Gentiopicroside ameliorates CCl 4 -induced liver injury in mice by regulating the PPAR-γ/Nrf2 and NF-κB/IκB signaling pathways

Abstract

Objective

This study explored the mechanisms by which gentiopicroside protects against carbon tetrachloride (CCl₄)-induced liver injury.

Methods

Male mice were randomly assigned to the control; CCl₄; bifendate 100 mg/kg; or gentiopicroside 25, 50, or 100 mg/kg groups. Both vehicle and drugs were administered intragastrically for 7 days. Mice were administered CCl₄ intraperitoneally 1 hour after the last drug dose. After 24 hours, we collected blood and liver samples for testing.

Results

Gentiopicroside significantly reduced serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities with corresponding reductions in hepatocyte denaturation and necrosis. Gentiopicroside enhanced superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and glutathione levels and reduced heme oxygenase 1 (HO-1) activity and malondialdehyde levels in the liver, and these effects were attributed to peroxisome proliferator-activated receptor (PPAR)-γ/nuclear factor erythroid 2-related factor 2 (Nrf2) activation. Meanwhile, gentiopicroside significantly downregulated HO-1 and upregulated SOD and GSH-Px at the mRNA level in the liver. Furthermore, gentiopicroside significantly suppressed serum tumor necrosis factor-α and interleukin-1β secretion, which was associated with the inhibition of nuclear factor-kappa B (NF-κB)/inhibitor of NF-κB (IκB).

Conclusions

Gentiopicroside ameliorated CCl₄-induced liver injury in mice via the PPAR-γ/Nrf2 and NF-κB/IκB pathways.

Keywords

Liver injury gentiopicroside peroxisome proliferator-activated receptor-γ nuclear factor erythroid 2-related factor 2 nuclear factor-kappa B inhibitor of nuclear factor-kappa B inflammatory response.

Introduction

The liver is the primary organ involved in the metabolism, detoxification, and clearance of substances in the body, including exogenous biological substances. Acute and chronic liver diseases represent a global problem, and exposure to environmental toxins, pesticides, and chemotherapeutics has significantly increased the risk of toxic liver injury.^1,2 The complex and diverse molecular mechanisms of toxicity-induced liver damage include inflammatory responses, reactive oxygen species production, and activation of the peroxisome proliferator-activated receptor γ (PPAR-γ)/nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-κB)/inhibitor of NF-κB (IκB) pathways.^2,3 Many compounds, including beneficial drugs, can damage liver cells through metabolic conversion into highly reactive forms and the formation of free radicals.³ Recently, Chinese herbal medicines have gained attention as raw materials for the development of healthy foods and medicines, offering a potential approach to natural medicines designed to protect and restore health.^4,5 Gentiopicroside (Gent; Figure 1), an iridoid glycoside, is one of the most crucial rhododendron tonic components of traditional Chinese medicines.⁶ Gent has been reported to possess anti-inflammatory,^7,8 anti-cancer,⁹ and anti-depressant properties.¹⁰ Recently, several studies reported that Gent has hepatoprotective effects. For instance, Gent significantly reduced the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice with carbon tetrachloride (CCl₄)-induced liver injury.^11,12 Gent also exerted hepatoprotective effects on cholestatic liver injury induced by α-naphthylisothiocyanate and hepatic obstruction in rodents.^13,14

Figure 1.

The chemical structure of gentiopicroside.

Studies identified inflammation as a major event in the pathogenesis of liver diseases, which is typified by elevated levels of inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β), and PPAR-γ and Nrf2 play regulatory roles in oxidative stress-mediated liver dysfunction.^4,14 However, the potential mechanisms underlying the role of Gent in liver injury remain elusive. Moreover, CCl₄ is a frequently employed hepatotoxin that provides an established approach to trigger liver injury, and CCl₄ is relatively well characterized regarding its histological, biochemical and molecular alterations. Moreover, CCl₄ is widely utilized to induce liver injury in experimental animal studies.^4,11 Thus, this study explored the potential mechanisms underlying the preventive effects of Gent on CCl₄-induced liver injury in mice.

Materials and methods

Drugs and reagents

Gent (MW: 356.32, batch No.: GR-135-180320) was purchased from Nanjing Guangrun Biotechnology Co., Ltd. (Nanjing, China). Bifendate tablets (batch No.: A02J180304) were purchased from Wanbond Pharmaceutical Group Co., Ltd (Taizhou, China). CCl₄ (MW: 153.82, batch No.: C11588428) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). ALT, AST, lactate dehydrogenase (LDH), heme oxygenase 1 (HO-1), glutathione (GSH), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and superoxide dismutase (SOD) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α (EMC102a.96) and IL-1β (EMC001b.96) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Total RNA extraction reagent, a cDNA synthesis kit, and SYBR mixture were purchased from Takara Co., Ltd (Dalian, China). Antibodies against p-NF-κB p65 (WL02169), NF-κB p65 (WL01273b), IκB-α (WL01936), p-IκB-α (WL02495), Nrf2 (WL02135), PPAR-γ (WL01800), and β-actin (WL01372) were purchased from Shenyang Wanlei Biotechnology Co., Ltd. (Shenyang, China). A bicinchoninic acid (BCA) protein concentration assay kit (081517171207), 30% Acr-Bis (29:1, 121217171212), 10% sodium dodecyl sulfate (SDS, 120916170706), ammonium persulfate (ST005), TEMED (ST728), an enhanced chemiluminescence (ECL) kit (P0018F), and protein molecular weight standard (P0072) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The remaining chemicals were of analytical grade.

Animals’ treatment and experimental design

Male Swiss mice (specific pathogen-free grade) weighing 20 ± 2 g were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. [Certificate No.: SCXK (Lu) 20140007, Jinan, China]. Mice were housed under standard housing conditions (22 ± 2°C, 50% ± 5% relative humidity, 12-hour/12-hour light/dark cycle) with ad libitum access to food and water. All animal experiments in this study were approved by the Animal Ethics Committee of Yantai Hospital Affiliated with Binzhou Medical University (grant No. AH-202007).

Mice were randomly divided into six groups (n = 10/group) as follows: control group, CCl₄ group; 100 mg/kg bifendate (BP100) group; and 25-, 50-, and 100-mg/kg Gent groups (Gent 25, 50, and 100). Mice in the control group were administered 0.9% saline. Mice in the BP100 group received 100 mg/kg bifendate, and those in the Gent groups received 25, 50, or 100 mg/kg Gent once per day for 7 days. One hour following the last dose, mice received an intraperitoneal injection of 0.2 mL of 0.2% CCl₄. After 24 hours, mice were anesthetized using 3% isoflurane inhalation anesthesia. Blood samples from the heart were taken using syringes, and the serum was extracted and stored for later analysis at −70°C freezer after being centrifuged at 3000 × g for 10 minutes. The liver and spleen tissues were either immediately removed or stored for later use at −70°C. The BPP00 group served as the positive control.

Determination of AST, ALT, and LDH activities

ALT, AST, and LDH activities in serum were assayed using commercial kits.

Measurement of liver and spleen indices

The liver and spleen were removed to assess their indices using the following formula: index (%) = tissue weight (g)/body weight (g) × 100%.

Histopathological examination

The mice were sacrificed, and livers were immediately removed for histological analysis. Liver samples (n = 5/group) were fixed in 10% phosphate-buffered formalin, excised, and embedded in paraffin. Each paraffin-embedded block was cut into 5-µm sections and stained with hematoxylin–eosin for histological observation.

Assessment of HO-1, SOD, and GSH-Px activities and GSH and MDA levels in the liver

Liver homogenate (25% w/v 0.01 mol/L phosphate buffer with 1.15% KCl, pH 7.4) was centrifuged at 10,000 × g for 20 minutes at 4°C. A portion of the supernatant was collected, and the activities of HO-1, SOD, and GSH-Px and levels of GSH and MDA were determined by ELISA, water-soluble tetrazolium salt, colorimetric, and TBA methods following the manufacturer's instructions.

Serum IL-1β and TNF-α assays

The serum levels of IL-1β and TNF-α were assayed using ELISA kits following the manufacturer’s instructions.

Quantitative reverse transcription-polymerase chain reaction

According to the manufacturer’s instructions, total RNA was isolated from liver samples using total RNA extraction reagent (Takara Co., Ltd.). Following verse transcription, complementary DNA (cDNA) samples were utilized in a 40-cycle real-time polymerase chain reaction consisting of 95°C for 30 s, followed by denaturation at 95°C for 5 s and annealing at 60°C for 45 s. Primers were used to amplify HO-1 (forward primer: 5′-AAGCCGAGAATGCTGAGTTCA-3′, reverse primer: 5′-GCCGTGTAGATATGGTACAAGGA-3′), SOD (forward primer: 5′-AACCAGTTGTGTTGTCAGGAC-3′, reverse primer: 5′-CCACCATGTTTCTTA GAGTGAGG-3′), GSH-Px (forward primer: 5'-AAT GTCGCGTCTCTCTGAGG-3′, reverse primer: 5′-TCCGAACTGATTGCACGGG-3′), and GAPDH cDNA (forward primer: 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse primer: 5′-TGTAGACCATGTAGTTGAGGTCA-3′). GAPDH was used as an internal reference.

Western blotting

All liver proteins were lysed using lysis buffer containing protease inhibitors. The protein concentration in the supernatant was detected using a BCA protein detection kit, and 30 μg of protein were separated by 10% SDS–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene difluoride membrane. Primary antibodies against p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, Nrf2, and PPAR-γ (all 1:1000) were mixed with 5% non-fat milk for blocking for 2 hours at room temperature. Membranes were then incubated with the primary antibodies in an appropriate manner at 4°C overnight, followed by incubation with rabbit or mouse horseradish peroxidase-coupled secondary antibodies for 1 hour at room temperature. The immunoreactive protein bands were visualized using the ECL assay (LAS4000, GE Healthcare, Chicago, IL, USA).

Statistical analysis

Data were presented as the mean ± standard deviation. One-way analysis of variance and Student’s t-test were used to compare more than two groups. P < 0.05 indicated statistical significance.

Results

Effects of Gent on serum ALT, AST, and LDH activities in mice with CCl₄-induced liver injury

The serum activities of ALT, AST, and LDH were significantly increased in CCl₄-treated mice compared with those in the control group (all P < 0.01, Table 1). Gent significantly decreased ALT, AST, and LDH activities in a dose-dependent manner (P < 0.05).

Table 1.

Effects of gentiopicroside on serum ALT, AST, and LDH activities in mice with CCl₄-induced liver injury.

Group	ALT (U/L)	AST (U/L)	LDH (U/L)
Control	31.8 ± 9.6	29.2 ± 6.1	7368.7 ± 1073
CCl₄	200.4 ± 9.3^##	114.3 ± 7.3^##	18,756.9 ± 4125^##
BP100	91.4 ± 53.5**	67.6 ± 20.3**	15,375.3 ± 3994**
Gent 25	187.3 ± 15.5*	66.7 ± 16.2**	17,104 ± 4156
Gent 50	165.7 ± 11.6**	53.1 ± 16.8**	11,352 ± 4154*
Gent 100	100.2 ± 72.8**	54.7 ± 18.5**	10,720 ± 3747**

Data are expressed as the mean ± standard deviation (n = 10). ^##P < 0.01 versus the control group, *P < 0.05, **P < 0.01 versus the CCl₄ group.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; CCl₄, carbon tetrachloride; BP100, 100 mg/kg bifendate; Gent 25, 25 mg/kg gentiopicroside; Gent 50, 50 mg/kg gentiopicroside; Gent 100, 100 mg/kg gentiopicroside

Effects of Gent on histopathological changes and liver and spleen indices in mice with CCl₄-induced liver injury

As indicated in Figure 2a, inflammatory cellular infiltration, hepatocellular necrosis, fragmented hepatic nuclei, and cell boundary disappearance were observed in the CCl₄ group. Liver histology injuries caused by CCl₄ were markedly attenuated by Gent, including mild inflammatory cellular infiltration and hepatocyte necrosis.

Figure 2.

Effects of gentiopicroside on histopathological changes and liver and spleen indices in mice with CCl₄-induced liver injury. (a) The histopathology of the liver (×100 magnification). The arrows indicate inflammatory cellular infiltration, hepatocellular necrosis, and fragmented hepatic nuclei. Scale bar = 50 μm. (b) Liver index. (c) Spleen index. Data are expressed as the mean ± standard deviation. ^##P < 0.01 versus the control group, ^*P < 0.05, ^**P < 0.01 versus the CCl₄ group. CCl₄, carbon tetrachloride; BP100; 100 mg/kg bifendate; Gent 25, 25 mg/kg gentiopicroside; Gent 50, 50 mg/kg gentiopicroside; Gent 100, 100 mg/kg gentiopicroside.

As indicated in Figure 2b and 2c, the liver and spleen indices were significantly higher in the CCl₄ group than in the control group (both P < 0.01). However, Gent significantly decreased both indices in mice with CCl₄-induced liver injury (both P < 0.05).

Effects of Gent on HO-1, SOD, and GSH-Px mRNA expression in mice with CCl₄-induced liver injury

As depicted in Figure 3, CCl₄ suppressed the mRNA expression of SOD and GSH-Px and enhanced that of HO-1 in the liver compared with the findings in control mice (all P < 0.01). Gent pretreatment significantly alleviated these changes (all P < 0.05).

Figure 3.

Effect of gentiopicroside on the mRNA expression of HO-1, SOD, and GSH-Px in mice with CCl₄-induced liver injury. (a) HO-1 mRNA expression in the liver. (b) SOD mRNA expression in the liver. (c) GSH-Px mRNA expression in the liver. Data are expressed as the mean ± standard deviation. ^##P < 0.01 versus the control group, ^*P < 0.05, ^**P < 0.01 versus the CCl₄ group. HO-1, heme oxygenase 1; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CCl₄, carbon tetrachloride; BP100; 100 mg/kg bifendate; Gent 25, 25 mg/kg gentiopicroside; Gent 50, 50 mg/kg gentiopicroside; Gent 100, 100 mg/kg gentiopicroside.

Effects of Gent on oxidative stress in mice with CCl₄-induced liver injury

As depicted in Figure 4, compared with the findings in the control group, liver HO-1 activity and MDA levels were significantly elevated in the CCl₄ group (both P < 0.01), and SOD and GSH-Px activities and GSH level were significantly decreased (all P < 0.01). Compared with the results in the CCl₄ group, SOD and GSH-Px activities and GSH levels were significantly increased in the Gent 50 and 100 groups (all P < 0.05), whereas HO-1 activity and MDA levels in the liver were decreased (both P < 0.01).

Figure 4.

Effects of gentiopicroside on oxidative stress in mice with CCl₄-induced liver injury. (a) HO-1 activity in the liver. (b) SOD activity in the liver. (c) GSH levels in the liver. (d) GSH-Px activity in the liver. (e) MDA levels in the liver. Data are expressed as the mean ± standard deviation. ^##P < 0.01 versus the control group, ^*P < 0.05, ^**P < 0.01 versus the CCl₄ group. CCl₄, carbon tetrachloride; HO-1, heme oxygenase 1; SOD, superoxide dismutase; GSH, glutathione; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; BP100; 100 mg/kg bifendate; Gent 25, 25 mg/kg gentiopicroside; Gent 50, 50 mg/kg gentiopicroside; Gent 100, 100 mg/kg gentiopicroside.

Effects of Gent on PPAR-γ and Nrf2 expression in mice with CCl₄-induced liver injury

As depicted in Figure 5, PPAR-γ and Nrf2 expression was significantly lower in the livers of CCl₄-treated mice than in control mice (both P < 0.01). Gent significantly increased the expression of PPAR-γ and Nrf2 (both P < 0.05).

Figure 5.

Effects of gentiopicroside on PPAR-γ and Nrf2 expression in mice with CCl₄-induced liver injury. (a) PPAR-γ expression in the liver. (b) Nrf2 expression in the liver. Data are expressed as the mean ± standard deviation. ^##P < 0.01 versus the control group, ^*P < 0.05, ^**P < 0.01 versus the CCl₄ group. PPAR, peroxisome proliferator-activated receptor; Nrf2, nuclear factor erythroid 2-related factor 2; CCl₄, carbon tetrachloride; BP100; 100 mg/kg bifendate; Gent 25, 25 mg/kg gentiopicroside; Gent 50, 50 mg/kg gentiopicroside; Gent 100, 100 mg/kg gentiopicroside.

Effect of Gent on inflammatory response in CCl₄-induced liver injury in mice

As depicted in Figure 6, compared with those in the control group, the levels of TNF-α and IL-1β in the serum of CCl₄-treated mice were significantly increased (both P < 0.01). Gent significantly inhibited the secretion of TNF-α and IL-1β in a dose-dependent manner (both P < 0.01).

Figure 6.

Effects of gentiopicroside on the inflammatory response in mice with CCl₄-induced liver injury. (a) TNF-α levels in serum. (b) IL-1β levels in serum. Data are expressed as the mean ± standard deviation. ^##P < 0.01 versus the control group, ^*P < 0.05, ^**P < 0.01 versus the CCl₄ group. CCl₄, carbon tetrachloride; TNF, tumor necrosis factor; IL, interleukin; BP100; 100 mg/kg bifendate; Gent 25, 25 mg/kg gentiopicroside; Gent 50, 50 mg/kg gentiopicroside; Gent 100, 100 mg/kg gentiopicroside.

Effects of Gent on the p-NF-κB p65/NF-κB p65 and p-IκB-α/IκB-α pathways in mice with CCl₄-induced liver injury

To examine the mechanistic basis of Gent-mediated hepatoprotection in mice with CCl₄-induced liver injury, we examined the expression of NF-κB p65, p-NF-κB p65, IκB-α, and p-IκB-α. The results revealed that the p-NF-κB p65/NF-κB p65 and p-IκB-α/IκB-α ratios were significantly increased in the CCl₄ group (both P < 0.01, Figure 7). By contrast, Gent significantly reduced both ratios. This suggests that Gent prevented liver injury by inhibiting NF-κB/IκB pathway activation (P < 0.05).

Figure 7.

Effects of gentiopicroside on the p-NF-κB p65/NF-κB p65 and p-IκB-α/IκB-α pathways in mice with CCl₄-induced liver injury. (a) p-NF-κB p65/NF-κB p65 ratio in the liver. (b) p-IκB-α/IκB-α ratio in the liver. Data are expressed as the mean ± SD. ^##P < 0.01 versus the control group, ^*P < 0.05, ^**P < 0.01 versus the CCl₄ group. CCl₄, carbon tetrachloride; NF-κB, nuclear factor-kappa B; IκB, inhibitor of NF-κB; BP100; 100 mg/kg bifendate; Gent 25, 25 mg/kg gentiopicroside; Gent 50, 50 mg/kg gentiopicroside; Gent 100, 100 mg/kg gentiopicroside.

Discussion

CCl₄-induced liver injury is widely used as an experimental liver injury model in mice. The metabolism of CCl₄ by cytochrome P450 enzyme results in the production of two highly toxic free radicals, namely trichloromethyl free radical and trichloromethyl peroxyl free radicals, which cause liver cell necrosis, induce inflammation, and further promote the development of liver oxidative stress.^14,15 In this study, we demonstrated that Gent significantly reduced CCl₄-induced hepatocyte death and pathological indicators of liver injury in mice. Additionally, Gent reduced oxidative stress in the liver and significantly inhibited CCl₄-induced inflammatory responses.

The results of transaminase analysis revealed that Gent significantly reduced the activities of ALT, AST, and LDH in a dose-dependent manner. Histopathological analysis revealed that Gent significantly improved hepatocyte inflammatory cellular infiltration and necrosis, indicating that Gent exhibited protective effects against CCl₄-induced liver injury in mice.

Oxidative stress and inflammatory responses have significant roles in the occurrence and development of CCl₄-induced liver injury. Injection with CCl₄ significantly induced disturbances in oxidative stress indices and necroinflammation in the liver, which might be attributable to the free radical metabolites of CCl₄ that induce oxidative stress and trigger the production of inflammatory mediators in the liver, leading to an inappropriate inflammatory response and alterations in liver functions.¹⁶ Oxidative stress has been extensively studied as a crucial factor involved in numerous liver diseases.¹⁷ CCl₄ metabolism drives free radical formation in the liver, and antioxidant defense mechanisms, such as the non-enzymatic antioxidant GSH and enzymatic antioxidants such as HO-1, SOD, and GSH-Px, play critical roles in preventing damage. Meanwhile, MDA is employed as a diagnostic modality for oxidative stress to assess the level of membrane lipid peroxidation.¹⁸ In this study, HO-1 and MDA levels were significantly higher in mice with CCl₄-induced liver injury, whereas those of SOD, GSH-Px, and GSH were lower. Nevertheless, the administration of Gent significantly enhanced SOD and GSH-Px activities and GSH levels, whereas HO-1 and MDA levels were reduced. An increasing number of studies have reported the significance of Nrf2 in the development of several liver diseases, including viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease.^19,20 PPAR-γ binds to a specific binding site, namely a peroxisome proliferative response element, to promote Nrf2 gene expression, and it possesses antioxidant properties.²¹ Nrf2 can regulate the oxidative defense system, and it is primarily involved in the synthesis of reducing factors and the promotion of superoxide decomposition.²¹ Anti-oxidative strategies targeting the PPAR-γ/Nrf2 pathway have produced promising results in alleviating liver injury.^7,8,23 In this study, PPAR-γ and Nrf2 expression in the livers of CCl₄-treated mice was significantly decreased. Gent administration significantly increased their expression.

In acute and chronic liver diseases, inflammation is occasionally associated with liver damage. CCl₄ is metabolized to free radicals by cytochrome P450 in the liver. Free radicals attack liver cells, causing parenchymal cell necrosis and promoting liver inflammation.^24,25 TNF-α and IL-1β are the key responsible mediators for the infiltration of inflammatory cells in the liver that in turn cause massive liver damage.²⁶ It has been reported that NF-κB plays an important role in CCl₄-induced liver injury by activating inflammatory signaling pathways.²⁷ The results in this study revealed that serum TNF-α and IL-1β levels were significantly increased in CCl₄-treated mice, and p-IκB-α and p-NF-κB p65 expression was also elevated. Following Gent administration, TNF-α and IL-1β secretion and p-IκB-α and p-NF-κB p65 expression were markedly reduced.

In conclusion, this study demonstrated the potent protective effect of Gent against CCl₄-induced hepatic inflammation and oxidative stress by inhibiting NF-κB/IκB activation and promoting PPAR-γ/Nrf2 activation. The molecular mechanism underlying the effects of Gent on liver injury and its clinical application need to be further studied.

Footnotes

Author contributions

Haitao Wang designed the study and wrote the original manuscript. Yun Zhang and Shiguang Pan performed the experiments and revised the manuscript. Shiming Yi analyzed the data. Jin Sun searched the literature. All authors read and approved the final manuscript.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declaration of conflicting interests

The authors declare there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

ORCID iD

Haitao Wang

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Gentiopicroside ameliorates CCl 4 -induced liver injury in mice by regulating the PPAR-γ/Nrf2 and NF-κB/IκB signaling pathways

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Drugs and reagents

Animals’ treatment and experimental design

Determination of AST, ALT, and LDH activities

Measurement of liver and spleen indices

Histopathological examination

Assessment of HO-1, SOD, and GSH-Px activities and GSH and MDA levels in the liver

Serum IL-1β and TNF-α assays

Quantitative reverse transcription-polymerase chain reaction

Western blotting

Statistical analysis

Results

Effects of Gent on serum ALT, AST, and LDH activities in mice with CCl4-induced liver injury

Effects of Gent on histopathological changes and liver and spleen indices in mice with CCl4-induced liver injury

Effects of Gent on HO-1, SOD, and GSH-Px mRNA expression in mice with CCl4-induced liver injury

Effects of Gent on oxidative stress in mice with CCl4-induced liver injury

Effects of Gent on PPAR-γ and Nrf2 expression in mice with CCl4-induced liver injury

Effect of Gent on inflammatory response in CCl4-induced liver injury in mice

Effects of Gent on the p-NF-κB p65/NF-κB p65 and p-IκB-α/IκB-α pathways in mice with CCl4-induced liver injury

Discussion

Footnotes

Author contributions

Data availability statement

Declaration of conflicting interests

Funding

ORCID iD

References

Effects of Gent on serum ALT, AST, and LDH activities in mice with CCl₄-induced liver injury

Effects of Gent on histopathological changes and liver and spleen indices in mice with CCl₄-induced liver injury

Effects of Gent on HO-1, SOD, and GSH-Px mRNA expression in mice with CCl₄-induced liver injury

Effects of Gent on oxidative stress in mice with CCl₄-induced liver injury

Effects of Gent on PPAR-γ and Nrf2 expression in mice with CCl₄-induced liver injury

Effect of Gent on inflammatory response in CCl₄-induced liver injury in mice

Effects of Gent on the p-NF-κB p65/NF-κB p65 and p-IκB-α/IκB-α pathways in mice with CCl₄-induced liver injury