Vinpocetine and Lactobacillus improve fatty liver in rats: role of adiponectin and gut microbiome

Abstract

Therapeutics that interfere with the damage/pathogen-associated molecular patterns (DAMPs/PAMPs) have evolved as promising candidates for hepatic inflammation like that occurring in non-alcoholic fatty liver disease (NAFLD). In the current study, we examined the therapeutic impact of the phosphodiesterase-1 inhibitor vinpocetine (Vinpo), alone or when combined with Lactobacillus, on hepatic abnormalities caused by a 13-week high-fat diet (HFD) and diabetes in rats. The results show that Vinpo (10 and 20 mg/kg/day) dose-dependently curbed HFD-induced elevation of liver injury parameters in serum (ALT, AST) and tissue histopathology. These effects were concordant with Vinpo’s potential to ameliorate HFD-induced fibrosis (Histological fibrosis score, hydroxyproline, TGF-β₁) and oxidative stress (MDA, NOx) alongside restoring the antioxidant-related parameters (GSH, SOD, Nrf-2, HO-1) in the liver. Mechanistically, Vinpo attenuated the hepatocellular release of DAMPs like high mobility group box (HMGB)1 alongside lowering the overactivation of the pattern recognition receptors including, toll-like receptor (TLR)4 and receptor for advanced glycation end-products (RAGE). Consequently, there was less activation of the transcription factor nuclear factor-kappa B that lowered production of the proinflammatory cytokines TNF-α and IL-6 in Vinpo-treated HFD/diabetes rats. Compared to Vinpo treatment alone, Lactobacillus probiotics as adjunctive therapy with Vinpo significantly improved the disease-associated inflammation and oxidative stress injury, as well as the insulin resistance and lipid profile abnormalities via enhancing the restoration of the symbiotic microbiota. In conclusion, combining Vinpo and Lactobacillus probiotics may be a successful approach for limiting NAFLD in humans.

Introduction

Non-alcoholic fatty liver disease (NAFLD) involves an abnormal fat deposition in the liver hepatocytes (> 5%) of patients who are not ingesting alcohol. This metabolic hepatic disorder has a high prevalence percentage ranging from 25 to 30% in adults (Younossi et al. 2018). The pathogenic sequence of NAFLD starts with simple fatty liver (steatosis) and may transfer to steatohepatitis (NASH), followed by fibrosis, cirrhosis (Lonardo et al. 2018) and hepatocellular carcinoma (HCC) (Younossi et al. 2018). Insulin resistance makes the liver susceptible to other factors that trigger hepatocellular damage. Hyperinsulinemia elicits a rise in hepatic de novo lipogenesis alongside adipose tissue lipolysis that synergistically drives higher transfer of free fatty acids toward the hepatocytes of the liver (Postic and Girard 2008). Oxidative damage, adipokines dysregulation, inflammatory mediators, and hepatic stellate cell activation are involved in promoting NAFLD progression (Yilmaz 2012).

The ester of vincamine known as vinpocetine (Vinpo) is extracted from the periwinkle plant. Vinpo has a cerebroprotective impact that can ameliorate brain disorders, including memory disturbances, cognitive impairment, stroke, and dementia (Zhao et al. 2011). Besides possessing a high safety profile, no substantial toxicity for Vinpo has been reported on long-term use so far (Zhang et al. 2018). Vinpo causes vasodilation by inhibiting the cyclic nucleotide phosphodiesterase-1 (PDE-1), voltage-gated sodium channels, and calcium channels, and raising oxygen and glucose consumption in the brain. Besides, Vinpo has antioxidant, anti-inflammatory, and antithrombotic effects (Essam et al. 2019).

The gut-liver axis (GLA) is a bidirectional functional relationship that exists between the gastrointestinal tract and the liver. Changes in one component, like the gut microbiota or gut barrier, will affect the liver condition due to the close functional and anatomical association (Mazzotti et al. 2016). Dysbiosis of the gut microbiota and the intestinal epithelial barrier has also been attributed to various diseases, including fatty liver. A former study showed that the intestinal microbiota of NAFLD patients has a lower diversity than those of healthy individuals (Saltzman et al. 2018). Previous studies reported the increase in abundance of Proteobacteria, Lachnospiraceae Enterobacteriaceae including Escherichia, and Bacteroidetes, in the overall population of fatty liver patients, while several studies find a reduction or no difference in Bacteroidetes, as well as a drop in Prevotella and Firmicutes (Gómez-Zorita et al. 2019). Otherwise, the gut microbiota can directly affect the body weight that can worsen the NAFLD (Jayakumar and Loomba 2019). Accordingly, it seems reasonable to find that probiotics can improve the health of the host by their capability to restore the normal symbiotic status of the gut microbiota and treat digestive issues when consumed in an adequate amount (El-Baz et al. 2020).

FDA-approved treatments for NAFLD are still lacking, and the precise mechanisms underlying the disease’s progression remain unclear. Considering this, we examined the underlying mechanism of Vinpo in treating fatty liver disease in this experimental animal model study and clarified the function of Lactobacillus probiotics as an adjuvant therapy by assessing the relative abundance of specific gut microbiota members linked to fatty liver disease.

Materials and methods

Chemicals and reagents

Vinpo was supplied as 5 mg of Vinporal tablet (Amyria Co., Egypt) and prepared as 0.5% w/v suspension in carboxymethyl cellulose (CMC). Lactobacillus was purchased in the form of Lacteal forte sachets (Rameda Pharma Co, Giza, Egypt). The sachet contained 1 × 10¹⁰ microbial cells of Lactobacillus delbrueckii and Lactobacillus fermentum (Garcia-Castillo et al. 2019). The components of the sachet were dispersed in water and each rat was ingested 0.5 ml of microbial suspension containing 2.7 × 10⁸ CFU/ml (Fooladi et al. 2015). Streptozotocin (STZ), CMC, cholesterol, and cholic acid were provided from Sigma Chemical Co. (St. Louis, Missouri, USA).

Animals and experimental design

Male adult Sprague Dawley rats (180–220 g) were accustomed to the environment for a week before the experiment began. Rats were housed under typical nutritional and environmental conditions for the duration of the experiment. The experimental protocol was approved by the Delta University, Faculty of Pharmacy’s research ethics committee (ethical approval number: FPDU11/2022). A high-fat diet (HFD) containing 10% sugar, 10% lard stearin, 2% cholesterol, and 0.5% cholic acid was administered for 13 weeks to rats. Rats were given an intraperitoneal injection of STZ (30 mg/kg in citrate buffer, 1 ml/kg) after four weeks. The levels of fasting blood glucose (FBG) were assessed seven days after STZ injection. Only diabetic rats—those with FBG readings exceeding 200 mg/dL—were allowed to complete the test. The HFD was left unaltered after STZ treatment, but sugar was taken out to prevent excessive hyperglycemia (El-Derany and El-Demerdash 2020). With the beginning of the 6th week of the HFD, the 8-week treatment phase of the study continued till the end.

The study layout consisted of 7 groups (n = 6–8): the first three groups were fed a normal chow diet and either given no treatment (Normal), oral 20 mg/kg of Vinpo suspended in CMC (Vinpo) or Lactobacillus as mentioned earlier (Lacto). The other four groups were fed with HFD and were administered either no treatment (HFD), oral 10 mg/kg of Vinpo (Vinpo 10 + HFD), 20 mg/kg of Vinpo (Vinpo 20 + HFD), or 20 mg/kg of Vinpo and Lactobacillus (Vinpo 20/Lacto + HFD).

Oral vinpocetine 20 mg/kg (Petric et al. 2023; Zhang et al. 2018) suspended in CMC (Vinpo) or Lactobacillus (El-Baz et al. 2023) as mentioned earlier (Lacto). The other four groups were fed HFD and either administered no treatment (HFD), vinporal 10 mg/kg (Vinpo 10 + HFD), Vinporal 20 mg/kg (Vinpo 20 + HFD) or vinporal 20 mg/kg and Lactobacillus (Vinpo 20/Lacto + HFD).

Animal sacrifice and sample collection

Following the experiment, rats were starved overnight. The following day, they were weighed and injected with thiopental to make them comatose. After blood samples were drawn via cardiac puncture and centrifuged at 3000 rpm for 20 min, the serum was separated and preserved at − 80 °C for analysis. Additionally, the livers of every animal were taken out, cleaned, weighed, and washed in ice-cold phosphate-buffered saline (PBS, pH 7.4). The liver tissues were divided into pieces and mashed (10% w/v) in 20 mM Tris-HCl (pH 7.4) with 1 mM EDTA and then centrifuged at 3000 g for 20 min at 4 °C. For upcoming biochemical studies, the supernatants were collected and kept at − 80 °C. Liver sections were preserved in 10% neutral buffered formalin solutions for histological examination. Moreover, 300 mg cecal faeces samples were weighed shortly after the animals were put down to collect stool samples. DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen Inc., cat. # 51504 Hilden, Germany) in accordance with the manufacturer’s recommendations. The acquired DNA was kept at − 20 °C and subjected to spectrophotometric analysis using a NanoDrop device (Jenway Nano, UK) to determine the DNA concentration. The remaining faeces samples were kept at − 80 °C until they were needed once more.

Biochemical assessments for liver injury and lipid in serum samples

The injury of the liver was assessed biochemically by estimating serum concentrations of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) by the commercially available kits (Spectrum Diagnostics, Cairo, Egypt). Triglycerides (TGs), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) serum concentrations were determined following the methods reported by Fossati and Prencipe (1982), Allain et al. (1974) and Lopes-Virella et al. (1977), respectively, utilizing kits purchased from Biodiagnostics (Cairo, Egypt).

Histopathological and immunohistochemical assessments of the liver samples

Samples of excised hepatic tissue were fixed in neutral buffered formalin (10%). Using customary histological techniques, the tissue was treated, and paraffin blocks were made. Necroinflammation and fibrosis changes were evaluated using the Ishak-modified HAI method in sections (5 μm thick) stained with hematoxylin-eosin and Mallory’s trichrome, respectively (Ishak et al. 1995). The following NAFLD activity scores (NAS) were used to assess the severity of hepatic steatosis, inflammation, and ballooning. Grades 0–3 for hepatocellular steatosis were assigned to the specimens: grade 0, where less than 5% of the hepatic parenchyma was affected by steatosis; grade 1, where 6–33% of the hepatic parenchyma were affected; grade 2, where 34–66% of the hepatic parenchyma were affected; and grade 3 when steatosis occupied more than 66% of the hepatic parenchyma). For hepatic ballooning, the specimens were divided into classes 0–2 (grade 0, no ballooning; grade 1, little balloon cells; and grade 2, numerous cells/notable ballooning). According to the amount of inflammatory cell infiltration, the specimens were grouped into classes 0–3 (grade 0, no infiltration; grade 1, 1–2 foci every 400× field; grade 2, 3–4 foci every 400× field; and grade 3, more than 4 foci every 400× field). Hepatic fibrosis was classified into stages 0–4 (stage 0: no fibrosis; stages 1 and 2: mild to moderate periportal and perisinusoidal fibrosis; stage 3: bridging fibrosis; and stage 4: cirrhosis).

Concerning liver immunohistochemical assessment, for the purpose of identifying target proteins, 5 μm paraffin-embedded sections were placed on coated glass slides, and endogenous peroxidases and non-specific protein binding were blocked. Slide sections were first treated with primary antibodies, followed by HRP-conjugated secondary antibodies and substrate/chromogen for color development. Nuclear factor-kappa B (NF-κB), TLR4, HMGB-1 and RAGE (Santa Cruz Biotechnology, CA, USA) are the primary antibodies utilized in this investigation. The ImageJ software (NIH, Bethesda, MD, USA) was used to perform quantifications of the immunohistochemical results.

Hepatic hydroxyproline determination

To determine how much collagen was deposited in the liver, the hydroxyproline content was measured as previously explained (Bergman and Loxley 1963). Briefly stated, a section of the liver was submerged in KOH (5%) for a whole night at 37 °C, hydrolyzed with NaOH (10 N), and then incubated with Chloramine-T for three hours at 25 °C. Ehrlich’s reagent was then added to the reaction mixture and heated to 60 °C for 20 min. The resulting color’s spectrophotometric absorbance at 550 nm was measured.

Enzyme-linked immunosorbent assay (ELISA)

Using ELISA kits, tissue lysates were used for the determination of TNF-α (RayBiotech Inc., Norcross, GA, USA), TGF-β1 (eBioscience, Vienna, Austria), Nrf-2 and HO-1 (Cloud-Clone Co., Houston, USA). On the other hand, adiponectin (Assaypro, St. Louis, MO, USA) and leptin (RayBiotech Inc., Norcross, GA, USA) were determined in serum. To make tissue lysates, 35 mg of tissue was minced in 0.315 ml of ice-cold lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.5% v/v Triton X-100), which also contained a protease inhibitor. The supernatants were then separated by centrifugation at 5000×g for 10 min at 4 °C. Eventually, 96-well plates were filled with tissue lysate supernatants for ELISA.

Assessment of oxidative stress and antioxidant parameters in the liver

Portions of liver samples (10% w/v) were minced in an ice-cold buffer (20 mM Tris-HCl, 1 mM EDTA, pH 7.4) by a Potter-Elvehjem homogenizer, followed by centrifugation at 3000×g for 20 min at 4 °C to collect supernatants.

Hepatic malondialdehyde content determination

The concentration of malondialdehyde (MDA) was calculated as previously mentioned (Gérard-Monnier et al. 1998). In a nutshell, 0.3 ml of liver homogenate sample was added to 0.65 ml of 10.3 mM 1-methyl-2-phenyl-indole in acetonitrile mixed with methanol containing 32 mM FeCl₃ (3:1) and vortexed. The samples were thoroughly mixed, sealed with a tight stopper, and incubated at 45 °C for 60 min after the addition of 0.15 ml of 37% (v/v) HCl. After being chilled on ice, the samples were centrifuged at 4000×g for 10 min, and the absorbance at 586 nm was determined spectrophotometrically. For the quantification of MDA, a standard curve made of 1,1,3,3-tetramethoxypropane was also used.

Hepatic nitric oxide content determination

According to a prior description (Miranda et al. 2001), the total nitrate/nitrite (NOx) products were calculated as a measure of nitric oxide (NO⁻) synthesis. 0.25 ml of 0.3 N NaOH was mixed with 0.5 ml of liver homogenate. For deproteinization, 0.25 ml of 5% (w/v) ZnSO4 was added after the mixture had been incubated for 5 min at room temperature. After centrifuging this mixture at 3000×g for 20 min at 4 °C, 0.4 ml of the supernatant was added to 0.3 ml of Vanadium chloride (8 mg/ml) in 1 M HCl and 0.3 ml of Griess reagent [0.15 ml of 2% (w/v) sulfanilamide in 5% (v/v) HCl and 0.15 ml of 0.1% (w/v) N-(1-naphthyl)-ethylendiamine dihydrochloride in distilled water]. Spectrophotometric measurements of the samples were made at 540 nm after 45 min of incubation at 37^oC. NaNO₃ (0–100 nmol/ml) was used to construct a standard curve for determination of NOx concentration in samples.

Hepatic reduced glutathione content determination

With a small alteration, hepatic reduced glutathione (GSH) was measured as stated previously (Moron et al. 1979). Summarily, 0.45 ml of liver homogenate was centrifuged at 1000×g for 5 min after the protein was precipitated with 0.05 ml of 50% (w/v) trichloroacetic acid. After 5 min at room temperature, the reaction mixture of 0.25 ml of supernatant, 1 ml of 0.2 M Tris-HCl (including 1 mM EDTA, pH 8.9), and 0.05 ml of 10 mM 5,50-dithiobis-(2-nitrobenzoic acid) in absolute methanol was measured spectrophotometrically at 412 nm. The calculations were done using a standard GSH curve (0–500 nmol/ml).

Hepatic superoxide dismutase activity determination

Superoxide dismutase (SOD) activity was assessed in a manner slightly different from that described earlier (Marklund and Marklund 1974). This technique is based on SOD’s capacity to prevent pyrogallol’s autoxidation. Briefly, 1.5 ml of 20 mM Tris-HCl (including 1 mM EDTA, pH 8.2) and 0.1 ml of 15 mM pyrogallol were combined with 0.1 ml of liver homogenate. After that, the increase in OD for the samples at 420 nm was observed for 3 min in order to calculate the change in OD per minute. By running a control under the same circumstances without any sample, the percentage of inhibition for the samples was calculated. One unit of SOD enzyme activity was defined as the amount of the enzyme that reduced the rate of pyrogallol autoxidation by 50%.

Real-time PCR test for measuring relative abundance of some selected gut microbiota

The relative abundances of Bifidobacteria spp., Lactobacillus spp., Bacteroides, Escherichia coli, Clostridium spp., Fusobacteria spp., Porphyromonas gingivalis, Providencia spp., and Prevotella intermedia were assessed by the quantitative real-time polymerase chain reaction (RT-PCR) technique. By utilizing adequate primers for 16 S rRNA housekeeping gene amplification, the total number of bacteria was calculated (​Table S1​). For quantitation of DNA, 40–80 ng of isolated fecal DNA was combined with 12.5 µl (2×) SYBR Green PCR master mix (Willowfort Co., Birmingham, UK.) and 1.5 µl of each forward and reverse primer (10 µM each). A final volume of 25 µl was created by adding 7.5 µl of nuclease-free water. In a RT-PCR system (QuantStudio, ThermoFisher Scientific, USA), RT protocol was started at 95 °C for 5 min, then 45 cycles of 95 °C for 20 s, annealing for 20 s (​Table S1​), followed by 40 s at 72 °C and 5 min at 72 °C as the final extension (Wong et al. 2017).

Statistical analysis

Values in each experimental group were presented as a mean ± SE. The one-way analysis of variance test ensued by Tukey’s multiple comparison test was used to analyze parametric data. For non-parametric data, the Kruskal–Wallis test and Dunn’s multiple comparison test were used. Statistical analyses were carried out by GraphPad Instat V 3.1 (GraphPad Inc., San Diego, CA, USA) with a cut-off level of statistical significance < 0.05.

Results

Vinpo and its combination with Lactobacillus ameliorated HFD-induced liver injury

The liver enzymes AST, ALT, and ALP significantly increased in rats fed HFD alone. Treatment with Vinpo prevented these parameters from rising. The addition of Lactobacillus improved the liver damage markers even further (Fig. 1A–C). However, there were no discernible differences in albumin levels between the groups (Fig. 1D). When liver sections from the group exposed to HFD/STZ were examined histopathologically, they revealed congested blood vessels, occluded sinusoids, diffuse hydropic degeneration with macro-vesicular steatosis. Portal fibrosis appears in some sections infiltrated with leukocytes. Vinpo therapy resulted in a dose-dependent milder pathological alteration. Histopathological alterations were further restored with the inclusion of Lactobacillus (Fig. 2A; Table S2).

Fig. 1

Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in serum aspartate transaminase (ALT, A), alanine transaminase (AST, B), alkaline phosphatase (ALP, C) and albumin (D). Bars are means ± SE (n = 6–8 per group). Statistical significances are presented as: # vs. Normal group; * vs. HFD group and § vs. Vinpo 10 + HFD

Fig. 2

Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in liver histopathology. Representative pictures of hematoxylin-eosin (A) and Mallory’s trichrome (B) stainings (Low magnification 100×; bar 100 µM, high magnification 400×; bar 50 µM) showing normal hepatocytes arranged in radiating plates around central vein (CV) with normal portal areas (PA) and sinusoids in normal group. A liver sections from HFD group showing congested blood vessels (red arrow), occluded sinusoids, diffuse hydropic degeneration with macro-vesicular steatosis (arrowheads). Portal fibrosis appears in some sections infiltrated with leukocytes (black arrows). Liver sections showing milder pathological changes in the treated groups including: few micro-vesicular steatosis (arrowheads), mild portal fibrosis (thin black arrow) and inflammation (yellow arrow) in Vinpo10 + HFD group, few micro-vesicular steatosis (arrowheads), mild portal inflammation (yellow arrow) in Vinpo20 + HFD group, very few micro-vesicular steatosis (arrowheads) in Vinpo20/Lacto + HFD group. B Normal liver sections showed no collagen deposition around CV or PA. Meanwhile, liver sections from HFD group showing excess bluish stained collagen deposition around CV or PA (black arrows). Liver sections from the treated groups showing decreased bluish stained collagen deposition around CV or PA (black arrows) in Vinpo10 + HFD group, much less bluish stained collagen deposition around CV or PA (black arrows) in Vinpo20 + HFD group, no excess bluish stained collagen deposition around CV or PA in Vinpo20/Lacto + HFD group

Vinpo and its combination with Lactobacillus resolved liver fibrosis in HFD/STZ induced NASH

The collagen accumulation was assessed histologically and biochemically in the liver. Histological analysis of Mallory’s trichrome stained liver sections showed excess bluish stained collagen deposition around central vein and portal areas in rats exposed to HFD/STZ. On the other hand, collagen deposition was prevented by Vinpo therapy, whether it was administered alone or in conjunction with Lactobacillus (Fig. 2B; Table S2). Biochemically, liver hydroxyproline level was greatly increased in rats when subjected to HFD and STZ compared to the control group. Rats co-treated with vinpocetine significantly decreased hydroxyproline level (Fig. 3A). Furthermore, rats exposed to HFD showed a significant elevation in hepatic TGF-β1 as compared to the control group. Treatment of rats with Vinpo induced a significant reduction in serum level of TGF-β1 as compared to non-treated rats (Fig. 3B). Lactobacillus addition significantly increases the inhibitory impact of Vinpo on TGF-β1 and hydroxyproline when compared to Vinpo treated groups alone.

Fig. 3

Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in hepatic tissue hydroxyproline (A) and TGF-β₁ (B). Bars are means ± SE (n = 6–8 per group). Statistical significances are presented as: # vs. Normal group; * vs. HFD group; § vs. Vinpo 10 + HFD and © vs. Vinpo 20 + HFD

Vinpo and its combination with Lactobacillus reverses HFD-induced lipid panel alterations

Comparing the NASH group to the normal group, there was a discernible rise in cholesterol and TG levels as well as a fall in HDL serum levels (Fig. 4A-C). The cholesterol levels in HFD-groups treated by Vinpo 10 or 20 mg/kg groups were lesser than those of the HFD/STZ group. When Lactobacillus is added, the inhibitory action is significantly increased. However, only simultaneous administration of Vinpo and Lactobacillus effectively reduced TG. None of the therapeutic groups’ HDL levels have differed noticeably from those of the NASH group.

Fig. 4

Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in serum cholesterol (A), triglycerides (TG, B) and high-density lipoprotein (HDL, C). Bars are means ± SE (n = 6–8 per group). Statistical significances are presented as: # vs. Normal group; * vs. HFD group; § vs. Vinpo 10 + HFD and © vs. Vinpo 20 + HFD

Vinpo and its combination with Lactobacillus restored HFD induced adiponectin and leptin alterations

To obtain insight into the protective mechanisms of Vinpo against hepatic steatosis in HFD-fed rats, we measured the levels of adiponectin and leptin to determine whether improvement in insulin resistance is a factor in the positive effect of Vinpo and Lactobacillus. When compared to the control group, the HFD group’s levels of leptin dramatically increased while Adiponectin greatly reduced (Fig. 5A and B). It’s interesting that Vinpo, especially at a dose of 20 mg, reversed these abnormalities brought on by HFD. These markers further improved when Lactobacillus and Vinpo 20 mg were given simultaneously.

Fig. 5

Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in serum adiponectine (A) and leptin (B). Bars are means ± SE (n = 6–8 per group). Statistical significances are presented as: # vs. Normal group; * vs. HFD group; § vs. Vinpo 10 + HFD and © vs. Vinpo 20 + HFD

Vinpo and its combination with Lactobacillus ameliorated HFD-induced oxidative stress and Nrf2/HO-1 expression

Next, we examined whether the protective effects of Vinpo and Lactobacillus against HFD/STZ-induced fatty liver involve reducing oxidative stress. As seen in ​Fig. 6​A–D, untreated HFD-rat livers experienced a considerable hepatic increase in MDA and nitrite, as well as NOx alongside a significant decrease in SOD and GSH, when compared to the normal counterparts. All treated groups demonstrated a statistically significant decline in MDA and restoration in GSH, compared to HFD-group. Furthermore, compared to the 10 mg/kg dose, the 20 mg/kg of Vinpo considerably reduces MDA. Only Vinpo 20 mg/kg treatment group and its combination with Lactobacillus showed a significant improvement in SOD compared to the HFD group. The addition of Lactobacillus to Vinpo had a significant effect on MDA and SOD as compared to the Vinpo alone treated group. Next, we evaluated the expression of Nrf-2 and HO-1 which confer cytoprotection against oxidative stress-induced cellular damage. The HFD group dramatically decreased HO-1 and NrF2 compared to the control group. In contrast to the HFD group, significant improvements in NrF2 and HO-1 were observed in the group treated with Vinpo 20 and to a greater extent when combined with Lactobacillus (Fig. 6E and F).

Fig. 6

Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in hepatic tissue malondialdehyde (MDA, A); superoxide dismutase (SOD, B); reduced glutathione (GSH, C); total nitrate/nitrite (NOx, D), Nrf-2 (E) and HO-1 (F). Bars are means ± SE (n = 6–8 per group). Statistical significances are presented as: # vs. Normal group; * vs. HFD group; § vs. Vinpo 10 + HFD and © vs. Vinpo 20 + HFD

Vinpo and its combination with Lactobacillus ameliorated HFD-induced inflammation

It was observed that hepatic inflammation was successfully induced by giving rats HFD and a single STZ injection. This was demonstrated by the significant increase in liver concentrations of the inflammatory cytokines TNF-α and IL-6 in HFD-untreated rats. However, Vinpo administration was able to reverse the inflammation that HFD had caused, as shown by the significantly reduced tissue levels of TNF-α and IL-6 when compared to the HFD group. When Lactobacillus was added to Vinpo, these effects were more prominent (​Fig. 7​A and B). We then investigated the potential mechanism that could be responsible for these elevated inflammatory cytokines. HMGB1, TLR-4, RAGE, and NF-κB immunohistochemistry evaluations were then performed to identify whether Vinpo protective effects involve a lessening of the release of DAMPs and activation of PRRs. While tissue hepatic sections from the NASH untreated HFD- group were shown to have more brown color than the controls, Vinpo treatment, especially at a dose of 20 mg/kg, significantly reduced the hepatic overexpression of HMGB1, TLR-4, RAGE, and NF-κB, as shown by the significant decrease in brown staining compared to in HFD-rat livers (Figs. 8 and 9, respectively). Co-administration of Lactobacillus further decreased brown staining indicating its additional protective impact.

Fig. 7

Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in hepatic tissue IL-6 (A) and TNF-α (B). Bars are means ± SE (n = 6–8 per group). Statistical significances are presented as: # vs. Normal group; * vs. HFD group; § vs. Vinpo 10 + HFD and © vs. Vinpo 20 + HFD

Fig. 8

A Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in hepatic high mobility group box 1 (HMGB1) protein immunostaining (×400) showing negative staining in normal group. Meanwhile, liver sections from HFD group showing significant positive brown cytoplasmic staining of many hepatocytes (black arrows). Liver sections from the treated groups showing decreased positive brown cytoplasmic staining that appears in some hepatocytes (black arrows) in Vinpo10 + HFD group, much less positive cytoplasmic staining that appears in few hepatocytes (black arrows) in Vinpo20 + HFD group, very mild positive brown cytoplasmic staining of very few hepatocytes (black arrows) in Vinpo20/Lacto + HFD group. IHC counterstained with Mayer’s hematoxylin. B Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in hepatic toll like receptor-4 (TLR-4) immunostaining (400×) showing negative staining in normal group. Meanwhile, liver sections from HFD group showing significant positive brown cytoplasmic staining in many hepatocytes (black arrows). Liver sections from the treated groups showing decreased positive brown cytoplasmic staining that appears in some hepatocytes (black arrows) in Vinpo10 + HFD group, much less positive cytoplasmic staining that appears in few hepatocytes (black arrows) in Vinpo20 + HFD group, very mild positive brown cytoplasmic staining that appears in very few hepatocytes (black arrows) in Vinpo20/Lacto + HFD group. IHC counterstained with Mayer’s hematoxylin

Fig. 9

A Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in hepatic receptors for advanced glycation end products (RAGEs) immunostaining (400×) showing negative staining in normal group. Meanwhile, liver sections from HFD group showing significant positive brown cytoplasmic staining of many hepatocytes (black arrows). Liver sections from the treated groups showing decreased positive brown cytoplasmic staining that appears in some hepatocytes (black arrows) in Vinpo10 + HFD group, much less positive cytoplasmic staining that appears in few hepatocytes (black arrows) in Vinpo20 + HFD group, very mild positive brown cytoplasmic staining of very few hepatocytes (black arrows) in Vinpo20/Lacto + HFD group. IHC counterstained with Mayer’s hematoxylin. B Effect of vinpocetine (Vinpo 10 and 20 mg/kg) and Lactobacillus (Lacto) treatments on HFD-induced changes in hepatic nuclear factor-kappa B (NF-κB) immunostaining (400×) showing negative staining in normal group. Meanwhile, liver sections from HFD group showing prominent positive brown nuclear staining of many von kupffer cells (black arrows). Liver sections from the treated groups showing decreased positive brown nuclear staining that appears in some von kupffer cells (black arrows) in Vinpo10 + HFD group, much less positive brown nuclear staining that appears in few von kupffer cells (black arrows) in Vinpo20 + HFD group, negative staining of von kupffer cells in Vinpo20/Lacto + HFD group. IHC counterstained with Mayer’s hematoxylin

Effect of Vinpo and Lactobacillus combination on gut microbiota composition

Across the examined groups in the current investigation, a change in gut microbiota composition was observed. A remarkable variation of the relative abundance of intestinal microbial flora, with NAFLD being associated with a significant decline in Bifidobacteria spp. and Lactobacillus spp. as compared to healthy controls. Bacteroides spp., Fusobacterium spp., E. coli, Clostridium spp., Providencia spp., Prevotella intermedia, and Porphyromonas gingivalis were significantly abundant compared to the normal healthy control group. In the current investigation, when compared to the positive control group, the administration of combination therapy of Vinpo and Lactobacillus probiotics significantly enhanced the restoration of the symbiotic gut microbiota (Fig. 10).

Fig. 10

Relative abundance (RA) of different microbial species in different groups of tested samples in the fatty liver animal model. RA is calculated for different groups of tested samples through targeted genes specific to different reference microbiota relative to the control housekeeping 16 S rRNA gene. a Bacteroides spp., b E. coli, c Clostridium spp., d Fusobacteria spp, e P. gingivalis., f Providencia spp., g Prevotella intermedia, h Bifidobacteria spp., i Lactobacillus spp., and j Distribution of the tested gut microbiota

Discussion

Current NAFLD management therapies are primarily focused on dietary and lifestyle changes, with discrepancies in the clinical results due to low patient compliance. So far, no pharmaceutical therapy or surgical methods have been approved for the treatment of NAFLD. Meanwhile, hypolipidemic medicines, anti-TNF, antioxidants, and diabetic medications are merely recommended for attenuating the progress of NAFLD (Mantovani and Dalbeni 2021). Dysregulation of the gut microbiome, or dysbiosis, is among the factors implicated in driving different pathways that aggravate the steatosis and inflammation cascades in the liver of NAFLD patients (Campo et al. 2019). Otherwise, the activation of the cAMP/PKA or cGMP-dependent signaling pathways in the liver can impair lipogenesis and glycogenesis via multiple mechanisms (Wahlang et al. 2018). Phosphodiesterase (PDE) inhibitors can be sustained in both pathways, which may be useful in the setting of fatty liver disease. Accordingly, we investigated whether the PDE-1 inhibitor Vinpo would ameliorate the HFD-induced steatosis and inflammation in rat livers, and if so, Vinpo would exhibit better efficacy when combined with Lactobacillus.

Vinpo significantly lowered the HFD-induced rise in serum ALT, AST, and ALP activities and improved the hepatic architecture of steatotic rats, especially at a dose of 20 mg/kg. On the lipid profile, Vinpo caused a mediocre decrease in the elevation caused by HFD in serum total cholesterol, but not TGs and HDL. Moreover, the HFD-induced decline of adiponectin and escalation of leptin were reversed by Vinpo at doses of 10 and, more pronouncedly, 20 mg/kg. Both adiponectin’s increase and leptin’s decrease mediated by Vinpo are reliable evidence for improving the insulin sensitivity and lipid metabolism in HFD rats (Forny-Germano et al. 2018). Interestingly, concomitant administration of Lactobacillus with Vinpo (20 mg/kg) exhibited the maximal efficacy in restoring these abnormalities elicited by HFD to an extent near the normal control group.

The anticipated manifestations of the fatty liver disease were evident in the rats by a marked rise in the indexes of inflammation (TNF-α and IL-6) and fibrosis (TGF-β1 and hydroxyproline). Meanwhile, these abnormalities were countered by treating the rats with Vinpo (20 mg/kg) alone and, more pronouncedly, when combined with Lactobacillus. The inflammation in fatty liver disease arises from the excessive generation of DAMPs “Damage-associated molecular patterns” from the dying hepatocytes. HMGB1 is a key DAMP and driver for the sterile inflammatory response upon release from necrotic hepatocytes. HMGB1 mediates chemoattraction of the inflammatory cells to the liver through stimulating TLR4 localized on Kupffer cells and other hepatic immune cells recruited in the setting of HFD-induced NAFLD in mice (Li et al. 2011; Shaker 2022). Besides TLR4, HMGB1 can stimulate the receptors for advanced glycation end products (RAGEs), which are expressed on hepatic DCs, neutrophils, monocytes, and, to a lesser extent, on Kupffer cells (Sims et al. 2010; Zhong et al. 2020). Stimulation of both TLR4 and RAGE localized on hepatic immune cells by HMGB-1 or lipopolysaccharides (LPS) will subsequently activate diverse downstream pathways like mitogen-activated protein kinases (MAPKs), NF-κB, interferon regulatory factor 1 (IRF1) and NADPH oxidase (Guimaraes et al. 2010; Yan et al. 2023; Zeng et al. 2004). Eventually, this will produce exuberant inflammatory mediators and ROS that aggravate the initial insult and amplify the hepatic inflammation.

Vinpo (20 mg/kg) alone or with Lactobacillus successfully lowered the higher nuclear translocation of the transcription factor NF-κB in the hepatic cells of the HFD-rats. Besides TLR4, NF-κB is translocated to the nucleus after stimulation of pattern recognition receptors like TLR9, IL-1R, and TNF-R. For instance, TLR9 senses the denatured DNA released from host dying hepatocytes and upregulates the inflammatory cytokines like IL-1β and TNF-α via activating the transcription factor NF-κB (Bamboat et al. 2010). Thus, the hepatocellular protection of the Vinpo and Lactobacillus combination will also decrease the release of HMGB1 and other DAMPs like the denatured DNA, leading to less activation of the NF-κB and upregulation of the proinflammatory cytokines.

The beneficial effects of Vinpo against the hepatocellular damage instigated by HFD were also mediated by countering the oxidative stress as evidenced by limiting the rise of the lipid peroxidation product MDA and the nitrite products in the liver. These data were concordant with the impact of Vinpo in restoring the HFD-induced depletion of the hepatic non-enzymatic antioxidant GSH and enzymatic antioxidant SOD. Activation and transcription of Nrf2 have a key role in HO-1 expression to protect hepatocytes from injury caused by oxidative stress alongside inducing antioxidant enzymes like SOD and GSH-peroxidase (Solano-Urrusquieta et al. 2020). Besides, Nrf2 negatively regulates genes that foster hepatic steatosis (Chambel et al. 2015; Galicia-Moreno et al. 2020). Based on these, Vinpo direct inducing effect on Nrf2 accounts for reversing the HFD-induced decrease of the hepatic content of HO-1 alongside those of GSH and SOD. Similarly, Lactobacillus promoted the beneficial impact of Vinpo on oxidative stress and antioxidant parameters.

Based on previous literature, NAFLD strongly correlates to the gut microbiome (Castillo et al. 2021; Lee et al. 2021). Our study investigated the NAFLD-associated gut microbial signatures and the augmentative role of Lactobacillus probiotics in enhancing the therapeutic effect of Vinpo. Lactobacilli, Bifidobacteria, and Streptococci can be naturally found in some foods, and are commonly used as commercial probiotics that control the abundance of gut gram-negative bacteria and are strongly involved in research trials for the treatment of NAFLD and NASH (Koopman et al. 2019). Our study recorded Lactobacillus spp. and Bifidobacterium spp. as health indicator gut taxa that significantly declined in abundance in the induced-fatty liver group. Their normal healthy levels were restored upon treatment with Vinpo. Simultaneously, Bacteroides spp., E. coli, Clostridium spp., Fusobacteria spp. P. gingivalis, Prevotella intermedia, and Providencia spp. were enriched in the induced-disease group and identified as proinflammatory gut microbiota members in NAFLD.

Previous studies demonstrated the richness of Fusobacterium and Escherichia in patients with NAFLD (Su et al. 2024). Fusobacteria phylum and Fusobacteriaceae family were previously reported for their increased abundance in NAFLD patients compared to healthy individuals, probably due to their implication in triggering the inflammatory signaling during the disease progression (Kim et al. 2019; Li et al. 2011; Shen et al. 2017). Our study reported a high abundance of Fusobacteria spp. among the NAFLD group compared to healthy and treated groups especially those receiving the combination of Lactobacillus and Vinpo. In general, E. coli is consistently identified as a disease indicator taxon associated with type-2 diabetes mellitus and NAFLD (El-Baz et al. 2021; Khalaf et al. 2022; Wang et al. 2016). Our findings were in line with the previously published work where the NASH group showed a significant increase in E. coli abundance that has been reduced in a dose-dependent pattern upon treatment with Vinpo. Interestingly, the coadministration of Lactobacillus spp. probiotics significantly diminished the proliferation of E. coli, restoring its level in healthy groups.

Moreover, the literature reported a reduction in Bacteroides fragilis abundance (Gómez-Zorita et al. 2019), nevertheless, in this study, Bacteroides spp. increased in abundance aligning with the published discrepancies regarding the taxon proliferation according to the measured species and the applied treatment (Fig. 10). Oxidative balance is crucial for mitochondrial function and regulates the metabolic pathways of antioxidants, especially glutathione. A decrease in intracellular glutathione can lead to oxidative stress (Mardinoglu et al. 2015). During dysbiosis, the microbiota consumes glycine, a glutathione precursor, leading to a deficiency in glutathione production and consequently, oxidative stress. Clostridium sensu stricto is notably abundant in mice with NASH. Treatment with glycine-based tripeptide DT-109 replenishes glutathione, reducing C. sensu stricto abundance, lipogenesis, and lipotoxicity while enhancing fatty acid oxidation (Rom et al. 2020). A previous study demonstrated that Lactobacillus rhamnosus GG (LGG) boosts the intracellular antioxidant pool and activates the Nrf2 antioxidant signaling pathway, preventing liver injury from acetaminophen and ethanol toxicity (Saeedi et al. 2020). These findings align with our study, which showed an increased abundance of Clostridium spp. that was reversed upon treatment with Vinpo and Lactobacillus probiotic combination.

In our study, Prevotella intermedia significantly increased in the NAFLD group compared to the healthy one, however, its proliferation was significantly reduced upon treatment with Vinpo at different concentrations where the highest level of significance was recorded upon the coadministration of Lactobacillus probiotics. Interestingly, P. gingivalis, a common oral pathogen worldwide was recently reported to be implicated in the development of NAFLD because of the contribution of the pathogen LPS to the activation of NF-κB and JNK signaling pathways (Ding et al. 2019). Furthermore, recent studies have identified P. gingivalis as a direct risk factor for NAFLD due to its disruption of metabolic and immunologic regulatory pathways (Liu et al. 2023). According to our study, the increased abundance of P. gingivalis was associated with the NAFLD group that significantly decreased by Vinpo alongside Lactobacillus spp. Providencia rettgeri was reported as one of the intestinal microbiota members that contributes to choline metabolism into trimethylamine and subsequently, trimethylamine N-oxide that reduces hepatic cholesterol and improves the blood lipid profile (Bluemel et al. 2016; Li et al. 2021). However, the abnormally increased choline metabolism could lead to liver steatosis due to reduced choline levels (Bluemel et al. 2016). This explains the association of increased abundance of Providencia spp. in our study with NAFLD diseased group that significantly reversed upon treatment with Vinpo.

The findings of this study align with the reported therapeutic effects of both Lactobacillus fermentum (Limosilactobacillus fermentum CQPC06) and Lactobacillus delbrueckii probiotics in the treatment of NAFLD, where Lactobacillus fermentum mitigated lipid metabolism issues and fat accumulation by regulating the expression of various proteins and genes and contributed to reducing ROS levels in the liver, as well as Lactobacillus delbrueckii improved the liver histology and reduced steatohepatitis (Mijangos-Trejo et al. 2023).

In conclusion, Vinpo dose-dependently mitigated the hepatic injury, steatosis, and inflammation elicited by HFD in rats. Our findings helped us understand the mechanisms behind the beneficial effects of Vinpo including insulin sensitivity improvement, attenuation of oxidative stress, and dampening of the sterile inflammatory response. Importantly, the reported microbial gut signatures in this study open the horizon for developing novel diagnostic lab testing for the early diagnosis of NAFLD and highlighting the importance of the concomitant administration of Lactobacillus spp. probiotics with Vinpo for augmented therapeutic action.