5-O-Demethylnobiletin Alleviates CCl₄-Induced Acute Liver Injury by Equilibrating ROS-Mediated Apoptosis and Autophagy Induction

Abstract

Polymethoxyflavanoids (PMFs) have exhibited a vast array of therapeutic biological properties. 5-O-Demethylnobiletin (5-DN) is one such PMF having anti-inflammatory activity, yet its role in hepatoprotection has not been studied before. Results from in vitro study revealed that 5-DN did not exert a high level of cytotoxicity on HepG2 cells at 40 μM, and it was able to rescue HepG2 cell death induced by carbon tetrachloride (CCl₄). Subsequently, we investigated acute liver injury on BALB/c mice induced by CCl₄ through the intraperitoneal injection of 1 mL/kg CCl₄ and co-administration of 5-DN at (1 and 2 mg/kg) by oral gavage for 15 days. The results illustrated that treatment with 5-DN attenuated CCl₄-induced elevated serum aminotransferase (AST)/alanine aminotransferase (ALT) ratio and significantly ameliorated severe hepatic damage such as inflammation and fibrosis evidenced through lesser aberrations in the liver histology of 5-DN dose groups. Additionally, 5-DN efficiently counteracted and equilibrated the production of ROS accelerated by CCl₄ and dramatically downregulated the expression of CYP2E1 vitally involved in converting CCl₄ to toxic free radicals and also enhanced the antioxidant enzymes. 5-DN treatment also inhibited cell proliferation and inflammatory pathway abnormally regulated by CCl₄ treatment. Furthermore, the apoptotic response induced by CCl₄ treatment was remarkably reduced by enhanced Bcl-2 expression and noticeable reduction in Bax, Bid, cleaved caspase 3, caspase 9, and apaf-1 expression. 5-DN treatment also induced the conversion of LC3 and promoted the autophagic flux. Conclusively, 5-DN exhibited hepatoprotective effects in vitro and in vivo and prevented liver fibrosis induced by CCl₄.

1. Introduction

Liver is the major organ where chemicals and drugs are processed and metabolized [1]. Damage to the liver poses a serious and fatal risk. Liver fibrosis has been one of the critical issues in the medical community because of the significant cases of morbidity and mortality worldwide. Medical negligence associated with these alterations leads to fibrosis, progressing to cirrhosis, carcinoma, liver failure, and death. The major causes of liver fibrosis include chronic ethanol consumption, non-alcoholic steatohepatitis, viral infections such as hepatitis B and hepatitis C, helminthes infection, excessive amount of iron and copper in the body, obstruction to the bile ducts, and drug-induced liver damage [2,3]. A fibrotic liver is distinguished by the excessive deposition of extracellular matrix (ECM) proteins namely glycoproteins, proteoglycans, and collagens [4,5]. The most commonly studied marker for fibrosis is the accumulation of collagens, and specifically the fibrillar type of collagens. As a result of the alteration in the collagen synthesis extending from the mRNA to the protein levels, there is a hyper elevated increase in the different types of collagen and a significant recognizable increase from basal level in type III collagen [6,7]. During stimulus to fibrogen or liver injury, the hepatic stellate cells (HSCs) go through transformation in the complex and activation, which contributes to excessive collagen synthesis and other aberrations normally seen in liver fibrosis. Studies have shown that HSC transitions from a quiescent vitamin A storing cell into an activated myofibroblast-like cell [8]. The molecular changes occurring in the cells include morphological alterations such as the appearance of cytoskeleton protein smooth muscle α-actin (α-SMA) and a drastic decrease of vitamin A and increased expression of the rough endoplasmic reticulum [9,10].

We used carbon tetrachloride (CCl₄), which is a well-known established chemical hepatotoxin to induce liver injury and fibrosis on BALB/c mice, and it has been extensively studied over the years [11]. CCl₄ causes injury to the liver through different mechanisms such as oxidative stress, inflammation, and programmed cell death [12,13]. CCl₄ is converted to tri-chloromethyl (CCl₃) free radical or tri-chloroperoxyl radical (CCl₃O₂) produced by the cytochrome P450 oxygenase system arising from the endoplasmic reticulum [14]. The newly formed CCl₃ free radical interacts with nucleic acids, lipids, and proteins, damaging the important cellular processes and consequently resulting in impaired lipogenesis leading to fatty acid degeneration and steatosis as well as transcribing low quantities of proteins. Furthermore, compounds formed by the addition of CCl₃ trigger mutations in DNA synthesis, and eventually, it leads to hepatocellular carcinoma. The formed CCl₃O₂ free radical through the oxygenation of CCl₃ triggers lipid peroxidation, resulting in the damage of polyunsaturated fatty acids (PUFAs). Consequently, the cell membrane becomes permeable, exposing the organelles susceptible to damage, thereby causing hepatic damage through inflammatory response, fibrosis, cirrhosis, and progression to hepatocellular carcinoma [15].

Flavonoids are a class of polyphenolic metabolites possessing a benzo-γ-pyrone structure and are abundantly found in plants. The word flavonoid comes from the Latin word flavus, which describes a natural compound that is yellow-colored by nature. There is an approximate estimation that there are around 9000 flavonoids found in different kinds of plants [16]. Flavonoids have been shown to possess several health-promoting effects because of their high antioxidant capacity, anti-bacterial, anti-inflammatory, anti-viral, anti-cancer, and hepatoprotective properties [17]. Different flavonoids extracted from citrus fruits have shown potent hepatoprotective effects on different liver disease models [18,19,20]. 5-DN is one such flavonoid commonly extracted from the citrus fruits and has been shown to possess anti-inflammatory and anticancer properties [21]. However, the hepatoprotective effect of flavonoid, 5-DN has not been studied before. Therefore, the present study was designed to evaluate the effect of flavonoid, 5-O-demethylnobiletin (5-DN) on CCl₄-induced hepatotoxicity. We observed that 5-DN significantly reduced the accumulation of collagen, and neutralized oxidative stress arising from the CCl₄ intermediates observed through ROS downregulation and by boosting antioxidant response. 5-DN was also able to prevent CCl₄-induced apoptosis and subsequently activated autophagy. Taken together, 5-DN has the potential to ameliorate liver fibrosis.

2. Results and Discussion

2.1. 5-O-Demethylnobiletin Protects against CCl4-Induced Cell Death in HepG2 Cells

We evaluated the toxicity of CCl₄ on HepG2 cells in a time-dependent and dose-dependent manner. The results illustrated the toxic nature of CCl₄ at a dose range (5–20 mM), and it was found to be highly cytotoxic. Cell viability massively plummeted at all-time points of 6, 12, 24, and 48 h (Figure 1A). In contrast, we observed that 5-DN was not toxic to HepG2 cells at (10, 20, 40 μM), showing a cell viability of 98%, 94.33%, and 90.66%, respectively, as shown in Figure 1B. Additionally, we co-treated HepG2 cells with CCl₄ and 5-DN and observed that 5-DN exerted a cytoprotective effect with increased dose (Figure 1C). Likewise, the CCl₄-treated group had an elevated expression of lactate dehydrogenase (LDH), which was significantly downregulated on CCl₄ and 5-DN co-treatment groups (Figure 1D). Evaluation of the morphological image revealed that CCl₄ drastically distorted the cellular morphology and caused cell death, which was observably prevented upon 5-DN treatment (Figure 1E). HepG2 cells treated with CCl₄ also had a higher expression of malondialdehyde (MDA) (Figure 1D), ROS generation, mitochondrial membrane damage, and a higher occurrence of apoptotic event, and these effects were efficaciously prevented on cells co-treated with 5-DN (Figure 1F–H). The expression of glutathione (GSH) was also found to be relatively higher in CCl₄ and 5-DN co-treatment groups in comparison to CCl₄-only treatment groups (Figure 1D). In summary, 5-DN rescued HepG2 cells from CCl₄ induced cell death and increased the expression of GSH, which is the chief antioxidant involved in the detoxification of xenobiotics and endogenous compounds.

2.2. 5-O-Demethylnobiletin Ameliorates CCl₄-Induced Collagen Deposition and Severe Hepatic Injury in Mice

CCl₄ is a well-known hepatotoxin that causes serious liver injury and cell death through death pathways such as apoptosis and necrosis [22], and prolonged exposure to CCl₄ leads to cirrhosis and hepatocellular carcinoma [23]. We evaluated the therapeutic effect of 5-DN on CCl₄-induced liver damage. To do this, BALB/c male mice were subjected to 3 times/week treatment with CCl₄ alone or in co-treatment with 1–2 mg/kg of 5-DN for 15 days. We measured the body weight and calculated the liver-to-body weight ratio to observe whether the CCl₄ group had a decreasing effect on body weight and liver weight (Figure 2A,B). Similarly, the estimation of important serum parameters such as AST and ALT revealed elevated levels in comparison to control and 5-DN treated group weight (Figure 2D,E). Histopathological analysis revealed that CCl₄ adversely aggravated liver through the loss in the architecture, massive mononuclear cellular infiltration, inflammation, regenerative nodules, and fibrous septa (Figure 2C). In addition, CCl₄ treatment groups had elevated levels of iron deposits (Figure 3A) evaluated in the hepatocytes as a soluble compound called ferritin or in an insoluble form known as hemosiderin. Coherently, we quantified the occurrence of collagen fiber deposits and found that CCl₄ treatment groups had a higher expression of fibrous septa that vastly extended from the portal veins to the hepatic lobules (Figure 3B,D). In addition, immunohistochemical staining for an important marker of hepatic stellate cell activation αSMA (Figure 3C,E) revealed that CCl₄ aggravated the liver homeostasis, which was observably attenuated by 5-DN treatment.

2.3. 5-O-Demethylnobiletin Ameliorates CCl4-Induced Liver Injury by Governing Inflammation and MAP Kinase Pathway

As we know, inflammatory stimuli activate the cells of the immune system through different intracellular signaling pathways for activating and releasing the inflammatory mediators such as cytokines tumor necrosis factor- alpha (TNF-α) and interleukin- 6 (IL-6) that act through different surface receptors such as Toll-like receptors (TLRs) and activate the mitogen-activated protein kinases (MAPK) [24], thereby leading to the activation of the nuclear factor kappa- light- chain- enhancer of activated B cells (NF-κB) pathway [25]. We observed an elevated concentration of pro-inflammatory cytokines, namely TNF-α and IL-6, after treatment with CCl₄ on the liver tissues, which was significantly neutralized after treatment with 5-DN (Figure 4A,B). In addition, IκBα and NF-κB-p65 were perceptibly reduced on 5-DN groups in comparison to CCl₄ treatment groups (Figure 4C). Similarly, inhibiting the MAP kinase pathway has been one molecular target for ameliorating inflammatory diseases [26]. The MAP kinase family markers such as extracellular signal-regulated kinase (ERK) and p38 were found to be upregulated. When these molecules are activated, they recruit Ras, which leads to the transcription of cell proliferative markers and other pro-fibrogenic factors. MAP kinase is critically and fundamentally involved in activating the hepatic stellate cells (HSCs) and for the synthesis of collagen. Suppressing these molecular markers is associated with inhibiting the HSCs proliferation [27,28,29]. Studies have also shown that MAP kinase is associated with the progression and generation of oxidative stress [24]. Consistent with the anti-inflammatory effects of 5-DN, we observed that the MAP kinase signaling pathway was also significantly inhibited, which was evaluated through immunoblotting analysis. The expressions of p-p38 and p-ERK 1/2 were significantly downregulated (Figure 4C), which were observably and noticeably upregulated on CCl₄ treatment groups. In conclusion, 5-DN treatment neutralized the CCl₄-induced inflammatory response and normalized the cell proliferation pathway.

2.4. 5-O-Demethylnobiletin Modulates CCl₄-Induced ROS Production by Downregulating Lipid Peroxidation, CYP2E1, and Boosting Antioxidant Enzymes

5-DN is a citrus flavonoid possessing high antioxidant activity, which directly participates in reducing the oxidative stress induced by CCl₄ [30]. An excessive amount of endogenous ROS generation creates an imbalance in the redox reaction, consequently increasing the oxidative stress, which resultantly damages the important biomolecules necessary for cell survival [31]. The toxic effects of CCl₄ arise from the conversion of CCl₄ molecule into a free radical trichloromethyl by the cytochrome P450 isozymes of the endoplasmic reticulum [32]. After the formation of trichloromethyl radical, it reacts with a molecular oxygen to form an even more highly toxic form known as the tri-chloromethoxy peroxyl radical [33]. The generated free radicals covalently bind to different proteins and lipids or by removing hydrogen atoms from the polyunsaturated fatty acids in lipids and thus initiate hepatic injury through the lipid peroxidation process. Thus, these unfolded chain of events result in the breakdown of the cellular membrane structure and disrupt the cellular energy process and protein synthesis [32]. As illustrated in Figure 5A, we observed that 5-DN significantly inhibited the production of ROS observed through H₂DCFDA staining images and histogram. In addition, the production of lipid peroxidation marker malondialdehyde was remarkably downregulated in 5-DN treatment groups in comparison to the elevated levels observed in the CCl₄ group (Figure 5E). Studies has shown MDA to be highly upregulated followed by a decrease in glutathione induced by the free radical metabolite carbon tetrachloride [34,35]. Likewise, as presented in Figure 5F,G, the CCl₄ group exhibited reduced levels of antioxidant enzymes SOD and GSH, which significantly differed to those in the control group and 5-DN group in liver tissues. Furthermore, immunostaining and Western blot analysis indicated that 5-DN remarkably downregulated the expression of CYP2E1 (Figure 5B–D), which is a key metabolizing enzyme for CCl₄ bio-activation, which was highly elevated in the CCl₄ group, manifesting enhanced ROS generation and tissue damage.

2.5. 5-O-Demethylnobiletin Attenuates CCl₄-Induced Liver Injury by Inhibiting Apoptosis

CCl₄ augments catastrophic liver damage by increasing the generation of ROS, genotoxicity, and DNA damage, thereby spurring necrotic and apoptotic events [36,37]. In order to enumerate whether CCl₄ administration to mice would trigger apoptosis, we evaluated the expression of apoptosis-associated markers. CCl₄ treatment significantly and markedly upregulated the protein expression of Bax, which is an important pro-apoptotic mitochondrial protein, and reduced the anti-apoptotic Bcl-2 expression. In addition, the expression of cleaved caspase 3, and caspase 9 was significantly reduced in the 5-DN treatment group in comparison to CCl₄ treatment groups. Apaf-1, another important marker during the caspase-dependent mitochondrial apoptotic pathway [38], was noticeably and remarkably upregulated following CCl₄ treatment, while 5-DN administration had lesser expression of an Apaf-1 protein (Figure 6A). We also performed immunofluorescence staining of cleaved caspase 3 on mice liver tissue (Figure 6C). The expression of cleaved caspase 3 was prominently higher in CCl₄ treatment groups evaluated through the intensity of green fluorescence observed on the cytoplasmic regions of the liver tissue. In addition, Transferase-Mediated dUTP Biotin Nick End Labeling (TUNEL) assay staining (Figure 6B) revealed that CCl₄ was able to drastically heighten the occurrence of apoptotic hepatocytes throughout the liver. The apoptotic hepatocytes were clearly and predominantly visible and located around the fibrous septa arising from the central veins and extending further. 5-DN treatment was able to significantly downregulate the expression of TUNEL-positive hepatocytes, indicating apoptotic blockade.

2.6. 5-O-Demethylnobiletin Promotes Autophagy in CCl₄-Induced Liver Injurys

In order to confirm our experimental findings, we evaluated the different markers involved in autophagy. 5-DN treatment increased the expression of Beclin-1 in mice co-treated with CCl₄ and 5-DN. In addition, 5-DN treatment had a higher conversion of LC3-I to LC3-II observed through Western blotting. Furthermore, we also performed immunohistochemistry for analyzing the concentration of LC3-II conversion by estimating LC3B. Since punctate LC3B staining is a major marker for substantiating the formation of autophagosomes and also as a confirmation for autophagy occurrence, we observed that 5-DN treatment groups had a prominently higher formation of punctate structure in comparison with the other groups (Figure 7B). These results revealed that 5-DN promoted autophagic flux in CCl₄-treated mice.

3. Materials and Methods

3.1. Chemicals and Reagents

5-O-demethylnobiletin was purchased from Med-Chemexpress (Cat. No.: HY-N1942, purity > 99%). DMEM, fetal bovine serum (Gibco), penicillin–streptomycin, trypsin, DAPI (4′,6-diamidino-2-phenylindole), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), direct red, hematoxylin and eosin, and di-chlorodihydro-fluorescein diacetate (H₂DCFDA) were purchased from Sigma-Aldrich, St. Louis, Missouri, USA. All other chemicals used for the experiment but not mentioned here were of the highest quality. The list of primary and secondary antibodies used for the study is also provided in the Supplementary Tables S1 and S2. The assay kits used for carrying out experiments for this study are mentioned in Supplementary Table S3.

3.2. Cell Viability Assay

For measuring cell survival, we referred to our previously published paper [39]. We evaluated the reduction of water-soluble yellow dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich: M2128) into an insoluble blue formazan product. Briefly, 96-well plates were used for seeding HepG2 cells at a density of 5 × 10³ cells/well and allowed to grow in a CO₂ incubator. After the cells were 70% confluent, varying doses of CCl₄ (5, 10, 20 mM) or 5-DN (10, 20, 40 μM) or in combination with CCl₄ and 5-DN were used to treat the cells. Following 24 h incubation or different time points for CCl₄-only treated groups, DMEM media was aspirated out and replaced with fresh 100 μL of DMEM in combination with 10 μL of MTT per well and was further incubated for 4 h at 37 °C. Later, the medium was aspirated out, and the insoluble formed formazan crystals were dissolved in 50 μL DMSO and incubated for 30 min in an ELISA plate shaker. The absorbance was measured at 540 nm using a microplate reader, and the percentage of cell proliferation was calculated as per the following formula: (absorbance of treated cells/absorbance of control cells) × 100. For performing LDH cytotoxicity assay, we followed the manufacturer’s protocol.

3.3. Cell Staining

HepG2 cells were cultured in DMEM media supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C under 5% CO₂ incubation condition. For performing the experiment, cells were treated with CCl₄ (20 mM) and 5-DN (20 and 40 μM) and incubated for 24 h. The cells were further used for fluorescence studies following the published protocols [40,41]. Acridine orange (AO) and ethidium bromide (EtBr) staining was followed for the detection of apoptotic cell formations and similarly JC-1 staining for mitochondrial membrane potential damage. After 24 h treatment with CCl4 and 5-DN, cells were washed with PBS and were fixed with cold 4% formaldehyde. Then, the fixed cells were washed again with PBS and treated with 1:1 ratio of AO/EtBr or JC-1 (20 μM) or H₂DCFDA and incubated for 30 min at 37 °C. The cells were washed again with PBS, and the intensity of the resulting fluorescence was detected using an Olympus BX50 fluorescence microscope.

3.4. Acute Liver Damage Induction and Treatment

Male BALB/c mice (5–6 weeks) weighing around 25–30 g were purchased from Orient Bio (Seoul, South Korea) and housed under room temperature 22 ± 2 °C, 55 ± 5% humidity with 12 h light–dark cycle condition in the animal breeding center of Advanced Bio convergence center of Pohang Technopark foundation, South Korea. All the mice were provided with free access to drinking water and rodent food. Before the start of the study, we also ascertained that the experiments related to animal use were conducted in accordance with the Pohang Technopark Laboratory Ethics Committee (approval number ABCC2018004) after obtaining approval on 30 June 2018.

After one week of acclimatization, healthy mice were randomly allocated into six mice per group. For an acute liver injury model (n = 6/group), all mice were intraperitoneally injected with CCl₄ (1 mL/kg) three times a week, except for control groups [42]. 5-DN mice groups were orally administered with 1 and 2 mg/kg every day until the end of the experiment. On the 15th day, mice were anesthetized with isoflurane, and the blood was drawn through cardiac puncture for biochemical analysis. Liver was weighed, measured, and collected for molecular and histological studies.

3.5. Serum Biochemical Examination

At the end of the experiment after 15 days, 1 mL of serum was collected per mouse through cardiac puncture. After blood collection, samples were incubated at room temperature for 30 min and then centrifuged at 14,000 rpm for 15 min. The supernatant was collected after centrifugation. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using the previously described method [43] employing a chemistry analyzer (Mindary, WA, USA). Biochemical parameters were repeated three times.

3.6. Malondialdehyde Assay

For measuring lipid peroxidation, we estimated the concentration of malondialdehyde (MDA), which is the final product during a lipid peroxidation event estimated by following the manufacturer’s protocol (Abcam, Cambridge, CB2 0AX, UK). Optical density was measured at 532 nm through an ELISA plate reader (TECAN 200 infinite PRO).

3.7. Quantitative Determination of Inflammatory Markers through ELISA Assay

For the estimation of proinflammatory cytokines from liver, we homogenized the 30 mg of liver tissues and dissolved them with 600 μL of RIPA buffer and protease and phosphatase cocktail inhibitors, which were sonicated and incubated on ice for 20 min. The liver tissue homogenates were further centrifuged at 13,000× g for 10 min. The supernatant was collected, and levels of IL-6, TNF-α (R&D Systems Quantikine ELISA kits), GSH, and SOD (Biovision, Milpitas, CA, USA) were measured from the supernatants by the ELISA technique as per the manufacturer’s protocol (R&D Systems Quantikine ELISA kits). The details of the assay kits used for the experiment is provided in the Supplementary Table S3.

3.8. Intracellular ROS Production by H₂DCHFDA Staining

For the detection of intracellular reactive oxygen species (ROS) production, we referred to a published paper [44], and the liver tissue paraffin embedded slides were incubated with different concentration of ice-cold methanol (5 min each) for proper permeabilization. After methanol treatment, PBST washing was given, and slides were immersed in acetone and incubated for a further 7 min. Later, distilled water washes (2–5 min) were given, and slides were blocked overnight at 4 °C in 3% BSA as a blocking agent. After that, we washed the slides with PDT 4 times (3 min each). After washing step, H₂DCFDA (10 μM) was treated to each of the slides and incubated for 30 min at 37 °C. The ROS production was analyzed using an epi-fluorescence microscope Nikon Eclipse TS200 (Nikon Corp., Tokyo, Japan Japan) at 200× magnification.

3.9. Hematoxylin and Eosin Staining

For performing H&E staining, we followed the protocol from our previously published paper [45]. The liver tissues were fixed with 4% paraformaldehyde solution for a few days. The liver tissues were processed for staining preparation by incubating them with different concentrations of ethanol and xylene and finally embedding in paraffin and sectioned at a thickness of 6 μM using a histocore rotary microtome. The slides were further allowed to dry for more than 6 h on a platform with a temperature adjusted to 5 °C. After drying the slides, they were immersed in xylene for deparaffinization. Next, the deparaffinized slides were dehydrated in different dilutions of EtOH (100, 90, 80, and 70%) for 3 min. The slides were washed on running tap water with care and immersed in hematoxylin for 15 min. Excessive stain was removed by washing it on running tap water. The slides were again immersed in a 1% HCl-EtOH solution for 30 s, washed with water and immersed on ammonia water for another 30 sec, and then washed again with water. Slides were immersed with eosin for 2 min to counter-stain, which was followed by a dehydration process of ethanol and xylene fixation in a reverse order performed at the beginning of the staining process. A coverslip was added onto the slides along with a few drops of mounting medium and analyzed under a light microscope (200× resolution).

3.10. Prussian Blue Stain

We followed the previously published paper for performing Prussian blue staining [46]. Liver tissue were stored in 10% paraformaldehyde, embedded in paraffin, and sectioned at 5 μM. The slides were de-paraffinized and hydrated in distilled water. Equal parts of mixed hydrochloric acid and potassium ferrocyanide were mixed and prepared. Slides were immersed in the prepared solution and incubated for 20 min. The slides were washed with distilled water, and the slides were dehydrated using different concentrations of alcohol (95% and 100%) for 3 min each. Later, the slides were dipped in xylene (2×) for a duration of 3 min. The slides were covered using a coverslip and a mounting medium. The slides were viewed under a microscope for the accumulation of iron deposits.

3.11. Picrosirius Red Staining for Detection of Collagen Histochemistry

For performing the detection of collagen histochemistry, we followed the Picrosirius red staining method. The paraffin-embedded slides were de-waxed in xylene solution and dehydrated in EtOH solution. Next, the slides were stained with hematoxylin (10 min) and washed under running tap water for another 10 min. The slides were incubated with direct red solution for 1 h. After Sirius red staining, the slides were washed with two changes of acidified water, which was followed by vigorous shaking to remove any remaining acidified water on them. Later, the slides were dehydrated by incubating in 100% ethanol for 5 min. Finally, the slides were cleared in xylene and covered with a coverslip after adding a few drops of mounting medium. The slides were viewed under a microscope for accumulation of collagen deposits.

3.12. Immunohistochemistry

For performing immunohistochemistry, the liver tissues were processed for tissue preparation as discussed earlier. Before the start of the staining process, the slides were heated by boiling for the antigen retrieval step. The sectioned slides (8 μM) were immersed in xylene (5 min, 2×). After xylene fixation, slides were dehydrated in 100%, 95%, 80%, 70%, and 50% EtOH (5 min each). After the end of the dehydration step, slides were washed with PBS (3 min, 3×) and were further incubated with a blocking buffer solution (SuperBlock, Thermofisher Scientific). After blocking, slides were treated with primary antibodies such as αSMA, CYP2E1, and LC3B [47] (Bioworld Technology) and incubated overnight at 4 °C. After primary antibody incubation, slides were washed with PBS and incubated with secondary antibody for 30 min at room temperature. After secondary antibody treatment on the slides, a few drops of DAB substrate solution were added onto each slides and were further incubated for 15 min. Following DAB incubation, slides were washed with distilled water and counter-stained by the nucleus using hematoxylin for 1 min. The slides were incubated with different concentrations of EtOH followed by xylene incubation following the protocol performed during the start of the experiment. Finally, slides were fixed using a mounting medium and allowed to air dry properly, removing unwanted bubbles from them. Then, the slides were observed under a microscope for data collection (Olympus BX51 Fluorescence Microscope).

3.13. Immunofluorescence Staining

For performing immunofluorescence staining, we referred to our previously published paper [48], paraffin-embedded liver tissues were dewaxed by incubating them in xylene and dehydrated using different concentration of EtOH. Next, we performed a heat- induced antigen retrieval step using a pH 9.5 buffer (0.1 M TRIS/HCl and 5% urea) and heating the coverslip at 95 °C for 10 min. The slides were pre-rinsed with PBS (5 min, 3×). Next, samples were incubated with PBS containing 0.1% triton X-100 (10 min) to help in the penetration of the antibody. Later, the slides were washed with PBS for 5 min. Next, the slides were incubated with 1% BSA containing glycine in PBST (PBS + 0.1% tween 20) for 30 min so as to block any unspecific antibody binding and to avoid high background staining. After blocking, the slides were incubated with cleaved caspase 3 [49] and dissolved in 1% BSA in PBST in a humidified chamber overnight at 4 °C. The next day, the slides were washed in PBS (5 min, 3×). After the washing step, the slides were further incubated with FITC-secondary antibody and dissolved in 1% BSA for 1–3 h at room temperature in a dark compartment to avoid loss of fluorescence. After the desired incubation, the FITC-secondary antibody solution was decanted, and the slides were washed again with PBS (5 min, 3×) in the dark. Later, the slides were counterstained with DAPI or Hoechst (DNA stain) at a concentration of 1 μg/mL for 15 min in the dark. The slides were rinsed with PBS and mounted with a coverslip by adding a few drops of mounting medium. The coverslips were later sealed using a nail polish to further prevent the slide from drying. Images were observed under a microscope (Olympus BX51 Fluorescence Microscope) at 200×.

3.14. Measurement of Apoptotic Hepatocytes Using Terminal Deoxynucleotidyl Transferase-Mediated dUTP Biotin Nick End Labeling (TUNEL) Assay

For the detection of apoptosis in paraffin-embedded liver tissues, we performed the experiment as per the manufacturer’s protocol using a TUNEL peroxidase detection kit (DeadEnd™ Colorimetric TUNEL System, Promega). The slides were washed in xylene (5 min, twice) and immersed in decreasing concentration of ethanol (100%, 95%, 85%, 70%, and 50%) for 3 min each. Next, slides were immersed in 0.85% NaCl (5 min) and washed with PBS (5 min). Then, the slides were immersed in 4% paraformaldehyde (15 min) and washed with PBS (5 min, twice). Next, for the permeabilization step, we added 100 μL of 20 μg/mL proteinase K solution followed by incubation at room temperature for 30 min. Later, slides were washed in PBS (5 min) and fixed again with 4% paraformaldehyde (5 min). Slides were washed in PBS (5 min, twice) and incubated in 100 μL equilibration buffer (10 min). Next, 100 μL of TdT reaction mix was added to the tissue sections on the slides and cover slides with plastic coverslips followed by incubation for 1 h at 37 °C. Later, the plastic coverslips were removed, and the slides were immersed in 2 × SSC for 15 min. Slides were re-washed in PBS (5 min, twice) and immersed in 0.3% hydrogen peroxide (5 min). The slides were washed in PBS (5 min), 100 μL of streptavidin HRP (1:500 in PBS) was added to each slide, and the slides were incubated for 30 min at room temperature. Then, the slides were again washed in PBS (5 min) and incubated in DAB solution until a light brown background appeared. Finally, the slides were washed several times in distilled water and mounted with a medium with coverslips and observed under a microscope.

3.15. Western Blotting

The liver tissue was homogenized following the previously mentioned protocol in Section 3.7. The concentration of the protein was measured through Bradford assay at 595 nm wavelength. Proteins were loaded onto an SDS-polyacrylamide gel along with a reference protein ladder [50]. After the completion of protein separation, the gel was transferred onto a PVDF membrane. The membranes were further incubated in 3% BSA prepared in TBST (1 h) for blocking of the membranes. After incubation, the membranes were washed with TBST (5 min, 3×) and were treated with primary antibody against the protein of interest. To visualize, the membranes were incubated for 3 h at 37 °C or overnight at 4 °C in a gel rocker. After that, membranes were washed with TBST (5 min, 3×) followed by treatment with secondary antibody (2 h) at room temperature in a gel rocker. The membranes were again washed with TBST (3 times, 5 min each). The membranes were visualized by using enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences Inc., Piscataway, NJ, USA).

3.16. Statistical Analysis

For determining the quantitative results of all the experiments performed in this study, the values are expressed as a mean ± standard deviation (SD) of the experiments. The statistical significance and the differences in the experimental groups were calculated by using one-way analysis of variance (ANOVA) with Tukey’s test comparing all pair of columns and Dunnett’s post-comparison test for multiple groups, where * represents p-values < 0.05, ** represents p-values < 0.01, and *** represents p-values < 0.001.

4. Conclusions

Our findings from this study clearly showed that 5-DN was able to attenuate CCl₄-induced fibrotic liver and effectively suppressed inflammatory rush on the liver tissue, by inhibiting of IκBα and NF-κB-p65 and the subsequent drop in the pro-inflammatory cytokine production (Figure 8). 5-DN also effectively downregulated the expression of CYP2E1 and inhibited apoptosis arising from CCl₄-induced hepatic damage and subsequently promoting the autophagic flux. Conclusively, our findings from this study point toward the very strong positive effects 5-DN had on acute liver injury, although further studies need to be carried out to validate the effectiveness of 5-DN on fibrosis.