Morin (98%) (Figure 1), paracetamol (99%), and silymarin (98%) were purchased from Sigma-Aldrich (Germany).

Figure 1

Adult albino rats weighing roughly 185 ± 5 g were obtained from the animal house at the National Institute of Cancer and maintained in an observational environment unders daily observation. They were housed in cages and provided free access to food and water.

Table 1

Groups	Group name	Treatment description
I	Normal 1 % tween 80	3 mL of 1 % tween 80, orally for 14 days
II.	Control Paracetamol	3 mL of 1 % tween 80, orally for 14 days
III	Paracetamol + Morin	Rats treated with Morin (50 mg/kg.b.w.) suspended in 1 % tween 80 by oral gavages for 14 days.
IV	Paracetamol + Morin	Rats treated with Morin (100 mg/kg.b.w.) suspended in 1 % tween 80 by oral gavages for 14 days.
V	Paracetamol + Silymarin	Rats treated with silymarin (100 mg/kg b.w.) suspended in 1 % tween 80 by oral gavages for 14 days.

The hepatoprotective activity of morin when administered for 2 weeks against paracetamol-induced hepatotoxicity was evaluated. The Animal Care and Use Committee of the Faculty of Applied Health Sciences Technology at O6U developed the criteria for this experiment. Table 1 illustrates the experimental study design.

The animals in all groups fasted for 18 hours on day 13, the day before the last treatment. Those categorized into groups II, III, IV, and V were administered with paracetamol (1 g/kg body weight) 1 hour after the last dose of morin therapy on day 1432.

Blood was drawn from the retroorbital vein of each animal and then placed in heparin-containing tubes. The heparinized blood was centrifuged at 1000 × g for 20 minutes, and the cholesterol33, triglyceride34, and HDL-C35 levels were measured in the separated plasma.

The rats were sacrificed, and their livers were collected to assess the biochemical parameters. The liver homogenate was prepared using 25% W/V saline and glass homogenizer. Four aliquots of the homogenate were made.

The first aliquot was used to calorimetrically estimate the ALT36, AST36, ALP37, LDH38, tumor necrosis factor-alpha (TNF-α)39, and protein thiol40 levels. The GSH level was calorimetrically measured in the second aliquot41 at 412 nm. The third aliquot was used to calculate the liver MDA level42 at 535 nm against a blank containing all reagents, except for the tissue homogenate. The fourth aliquot was minced with physiological saline and rinsed in ice-cold water. The supernatant was used to evaluate the SOD43 and CAT44 levels.

Table 2

Genes	Primer sequences
NF-κB	F: 5′-CATGAAGAGAAGACACTGACCATGGAAA-3’
	R: 5′-TGGATAGAGGCTAAGTGTGACACG-3’
NOX-2	F: 5′-TGAACAACAGCACTCACCAATGCC-3′
	R: 5′-AGTTGTTGAACCAGGCAAAGGCAC-3′
IL-6	F: 5'-GCCCTTCAGGAACAGCTATGA-3'
	R:5'-TGTCAACAACATCAGTCCCAAGA-3'
HO-1	F:5′-TGCTAGCCTGGTGCAAGATA-3′
	R:5′-GCCAACAGGAAGCTGAGAGT-3′
β-Actin (internal control for qRT-PCR)	F: 5′-GGCTGTATTCCCCTCCATCG-3’
	R:5′-CCAGTTGGTAACAATGCCATGT-3’

The total liver RNA was obtained using the TRIzol technique in accordance with the manufacturer’s instructions (Life Technologies Corp., Grand Island, NY). Thereafter, 1 µg RNA was combined with 0.5 mmol/L dNTP, 10 nmol/L dithiothreitol, 25 pg primer oligo (dT), and 200 units reverse RNase H superscript II in reaction buffer. The reactions were incubated for one cycle at 42°C for 2 minutes and then for another 50 minutes at 42°C, after which they were heated for 15 minutes at 70°C and chilled at 4°C. Table 2 illustrates the primers for NF-κB, NADPH oxidase-2 (NOX-2), interleukin-6 (IL-6), and heme oxygenase-1 (HO-1). β-actin mRNA was employed as a housekeeping gene45.

The liver tissues were divided into very thin slices (4 μm) and then fixed in 10% formaldehyde solution46. A light microscope was used to evaluate the histological alterations on H&E-stained slides.

The results were reported as means ± SDs. All data were statistically analyzed using SPSS version 1847 and one-way analysis of variance, with the statistical significance level set at P < 0.001.

Table 3

No.	Groups	Cholesterol (mg/dl)	Triglycerides (mg/dl)	HDL-C (mg/dl)
(I)	Normal 1 % tween 80	153.2 ± 13.50	101.3 ± 6.85	34.12 ± 3.90
(II)	Control Paracetamol (1 g/kg.b.w)	261.7 ± 14.50a	144.2 ± 9.61a	23.29± 4.97a
(III)	Paracetamol + Morin (50 mg/kg.b.w.)	184.2 ± 8.63ab	121.2 ± 7.47ab	29.48 ± 3.99ab
(IV)	Paracetamol + Morin (100 mg/kg.b.w.)	165.8 ± 14.95b	110.9 ± 11.55b	31.86± 3.67b
(V)	Paracetamol + Silymarin (100 g/kg b.w.)	157.6 ± 17.65bc	108.5 ± 8.27b	33.03± 3.43b

Paracetamol was given orally as a single dose of 1g/kg.b.w. to 18h fasted animals. It was given to all groups except the normal one. Morin and silymarin were orally given daily for 2 weeks and the last dose of each was given 1 h before paracetamol administration. Values represent the mean ± SE (n = 6). Data shown are mean ± standard deviation of number of observations within each treatment. a: significant with Group I; b: significant with Group II; c: significant with Group III. Data are significantly different at P ≤ 0.001.

Table 4

No.	Groups	ALT (U/g tissue)	AST (U/g tissue)	ALP (U/g tissue)	LDH (U/g tissue)
(I)	Normal 1 % tween 80	63.26 ± 6.24	129.0 ± 6.15	206.08 ± 12.77	312.1 ± 19.25
(II)	Control Paracetamol (1 g/kg.b.w)	103.1 ± 9.29a	183.4 ± 8.40a	276.89 ± 10.45a	422.3 ± 14.19a
(III)	Paracetamol + Morin (50 mg/kg.b.w.)	82.26 ± 7.13b	137.19 ± 9.01b	225.02 ± 8.06ab	340.6 ± 15.22b
(IV)	Paracetamol + Morin (100 mg/kg.b.w.)	68.58 ± 4.33bc	134.5 ± 5.18b	211.99 ± 12.53b	311.9 ± 20.65b
(V)	Paracetamol + Silymarin (100 g/kg b.w.)	64.62 ± 5.86bc	129.8 ± 11.41b	208.53 ± 8.92b	308.8 ± 16.89bc

Paracetamol was given orally as a single dose of 1g/kg.b.w. to 18h fasted animals. It was given to all groups except the normal one. Morin and silymarin were orally given daily for 2 weeks and the last dose of each was given 1 h before paracetamol administration. Values represent the mean ± SE (n = 6). Data shown are mean ± standard deviation of number of observations within each treatment. a: significant with Group I; b: significant with Group II; c: significant with Group III. Data are significantly different at P ≤ 0.001.

Table 5

No.	Groups	Protein thiols (nmol sulfhydryl/mg protein)	GSH (nmol/gm tissue)	SOD (U/gm tissue)	CAT (U/g tissue)
(I)	Normal 1 % tween 80	78.96 ± 5.57	26.41 ± 3.09	30.53 ± 5.65	99.81 ± 8.27
(II)	Control Paracetamol (1 g/kg.b.w)	52.65 ± 7.57a	11.75 ± 1.68a	10.86 ± 2.49a	35.92 ± 4.46a
(III)	Paracetamol + Morin (50 mg/kg.b.w.)	64.75 ± 5.47ab	20.88 ± 2.63ab	24.29 ± 3.42ab	79.95 ± 7.23ab
(IV)	Paracetamol + Morin (100 mg/kg.b.w.)	73.44 ± 6.99b	22.37 ±3.13b	27.36 ± 3.27b	88.98 ± 8.44b
(V)	Paracetamol + Silymarin (100 g/kg b.w.)	79.36 ± 7.41bc	24.11 ±2.99 b	29.13 ±2.63b	92.26 ±11.50b

Paracetamol was given orally as a single dose of 1g/kg.b.w. to 18h fasted animals. It was given to all groups except the normal one. Morin and silymarin were orally given daily for 2 weeks and the last dose of each was given 1 h before paracetamol administration. Values represent the mean ± SE (n = 6). Data shown are mean ± standard deviation of number of observations within each treatment. a: significant with Group I; b: significant with Group II; c: significant with Group III. Data are significantly different at P ≤ 0.001.

Table 6

No.	Groups	Hepatic MDA (nmole/g tissue)	Plasma TNF-α (Pg/mL)
(I)	Normal 1 % tween 80	73.82 ± 6.93	36.64 ± 4.97
(II)	Control Paracetamol (1 g/kg.b.w)	154.0 ± 11.84a	122.8 ± 10.28a
(III)	Paracetamol + Morin (50 mg/kg.b.w.)	81.56 ± 7.92b	50.20± 6.0ab
(IV)	Paracetamol + Morin (100 mg/kg.b.w.)	76.07 ± 7.68b	43.25± 4.38b
(V)	Paracetamol + Silymarin (100 g/kg b.w.)	72.53 ± 5.10b	40.91± 5.61b

Paracetamol was given orally as a single dose of 1 g/kg.b.w. to 18 h fasted animals. It was given to all groups except the normal one. Morin and silymarin were orally given daily for 2 weeks and the last dose of each was given 1 h before paracetamol administration. Values represent the mean ± SE (n = 6). Data shown are mean ± standard deviation of number of observations within each treatment. a: significant with Group I; b: significant with Group II. Data are significantly different at P ≤ 0.001.

Figure 2

Figure 3

Table 7

No.	Groups	Congestion	Lymphatic infiltration	Necrosis	Degenerated hepatocytes	Pyknosis, karyolysis	Fatty degeneration (steatosis)
(I)	Normal (1 % tween 80)	-	-	-	-	-	-
(II)	Control Paracetamol (1 g/kg.b.w)	++	++	++	+++	+	+++
(III)	Paracetamol + Morin (50 mg/kg.b.w.)	+	+	+	++	+	++
(IV)	Paracetamol + Morin (100 mg/kg.b.w.)	+	+	-	+	-	-
(V)	Paracetamol + Silymarin (100 g/kg b.w.)	+	+	-	+	+	-

(-) indicates normal, (+) indicates mild, (++) indicates moderate, (+++) indicates high

Figure 4

The plasma TC, TG, and HDL-C levels in the different groups of treated rats are shown in Table 3. The plasma TC and TG levels increased significantly by 70.82% and 42.34%, respectively, while the plasma HDL-C level decreased significantly by 31.74% in the paracetamol-treated rats compared with that in the control rats. Furthermore, morin treatment (50 mg) significantly reduced (P < 0.001) the plasma TC and TG levels by 36.64% and 15.95%, respectively, and significantly increased (P < 0.001) the plasma HDL-C level by 26.57%. Meanwhile, compared with paracetamol treatment, morin treatment (100 mg) significantly reduced the plasma TC and TG levels by 36.64% and 23.09%, respectively, and significantly increased the plasma HDL-C level by 36.76%. Silymarin treatment (100 mg) significantly decreased the plasma TC and TG levels by 39.77% and 24.75%, respectively, and significantly increased the plasma HDL-C level by 41.82% compared with paracetamol treatment (P < 0.001). Compared with the control treatment, oral paracetamol (1 g) administration substantially increased the liver ALT, AST, ALP, and LDH levels by 62.97%, 42.17%, 34.36%, and 35.31%, respectively, indicating the presence of acute liver injury (P < 0.001). Compared with paracetamol treatment, morin treatment (50 mg) significantly reduced the liver ALT, AST, ALP, and LDH levels by 20.24%, 25.19%, 18.73%, and 19.35%, respectively, while morin treatment (100 mg) significantly reduced the liver ALT, AST, ALP, and LDH levels by 33.48%, 26.66%, 23.46%, and 26.14%, respectively (Table 4).

Silymarin treatment (100 mg/kg body weight) reduced the liver AST, ALT, and ALP levels by 37.32%, 29.23%, 24.68%, and 26.87%, respectively, compared with paracetamol treatment.

Table 5 shows that the liver protein thiol, GSH, SOD, and CAT levels significantly decreased (P < 0.001) by 33.32%, 55.50%, 64.42%, and 64.01%, respectively, in the paracetamol (1 g)-treated rats compared with those in the control rats. Meanwhile, the liver protein thiol, GSH, SOD, and CAT levels increased by 22.98%, 77.70%, 104.81%, and 122.58%, respectively, in the paracetamol (1 g)- and morin (50 mg)-treated rats compared with those in the paracetamol-treated rats (P < 0.001). Morin treatment (100 mg) among the paracetamol-treated rats significantly increased the liver protein thiol, GSH, SOD, and CAT levels by 39.48%, 90.38%, 151.93%, and 147.72%, respectively, compared with paracetamol treatment alone (P < 0.001). Silymarin treatment (100 mg) significantly increased the liver protein thiol, GSH, SOD, and CAT levels by 50.73%, 105.19%, 160.23%, and 156.85%, respectively, compared with paracetamol treatment.

Table 6 shows the effect of morin and silymarin on the liver malondialdehyde (MDA) and plasma TNF-α levels among the rats. The liver MDA and TNF-α levels (P < 0.001) in the paracetamol (1.0 g)-treated rats significantly increased by 108.62% and 235.15%, respectively, compared with those in the control rats. Further, morin treatment (50 mg) significantly decreased the liver MDA and TNF-α levels by 47.03% and 59.12%, respectively, compared with paracetamol treatment (P < 0.001).

The liver MDA and TNF-α levels significantly decreased by 50.60% and 64.78%, respectively, in the morin-treated rats compared with those in the paracetamol-treated rats (P < 0.001).

However, silymarin administration significantly decreased the liver MDA and TNF-α levels by 52.90% and 66.69%, respectively, compared with paracetamol administration alone (P < 0.001).

Figure 3 shows significantly increased (P < 0.001) liver NF-κB, NOX-2, and IL-6 levels (by 618.6%, 516.83%, and 523.76%, respectively) as well as a significantly decreased (P < 0.001) liver HO-1 level (by 77.23%) in the paracetamol (1 g)-treated rats compared with those in the control rats (P < 0.001), suggesting the presence of acute liver inflammation. Morin administration at 50 mg/kg body weight significantly decreased the expression of liver NF-κB, NOX-2, and IL-6 by 63.44%, 53.29%, and 65.5%, respectively, and significantly increased (P < 0.001) that of liver HO-1 by 204.34% compared with paracetamol administration (P < 0.001).

Morin administration at 100 mg significantly decreased the expression of liver NF-κB, NOX-2, and IL-6 by 63.16%, 57.14%, and 67.31%, respectively, and significantly increased (P < 0.001) that of liver HO-1 by 226.09% compared with paracetamol administration (P < 0.001).

Further, the silymarin (100 mg)-treated rats showed significantly decreased liver NF-κB, NOX-2, and IL-6 levels (by 66.30%, 58.42%, and 65.39%, respectively) and significantly increased (P < 0.001) liver HO-1 levels (by 208.70%) compared with the paracetamol-treated rats (P < 0.001).

The results of the agarose gel electrophoresis of NF-κB, NOX-2, IL-6, HO-1, and β-action via real-time PCR are presented in Figure 3.

In the histopathological evaluation of the liver sections, the control rats had a typical central vein and hepatocyte arrangement as well as a normal structure with no histological abnormalities. The nuclei of the hepatocytes were visible as dark red structures within the cells, whereas the cytoplasm was stained red. The sinusoids and typical portal area as well as the hepatic strands were observed from the margin of the lobule to the central vein. The number of binucleated and Kupffer cells was evaluated (Table 7 and Figure 4a).

The liver sections of the paracetamol-treated rats showed an altered lobular shape and a nuclear disintegration in certain locations as well as disarrangement of normal hepatic cells, necrosis, and fatty degeneration. Enlargement and congestion of the hepatic central vein were observed, and lymphocyte infiltration was found in the portal area (Table 7 and Figure 4b). Furthermore, the hepatocytes revealed some pathological alternation; moderate hepatocyte deterioration was observed. Moderate fatty degeneration was seen, along with mild mononuclear cell invasion and pyknosis/necrotic alterations in the paracetamol- and morin (50 mg/kg body weight)-treated rats compared with that in the paracetamol-treated control rats (Table 7 and Figure 4 c).

Histological examination of the hepatocytes of the groups showed signs of progress. The active euchromatic nuclei in the hepatocytes appeared to be in outstanding condition, while the hepatocytes and portal components appeared to be in good condition. Mild infiltrations of inflammatory cells were identified adjacent to the portal triads in the hepatic sections. The observed congestion was mild, and no evidence of fatty degeneration was found. The paracetamol- and morin-treated rats and silymarin-treated rats showed no necrotic changes compared with the paracetamol-treated rats (Table 7 and Figure 4d). The hepatic histology appeared normal, with the hepatic cords correctly positioned around the sinusoids. The observed congestion was minimal, and there was no evidence of fatty degeneration, necrosis, or invasion of mononuclear cells. There were a few liver cells that had deteriorated. The blood vessels were blocked to a modest degree in the silymarin-treated rats compared with those in the paracetamol-treated rats (Table 7 and Figure 4 e).

Paracetamol promotes hepatotoxicity by activating metabolic pathways. It is bio-transformed into NAPQI by the cytochrome P2E1 system in the endoplasmic reticulum. When NAPQI is combined with cellular lipids and proteins, mitochondrial dysfunction, lipid peroxidation, and oxidative stress occur48. The loss of Ca²⁺ homeostasis eventually leads to cell death. The potential of a drug to suppress the aromatase activity of cytochrome P450, leading to liver regeneration, is known as a hepatoprotective action49, 50. In our study, oral paracetamol administration significantly increased the TC and TG levels and decreased the plasma HDL-C level, indicating the presence of liver toxicity. Morin and silymarin treatment reduced the plasma TC and TG levels while considerably increasing the HDL-C level. Generally, lipid deposition in the liver can occur because of an excessive supply of lipids or lipid deposition interference. Lipid-lowering drugs have been reported to have antioxidant properties that prevent lipid peroxidation, TC and TG elevation, and HDL-C induction by paracetamol51. Omidi et al. suggested polyphenols as beneficial agents in improving the lipid profile levels52.

In the present study, the liver ALT, AST, ALP, and LDH levels dramatically increased in the paracetamol-treated rats. This elevation may be attributed to the disruption of the liver cells as a result of necrosis53.

The liver cell damage induced by paracetamol overdose leads to NAPQI overproduction, hepatocyte necrosis, and liver enzyme level elevation54.

Our results indicate that morin and silymarin significantly reverse the enhancement of liver enzyme activity. The hepatoprotective activity of morin has been identified in certain toxicant in vitro and in vivo models of induced liver fibrosis55, 56, 57, while that of polyphenols is common in experimental and clinical studies. Morin and silymarin are used as a hepatoprotective and antioxidant drug58.

Our study findings agree with those of Asmah et al.59 that polyphenols have a hepatoprotective impact and considerably reduce the liver enzyme levels in animal models.

The current work investigated paracetamol-induced liver necrosis in vivo. Endogenous reactive oxygen species are produced by NAPQI linked to the thiol group of GSH, resulting in lipid peroxidation and cell death60. Our analyses revealed that the paracetamol-treated rats had lower protein thiol, GSH, SOD, and CAT levels.

Oral morin treatment significantly improved the liver protein thiol, GSH, SOD, and CAT levels. Morin has a wide range of antioxidant activities because it has a hydroxyl group (-OH) and a double bond between the C2 and C3 atoms that activates the double bond61. Its anti-lipid peroxidation potential is caused by the presence of 2 -OH at the 2′ and 4′ locations of the B ring62, 63, 64. Furthermore, the antiradical action of related polyphenols is thought to be predominantly due to -OH61.

The free radical scavenging activity of morin against ABTS⁺ and cytoprotective activity against ABTS⁺ radicals in tissue fibroblasts were evaluated and found to increase the superoxide dismutase activity and reduce intracellular ROS generation.

Herein, paracetamol-induced lipid peroxidation was detected in the rats, as demonstrated by the increased MDA levels and decreased GSH, SOD, and CAT levels in the liver tissues, all of which are key antioxidant indicators. MDA immunohistochemistry was found to be associated with hepatocyte necrosis in the central zone at early time points. Attenuation of inflammatory markers is mediated by a similar mechanism65. We found an elevation of the liver TNF-α levels that indicated the presence of oxidative stress and inflammation in the liver of the paracetamol-treated rats. However, oral morin and silymarin administration to the paracetamol-treated rats significantly decreased the MDA and TNF-α levels compared with paracetamol administration only. The current results on morin and silymarin treatment could be attributed to their antioxidant and membrane-stabilizing capabilities as well as a probable influence on paracetamol metabolism, as suggested by our in vivo findings on phase I bio-transformation enzymes involved in paracetamol bioactivation60, 61.

Morin enhanced the hemodynamic profile and inhibited the inflammatory marker pathways in a dose-dependent manner66.

We observed that the paracetamol-treated rats showed increased NF-κB, NOX-2, and IL-6 levels and decreased HO-1 levels. However, morin and silymarin administration depleted the expression of NF-κB, NOX-2, and IL-6 and alleviated the significant increase in the OH-1 level compared with paracetamol administration only.

Morin downregulated HO-1, which is linked to decreased Nrf2 expression and phosphorylation and improved Keap1 expression67. Morin administration in chemotherapy also had cytoprotective effects68.

The effect of morin in suppressing LXR signaling may help reduce liver steatosis and other metabolic disorders69. Morin can also improve the degree of gene expression of inflammatory mediators and controlled liver function induced by anticancer medication70. All biochemical parameters, especially NF-κB, NOX-2, IL-6, and HO-1, proved the antioxidant and anti-inflammatory mechanism of morin in the amelioration of paracetamol-induced toxicity.

To our knowledge, our study may be the first to evaluate the regulatory activity of morin for NF-κB, NOX-2, IL-6, and HO-1 in a model of paracetamol-induced liver fibrosis.

Morin protects against paracetamol-induced liver fibrosis by ameliorating the antioxidant and inflammatory biomarker levels. Its scavenging activity improves the expression of NF-κB, NOX-2, IL-6, and HO-1 in the liver.

None.

All authors equally contributed to this work. All authors read and approved the final manuscript.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.