of the gastrointestinal wall and ameliorate insulin secretion from beta cells and hence diminishing the amount of adipose tissues in the body.³⁹ L. reuteri is considered one of these effective probiotics, which improve IR, increases liver β-oxidation, and subsequently decreases the adipose and liver weights, which may clarify the diminish in liver fat accumulations.^18,48 Similarly, administration of antibiotics could improve IR with lowering of lipogenesis and hepatic steatosis.⁴⁹ In the current study, treatment with L. reuteri and MTZ in combination with MTF achieved a superior effect than using each treatment alone. The anti-inflammatory effect of L. reuteri could be attributed to decreased TNF-α expression as well as stimulation of the production of satiety-inducing peptides.⁵⁰ Moreover, L. reuteri suppresses IL-8 secretion, which is a potent neutrophil-activating chemokine, and is released from intestinal epithelial cells in response to several pathogenic bacteria.⁵¹ Herein, treatment with L. reuteri + MTZ alone or in combination with MTF succeeded to modulate gut dysbiosis as appeared by normalization of faecal contents of Firmicutes and Bacteroidetes as well as acetate, butyrate and propionate. Furthermore, the combined regimen possessed outstanding effect in inducing hepatic autophagy, as illustrated by increasing the hepatic p-AKT and mTOR contents and an increase in the number of positive LC-3II cells versus each treatment alone. This was accompanied with prevention of NAFLD development as shown histopathologically by reversal of steatosis, inflammation and vacoulation with normal hepatic architecture. The increment in autophagy has important metabolic effects such as enhancing insulin sensitivity, reducing hepatic lipid accumulation, providing a cytoprotective effect against cytokines and oxidant induced-injury and decreasing macrophage activation leading to a decrease in inflammation.⁵² This denotes that therapies designed to increase autophagy may be an effective treatment for this disease.

Conclusions

Gut–liver axis plays a pivotal role in the pathogenesis of NAFLD, mainly through the crosstalk of the intestinal microbiota with the host immune system modulating inflammation, insulin resistance and intestinal permeability. Therefore, finding novel mechanisms for the pathogenesis of NAFLD, including the gut microbiota in particular, could affirm new research areas to develop new therapeutic targets. Our study is an attempt to shed light on significance of gut dysbiosis modulation and autophagy as potential therapeutic targets for treatment of HFD-induced hepatic steatosis, inflammation and injury. More precisely, it could be concluded that early treatment with L. reuteri + MTZ in combination with MTF could prevent the progression of NAFLD, steatosis and liver injury through targeting of gut dysbiosis and p-AKT/mTOR//LC-3II pathways. Therefore, concurrent administration of both medications may have a therapeutic value in NAFLD patients with simple steatosis conferring a protection against NAFLD progression and complications.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work is supported by the internal research project 117/A (PI: SH Seif el-Din) for basic and applied research, a grant from Theodor Bilharz Research Institute.

ORCID iDs

Sayed H Seif el-Din

NM El-Lakkany

OA Hammam

References

Masarone

Federico

Abenavoli

, et al. Non-alcoholic fatty liver: epidemiology and natural history. Rev Recent Clin Trials 2014; 9: 126–133.

Younossi

Koenig

Abdelatif

, et al. Global epidemiology of non-alcoholic fatty liver disease-meta analytic assessment of prevalence, incidence and outcomes. Hepatology 2015; 64: 73–84.

Younossi

Anstee

Marietti

, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 2017; 15: 11–20.

Chalasani

Younossi

Lavine

, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018; 67: 328–357.

Diehl

. Cytokine regulation of liver injury and repair. Immunol Rev 2000; 174: 160–171.

Buzzetti

Pinzani

Tsochatzis

. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism 2016; 65: 1038–1048.

Singh

Kaushik

Wang

, et al. Autophagy regulates lipid metabolism. Nature 2009; 458: 1131–1135.

Ganz

Szabo

. Immune and inflammatory pathways in NASH. Hepatol Int 2013; 7: 771–781.

Aron-Wisnewsky

Vigliotti

Witjes

, et al. Gut microbiota and human NAFLD: disentangling microbial signatures from metabolic disorders. Nat Rev Gastroenterol Hepatol 2020; 17: 279–297.

10.

Saltzman

Palacios

Thomsen

, et al. Intestinal microbiome shifts, dysbiosis, inflammation, and non-alcoholic fatty liver disease. Front Microbiol 2018; 9: 61.

11.

Alisi

Bedogni

Baviera

, et al. Randomised clinical trial: the beneficial effects of VSL#3 in obese children with non-alcoholic steatohepatitis. Aliment Pharmacol Ther 2014; 39: 1276–1285.

12.

Raman

Ahmed

Gillevet

, et al. Fecal microbiome and volatile organic compound metabolome in obese humans with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol 2013; 11: 868–875.

13.

Lee

. Effect of metformin on metabolic improvement and gut microbiota. Appl Environ Microbiol 2014; 80: 5935–5943.

14.

Brunkwall

Orho Melander

. The gut microbiome as a target for prevention and treatment of hyperglycaemia in type 2 diabetes: from current human evidence to future possibilities. Diabetologia 2017; 60: 943–951.

15.

Ahmed

Salem

Seif El-Din

, et al. Gut microbiota modulation as a promising therapy with metformin in rats with non-alcoholic steatohepatitis: role of LPS/TLR4 and autophagy pathways. Eur J Pharmacol 2020; 887: 173461.

16.

El-Lakkany

Seif El-Din

Sabra

, et al. Co-administration of metformin and N-acetylcysteine with dietary control improves the biochemical and histological manifestations in rats with non-alcoholic fatty liver. Res Pharm Sci 2016; 11: 374–382.

17.

Woo

, et al. Metformin ameliorates hepatic steatosis and inflammation without altering adipose phenotype in diet-induced obesity. PLoS One 2014; 9: e91111.

18.

Hsieh

Lee

Chai

, et al. Oral administration of Lactobacillus reuteri GMNL-263 improves insulin resistance and ameliorates hepatic steatosis in high fructose-fed rats. Nutr Metab (Lond) 2013; 10: 35.

19.

Freund

Muggia-Sullam

La France

, et al. A possible beneficial effect of metronidazole in reducing TPN-associated liver function derangements. J Surg Res 1985; 38: 356–363.

20.

Haak

Lankelma

Hugenholtz

, et al. Long-term impact of oral vancomycin, ciprofloxacin and metronidazole on the gut microbiota in healthy humans. J Antimicrob Chemother 2019; 74: 782–786.

21.

Matthews

Hoskerm

Rudenski

, et al. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: 412–419.

22.

Lowry

Rosebrough

Farr

, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275.

23.

Ellman

. Tissue sulfhydryl groups. Arch Biochem Biophys 1959; 82: 70–77.

24.

Ohkawa

Ohishi

Yagi

. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–358.

25.

Lee

Park

, et al. Fermentation of rice using amylolytic Bifidobacterium. Int J Food Microbiol 1999; 50: 155–161.

26.

Layton

McKay

Williams

, et al. Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water. Appl Environ Microbiol 2006; 72: 4214–4224.

27.

Guo

Xia

Tang

, et al. Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs. Lett Appl Microbiol 2008; 47: 367–373.

28.

Yang

Chen

Yang

, et al. Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in mouse feces. Appl Environ Microbiol 2015; 81: 6749–6756.

29.

Livak

Schmittgen

. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402–408.

30.

Kleiner

Brunt

Van Natta

, et al. Design and validation of a histopathological scoring in non-alcoholic fatty liver disease. Hepatology 2005; 41: 1313–1321.

31.

Jasirwan

COM

Lesmana

CRA

Hasan

, et al. The role of gut microbiota in non-alcoholic fatty liver disease: pathways of mechanisms. Biosci Microbiota Food Health 2019; 38: 81–88.

32.

Del Campo

Gallego

Grande

. Role of inflammatory response in liver diseases: therapeutic strategies. World J Hepatol 2018; 10: 1–7.

33.

Chakaroun

Massier

Kovacs

. Gut microbiome, intestinal permeability, and tissue bacteria in metabolic disease: Perpetrators or bystanders? Nutrients 2020; 12: 1082.

34.

Lau

Carvalho

Freitas

. Gut microbiota: association with NAFLD and metabolic disturbances. Biomed Res Int 2015; 2015: 979515.

35.

Rau

Rehman

Dittrich

, et al. Fecal SCFAs and SCFA-producing bacteria in gut microbiome of human NAFLD as a putative link to systemic T-cell activation and advanced disease. United Eur Gastroenterol J 2018; 6: 1496–1507.

36.

Xiao

Guo

Fung

, et al. Garlic-derived S-allylmercaptocysteine ameliorates non-alcoholic fatty liver disease in a rat model through inhibition of apoptosis and enhancing autophagy. Evid Based Complement Alternat Med 2013; 2013: 642920.

37.

Chakraborti

. New-found link between microbiota and obesity. World J Gastrointest Pathophysiol 2015; 6: 110–119.

38.

Allin

Nielsen

Pedersen

. Mechanisms in endocrinology: gut microbiota in patients with type 2 diabetes mellitus. Eur J Endocrinol 2015; 172: 167–177.

39.

Sohail

Althani

Anwar

, et al. Role of the gastrointestinal tract microbiome in the pathophysiology of diabetes mellitus. J Diabetes Res 2017; 2017: 9631435.

40.

Goffredo

Mass

Parks

, et al. Role of gut microbiota and short chain fatty acids in modulating energy harvest and fat partitioning in youth. J Clin Endocrinol Metab 2016; 101: 4367−4376.

41.

Boursier

Mueller

Barret

, et al. The severity of non-alcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota. Hepatology (Baltimore, MD) 2016; 63: 764–775.

42.

Sobhonslidsuk

Chanprasertyothin

Pongrujikorn

, et al. The association of gut microbiota with non-alcoholic steatohepatitis in Thais. BioMed Res Int 2018; 2018: 9340316.

43.

Foretz

Hébrard

Leclerc

, et al. Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state. J Clin Invest 2010; 120: 2355–2369.

44.

Lin

Yang

Chuckaree

, et al. Metformin reverses fatty liver disease in obese, leptin-deficient mice. Nat Med 2000; 6: 998–1003.

45.

de la Cuesta-Zuluaga

Mueller

Corrales-Agudelo

, et al. Metformin is associated with higher relative abundance of mucin-degrading Akkermansia muciniphila and several short-chain fatty acid-producing microbiota in the gut. Diabetes Care 2017; 40: 54−62.

46.

Gorbach

. Probiotics and gastrointestinal health. Am J Gastroenterol 2000; 95: S2–4.

47.

Yadav

Lee

Lloyd

, et al. Beneficial metabolic effects of a probiotic via butyrate-induced GLP-1 hormone secretion. J Biol Chem 2013; 288: 25088–25097.

48.

Poutahidis

Kleinewietfeld

Smillie

, et al. Microbial reprogramming inhibits Western diet-associated obesity. PLoS One 2013; 8: e68596.

49.

Membrez

Blancher

Jaquet

, et al. Gut microbiota modulation with norfloxacin and ampicillin enhances glucose tolerance in mice. FASEB J 2008; 22: 2416–2426.

50.

Dahiya

Puniya

. Isolation, molecular characterization and screening of indigenous lactobacilli for their abilities to produce bioactive conjugated linoleic acid (CLA). J Food Sci Technol 2017; 54: 792–801.

51.

Ceponis

Botelho

Richards

, et al. Interleukins 4 and 13 increase intestinal epithelial permeability by a phosphatidylinositol 3-kinase pathway. Lack of evidence for STAT 6 involvement. J Biol Chem 2000; 275: 29132–29137.

52.

Czaja

. Function of autophagy in non-alcoholic fatty liver disease. Dig Dis Sci 2016; 61: 1304–1313.