Hepatoprotective effect of the flavonoid fustin in a rat model of paracetamol-induced acute liver damage

Paracetamol (PCM)-induced toxicity is a well-established pharmacological model. Cotinus coggygria is a medicinal plant rich in polyphenols, including the flavonoid fustin. The aim of the present study was to evaluate the effects of the flavonoid fustin isolated from Cotinus coggygria heartwood in a model of PCM-induced liver damage. Male Wistar rats (n=48) were allocated to four groups: Control, PCM, F5+PCM, F10+PCM. The rodents were treated daily orally for nine consecutive days as follows: groups F5+PCM and F10+PCM – with fustin (suspended in a vehicle) at doses of 5 and 10 mg/kg, respectively; groups Control and PCM – with the vehicle. PCM was injected intraperitoneally (1.0 g/kg) on day 7. At the end of the experiment, serum and liver samples were prepared. PCM caused a severe liver damage confirmed by histopathological, immunohistochemical and biochemical indices. Compared to PCM group, in F5+PCM and F10+PCM groups, the hepatic necrosis, steatosis, ballooning degeneration, inflammation and expression of NF-κB were significantly reduced. Fustin treatment resulted in a significant reduction of serum activities of alanine aminotransferase and gamma-glutamyl transferase to levels that did not differ from the control values. The present study demonstrated a hepatoprotective effect of the flavonoid fustin in a rat model of acute paracetamol-induced toxicity.


Introduction
Drug-induced liver injury remains the most common cause of acute liver failure [1].Paracetamol (acetaminophen) is a drug often used as an antipyretic and analgesic, which can lead to liver damage if taken in overdose.Paracetamol (PCM) is metabolized in the liver mainly by conjugation (sulfation and glucuronidation) to give non-toxic metabolites that are excreted by the kidneys.At doses above the therapeutic ones, these metabolic pathways are saturated and another metabolic pathway is involved: oxidation by the cytochrome P450 monooxygenase system to the toxic metabolite N-acetyl-pbenzoquinoneimine (NAPQI).Its detoxication consumes endogenous glutathione to form a harmless conjugate.When glutathione is depleted, the remaining toxic metabolite binds to the sulfhydryl groups of proteins, leading to cell necrosis and lipid peroxidation [2].The oxidation of paracetamol by cytochrome P450 generates hydrogen peroxide and a superoxide radical as well.An overdose of paracetamol also causes a decrease in the activities of the antioxidant enzymes catalase and glutathione peroxidase [2,3] as well as inflammation and abnormal immune response [4,5,6].
Cotinus coggygria (smoke tree) is a medical plant with health-promoting biological activities proven in experimental and clinical studies [7,8,9] and attributed to its phytochemical components such as tannins, flavonoids, phenolic acids and essential oils [10].Among these components, dihydroflavonol fustin is the least studied.Fustin and sulfuretin are the main phenolic components of smoke tree heartwood [11].Our previous experiment has shown that aqueous Cotinus coggygria leaf extract could protect the liver from the toxic effect of PCM [8].A review of the database shows that fustin has pronounced antioxidant, cytoprotectivе and anti-inflammatory properties [12,13,14].Currently, no evidence exists about the effect of fustin in acute liver damage in rats.
The aim of the present study was to evaluate the effects of the natural flavonoid fustin isolated from Cotinus coggygria heartwood in rats exposed to acute PCM overdose.

Plant material and extraction
The heartwood from the Eurasian Smoke Tree (Cotinus coggygria Scop.) was collected at the Protected Area "Deliblato Sand", Vojvodina province, Serbia, in May 2022.Plant material was identified by Prof. Milan Veljic, Faculty of Biology, University of Belgrade, and compared to Herbarium specimen BEOU 17422 of the Institute of Botany and Botanical Garden in Belgrade, Serbia.The heartwood was air-dried and milled to a fine powder.One kg of wood powder was extracted three times with 10L of methylene chloride/methanol 1:1 for 24 h at room temperature.After evaporation, 76 g of crude extract was fractionated by Silica-gel column chromatography (CC).

Ethical Statement
This animal study was approved by Bulgarian Food Safety Agency -approval: Protocol № 23/April 15, 2021; Permission № 305/June 28, 2021.Procedures of animal treatment and experiments were conducted in accordance with the national and international laws and policies (EU Directive 2010/63/EU for animal experiments).

Experimental animals and treatment
In the experiment, 48 male Wistar rats (225 ± 25 g) were used.The animals were kept in plastic cages at an average room temperature of 22 ± 1ºC, exposed to 12-hours light /dark cycle (light 7:00-19:00).They had free access to food and drinking water with the exception of the period of 24 hours before PCM administration when all experimental groups were deprived of food.
The animals were randomly divided in four experimental groups each consisting of 12 rats: Control, PCM, F5+PCM, F10+PCM.Fustin was prepared as a suspension in а vehicle (50 μl of Tween 80 per 10 ml distilled water).From day 1 to day 9, the rats were treated once daily by direct stomach intubation as follows: Control and PCM rats received the vehicle (10 ml/kg).F5+PCM and F10+PCM animals received fustin at doses of 5 mg/kg and 10 ml/kg, respectively, as a 10 ml/kg suspension.These doses of fustin have been shown to exert anti-inflammatory and gastroprotective effects in our previous investigations [12,16].

Induction of acute liver damage
On the 7 th day of the experiment, 1 hour after the oral treatment, the rats from groups PCM, F5+PCM and F10+PCM were injected intraperitoneally with PCM (1.0 g/kg) in the form of a suspension with 2 % Tween 80 at a volume of 4.0 ml/kg.Control rats were applied 4 ml/kg of the vehicle.On day 9 th , 48 hours after PCM administration, the animals were anaesthetized with diethyl ether 2 hours after the last oral treatment.Both literature data and our previous experience show that a such a single dose of paracetamol causes a severe damage to the liver of rats after 48 hours [8,17].
Blood was collected from the sublingual veins.It was centrifuged at 2000 rpm for 10 min and serum was obtained for the biochemical analyses.

Histopathological investigation
Histological specimens from the liver were prepared at the Histopathology Laboratory, St. Marina University Hospital -Varna.Liver samples were fixed in 10% buffered formalin for at least 24 h before processing.The fixed tissues were embedded into paraffin.The deparaffinized and rehydrated sections thick 4 μm were stained with hematoxylin and eosin.The degree of hepatocellular changes was scored based on the grading system shown in Table 1.

Immunohistochemical study
The deparaffinized and rehydrated sections were treated in 1% hydrogen peroxide for peroxidase activity inhibition for 5 min.Subsequently the sections were incubated for 24 hours at room temperature with the polyclonal antibody against NF-κB -a rabbit anti-NF-kB-p100 polyclonal antibody (E-AB-32222; Elabscience, USA), diluted 1:200.NF-κB expression was determined by universal highly sensitive visualization system for antibody detection EnVision FLEX.
Negative controls were incubated with nonimmune sera instead of the primary antibody.Saturation index of immune deposits was determined semi-quantitatively in 50 cells of each probe using the following score: 1 -no cytoplasmic staining, 2 -weak cytoplasmic staining, 3 -moderate cytoplasmic staining, 4 -strong cytoplasmic staining.The average intensity of the immune reaction was verified in the following way: number of cells of each type x corresponding coefficient (1, 2, 3 or 4) x total number of cells -1 .

Statistical analysis
The processing and analysis of the data were performed with the statistical software GraphPad Prism using one-way variation analysis (one-way ANOVA) followed by Dunnett's multiple comparisons post-test.Results are presented as mean ± standard error (mean ± SEM).Statistical significance was assumed at a value of p < 0.05.

Histopathological examination of rat liver
The liver of the control group showed normal appearance and preserved hepatic architecture (Figure 1A).PCM induced extensive and confluent necrosis, degenerative changes of the hepatocytes predominantly in the centrilobular and partly in the intermediate zones of the liver lobules and increased number of apoptotic bodies (Figure 1B).Hepatocytes with ballooning and fatty degeneration were observed in close proximity with central veins and intermediate zones (Figure 1B).The livers of the animals from F5+PCM and F10+PCM groups were with more preserved architecture comparison to PCM group, with mild portal and lobular inflammation and ballooning degeneration, without extensive necrosis, with no or a few apoptotic bodies (Figure 1C, Figure 1D).The grading system of hepatocellular damage showed that the scores in fustin-treated groups were significantly reduced compared to PCM group (Figure 2): the steatosis (p < 0.001 for both fustin doses), the ballooning degeneration (p < 0.001 for both fustin doses), the portal inflammation (p < 0.001 for both fustin doses), the zonal necrosis (p<0.05) for both fustin doses), the degree of confluent necrosis (p < 0.01 for fustin 10 mg/kg) and the apoptosis (p < 0.001 for fustin 5 mg/kg and p < 0.01 for fustin 10 mg/kg).

Immunohistochemical investigation
The immunohistochemical expression of NF-κB in the control group is shown on figure 3 and figure 4A while on figure 4B is visible the increased strong expression NF-κB in group PCM.In fustin-treated rats, the expression of NF-kB was reduced significantly in comparison with PCM group (Figure 3, Figure 4C, Figure 4D).

Activities of ALT and GGT
The activities of liver enzymes are presented on figure 5A and figure 5B.The administration of PCM caused a significant elevation of serum ALT and GGT (p < 0.001 compared to Control).Fustin at the tested doses prevented the increase of the enzyme activities.Thus, in rats belonging to groups F5+PCM and F10+PCM, the levels of ALT and GGT did not differ significantly from the control value (Figure 5A, Figure 5B).

Discussion
PCM-induced hepatotoxicity is a well-established pharmacological method to assess the protective effects of new investigational substances [2].At therapeutic doses, PCM is considered effective and safe but in overdose it may cause severe liver injury, liver failure and even death.In the present study, the toxic dose of PCM (1.0 g/kg) caused a significant elevation of serum ALT and GGT activities, induced histopathological changes as hepatic necrosis steatosis, ballooning degeneration, inflammatory infiltration around the central veins and increased the expression of NF-κB.The molecular basis of acute liver injury induced by the toxic PCM metabolite (NAPQI) is an increase in oxidative stress and inflammation, as well as activated apoptosis and cell necrosis [18].Oxidative damage is an important mechanism of PCM-induced acute liver injury [19].The PCM reactive metabolite NAPQI is responsible for production of reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, peroxide radicals, hypochlorous acid, peroxynitrite, which cause lipid peroxidation, protein oxidation and hepatocyte DNA damage [19].
In addition, ROS also lead to an inflammatory response and activate the innate immune cells [5].The ROS cause activation of the transcription factor NF-κB, an important mediator of inflammatory responses which plays a significant role in the development of NAPQI-induced liver injury [18].It is known that natural substances are able to prevent NF-κB activation, likely by inhibition of oxidative stress [20].
Therefore, it can be expected that compounds with free radical scavenging and/or antioxidant activities could be protective against PCM-induced liver damage.Literature data show that natural flavonoids improved the hepatic histopathological changes mainly by ROS scavenging as well as by reducing the production of inflammatory cytokines as a result of their effects on the pathways of NF-κB and phosphatidylinositol 3-kinase/protein kinase [21].
In the present experiment, fustin treatment resulted in a significant reduction of serum activities of ALT and GGT to levels that did not differ significantly from the control values.It reduced the hepatic necrosis, steatosis, ballooning degeneration, inflammation and expression of NF-κB.Fustin, as a typical flavonoid, has been shown to possess strong antioxidant properties manifested by reduction of malondialdehyde level as well as enhancing the activities of superoxide dismutase, catalase and reduced glutathione (GSH) [14].Glutathione activity promotion is able to prevent damage to liver cell components caused by ROS, free radicals, peroxides, and lipid peroxides [13,14].Apart from that, detoxification of the toxic metabolite NAPQI is facilitated by glutathione.Glutathione is conjugated with NAPQI and the resulting conjugate is excreted in the urine [19].The ability of fustin to maintain the level of GSH [13] most likely contributes to its hepatoprotective effect demonstrated in this experiment of PCM-induced liver damage.
A number of polyphenols are known to owe their liver protective properties to modulation of proinflammatory cytokines [23].According to the present experiment, fustin significantly decreased the expression of the inflammatory mediator NF-κB in the liver.
The mechanisms behind the described hepatoprotective effect of fustin proven by biochemical, histopathological and immunohistochemical stadies might be a reduction of oxidative stress and enhancement of endogenous antioxidants [14] as well as suppression of the inflammatory signaling pathway of NF-κB.

Conclusion
This study demonstrated a hepatoprotective effect of the flavonoid fustin isolated from Cotinus coggygria heartwood in a rat model of acute paracetamol-induced toxicity.

Figure 1 .
Figure 1.Microscopic appearance of liver in a rat model of paracetamol (PCM)-induced acute liver damage and fustin treatment: (A) Control group (preserved architectonics); (B) group PCM (hepatic necrosis -black arrow, fatty changes -white arrow, ballooning degeneration -red arrow and inflammatory infiltration around the central veins observed -yellow arrow); (C) F5+PCM group (less marked ballooning degeneration, reduced number of cells with apoptosis and steatosis, reduced confluent necrosis compared to PCM group); (D) F10+PCM group (moderately expressed ballooning degeneration, less pronounced lobular and portal inflammation, confluent necrosis covering a smaller area of the liver parenchyma compared to PCM group); staining with hematoxylin and eosin; magnification А x 320.B, C, D x 200.

Table 1 .
A classification system used to determine the extent of hepatocellular changes in rats exposed to an acute paracetamol overdose; HPF -high power field