Nrf2/HO‐1, NF‐κB and PI3K/Akt signalling pathways decipher the therapeutic mechanism of pitavastatin in early phase liver fibrosis in rats

Abstract Liver fibrosis is a common chronic hepatic disease. This study aimed to investigate the effect of pitavastatin (Pit) against thioacetamide (TAA)‐induced liver fibrosis. Rats were divided into four groups: (1) control group; (2) TAA group (100 mg/kg, i.p.) three times weekly for 2 weeks; (3 and 4) TAA/Pit‐treated group, in which Pit was administered orally (0.4 and 0.8 mg/kg/day) for 2 weeks following TAA injections. TAA caused liver damage manifested by elevated serum transaminases, reduced albumin and histological alterations. Hepatic malondialdehyde (MDA) was increased, and glutathione (GSH) and superoxide dismutase (SOD) were decreased in TAA‐administered rats. TAA upregulated the inflammatory markers NF‐κB, NF‐κB p65, TNF‐α and IL‐6. Treatment with Pit ameliorated serum transaminases, elevated serum albumin and prevented histopathological changes in TAA‐intoxicated rats. Pit suppressed MDA, NF‐κB, NF‐κB p65, the inflammatory cytokines and PI3K mRNA in TAA‐intoxicated rats. In addition, Pit enhanced hepatic antioxidants and boosted the nuclear factor erythroid 2–related factor 2 (Nrf2) and heme oxygenase‐1 (HO‐1) mRNA. Moreover, immunohistological studies supported the ability of Pit to reduce liver fibrosis via suppressing p‐AKT expression. In conclusion, Pit effectively prevents TAA‐induced liver fibrosis by attenuating oxidative stress and the inflammatory response. The hepatoprotective efficacy of Pit was associated with the upregulation of Nrf2/HO‐1 and downregulation of NF‐κB and PI3K/Akt signalling pathways.

such injuries drive inflammation, and liver fibrosis becomes inevitable sequelae of all chronic liver diseases. 11,12iterated liver insults lead to cellular damage, activation of myofibroblasts and persistent inflammation. 13As a result, the wound healing response becomes abnormal, and an increased amount of reactive oxygen species (ROS) is produced, which in turn, triggers the massive generation of inflammatory mediators, such as chemokines, cytokines, and multiple growth factors. 14Increased numbers of immune and inflammatory cells may be attracted by chemokines; once there, cytokines and growth factors can bind to their specific receptor, stimulating the production of several transcription factors and proteins. 15Consequently, regulatory pathways that support normal liver functions, cell development, proliferation and differentiation will also be disrupted.Collagen, elastin, and glycoproteins, among other extracellular matrix components (ECMs), will be made in large quantities, deposited and regenerated in the peri-sinusoidal area. 14The aberrant overexpression of the enzymatic breakdown 'matrix metalloproteinases (MMPs)' and their corresponding antagonists causes matrix remodelling to begin.Moreover, the expression of genes that govern the production of interleukins, enzymes and growth factors is constantly upregulated. 15This causes a vicious cycle of liver damage and repair that finally results in the disruption of liver processes, chronic inflammation and, ultimately, liver fibrosis. 3,16,17 untreated, fibrosis can lead to cirrhosis, hepatic failure and hepatocarcinoma and potentially cause mortality.This process typically takes decades (about 20-30 years), although it can advance quickly, as in the case of biliary atresia, drug-induced liver damage, HIV/HCV coinfection, or HCV infections following liver transplantation. 18A great deal of work has been done to understand the pathophysiology of fibrosis, which has led to the identification of prospective targets for antifibrotic drugs that could either slow down or reverse fibrosis. 19tavastatin is a cholesterol-lowering agent (statin) that was approved in 2009.It is also one of the foundations for treating and preventing atherosclerotic cardiovascular disease. 20Similar to other members of its class (statins), Pit works by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in a competitive manner; it prevents HMG-CoA from being converted to mevalonic acid, the cholesterol precursor. 21More than 200 million users around the globe receive statins and have experienced positive effects, including lowering cardiovascular events and mortality. 22A great deal of recent evidence has shown that statin administration in patients with preexisting chronic liver conditions such as fibrosis, cirrhosis and hepatocellular carcinoma exerts no harmful effect on the liver. 23[24] Some studies postulated that the beneficial action of statins, known as pleiotropic effects, would return to the inhibitory effect on cell proliferation, the anti-inflammatory action or the improvement in endothelial function, and the vaso-protective effect. 25,26Herein, we conducted a study to decipher the cell signalling pathway underlying the therapeutic effect of Pit in liver fibrosis; and to delineate a specific mechanism of action to statins in chronic liver diseases.

| Animals
Wistar rats weighing 150-200 g and 5 months old were acquired from the Animal House Colony at the National Research Centre (NRC, Egypt).Twenty-four rats were housed on a 12-h light/dark cycle at ambient temperature (25°C).The animals were treated in accordance with national and international ethical standards.All the experiments followed the ethical guidelines established by the NRC's Committee on Animal Care and Use's ethics committees (Reg.No. 1041112022).

| Chemicals
For induction of thioacetamide (TAA) toxicity, TAA was bought from Sigma-Aldrich (St Louis, MO, USA).For Pit treatments, Pit was acquired from Western pharmaceutical industries, Egypt.Every other chemical used in the experiments had the highest purity and analytical grade.Freshly suspended Pit was orally administered at 0.4 and 0.8 mg/kg in a 1% Tween 80 solution.

| Experimental design
The rodents were split into four groups, each containing six animals; one was assigned as the negative control, one positive control, in addition to two Pit treatment groups.Rats in Group 1 (negative control group) were given an intraperitoneal (IP) injection of saline three times a week for two consecutive weeks.Rats in Group 2 (TAA group) received an intraperitoneal (IP) injection of TAA (100 mg/kg) three times a week for two consecutive weeks to cause liver fibrosis. 27Rats in groups three and four were given Pit orally '0.4 and 0.8 mg/kg' 28 every day for 2 weeks following the TAA injection.

| Preparation of serum samples
The serum sampling was performed as per Metwaly et al.; the rats were deprived of food overnight.Subsequently, blood samples were taken, and the serum was separated by spinning the samples in a centrifuge at 3000 rpm and 4°C for 5 min.The resulting serum was then stored at −80°C for future use in the analysis of biochemical parameters. 29

| Liver tissue collection
Liver tissues were gathered and rinsed with a cold saline solution.They were divided into three portions.The initial portion was immediately frozen in liquid nitrogen and stored at −80°C for extracting mRNA.
The second portion was homogenized with a mixture of 100 mg of tissue and 1 mL of iced 0.5% potassium chloride.This was followed by 1 min sonication and 10 min centrifugation at 3000 rpm at 4°C.Subsequently, the resulting supernatant was separated and preserved at −80°C.This portion was intended for assessing hepatic glutathione (GSH) and MDA levels using colorimetric kits, as well as for measuring NFκB, p-NFκB, TNFα, IL-6, Nrf2, and HO-1 levels using enzymelinked immunosorbent assay (ELISA).The last portion was fixed in 10% buffered formalin, which was necessary for conducting further histopathological and immune-histochemical examinations.

| Liver function tests
To evaluate liver function, serum ALT, AST, and albumin levels were measured colorimetrically using commercial kits (Bio-diagnostic® kits Cat# AS 10 61 and AL 10 31, Cairo, Egypt).

| Histopathological and immune-histochemical examinations
According to the procedure of El-Said et al., the tissue was embedded in 10% buffered formalin, liver tissues were fixed for 24 h.Then, dehydrated in different grades of alcohol, cleared in xylene, and embedded in paraffin wax.Using hematoxylin and eosin stain, the paraffin sections (4 μm) were stained.To avoid bias, the cells were examined by a blinded pathologist using a light microscope. 30

| Immune-histochemical examination of p-AKT expression
The other paraffin section from each group was used for immunohistochemical detection of the expression of p-AKT in various experimental groups using avidin-biotin-peroxidase according to the method described by. 31r the purpose of identifying a bound antigen and antibody, liver cuts were treated with antibodies for p-AKT (Abcam, Cambridge, MA, USA) at a dilution of 1:200 (v/v) and (Vactastain ABC peroxidase kit, Vector Laboratories, Burlingame, USA).Chromagen 3, 3-diaminobenzidine tetrahydrochloride was used to visualize each marker's expression (DAB, St Louis, MO, USA).

| Statistical analysis
Before the statistical analysis, data values were checked for normality using the Shapiro test.The data are presented as means ± S.E.Data were processed by one-way anova followed by the Tukey-Kramer post hoc test.GraphPad Prism software (version 9, CA, USA) was employed to perform the statistical analysis and establish the represented graphs.The significance level was set to p < 0.05 for all statistical tests. 32

| Thioacetamide-induced alterations in sera parameters in rats
Liver damage, as revealed by ALT and AST activity (Figure 1A,B), by 5.4-and 6.5-fold in relation to the negative control, and a reduction in albumin level (Figure 1C) by 57%, as a consequence of TAA exposure (Figure 1C).Pit administration at both 0.4 and 0.8 mg/kg reduced ALT and AST activity by about 75% and elevated the serum albumin level by 36% and 57%, respectively, compared to the TAA group.

| Inhibition of the TAA-Induced oxidative stress by pitavastatin treatment
The degree of lipid peroxidation was evaluated by detecting malondialdehyde (MDA), a result of lipid peroxidation.TAA-intoxicated rats had a 3.8-fold rise in MDA levels, while Pit (0.4 or 0.8 mg/ kg) treatment suppressed this increase by 43% and 58% relative to the TAA group (Figure 2A).Liver homogenates were tested for GSH level (Figure 2B) and superoxide dismutase (SOD) activity (Figure 2C), an intracellular antioxidant enzyme.TAA caused a decline in both the level of GSH and activity of SOD by 81% and 77% relative to the control group.While the rats given Pit (0.4 or 0.8 mg/kg) recovered the level GSH by 3.3-and 3.7-fold and the activity of SOD by 3.0-and 3.4-fold relative to the TAA group, respectively.

| Pitavastatin activates Nrf2/HO-1 signalling in TAA-Intoxicated rats
TAA downregulated the hepatic Nrf2 (Figure 4A) levels compared with the control group by 78%.Pit (0.4 or 0.8 mg/kg) upregulated hepatic Nrf2 by 3.3-and 3.7-fold relative to the TAA group.To confirm the Pit (0.4 or 0.8 mg/kg) activation in TAA-treated rats, HO-1, the downstream of Nrf2, was estimated, which showed a prominent rise by 2.2-and 2.8-fold relative to the TAA group (Figure 4B).

| Effect of pitavastatin on Nrf2 and PI3K mRNA expression
The mRNA liver content of Nrf2 (Figure 5A) and PI3K (Figure 5B) showed that TAA caused a significant decrease in the Nrf2 and a significant increase in the PI3K by 80% and 2.8-fold relative to the control group.Pit (0.4 or 0.8 mg/kg) administration upregulated the mRNA of Nrf2 by 3.3-and 3.7-fold and downregulated PI3K by 53% and 61% compared to the TAA group.

| Histopathological findings
Livers of control rats showed normal histological structure, normal central veins, portal areas, and hepatic cords (Figure 6A).Livers of TAA-cirrhotic model rats revealed capsular corrugation and marked parenchymal fibroplasia with portal-to-portal bridging fibrosis (Figure 6B).The portal areas showed increased fibrous tissue proliferation that sends septa extending peripherally, dividing the parenchyma into pseudolobules, some of which are hyperplasic regenerated F I G U R E 1 Thioacetamide-induced alterations in the serum of Wistar rats (A) alanine, (B) aspartate aminotransferases, and (C) albumin.Each bar represents the mean ± SE of six rats.* versus normal control group, @ versus TAA group, # versus Pit (0.4 mg/kg) at p < 0.05.Pit, pitavastatin; TAA, thioacetamide.lobules.Along those sepat, there are proliferated bile ducts, few lymphoplasmacytes, and congested vessels.The hepatic cells within those regenerated nodules are larger in size with large magenta nuclei that sometimes-compressed other vacuolated hepatic cells (Figure 6C).Regarding rats treated with Pit following TAA administration, examination of their livers showed that Pit could markedly retract hepatic fibroplasia, particularly with the higher dose group.
No pseudo-lobulation was noticed in both groups.With 0.4 mg/kg Pit administration, only mild fibrous proliferation is observed in scattered portal triads with or without incomplete peripheral extension accompanied by mild to moderate degrees of hepatocellular degenerative changes and scattered necrotic cells (Figure 6D).On the other hand, administering Pit at a dose of 0.8 mg/kg markedly curbed fibrous proliferation within the portal areas and hepatic parenchyma.
Only some hepatocellular vacuolar degeneration and scarce necrotic cells were the only pronounced lesions (Figure 6E).

| Immunohistochemistry findings
Regarding the p-Akt immune expression, the livers of control rats showed nil expression of p-Akt within the hepatic parenchymal cells (Figure 7A).While in TAA fibrotic model rats, a marked increase in the expression of p-Akt was noticed compared with the other experimental groups (Figure 7B).However, Pit administration at both doses (Figure 7C,D) markedly decreased the immune expression of p-Akt among the livers of the treated rats.

| DISCUSS ION
Statins have been recently prescribed for all chronic liver illnesses, and further hepatoprotective benefits are under clinical investigation. 33Pit, a third-generation statin, is of special interest in the treatment of chronic liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. 21,26,34,35It has the highest bioavailability among all statins (more than 60%); therefore, it is highly effective in low doses and will be readily absorbed with minimal side effects. 36e possible hepatoprotective effects of Pit and the underlying mechanisms are being assessed in the current study.TAA-induced liver fibrosis is a reliable model that exactly mimics the biochemical and histological changes of human liver fibrosis. 6 was employed in the present study as it is associated with lower mortality rates than other models.ALT and AST are cytoplasmic in F I G U R E 2 Effect of pitavastatin on (A) malondialdehyde, (B) glutathione, and (C) superoxide dismutase activity in rat livers intoxicated with TAA.Each bar represents the mean ± SE of six rats.* versus normal control group, @ versus TAA group, # versus Pit (0.4 mg/kg) at p < 0.05.Pit, pitavastatin; TAA: thioacetamide.8][39][40][41] Our results indicated that, unlike all other statins, Pit undergoes minimal hepatic metabolism 42 and does not cause any elevation in aminotransferase levels.Infect, Pit is a highly lipophilic agent that undergoes glucuronic acid conjugation, and a recent meta-analysis study demonstrated that only hydrophilic statins result in the risk of aminotransferase elevation. 43Therefore, we can state that the high-dose (0.8 mg/kg) F I G U R E 3 Pitavastatin mitigated inflammation in TAA-intoxicated rats.Pitavastatin decreased hepatic (A) NF-κB, (B) p-NF-κB, (C) TNFα and (D) IL-6 in TAA rats.Each bar represents the mean ± SE of six rats.* versus normal control group, @ versus TAA group, # versus Pit (0.4 mg/kg) at p < 0.05.Pit, pitavastatin; TAA, thioacetamide.

F I G U R E 4
Pit-activated hepatic (A) Nrf2/ (B) HO-1 signalling in TAA-intoxicated rats.Each bar represents the mean ± SE of six rats.* versus normal control group, @ versus TAA group, # versus Pit (0.4 mg/kg) at p < 0.05.Pit, pitavastatin; TAA, thioacetamide.administration of Pit, which showed better control of aminotransferase, was associated with no signs of toxicity in liver fibrosis.In line with our data, the safety of statins for patients with liver dysfunction has also been reported in several clinical trials. 44r results indicate that Pit (0.8 mg/kg) can reverse liver fibrosis by targeting two main pathways-NF-κB and PI3K/Nrf2/ HO-1-that capture inflammation, oxidative stress, and proliferation.Kupffer cells are an important contributor to HSC/HMF activation and liver fibrosis.They function to sense and remove pathogens and dangerous molecules via pattern-recognition receptors (PRRs), which detect danger signals, including lipopolysaccharide, chemical insults, and carcinogens. 45Upon TAA recognition, various inflammatory cytokines and chemokines were released by Kupfer cells, which stimulated HSCs and started the inflammation response. 6A also activated NF-κB, which regulates the inflammatory response in HSCs.This was further confirmed by the high content of Ser536-phosphorylated p65 (the active form of NF-κB).Accordingly, several NF-κB-dependent genes, including IL-6 and TNFα, were upregulated in the TAA group.Our results demonstrated that Pit exerts a potent anti-inflammatory effect, most probably via binding to NF-κB, curbing the formation of its active form, pNF-κB, and, in turn, inhibiting the downstream inflammatory response of NF-κB as represented in the reduced liver content of IL-6 and TNFα. 46idative stress is a common sequela of inflammation; ROS are highly generated in response to persistent inflammatory mediators.Nrf2 regulates the liver resistance to oxidants 47 ; it mediates the expression of antioxidant-responsive elements (ARE), which in turn, the several downstream antioxidant protective genes such as HO-1, SOD, and reduced glutathione (GSH). 48Herein, Pit played a dual function in preventing liver fibrosis: first, it greatly enhanced Nrf2 content and expression, allowing Nrf2 to inhibit the activation of hepatic stellate cells.Second, Pit increased the DNA-binding activity of Nrf2 and induced the expression (measured as liver content) of its target genes, HO-1, SOD, and GSH.Additionally, Pit-owing to its fundamental lipid-lowering effect-reduces the levels and oxidation of low-density lipoprotein; therefore, MDA, a lipid peroxidation byproduct, was significantly decreased upon Pit administration.Collectively, this confirms the cellular protective effect of Pit in liver fibrosis. 49It is worth noting that the MDA data presented in Figure 2A exhibited large error bars, particularly in the TAA group.This could be attributed to inherent individual variability in the degree of lipid peroxidation induced by TAA administration.Importantly, despite the variability, pitavastatin treatment at both doses still significantly reduced MDA levels compared to the TAA group. 35,37,50,51The lack of dose-dependent difference in MDA suppression between the two pitavastatin doses could suggest a potential ceiling effect on lipid peroxidation inhibition even at the lower 0.4 mg/kg dose.However, further studies with larger sample sizes may be warranted to fully evaluate the dose-response relationship.
The PI3K/AKT signalling pathway is intimately connected to the activation of hepatic stellate cells and the production of ECM. 52,53hibition of the PI3K/AKT signalling has been shown to be effective in preventing liver damage, enhancing liver function, and reducing collagen synthesis and deposition. 54,55Therefore, one of the current approaches for treating liver fibrosis is to reduce PI3K/AKT activity.
According to our findings, TAA increased the expression of PI3K and p-AKT, which is consistent with other research. 56,57In line with our findings in the rat model of TAA-induced hepatic fibrosis, the PCR and immunohistochemical analysis showed that Pit efficiently suppresses the PI3K/AKT signal pathway. 58These findings showed that the suppression of the PI3K/AKT signalling pathway by Pit could reduce hepatic fibrosis.Additionally, NF-κB signalling is governed by PI3K/AKT. 59Crosstalk between the PI3K/AKT and NF-κB pathways may have occurred in the current investigation because both of their activity was suppressed.
Beyond its lipid-lowering role, our study showed that Pit has beneficial pleiotropic effects that target key processes in the patho-

F I G U R E 5
Effect of pitavastatin on Nrf2 and PI3k mRNA expression.Each bar represents the mean ± SE of six rats.* versus normal control group, @ versus TAA group, # versus Pit (0.4 mg/kg) at p < 0.05.Pit, pitavastatin; TAA, thioacetamide.F I G U R E 6 Effect of pitavastatin on histopathology findings.Hematoxylin and eosin stained liver sections.(A) liver of control rat showing normal parenchymal cells, portal triad (dotted arrow), and central vein (arrow).(B, C) liver of TAA administrated rat showing portal to portal bridging fibrosis (dotted arrow) with pseudolobulation (PL) and increased fibrous proliferation in portal areas, few lymphoplasmacytes (long arrow), proliferated bile ducteols (short arrow), vacuolated hepatocytes (insert) and few apoptotic cells.Pit (D) (0.4 mg/kg) and (E) (0.8 mg/kg) treated rats showing marked retraction of fibrous proliferation in portal areas (dotted arrow) and in hepatic parenchyma with only mild incomplete peripheral extension of fibrous septa (arrow) in the low dose treated rat.
physiology of liver fibrosis.It acts on inflammation by decreasing the production of NF-kB, and hence the release of proinflammatory cytokines such as TNFα and IL-6, and it decreases the level of oxidative stress by stimulating the Nrf2/HO-1 pathways.F I G U R E 7 Photograph of liver slices stained with antibodies.Normal control (A), TAA group (B), and Pit-treated groups 0.4 or 0.8 mg/kg (C,D).