Kaempferol Suppresses Carbon Tetrachloride-Induced Liver Damage in Rats via the MAPKs/NF-κB and AMPK/Nrf2 Signaling Pathways

Oxidative stress plays a critical role in the development of liver disease, making antioxidants a promising therapeutic approach for the prevention and management of liver injuries. The aim of this study was to investigate the hepatoprotective effects of kaempferol, an antioxidant flavonoid found in various edible vegetables, and its underlying mechanism in male Sprague-Dawley rats with carbon tetrachloride (CCl4)-induced acute liver damage. Oral administration of kaempferol at doses of 5 and 10 mg/kg body weight resulted in the amelioration of CCl4-induced abnormalities in hepatic histology and serum parameters. Additionally, kaempferol decreased the levels of pro-inflammatory mediators, TNF-α and IL-1β, as well as COX-2 and iNOS. Furthermore, kaempferol suppressed nuclear factor-kappa B (NF-κB) p65 activation, as well as the phosphorylation of Akt and mitogen-activated protein kinase members (MAPKs), including extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 in CCl4-intoxicated rats. In addition, kaempferol improved the imbalanced oxidative status, as evidenced by the reduction in reactive oxygen species levels and lipid peroxidation, along with increased glutathione content in the CCl4-treated rat liver. Administering kaempferol also enhanced the activation of nuclear factor-E2-related factor (Nrf2) and heme oxygenase-1 protein, as well as the phosphorylation of AMP-activated protein kinase (AMPK). Overall, these findings suggest that kaempferol exhibits antioxidative, anti-inflammatory, and hepatoprotective effects through inhibiting the MAPK/NF-κB signaling pathway and activating the AMPK/Nrf2 signaling pathway in CCl4-intoxicated rats.


Introduction
Oxidative stress is implicated in the pathogenesis of various pathological conditions, including hepatic injuries [1][2][3]. The liver is susceptible to oxidative stress induced by several stimuli, such as alcohol, malnutrition, drugs, toxic substances, and hepatitis viruses. Oxidative stress causes hepatic injury by altering the structure or function of biomolecules and modulating molecular signaling pathways essential for regulating normal hepatic functions [4]. Hence, attenuating oxidative stress by administering antioxidants could be a rational therapeutic approach for the prevention or management of oxidative stress-related hepatic injuries [5]. Dietary phytochemicals have been shown to possess potent antioxidant properties [6,7]. Among the phytochemicals, flavonoids are considered the most active components present in various foods, vegetables, and herbal medicines. Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, Figure 1) is a flavonoid abundantly found in many edible and dietary sources, such as onion, pumpkin, cauliflower, carrot, broccoli, and black tea [8,9]. Approximately 17% of flavonoids in a regular diet are attributed to kaempferol [9]. In vitro, in vivo, and clinical studies have demonstrated that Numerous studies have reported that plant extracts containing kaemp Miconia albicans, Onosma bracteata, Physalis peruviana, and Solanum elaeagnif protective effects in various experimental liver-injury models [13][14][15][16]. Howe tracts contain various phytochemicals in addition to kaempferol, such as qu enin, and rutin, and those compounds have been shown to exhibit hepato fects. Therefore, it is essential to evaluate the hepatoprotective activity of kae cifically, not of crude extracts. Recent studies have reported kaempferol's h tive properties against: acetaminophen-induced liver damage by the upregu tion of silent information regulator 1 (SIRT1) signaling, alcoholic liver injury ing the activity and expression of CYP2E1 and by enhancing the protective oxidative defense system, and drug-induced hepatotoxicity in mice by inhib activity [17][18][19]. Nevertheless, further research is necessary to elucidate the a nisms underlying kaempferol's hepatoprotective properties as a modulator pathways related to oxidative stress-induced liver inflammation. Our exper that the oral administration of kaempferol has antioxidative, anti-inflam hepatoprotective effects on acute hepatic injury induced by carbon tetrachlo rats. This paper aims to explore the molecular mechanisms of kaempferol in oxidative stress and related signaling pathways. Specifically, we investigate nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPK phosphatidylinositide 3-kinases (PI3K), protein kinase B (Akt), AMP-activat nase (AMPK), and nuclear factor-E2-related factor (Nrf2), in the acute live duced by CCl4 in rats.

Antioxidative Activities of Kaempferol against FeSO4/H2O2-Induced Lipid Pero DPPH Radicals
Kaempferol was shown to possess potent antioxidant properties as dem its ability to inhibit FeSO4/H2O2-induced lipid peroxidation in rat liver homo well-known antioxidant, butylated hydroxytolune (BHT), was used as a pos and the IC50 of kaempferol was determined to be 9.69 μM, which was slightl Numerous studies have reported that plant extracts containing kaempferol, such as Miconia albicans, Onosma bracteata, Physalis peruviana, and Solanum elaeagnifolium, exhibit protective effects in various experimental liver-injury models [13][14][15][16]. However, these extracts contain various phytochemicals in addition to kaempferol, such as quercetin, apigenin, and rutin, and those compounds have been shown to exhibit hepatoprotective effects. Therefore, it is essential to evaluate the hepatoprotective activity of kaempferol specifically, not of crude extracts. Recent studies have reported kaempferol's hepatoprotective properties against: acetaminophen-induced liver damage by the upregulation/activation of silent information regulator 1 (SIRT1) signaling, alcoholic liver injury by attenuating the activity and expression of CYP2E1 and by enhancing the protective role of anti-oxidative defense system, and drug-induced hepatotoxicity in mice by inhibiting CYP2E1 activity [17][18][19]. Nevertheless, further research is necessary to elucidate the action mechanisms underlying kaempferol's hepatoprotective properties as a modulator of signaling pathways related to oxidative stress-induced liver inflammation. Our experiments found that the oral administration of kaempferol has antioxidative, anti-inflammatory, and hepatoprotective effects on acute hepatic injury induced by carbon tetrachloride (CCl 4 ) in rats. This paper aims to explore the molecular mechanisms of kaempferol in modulating oxidative stress and related signaling pathways. Specifically, we investigated the role of nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs), as well as phosphatidylinositide 3kinases (PI3K), protein kinase B (Akt), AMP-activated protein kinase (AMPK), and nuclear factor-E2-related factor (Nrf2), in the acute liver damage induced by CCl 4 in rats.

Antioxidative Activities of Kaempferol against FeSO 4 /H 2 O 2 -Induced Lipid Peroxidation and DPPH Radicals
Kaempferol was shown to possess potent antioxidant properties as demonstrated by its ability to inhibit FeSO 4 /H 2 O 2 -induced lipid peroxidation in rat liver homogenates. The well-known antioxidant, butylated hydroxytolune (BHT), was used as a positive control, and the IC 50 of kaempferol was determined to be 9.69 µM, which was slightly higher than the IC 50 of BHT at 8.66 µM ( Table 1). The free-radical scavenging activity of kaempferol against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was also evaluated using Trolox as a positive control. Kaempferol exhibited high scavenging activity with an IC 50 of 21.87 µM, while the IC 50 of Trolox was 23.71 µM (Table 2).

Protective Effect of Kaempferol on t-Butyl Hydroperoxide (t-BHP)-Induced HepG2
Cell Damage t-BHP is a pro-oxidant agent that generates several reactive free radicals, which can cause cytotoxicity by disrupting normal cellular functions [20]. HepG2 cells are a wellestablished in vitro model for studying xenobiotic metabolism and liver toxicity, as they retain many specialized functions of normal human hepatocytes [21]. After exposure to 300 µM t-BHP for three hours, HepG2 cells showed a significant decrease in viability, with only 46% surviving compared to untreated cells. However, pretreatment with kaempferol provided a concentration-dependent protective effect against t-BHP-induced cell damage in HepG2 cells, with an EC 50 of 45.8 µM (Figure 2). These results suggest that kaempferol has a hepatoprotective effect against t-BHP-induced cytotoxicity.

Changes in Body and Liver Weights and Serum Parameters in CCl 4 -Intoxicated Rats
The administration of CCl 4 resulted in a significant increase in liver weight and liver/body weight ratio compared to the control group (Table 3). However, treatment with kaempferol effectively restored the enlarged liver induced by CCl 4 to normal levels. To assess whether kaempferol protects the liver from CCl 4 -induced injury, we performed biochemical analyses of serum parameters ( Figure 3). Our results showed that aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were significantly increased in CCl 4 -intoxicated rats compared to the control group. However, kaempferol treatment dose-dependently reduced the levels of AST and ALT, indicating its in vivo hepatoprotective effects.

Changes in Body and Liver Weights and Serum Parameters in CCl4-Intoxicate
The administration of CCl4 resulted in a significant increase in live liver/body weight ratio compared to the control group (Table 3). However, t kaempferol effectively restored the enlarged liver induced by CCl4 to nor assess whether kaempferol protects the liver from CCl4-induced injury, we ochemical analyses of serum parameters ( Figure 3). Our results showed aminotransferase (AST) and alanine aminotransferase (ALT) activities wer increased in CCl4-intoxicated rats compared to the control group. Howeve treatment dose-dependently reduced the levels of AST and ALT, indicati hepatoprotective effects.    mide (MTT) assay. Values are mean ± standard error (n = 3). *** p < 0.001 vs. ### p < 0.001 vs. the t-BHP treated cells.

Changes in Body and Liver Weights and Serum Parameters in CCl4-Int
The administration of CCl4 resulted in a significant increase i liver/body weight ratio compared to the control group (Table 3). How kaempferol effectively restored the enlarged liver induced by CCl4 t assess whether kaempferol protects the liver from CCl4-induced injur ochemical analyses of serum parameters ( Figure 3). Our results sho aminotransferase (AST) and alanine aminotransferase (ALT) activitie increased in CCl4-intoxicated rats compared to the control group. H treatment dose-dependently reduced the levels of AST and ALT, in hepatoprotective effects.

Liver Histopathology
The effect of kaempferol on CCl 4 -induced histopathological alterations in the liver was evaluated by examining Hematoxylin and Eosin (H&E)-stained liver sections. The typical lobular architecture with central veins and radiating hepatic cords was disrupted, and there was sub-massive necrosis, vacuolization, and macrovesicular fatty changes in hepatocytes of CCl 4 -intoxicated rats ( Figure 4B). However, the administration of kaempferol dose-dependently improved these pathologic changes and led to the restoration of normal cell integrity and hepatic architecture ( Figure 4C,D). The quantitative analysis of the necrotic area observed on H&E-stained liver sections further supported kaempferol's hepatoprotective activity against CCl 4 -induced liver damage. kaempferol dose-dependently improved these pathologic changes and led to the restora tion of normal cell integrity and hepatic architecture ( Figure 4C,D). The quantitative anal ysis of the necrotic area observed on H&E-stained liver sections further supported kaempferol's hepatoprotective activity against CCl4-induced liver damage.

Effects of Kaempferol on CCl4-Intoxicated Liver Inflammatory Mediators
Reverse Transcription Polymerase Chain Reaction (RT-PCR) was utilized to measur the mRNA expression levels of the pro-inflammatory cytokine tumor necrosis factor (TNF) α and Interleukin (IL)-1β in the liver. The results were quantified by normalizing against th housekeeping gene GAPDH's mRNA expression. Kaempferol pretreatment significantly suppressed the CCl4-induced elevation of TNF-α and IL-1β expressions ( Figure 5A,D). Th mRNA expression levels of pro-inflammatory cyclooxygenase (COX)-2 and inducible nitri oxide synthase (iNOS), along with their protein levels in the liver, were measured through RT-PCR and Western blot analysis, respectively. Kaempferol administration significantly reduced the mRNA expression of COX-2 and iNOS ( Figure 5B,E) as well as the correspond ing protein levels in the CCl4-intoxicated rat liver ( Figure 5C,F).

Effects of Kaempferol on CCl 4 -Intoxicated Liver Inflammatory Mediators
Reverse Transcription Polymerase Chain Reaction (RT-PCR) was utilized to measure the mRNA expression levels of the pro-inflammatory cytokine tumor necrosis factor (TNF)α and Interleukin (IL)-1β in the liver. The results were quantified by normalizing against the housekeeping gene GAPDH's mRNA expression. Kaempferol pretreatment significantly suppressed the CCl 4 -induced elevation of TNF-α and IL-1β expressions ( Figure 5A,D). The mRNA expression levels of pro-inflammatory cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), along with their protein levels in the liver, were measured through RT-PCR and Western blot analysis, respectively. Kaempferol administration significantly reduced the mRNA expression of COX-2 and iNOS ( Figure 5B,E) as well as the corresponding protein levels in the CCl 4 -intoxicated rat liver ( Figure 5C,F). kaempferol dose-dependently improved these pathologic changes and led to the restoration of normal cell integrity and hepatic architecture ( Figure 4C,D). The quantitative analysis of the necrotic area observed on H&E-stained liver sections further supported kaempferol's hepatoprotective activity against CCl4-induced liver damage.

Effects of Kaempferol on CCl4-Intoxicated Liver Inflammatory Mediators
Reverse Transcription Polymerase Chain Reaction (RT-PCR) was utilized to measure the mRNA expression levels of the pro-inflammatory cytokine tumor necrosis factor (TNF)α and Interleukin (IL)-1β in the liver. The results were quantified by normalizing against the housekeeping gene GAPDH's mRNA expression. Kaempferol pretreatment significantly suppressed the CCl4-induced elevation of TNF-α and IL-1β expressions ( Figure 5A,D). The mRNA expression levels of pro-inflammatory cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), along with their protein levels in the liver, were measured through RT-PCR and Western blot analysis, respectively. Kaempferol administration significantly reduced the mRNA expression of COX-2 and iNOS ( Figure 5B,E) as well as the corresponding protein levels in the CCl4-intoxicated rat liver ( Figure 5C,F). , and iNOS (E) in rat liver intoxicated with CCl4 was assessed using RT-PCR. Western blot analysis was performed to measure COX-2 (C) and iNOS (F) protein levels. Values are mean ± SE (n = 3). ** p < 0.01 and *** p < 0.001 vs. the control group, and ## p < 0.01 and ### p < 0.001 vs. the CCl4 group.

Effects of Kaempferol on MAPK/NF-κB Signaling Pathway in CCl 4 -Induced Liver Damages
Kaempferol's effect on NF-κB activation was investigated by examining the translocation of NF-κB p65 from the cytosol to the cell nuclei. The protein levels of NF-κB in the nuclei and cytosol fractions were measured using Western blot analysis, with each fraction's protein levels quantified by image analysis and normalized against histone H1 and GAPDH, respectively. As shown in Figure 6A, CCl 4 -intoxicated rat liver exhibited an increase in nuclear NF-κB p65 protein and a decrease in cytosolic NF-κB protein. However, kaempferol treatment significantly reduced the levels of translocated NF-κB in the nuclei fraction.
To investigate the molecular mechanism of NF-κB activation the involvement of MAPK members was examined. MAPKs are lation and transmit stimuli to a downstream target NF-κB. Weste formed to measure the phosphorylated ERK1/2 ( Figure 6B), JN ( Figure 6D) protein levels. The results showed that the phospho ERK1/2, JNK, and p38 MAPKs were elevated in the CCl4 kaempferol treatment significantly decreased the phosphory ERK1/2, JNK, and p38 MAPKs, indicating that kaempferol's pro duced liver injury may be associated with the inhibition of NF-κB pathway. Figure 6. Effects of kaempferol on CCl4-induced NF-κB p65 activation and p-38 (D) phosphorylation. Western blotting was performed to detec p65 in the nuclear and cytosol and phosphorylated ERK, JNK, and p-38 total ERK, JNK, and p-38 were used as loading controls, respectively. V *** p < 0.001 vs. the control group, and # p < 0.05, ## p < 0.01, and ### p < 0.0

Effect of Kaempferol on Oxidative Status in CCl4-Intoxicated Rat'
To assess the oxidative status, we measured the levels of (ROS), lipid peroxidation, and intracellular antioxidants in liver ROS level was determined using a 2',7'-dichlorofluorescein di while the lipid peroxidation product MDA was measured to exa As shown in Figure 7A,B, CCl4 treatment resulted in a significa and MDA amounts in the rat liver, indicating oxidative stress. H plements dose-dependently suppressed these levels, highlightin erties. We also measured the levels of total SH ( Figure 7C) and n To investigate the molecular mechanism of NF-κB activation in CCl 4 -intoxicated rats, the involvement of MAPK members was examined. MAPKs are activated by phosphorylation and transmit stimuli to a downstream target NF-κB. Western blot analysis was performed to measure the phosphorylated ERK1/2 ( Figure 6B), JNK ( Figure 6C), and p38 ( Figure 6D) protein levels. The results showed that the phosphorylated protein levels of ERK1/2, JNK, and p38 MAPKs were elevated in the CCl 4 -treated rats. However, kaempferol treatment significantly decreased the phosphorylated protein levels of ERK1/2, JNK, and p38 MAPKs, indicating that kaempferol's protective effect on CCl 4 -induced liver injury may be associated with the inhibition of NF-κB activation via the MAPK pathway.

Effect of Kaempferol on Oxidative Status in CCl 4 -Intoxicated Rat's Liver
To assess the oxidative status, we measured the levels of reactive oxygen species (ROS), lipid peroxidation, and intracellular antioxidants in liver homogenates. The total ROS level was determined using a 2',7'-dichlorofluorescein diacetate (DCFDA) probe, while the lipid peroxidation product MDA was measured to examine lipid peroxidation. As shown in Figure 7A,B, CCl 4 treatment resulted in a significant increase in ROS levels and MDA amounts in the rat liver, indicating oxidative stress. However, kaempferol supplements dose-dependently suppressed these levels, highlighting its antioxidative properties. We also measured the levels of total SH ( Figure 7C) and non-protein SH (glutathione, GSH) ( Figure 7D), which are important endogenous antioxidants. In the CCl 4 -treated rat liver, these levels were reduced compared to the control group, but kaempferol prevented the reduction and restored the values almost to the extent of those of the control group, indicating its protective effects against oxidative damage. t. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW rat liver, these levels were reduced compared to the control group, but kae vented the reduction and restored the values almost to the extent of those group, indicating its protective effects against oxidative damage. Values are mean ± SE of n = 6 rats/group. * p < 0.05, *** p < 0.001 vs. the control group, and # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the CC

Effects of Kaempferol on Nrf2 Activation and the PI3K/Akt and AMPK Signal
We investigated the effect of kaempferol on Nrf2 activation by analyzin levels of Nrf2 in the nuclear and cytoplasmic fractions of liver samples using analysis. We found that the ratio of Nrf2 protein in the nuclear-to-cytoplasm CCl4-treated rat livers was significantly lower than that of the control group treatment restored the ratio to near-control levels ( Figure 8A). Additionally, the protein levels of heme oxygenase (HO)-1, a downstream target gene of sults show that kaempferol supplementation increased the protein levels of intoxicated rat livers, confirming the activation of Nrf2 by kaempferol (Fi further investigated the molecular mechanisms underlying kaempferol-ind tivation by examining the protein levels of PI3K ( Figure 8C), phosphorylate 8D), and phosphorylated AMPK ( Figure 8E). We found that the protein leve phosphorylated Akt were increased in CCl4-intoxicated rat livers, and kaem ment suppressed these increases. In contrast, the reduced levels of phosphor in CCl4-intoxicated rat livers were restored by kaempferol supplementation Values are mean ± SE of n = 6 rats/group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the control group, and # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the CCl 4 group.

Effects of Kaempferol on Nrf2 Activation and the PI3K/Akt and AMPK Signaling Pathways
We investigated the effect of kaempferol on Nrf2 activation by analyzing the protein levels of Nrf2 in the nuclear and cytoplasmic fractions of liver samples using Western blot analysis. We found that the ratio of Nrf2 protein in the nuclear-to-cytoplasmic fraction of CCl 4 -treated rat livers was significantly lower than that of the control group. Kaempferol treatment restored the ratio to near-control levels ( Figure 8A). Additionally, we examined the protein levels of heme oxygenase (HO)-1, a downstream target gene of Nrf2. Our results show that kaempferol supplementation increased the protein levels of HO-1 in CCl 4 -intoxicated rat livers, confirming the activation of Nrf2 by kaempferol ( Figure 8B). We further investigated the molecular mechanisms underlying kaempferol-induced Nrf2 activation by examining the protein levels of PI3K ( Figure 8C), phosphorylated Akt ( Figure 8D), and phosphorylated AMPK ( Figure 8E). We found that the protein levels of PI3K and phosphorylated Akt were increased in CCl 4 -intoxicated rat livers, and kaempferol treatment suppressed these increases. In contrast, the reduced levels of phosphorylated AMPK in CCl 4 -intoxicated rat livers were restored by kaempferol supplementation. Figure 8. Effects of kaempferol on Nrf2 activation (A), HO-1 production (B), and PI3K level phosphorylated Akt (D) and AMPK (E) in CCl4-intoxicated rat liver. Western blotting w formed to detect these protein levels and quantified by image analysis. Histone H1, β-act Akt, and AMPK were used as loading controls. Values are mean ± SE (n = 3). ** p < 0.01 an 0.001 vs. the control group and # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the CCl4 group.

Discussion
Oxidative stress occurs when there is an imbalance between the production and the body's ability to detoxify and repair the damage caused by these reactiv cules. This stress is a key risk factor in the development of liver diseases. To inve the mechanisms of hepatic injury and fibrosis, the hepatotoxicant CCl4 is commonl CYP2E1 converts CCl4 to a CCl3 radical, which reacts with molecular oxygen to g CCl3OO• radicals. These highly reactive radicals can damage the hepatic endoplas ticulum's phospholipids, initiating a chain reaction of lipid peroxidation [22]. This results in membrane damage, a primary cause of CCl4-induced hepatocellular inju Hepatocyte necrosis induced by CCl4 can be prevented by antioxidants that sc CCl3 and lipid peroxy radicals. Kaempferol, a natural flavonoid found in many pla been widely recognized for its potent antioxidant properties in numerous studies In our experiments, we observed that kaempferol exhibited antioxidant activity co ble to that of Trolox, a well-known antioxidant, in terms of its ability to scavenge radicals. Furthermore, kaempferol inhibited FeSO4/H2O2-induced lipid peroxidati similar extent as BHT, a known antioxidant.
In this study, we evaluated the protective effects of kaempferol against liver induced by a single dose of CCl4 in rats. To assess the extent of liver injury, we me the liver-to-body weight ratio and serum biochemical parameters, and we evaluat tissue morphology using H&E staining. Our results showed that CCl4 intoxicat duced significant liver damage, as evidenced by changes in the aforementioned p ters. However, pretreatment with kaempferol at doses of 5 and 10 mg/kg in CCl4 cated rats resulted in significant and dose-dependent improvements in these patho alterations, suggesting a hepatoprotective effect of kaempferol against a CCl4-i liver injury.
We evaluated the effect of kaempferol on CCl4-induced oxidative stress in liver, as assessed by increased ROS production and decreased levels of total SH an protein SH. Our results show that kaempferol administration effectively attenuate Figure 8. Effects of kaempferol on Nrf2 activation (A), HO-1 production (B), and PI3K level (C), and phosphorylated Akt (D) and AMPK (E) in CCl 4 -intoxicated rat liver. Western blotting was performed to detect these protein levels and quantified by image analysis. Histone H1, β-actin, total Akt, and AMPK were used as loading controls. Values are mean ± SE (n = 3). ** p < 0.01 and *** p < 0.001 vs. the control group and # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the CCl 4 group.

Discussion
Oxidative stress occurs when there is an imbalance between the production of ROS and the body's ability to detoxify and repair the damage caused by these reactive molecules. This stress is a key risk factor in the development of liver diseases. To investigate the mechanisms of hepatic injury and fibrosis, the hepatotoxicant CCl 4 is commonly used. CYP2E1 converts CCl 4 to a CCl 3 radical, which reacts with molecular oxygen to generate CCl 3 OO• radicals. These highly reactive radicals can damage the hepatic endoplasmic reticulum's phospholipids, initiating a chain reaction of lipid peroxidation [22]. This process results in membrane damage, a primary cause of CCl 4 -induced hepatocellular injury.
Hepatocyte necrosis induced by CCl 4 can be prevented by antioxidants that scavenge CCl 3 and lipid peroxy radicals. Kaempferol, a natural flavonoid found in many plants, has been widely recognized for its potent antioxidant properties in numerous studies [8][9][10][11][12]. In our experiments, we observed that kaempferol exhibited antioxidant activity comparable to that of Trolox, a well-known antioxidant, in terms of its ability to scavenge DPPH radicals. Furthermore, kaempferol inhibited FeSO 4 /H 2 O 2 -induced lipid peroxidation to a similar extent as BHT, a known antioxidant.
In this study, we evaluated the protective effects of kaempferol against liver injury induced by a single dose of CCl 4 in rats. To assess the extent of liver injury, we measured the liver-to-body weight ratio and serum biochemical parameters, and we evaluated liver tissue morphology using H&E staining. Our results showed that CCl 4 intoxication induced significant liver damage, as evidenced by changes in the aforementioned parameters. However, pretreatment with kaempferol at doses of 5 and 10 mg/kg in CCl 4 -intoxicated rats resulted in significant and dose-dependent improvements in these pathological alterations, suggesting a hepatoprotective effect of kaempferol against a CCl 4 -induced liver injury.
We evaluated the effect of kaempferol on CCl 4 -induced oxidative stress in the rat liver, as assessed by increased ROS production and decreased levels of total SH and non-protein SH. Our results show that kaempferol administration effectively attenuated CCl 4 -induced oxidative stress. Moreover, kaempferol was found to mitigate the high level of lipid peroxidation observed in CCl 4 -intoxicated rats, which indicates an imbalance between oxidative stress and antioxidant defense systems. These findings are consistent with the in vitro results, wherein kaempferol demonstrated significant hepatoprotective effects against t-BHP-induced oxidative stress on HepG2 cells, a commonly used cell line in liver research. Overall, our findings suggest that kaempferol possesses potent antioxidative activity, which is capable of protecting against oxidative stress and lipid peroxidation in both in vitro and in vivo systems.
This study aims to elucidate the molecular mechanisms underlying the hepatoprotective effects of kaempferol against CCl 4 -induced liver injury in rats. Specially, the study focuses on how kaempferol modulates the MAPKs/NF-κB and AMPK/Nrf2 signaling pathways, leading to anti-oxidative, anti-inflammatory, and hepatoprotective effects in CCl 4 -intoxicated rats.
Inflammation and elevated cytokines such as TNF-α and IL-1β are known to follow hepatocellular injury and are associated with the pathogenesis of liver diseases, in part, through the activation of the NF-κB signaling pathway. The production of pro-inflammatory mediators such as COX-2 and iNOS proteins, as well as TNF-α and IL-1β cytokines, is also regulated by this pathway [23]. The present study demonstrated that kaempferol supplementation effectively suppressed NF-κB activation in the liver and improved the profile of pro-inflammatory mediators, indicating that kaempferol exerts anti-inflammatory effects against CCl 4 -induced liver injury by inhibiting NF-κB activation.
The MAPK signaling pathway plays a crucial role in regulating diverse cellular processes, such as cell proliferation, differentiation, apoptosis, and stress responses [24]. Its three key members, ERK, JNK, and p38, can be activated by various stimuli, such as inflammatory cytokines and ROS, leading to the downstream activation of transcription factors, such as NF-κB, and the modulation of gene expression and cellular responses. In this study, we aimed to investigate the impact of kaempferol on the activation of MAPKs and the NF-κB pathway in a rat model of CCl 4 -induced liver injury. Our results demonstrate that CCl 4 exposure led to the activation of ERK, JNK, and p38 MAPKs in the liver, but treatment with kaempferol attenuated this response. Notably, the kaempferol-mediated suppression of MAPK activity was associated with an inhibition of NF-κB signaling and reduced levels of pro-inflammatory mediators, such as TNF-α, IL-1β, COX-2, and iNOS. These findings suggest that kaempferol exerts its anti-inflammatory effects by interfering with the MAPK/NF-κB signaling axis.
The Nrf2 signaling pathway plays a crucial role in protecting cells from oxidative stress by regulating the expression of various cytoprotective and detoxifying enzymes, which helps maintain cellular redox homeostasis [25]. One of the key functions of Nrf2 is the activation of the transcription of target genes that encode defense enzymes, such as HO-1 and GSH synthase/peroxidase, which can mitigate the damaging effects of oxidative stress. In this study, we investigated the effect of kaempferol on the Nrf2 pathway in CCl 4 -treated rats. Our results show that kaempferol treatment increased the translocation of Nrf2 into the nuclei and upregulated the expression of HO-1 in the liver. This suggests that kaempferol may enhance the cell defense system against oxidative stress by activating the Nrf2 pathway and promoting the expression of Nrf2 target genes, such as superoxide dismutase (SOD) and catalase, thereby reducing oxidative damage [26]. The improved oxidative status observed in CCl 4 -treated rats after kaempferol administration is likely due to a combination of Nrf2 activation and the direct antioxidative activity of kaempferol. Therefore, our findings suggest that kaempferol's ability to activate the Nrf2 pathway and upregulate the expression of antioxidant enzymes may contribute to its overall antioxidant and hepatoprotective effects.
The regulation of Nrf2 by the PI3K/Akt pathway in response to cellular stress is wellestablished. Previous studies have shown that kaempferol activates the PI3K/Akt pathway to protect against oxidative stress in various models, such as Zearalenone-induced oxidative stress [27], isoproterenol-induced heart failure [28], and myocardial ischemia/reperfusion injury [29], by activating Nrf2. However, the specific effects of kaempferol on Akt can vary depending on cell type, tissue, and the type of damage. Other studies have demonstrated that kaempferol exerts its anti-inflammatory effects in cardiac fibroblasts [30] and reduces inflammation in an LPS-induced acute lung-injury model [31] by inhibiting Akt phospho-rylation. It is worth noting that Akt activation has also been linked to NF-κB activation in other studies [32,33]. In our study, we found that CCl 4 treatment increased the levels of PI3K and phosphorylated Akt in the liver, while kaempferol treatment reduced Akt phosphorylation. These findings suggest that Akt activation in this model is likely related to NF-κB activation, rather than Nrf2 activation. Our results indicate that the protective effects of kaempferol in the CCl 4 -induced liver injury model may be mediated through the suppression of Akt phosphorylation, leading to the inhibition of NF-κB activation. These findings provide further insights into the complex interplay between different signaling pathways involved in oxidative stress and inflammation.
The serine/threonine protein kinase AMPK has been widely reported as playing a critical role in regulating cellular stress and energy homeostasis [34]. Studies suggest that AMPK activation has a protective effect against oxidative stress and inflammation in various cell types, tissues, and organs. This protective potential is closely linked to the activation of Nrf2 signaling [35,36]. For instance, Velagapudi et al. demonstrated that kaempferol can activate the AMPK/Nrf2/HO-1 pathway in BV-2 microglia, inhibiting neuroinflammation [37]. Similarly, Du et al. found that kaempferol prevented Angiotensin II-induced cardiac fibrosis and dysfunction by modulating the AMPK/Nrf2 pathway [38]. In this study, we observed that kaempferol administration led to an upregulation of AMPK phosphorylation in CCl 4 -intoxicated rats, indicating the involvement of the AMPK/Nrf2 signaling pathway in the protection against oxidative stress and inflammation induced by CCl 4 .

DPPH Assay and Antioxidative Activity against FeSO 4 /H 2 O 2 -Induced Lipid Peroxidation
The radical scavenging activity of kaempferol was measured against stable DPPH free radicals using a published method [39]. To evaluate its effect on lipid peroxidation, rat liver homogenates (7.5 mg protein/mL) were treated with the Fenton reaction, which comprised 0.1 mM FeSO 4 and 3 mM H 2 O 2 , along with various concentrations of kaempferol or BHT. The level of lipid peroxidation was determined as previous [40].

HepG2 Cell Damage Induced by t-BHP
The HepG2 human hepatocellular carcinoma cell line was obtained from the Korea Cell Line Bank (Seoul, Republic of Korea) and was maintained in a DMEM medium supplemented with 10% fetal bovine serum, 1% glutamine, 0.01% penicillin, and 0.01% streptomycin at 37 • C with 5% CO 2 . Cells were seeded at a density of 2.0 × 10 4 cells per well in 96-well plates and allowed to attach for 24 h. After serum starvation, cells were treated with various concentrations of kaempferol (10, 20, 50, and 100 µM) for 4 h, followed by exposure to 300 µM t-BHP for 3 h. Cell viability was assessed using the MTT assay, and the results were expressed as percentages of the viability of untreated cells.

Animals and Induction of Acute Liver Injury with CCl 4
Male Sprague-Dawley rats weighing 140-160 g were obtained from Samtako (Osan, Republic of Korea). The animal protocol used in this study was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (Approval Number PNU 2016-1417) in accordance with ethical issues and scientific care. The rats were randomly divided into four groups (n = 6): Control, CCl 4 , KA5, and KA10. To induce liver injury, a single intraperitoneal (i.p.) injection of 25% (w/v) CCl 4 (0.6 g/kg body weight) in olive oil was administered. Kaempferol (Sigma cat #K0133), suspended in a 0.5% sodium carboxymethylcellulose (CMC) solution was administered by oral gavage. The CCl 4 group received CCl 4 and CMC, while the KA5 and KA10 groups received CCl 4 and kaempferol at 5 and 10 mg/kg body weight/day, respectively. The control group received olive oil and CMC. Kaempferol was treated twice, once at 16 h and once at 30 min before CCl 4 intoxication. After 24 h of CCl 4 injection, all rats were sacrificed under anesthesia, and blood samples were obtained from the inferior vena cava for biochemical analyses. The livers were excised and frozen in liquid nitrogen for further analysis.

Liver Histology and Biochemical Analysis of Serum Parameters
Liver specimens were prepared for H&E staining following the methods described in our previous study [41]. The area of necrosis observed on the H&E-stained sections was quantitatively analyzed using Image J software (NIH, Bethesda, MD, USA). Serum levels of AST and ALT were measured using commercial kits (Asan Chemical Co., Cheonan, Republic of Korea) according to the manufacturer's instructions.

MDA and ROS Level in Liver Tissues
To determine the levels of MDA in the liver, we followed the method described in our previous report [20,40]. The concentration of MDA was calculated based on the absorbance at 532 nm of the supernatant, using MDA tetrabutylammonium as a standard. We assessed the level of ROS by performing a fluorometric assay with DCFDA. The esterase and ROS (such as ·O 2 − , ·OH, and H 2 O 2 ) in the sample oxidized DCFDA to the fluorescent 2',7'dichlorofluorescin, and we measured the change in fluorescence intensity with excitation and emission wavelengths set at 485 and 530 nm, respectively.

Total SH and Non-Protein SH Contents in Liver Tissues
To measure total SH, the liver tissue homogenate was mixed with 100 µL of 0.01 M 5,5-dithio-bis-2-nitrobenzoic acid, 4 mL methanol, and 1 mL 0.2 M Tris buffer (pH 8.2) and incubated at 25 • C for 15 min. After centrifugation at 1250× g for 30 min, the resulting supernatant was analyzed at 412 nm [42]. To determine non-protein SH, the homogenates were treated with trichloroacetic acid and centrifuged. One hundred µL of the resulting supernatant was mixed with 0.05 mL of 0.01 M NaNO 2 , 0.45 mL of 0.1 M H 2 SO 4 , and the mixture was allowed to stand for 5 min. Then, the sample was added to a solution containing 0.2 mL of 0.5% ammonium sulfamic acid, 0.1 mL of 1% mercuric chloride, and 0.9 mL of 3.4% sulfanilamide. Then, 1 mL of 0.1% N-naphthyl ethylenediamine in 0.4 M hydrochloric acid was added to the mixture. After 5 min, the mixture was measured at 540 nm using GSH as a standard.

Statistical Analyses
Statistical analysis was performed using a one-way analysis of variance followed by Tukey's multiple comparison test, and the results are presented as the mean ± standard error of the mean (SEM) from the indicated number of replicates. A p-value of less than or equal to 0.05 was considered statistically significant.

Conclusions
In conclusion, the results of this study demonstrate the significant potential of kaempferol as a protective agent against CCl 4 -induced acute liver damage. The observed inhibitory effects of kaempferol on NF-κB activation, pro-inflammatory cytokine expression (TNF-α and IL-1β), and protein production (COX-2 and iNOS) were associated with the suppression of upstream kinases such as ERK, JNK, p38 MAPKs, and Akt. Moreover, the administration of kaempferol effectively improved the oxidative balance in the livers of CCl 4 -intoxicated rats, likely through Nrf2 activation via AMPK phosphorylation. Overall, these findings suggest that kaempferol exerts antioxidative, anti-inflammatory, and hepatoprotective effects by modulating the MAPK/NF-κB and AMPK/Nrf2 signaling pathways in CCl 4intoxicated rats. These results support the potential of kaempferol as a therapeutic agent for mitigating liver inflammation induced by oxidative stress or hepatotoxins.