1. Introduction

Nonalcoholic steatohepatitis (NASH), a liver disease, occurs as a result of chronic oxidative and inflammatory stress. Several serious complications are associated with NASH, including hepatic cirrhosis [1, 2], hepatic failure [3], and hepatocellular carcinoma (HCC) [4, 5]. The clinical adverse effects related to NASH are regeneration of liver inhibition, infection, and others. To prevent and treat NASH and its complications, novel therapeutic methods are essential. There is no drug approved by the US Food and Drug Administration (FDA) to treat NASH without any negative effects [6]. NASH progression can still be controlled primarily with drug therapy. The commonly used drugs to treat NASH are peroxisome proliferators activated receptor (PPAR)-α and γ agonist [7], pioglitazone, [8] and vitamins [9], but these have many side effects.

A substantial amount of research has been conducted on the liver–gut axis in relation to NASH onset and progression [10]. Scientists may be able to mitigate the negative effects of this disease on the liver and general health by addressing gut health. During the metabolism process, the liver and intestinal environment are intimately connected via the portal circulation and packed two-way communication. The development of NASH is associated with increased intestinal permeability resulting from inflammation of intestinal tight junctions (TJs) [11]. TJs including occluding, claudin, and the occluding zone (ZO) regulate the permeability of intestinal mucosa [12]. There is an increase in intestinal permeability due to NASH which causes movement into the portal vein of bacterial endotoxins and contributes to liver inflammation [13–17]. As a result of de novo fatty acid synthesis, excess oleic acid (OA) also accumulates in the liver, causing hepatic steatosis. Whereas, the PPAR-α activation ameliorates NASH in hepatic and intestinal tissue through gene expression regulation.

Synbiotic, the combination of prebiotics and probiotics have significant anti-inflammatory and antioxidative properties. The garlic extract is rich in eight bioactive compounds, and thus, rich in various mineral ions and vitamins [18–21]. Anthocyanin in aged garlic extract (AGE) protects cells from inflammatory and diabetes-induced injuries through patatin-like phospholipase domain-containing protein 3/sterol regulatory element binding protein-1c (PNPLA3/SREBP-1c) pathway via modulating the PPAR-α levels [22]. The other phenolic compounds modulate the NF-κB, TGF-β, TNF-α, IL-1β, IL-6, and IL-32. The AGE can be used as a prebiotic and in turn, affects the intestinal microflora. Lactobacillus plantarum, a probiotic, improves the intestinal microflora distribution [23].

Furthermore, inflammation and lipid metabolism may make fibrosis, atherosclerosis, and gut dysbiosis worse [23, 24], which in turn might facilitate the development of NASH [25]. The high levels of PNPLA3 and SREBP-1c lead to the NASH risk. Similarly, low levels of PPAR-α also affect the NASH progression, which was treated by seed extract of chicory through modulating the PPAR-α level [26], where activation of PPAR-α inhibited the SREBP-1c pathway [27]. PNPLA3 overexpression has been found in gut and liver tissue such as epithelial cells, enterocytes villi, paneth cells, and hepatocytes and has higher expression in insulin-resistant animal models [28].

Keeping the above described concept in vision, the present study examines oxidative stress, colonic barrier activity, fat accumulation, and inflammation associated with NASH progression in epithelial cells and Wistar rat models. The synbiotic used is unique and few data or no data on this combination is analyzed or reported on the NASH rat model. Results obtained in the current study suggest synbiotic may function as a therapeutic agent for NASH through PNPLA3 lead SREBP1-c pathway, investigated molecularly [29].

2. Materials and Methods

2.8. Methionine and Choline-Deficient High-Fat Diet (MCDHFD)-Induced NASH Model Development

The rats were fed with an MCDHFD (Table S3). In a 9-week study, the MCDHFD diet caused damage to the epithelial and intestinal barriers in the liver and gut and led to fat droplet accumulation in the liver. The rat NASH model was then assessed by a scoring system of histological features that scored the complete lesion spectrum of NASH [36, 37]. The synbiotics (L. plantarum 10⁹ CFU/day and 180 mg/kg of AGE) were administered orally to the required group, respectively [19]. While PPAR-α inhibitor (2 mM OA and 25 µg/mL of GW6471) was injected intraperitoneal into 12 rats. Following 40–60 mg/kg of pentobarbital as anesthetic compound, all fasting (12 h) rats underwent peritoneal blood (1–2 mL) collection before sacrifice from vena cava. Plasma was collected following centrifugation in Minispin-plus-Eppendorf, Hamburg, Germany, for 10 min at 16,000 relative centrifugal force (RCF), and the samples were then stored at −80°C for later use. The tissue samples of 20–30 mg were homogenized using a tissue homogenizer of Remi (RPM 8000 RQ-127AD) in PBS (150 μl) of pH 7.4.

The animals were euthanized according to the animal ethical sacrifice protocol [19]. There were five experimental groups (six rats/group): the control group (Control), NASH group (NM), PPAR-α antagonist (PA), synbiotic model (NM + SM), and PPAR-α antagonist + synbiotic (PASM). Except control group all were provided with NASH inducing diet as a base (Figure 1).

3. Results

3.1. In Vitro

3.1.1. Effect of and/or Synbiotic Intervention on the Growth of HepG2 Cells

Hepatocellular uptake of plasma fatty acids and de novo biosynthesis in the liver contribute to liver fatty acid accumulation as proven already. To understand the basis of NASH progression due to MCDHFD, HepG2 cells were considered. A morphological examination of HepG2 cells was conducted after MCDHFD and PPAR-α antagonist treatment. The untreated HepG2 cells show their characteristic epithelial morphology (Figure 2A), while the PPAR-α antagonist-treated cells (Figure 2C) show constricted cell membranes and distorted cell shape. The untreated HepG2 cells show its characteristic epithelial morphology, whereas cells of the disease model (Figure 2B) show the affected growth rate of HepG2 cells after exposure to PPAR-α antagonist. Whereas, cotreated cells prevented MCDHFD or PPAR-α antagonist-induced morphological changes. This may be the effect of synbiotic on the cell cycle as a stimulator.

3.1.2. Synbiotic Improves the Morphology and Nuclear Condensation of HepG2 and Ameliorates Against PPAR-α Antagonist-Induced Damage

The normoxic PA injury was accompanied by a significant inner mitochondrial membrane and nuclear damage, enhanced production of ROS, and apoptosis. The nucleus of cells is fragmented and undergoing chromatin condensation leads to apoptosis. Therefore, observed dense granular spot inside the nuclei of the disease model (Figure 3A, a2), whereas in cells treated with PPAR-α antagonist (Figure 3A, a5) and synbiotic combination (Figure 3A, a4) depicts a reduced number of cells, the nuclei fragmentation, reduced chromatin condensation, and less are in early apoptotic phase. These changes in HepG2 cells were detected by fluorescence DAPI stain (Figure 3B). According to these observations as compared to cells of control group (Figure 3A, a1), PPAR-α antagonists (Figure 3A, a3) can damage hepatocyte morphology and modulate lipogenesis defects, particularly in the liver.

3.1.3. Synbiotic Alleviate the Formation of Lipid Droplets-Induced ROS in HepG2 Cells

Liver is the organ responsible for fat depots. As determined by the ORO staining method, treating HepG2 cells exposed to PPAR-α antagonist with synbiotic (Figure 4A, a5) or synbiotic (Figure 4A, a4) regulates the lipolysis effectively within the cells as compared to disease model cells (Figure 4A, a2 and a3). HepG2 cells were analyzed microscopically and spectrophotometrically quantified the lipid through the isopropanol extraction method to determine the total lipid accumulation and each group was assayed microscopically at 570 nm. The stained cells of the control group (Figure 4A, a1) generated an absorbance of 0.05 ± 0.002, whereas the absorbance by the PA model was 0.3 ± 0.004, while for cotreatment the value was 0.5 ± 0.010 (Figure 4B). The images obtained signify that the HepG2 cell morphology has been restored when administered with synbiotic.

3.1.4. Synbiotic Reduces PPAR-α Antagonist-Mediated ROS and Inflammation in HepG2 Cells

ROS was measured through both fluorescence microscopy using 2^′-7^′-dichlorodihydrofluorescein diacetate (DCFDA) and the spectrofluorometry method (Figure 5A,B, respectively). The fluorescence microscopic image of cells shows that the green color intensity was seen to be increased 2.5-folds in the disease model (Figure 5A, a2 and a3) as compared to treated (Figure 5A, a4 and a5) and untreated cells (Figure 5A, a1).

3.1.5. Effect of and/or Synbiotic Intervention on the Growth, Morphology, and Barrier Function of Caco2 Cells

To observe the morphological changes, Caco2 cells were treated with MCDHFD (Figure 5A, a2). The untreated cells show normal characteristic epithelial morphology (Figure 6A, a1), while the PPAR-α antagonist-treated cells (Figure 6A, a2) show constricted cell membranes and distorted cell morphology. Similarly, demorphed and reduced number cells can be observed in PA treated group (Figure 6A, a3). The positive effect on the cell cycle can be seen in cells treated with synbiotic (Figure 6A, a4 and a5). The transition of phases may be induced by the synbiotic. The results obtained suggest that synbiotic combination in Caco2 cells prevents the low level of PPAR-α activity (Figure 6A, a5).

3.1.6. Synbiotic Ameliorate the Viability of Caco2 Cells

To explore cytotoxic effects of NASH condition on Caco2 cells and on the nucleus specifically, DAPI stain was used. The nucleus of cells is fragmented and undergoing chromatin condensation leading to apoptosis in the disease model group. Therefore, observed dense granular spots inside nuclei. The reduced number of cell nuclei fragmentation, reduced chromatin condensation, and fewer cells were in the early apoptotic phase in the SM group (Figure 7A, a1) as compared to the NASH model (Figure 7A, a2) or PPAR-α antagonist group (Figure 7A, a3).

3.1.7. Synbiotic Alleviate the Lipid Accumulation Leading to Barrier Dysfunction in Caco2 Cells

To estimate the PPAR-α antagonist impact on Caco2 cells, ORO staining was carried out in the current study. ORO stained Caco2 cells showing fat droplets were formed (Figure 8A, a2 and a3). The fat globules are stained red in the capillary lumen and are indicative of lipid storage dysfunction. Assessment of lipid accumulation is carried out through the number and diameter of the droplets formed of different diameter graphically (Figure 8B). The lipid accumulation was significantly increased in the PA group (Figure 8A, a3) as compared to the control group (Figure 8A, a1). Visible down regulation of deregulated lipogenesis in SM and PASM treated groups (Figure 8A, a4 and a5).

3.1.8. Synbiotic Intervention Reduces MCDHFD and PPAR-α Antagonist-Induced ROS Generation and Inflammatory Stress in Caco2 Cells

Synbiotic-affected intestinal oxidative stress can be detected by measuring ROS generation in Caco2 cells. A significant increase in ROS generation was observed following PPAR-α antagonist stimulation (Figure 9A, a3). However, compared to a PPAR-α antagonist treated group, synbiotic combination significantly lowered the ROS accumulation (Figure 9A, a5). This could be happening due to the anti-oxidative properties of AGE on cells, protecting cells from ROS-mediated damages. Similarly, in the disease model cells (NM), there were dense granular stained cells (Figure 9A, a2). Whereas, in cells treated with synbiotic combination (SM), there were fewer fragmented/stained cells, reduced chromatin condensation, and fewer cells in the early apoptotic phase (Figure 9A, a4).

3.1.9. Synbiotic Treatment Reduces the Effects of PPAR-α Antagonist on Lipid Regulator Genes Responsible for ROS Generation and Inflammatory Stress in HepG2 Cells

PPAR-α, PNPLA3, and SREBP-1c levels were assessed to determine the effect of synbiotic on hepatic cells. MCDHFD and PPAR-α antagonist promotes aberrant metabolism of lipids in the liver and accrues fatty acid by hepatocellular uptake through de novo biosynthesis and plasma. The mRNA expression (Figure 10) reveals that synbiotic combination effectively and relatively suppresses the expression of causative genes in HepG2 cells.

3.1.10. Synbiotic Improves the PPAR-α Antagonist Induced on Dysfunction on Permeability and TJ in Caco2 Cells

The transcription of occluding and ZO-1 in PPAR-α antagonist-treated Caco2 cells was lowered significantly, depicting that the TJs are crucial for gut health. The results of in vitro mRNA expression (Figure 11) indicate that the synbiotic effectively increases the expression of TJs and lowers NASH-mediated inflammation.

3.1.11. Synbiotic Intervention Downregulate the Level of Pro-Inflammatory Cytokines in Hepatic Cells

The MCDHFD in vitro model disrupts the release of inflammatory and pro-inflammatory cytokines in HepG2 cells. This resulted in phenotypic alteration and metabolism in cells. Our results reveal that MCDHFD-fed cells induce high expression of cytokines (Figure 12). Whereas administration with synbiotic lowers the level of pro-inflammatory cytokines such as TNF-α.

3.2. In Vivo, Synbiotic Interventions Demonstrated a Positive Effect on Liver Tissue

The Table 1 provides data on body weight and liver index for different groups (Control, NM, PA, SM, and PASM). The average body weight for the control group is 415.3 g, while the other groups have slightly lower weights. The liver index, which represents the percentage of liver weight relative to body weight, is also slightly different among the groups, with the control group having the lowest index at 2.4%.

Table 1. Rat body weight and rat liver tissue weight.

Parameters	Control	NM	PA	SM	PASM
Body weight (g)	415.3 ± 43.5	250 ± 20	236 ± 0.7	361 ± 9	358 ± 30.7
Liver index (%)	2.4 ± 0.5	2.51 ± 0.5	2.7 ± 0.5	3.5 ± 0.4	3.1 ± 0.5

NASH models were determined based on earlier reports [39]. The fibrosis activity with a score ≥5 is diagnosed as NASH and a score ≤3 was diagnosed as no NASH condition. Each rats NASH score was more than 8, as depicted in Table 2.

Table 2. The score of the study set.

Events	Gradation	Percentage of occurrence (%; n = 30)	Score
Hepatic steatosis gradation	Liver parenchymal steatosis	—	—
	<5%	0	0
	5%–35%	2	1
	>36%–62%	29	2
	>63%	69	3
Fibrosis stage	None	43	0
	Phase 1, pericellular/sinusoidal; zone 3	7	1
	Phase 2, perisinusoidal and portal; zone 3	23	2
	Porto-central (panacinar) fibrosis	4	3
	Cirrhosis	5	4
Nonzonal patches (contiguous patches)	Microvesicles	81	0
	Not present	19	1
	Present	—	—
Nodular inflammatory foci	Foci absent	0	0
	<2/200 × field	1	1
	2−4/200 × field	57	2
	>4/200 × field	42	3
Large lipogranulomas	Absent	71	0
	Present	29	1
Micrograulomas	Absent	61	0
	Present	39	1
Liver injury and ballooning	No liver injury	23	0
	No ballooning	58	1
	Few prominent ballooning of cells present	19	2
Portal inflammation	Absent	51	0
	Greater than minimal	49	1
Macrophages activation	None/few	91	0
	Many	15	1
Hyaline of Mallory bodies	None/few	65	0
	Many	35	1
Glycogenated cell’s nuclie	Absent	51	0
	Present	49	1
Acidophil cells	Absent	89	0
	Many	11	1
Giant mitochondria	None/few	73	0
	Many	26	1
Diagnostic classification	Not staetohepetitis	0	0
	Boaderline	1	1
	Prominent steatohepetistis	99	2

3.2.1. Synbiotic Reduced Lipogenesis and Accumulation in Rat Hepatic Tissue

Liver being the crucial organ for fatty acid regulation and metabolism, maintains fatty acid secretion and oxidation within cell and plasma. Therefore, an enhanced level of triglycerides in the system is indicative of fat depots. To assess such findings associated with histological feature changes during NASH condition were observed through confocal imaging and were indicated by arrows. The image shows macrovesicular steatosis, hepatocytes inflammation, and apoptotic bodies’ formation. Figure 13 represents three different groups and the comparative changes in histology (Figure 13A,B). The lipid accumulation within the hepatic cells was reduced in the PASM group (Figure 13E) as compared to the PA (Figure 13C). In the PPAR-α antagonist group macrovesicular steatosis can be observed with scattered and distant hepatocytes. The regain of morphology may be due to the efficacy of synbiotic (Figure 13D,E) as a therapeutic agent that can help in lipolysis process through the bioactive compounds present in the AGE.

3.2.2. Rat Colonic Epithelium Lipid Accumulation Reduced Through Synbiotic Intervention

In the PPAR-α antagonist group (Figure 14C), the ORO staining revealed the development of lipid droplets in the colon bilayer along with crypts of Lieberkuhn. In an anomalous shape, TJs are disrupted. Comparing the control group (Figure 14A) with the NM group (Figure 14B), synbiotic treatment (Figure 14D) shows improvements in epithelial barrier function and reduced lipogenesis. The antioxidative and anti-inflammatory properties of the prebiotic and synergistic effect of probiotic may be responsible for the reduced effect of PPAR-α antagonist in the PASM group (Figure 14E).

3.2.3. Synbiotic Effect on MCDHFD and PPAR-α Antagonist-Induced Decrease in Hepatic Cell Dimensions, Hepatocytes Mass, and Lipid Aggregation in Wistar Rat Model

The histologic characteristics of NASH model (Figure 15A, a2) and PPAR-α antagonist (Figure 15A, a3) shown here are indicated by the arrow. The changes reported as compared to the control group (Figure 15A, a1) are steatosis of macrovesicles hepatocyte ballooning, inflammation, and clear fibrosis. Furthermore, Mallory bodies and apoptotic bodies formation were also reported (Figure 15A, a1 and a2). H/E staining of liver tissue of PA rats (Figure 15A, a3) shows Mallory body formation due to hepatocyte degeneration and intralobular inflammatory infiltrations of mononuclear cells, whereas in the PASM (Figure 15A, a5) and SM group (Figure 15A, a4), treated hepatocytes microvesicular degeneration and diffusion was reduced.

3.2.4. Synbiotic Ingestion in Wistar Rats Regulates MCDHFD Induces Modifications in Colon Tissue and Integrity

The lipid droplets and colonic epithelium assayed through H/E staining in the NM and PA (Figure 16B,C) show colon epithelial layer disruption and regained morphology of epithelial barrier in the SM treatment group (Figure 16D,E). The anomaly in epithelium, crypts distortion reduction in the crypts of Lieberkuhn, and alteration in colon mucosa are indicatives of PPAR-α antagonist-affected gut barrier function with respect to the histology of normal group (Figure 16A). This suggests that the combination rectifies the defects caused by antagonist.

3.2.5. The Effects of Synbiotic on Lipid Profiles in Rodents With NASH

The lipid profile analysis showed that PA rat showed elevated levels of colon malondialdehyde (MDA), fecal TG, and mesenteric translocation (Figure 17A, B and –C) as compared to controls. Conversely, HDL-C levels in the serum were low. The treated groups with synbiotic combination comparatively showing reduced levels of lipid profiles. The lipid profile research also reveals a statistically discernible difference in the levels of serum, hepatic triglycerides, and rat liver MDA, sample between the groups (Figure 18A, B and –C, respectively).

3.2.6. Synbiotic Regulate the PPAR-α Antagonist-Induced Lipid Regulatory Genes, Pro-Inflammatory, and Other Related Cytokines Responses of Hepatic Tissue and Improves Anti-Inflammatory Activities

In NASH condition led by PPAR-α antagonist disrupts the release of lipid regulatory genes, and pro-inflammatory and inflammatory cytokines from liver and gut tissue resulting in the deregulation of hepatic metabolism and phenotypic changes [6, 11]. The rat NM and PA models revealed an increase of PNPLA3, SREBP-1c, and decrease of PPAR-α expression (Figure 19A, a1–a3). Similarly, the apoptotic gene regulators expression also improves in the presence of SM (Figure 19B, b1 and b2). Moreover, after synbiotic intervention, there was a simultaneous downregulation registered of cytokines such as IL-6 and TGF-β (Figure 19C, c1 and c2).

3.2.7. Synbiotic Alleviate the Protein Levels of PNPLA3 and TGF-β Suggesting Improved Lipid Regulation and Epithelial–Mesenchymal Transition/Changes (EMT) in Hepatic Cells

Rodents with NASH condition and treated with PPAR-α antagonist showed histopathological changes and the epithelial nature of hepatic tissue changes to fibrotic and elongated in phenotype (Figure 15B). Possibly due to overexpression of PNPLA3 parenchymal and nonparenchymal cell interactions starts and may affect features of metabolic disorder through lipid accumulation and modulating fat content in NASH condition. This causes a cascade of signaling that is initiated by high amounts of TGF-β and low levels of PPAR-α. These EMT affect the morphology of liver cells. This upregulation of TGF-β induced by PPAR-α antagonist activates the downstream transcription factors signaling causing various modifications in hepatic tissue. It is reported previously that TGF-β plays an inevitable role in NASH pathogenesis. TGF-β induces fibroblast marker up regulation such as fibronectin-1, alpha-smooth muscle actin (α-SMA), and epithelial E cadherin downregulation. Synbiotic treatment shows downregulation of PNPLA3 and TGF-β through the PPAR-α pathway demonstrated in protein profile analysis (Figure 20A,B).

3.2.8. Functional Pathway Enrichment Analysis of AGE

KEGG has been used for performing analysis of bioactive agents associated with NASH condition targets to explore their functions and efficacy inside the human body (Figure 21).

3.2.9. AGE Possess Inhibition Affinity for PNPLA3 as a Target Protein for NASH Prevention

Several pro-inflammatory cytokines are produced by irregular diet, leading to oxidative stress in the body. PNPLA3 inhibition prevents different cytokines and inflammatory reactions, which may play a role as a protective compound that attenuate the NASH progression. In the field of computational biology, docking is a method which may be used to predict ligands’ experimental binding modes and their relative affinity when bound to target receptors through their binding pockets. C-D-T network interactions and binding energy were analyzed using molecular docking for bioactive components from AGEs. The bond affinities of S-allyl cysteine sulfoxide, S-allyl-L-cysteine diallyl-disulfide, allicin, Z-ajoene, diallyl sulfide, diallyl tri-sulfide, and E-ajoene with PNPLA3 were −5.4, −5.2, −4.0, −4.3, −4.6, −3.9, −3.7, and −4.8 kcal/mole, respectively. These eight bioactive components of AGE expressed binding affinity for PNPLA3, suggesting that AGE as a compound may target PNPLA3 as a core protein to reduce the NASH progression as shown (Figure 22).

4. Discussion

Pathogenesis of progressive NASH is primarily driven by oxidative stress and inflammation [35]. A high fat diet, as well as abnormal metabolism and hereditary variables such as polymorphism genes, may all contribute to the advancement of NASH, gut dysbiosis, lining eruption, and microbial translocation [39]. Persistent excessive lipid metabolism deregulation has an adverse effect on liver and colon tissue and affects host health negatively [40]. There is an absence of FDA-approved pharmacological or dietary treatment available to prevent or cure NASH. It may be prevented by liver transplant. There are few treatments available till the fibrosis stage. One tailored therapy on L. rhamnosus, as a probiotic improves intestinal integrity [41]. This investigation on the prevention of NASH progression by AGE and L. plantarum MTCC 2621 is the first to demonstrate the preventive efficacy of this combination. SREBP-1c is a crucial gene implicated in NASH development and progression, cell cycle control, apoptosis, lipid metabolism, and transcription regulation, among other functions and has widely been associated with steatosis in many studies [41, 42]. PPAR-α has an important role in various activities of physiology such as glucose homeostasis, lipid metabolism, and anti-inflammatory action [43]. Similarly, PNPLA3 is associated with hepatic lipid accumulation and eventually contributes to the NASH progression. Meanwhile, there is no direct link between SREBP-1c association with cholesterol homeostasis still the levels of cholesterol get modulated in the NASH condition and PPAR-α activation/deactivation [44, 45].

This article describes a series of experiments to elucidate the function of PPAR-α and PNPLA3 in the pathogenesis of NASH, which results in hepatic tissue damage. In vitro studies were conducted on synbiotic combinations that showed no cytotoxicity on HepG2 cells with 180 mg/kg concentration of AGE and 10⁹ CFU/mL concentration of probiotic till 48–72 h. At the cellular level, synbiotic combination enhanced the growth rate of HepG2 cells treated with PPAR-α antagonist concerning other groups with no AGE or synbiotic combination as demonstrated (Figure 2; p < 0.001). To study such intracellular oxidative stress, a lipophilic and nonfluorescent compound DCFDA has been used where PPAR-α antagonist model cells showed more prominent green fluorescent as compared to control and cosupplementation of synbiotic resulted in a significant decrease in oxidative stress as apparent by weak fluorescence with respect to NM group. The apoptosis term refers to peculiar cell death (Figures 5 and 9). In this study, nuclear morphology was examined using DAPI dye to assess apoptotic changes. The cells treated with PPAR-α antagonist show visual evidence of nuclear condensation with respect to cells of the normal group. A significant inhibitory effect of synbiotic treatment was observed on hepatic and colon cell apoptosis. The rate of apoptosis in the PASM group was reduced than in the PA (p < 0.001) for NASH treatment (Figures 3 and 7).

To investigate whether the synbiotic combination protects against NASH mediated by PPAR-α antagonist condition by mediating intestinal barrier disruption of the mucosal membrane the permeability of Caco-2 cells and rat colons was examined. Regained percentage of TEER (%) and intestinal barrier function indicates the positive effect of the synbiotic combination (Figure 6B). ZO-1 is required for the integrity of transmembrane proteins such as occluding, where occluding supports the structural integrity and membrane barrier function [45]. In support of previous research, Occludin and ZO-1, components of TJ assembly, were reduced in the disease model. However, in the treated gut tissue, this function was regained (Figure 10A).

ROS produced during lipid metabolism dysfunction leads to hepatic apoptosis. To verify the same, Bcl-2 and caspase3 could be good apoptotic biomarkers. The reduction in the ROS formation in synbiotic-treated cells unravels the preventive effect of the combination on cells (Figures 17 and 18). Studies in previous reports stated that n = 3 chain polyunsaturated fatty acid consumption lowered the ratio of SREBP1-c to PPAR-α [46]. This ratio change contributes to fatty acid oxidation and steatosis [47]. In this study, PA rats resulted in increased fat mass, leaky gut, and liver inflammation, as well as insulin resistance, which is linked to fatty acid overflow and the buildup of hepatic fat. In addition to gut barrier dysfunction, AMPs production, immune function, and TJ accessibility inhibit bacterial translocation simultaneously [48, 49]. The increased levels TGF-β modulates intracellular oxidative pathways in vascular tissues such as the liver to affect cell adhesion of cells, proliferation, and differentiation leading to apoptosis [50, 51].

Moreover, the treatment with PPAR-α antagonist on rats’ liver resulted in oxidative stress elevation, which leads to lipid peroxidation. Lipid peroxidation results in the MDA formation and oxidative stress marker in the hepatic cells. The exacerbated MDA levels were measured. Clear amelioration is visible after synbiotic treatment. Further, these reduced levels of MDA in the tissue were measured through fluorescence spectroscopy. The confocal images of the liver and gut stained with ORO correlated with restored membrane and barrier function along with mRNA expressions, which is crucial for the prevention of NASH and translocation of bacteria (Figures 12 and 13).

PASM was found to express lower levels of inflammatory cytokine mRNA compared to the PA group (p < 0.01; Figure 19A, a2), suggesting the cumulative effect of the symbiotic combination also improved the PPAR-α levels. Lactobacillus plantarum administration may contribute to the modulations associated with the human gastrointestinal tract. Moreover, to support the theory results of protein levels of PNPLA3 and SREBP1-c also have been checked. The clear drop in the protein levels of PNPLA3 and SREBP1-c suggests the role of AGE in targeting the said pathway (Figure 20).

The KEGG enrichment analysis resulted in the metabolic pathways (hsa01100) being the most affected in the presence of AGE. Other signaling pathway may be affected by the presence of AGE. SM reduces the inhibition of PPAR-α and delays the progression of NASH by reducing inflammatory responses. In molecular docking, ligands binding affinities obtained were −5.4, −3.7, −4.0, −3.7, −4.2, −4.8, −5.2, and −4.8 kcal/mole, of S-allyl cysteine sulfoxide, diallyl sulfide, diallyl-disulfide, diallyl trisulfide, allicin, E-ajoene, Z-ajoene, and S-ally, respectively. S-allyl cysteine sulfoxide binds and downregulates PNPLA3 proteins more efficiently. All eight bioactive components are depicted (Figure 22) with their hydrogen and hydrophobic interactions and distances from the PNPLA3 receptor. The negative binding affinity of these components indicates that they can bind to the PNPLA3 core protein.

5. Conclusion

In conclusion, the current study suggests that the synbiotic has synergistic preventive effects on the development and progression of NASH. The synbiotic combination used can reduce liver tissue injuries and gut dysbiosis mediated by the high expression of PNPLA3 and SREBP-1c and improves the activity of PPAR-α (Figure 23). The results enhanced the antioxidant enzyme levels to eradicate the free radicals, protected hepatocytes, and lowered lipid peroxidation. In the form of food additives, clinical translation of this study would be beneficial to NASH human subjects.

Ethics Statement

The Institutional Animal Ethics Committee (IAEC), R. P. College of Pharmacy, Charotar University of Science and Technology (Changa, Anand, India) with approval letter ID: RPCP/IAEC/2023-24/R15 sanction the study, which complied with CPCSEA guidelines.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Conceptualization, methodology, investigation, visualization, project drafting, formal analysis, software and data validation, data curation, writing–review and editing: Dixa Sharma. Supervision, funding, resources, administration: Palash Mandal. Writing–review: Dhara Patel. All authors have read and approved the final manuscript.

Funding

The authors received no specific funding for this work.

Supporting Information

Additional supporting information can be found online in the Supporting Information section.