Aloperine attenuates carbon tetrachloride-induced mouse hepatic injury via Nrf2/HO-1 pathway

Purpose: To investigate whether aloperine pretreatment ameliorates acute liver injury in carbon tetrachloride (CCl4)-treated mice. Methods: Mice were injected with CCl4 and orally administered aloperine. Blood samples and liver tissues were used for histopathological and biochemical analyses, respectively. Protein expression levels were determined by western blotting. Results: Histopathological analysis indicate that aloperine pretreatment significantly alleviated CCl4induced mouse hepatic injury. CCl4 treatment induced the upregulation of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine amino transferase (ALT), and total bilirubin (p < 0.05). However, these alterations were significantly inhibited by aloperine treatment. Moreover, aloperine pretreatment markedly decreased (p < 0.05) the CCl4-induced expression of oxidative stress biomarkers, including malondrialdeline (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). Compared to the control group, the protein levels of Nrf2, HO-1, iNOS, and COX-2 were significantly increased in the CCl4 group, while Nrf2 and HO-1 were upregulated. Furthermore, iNOS and COX-2 were downregulated in mouse liver in CCl4 + aloperine group compared to CCl4 group in a concentration-dependent manner (p < 0.05). Conclusion: Aloperine pretreatment appears to markedly upregulate Nrf2 and HO-1 and downregulate iNOS and COX-2 to suppress hepatic injury in mice. Thus, aloperine is a promising treatment for acute liver injury.


INTRODUCTION
Environmental toxins often disturb hepatic metabolic function and increase the expression of liver enzymes, leading to liver fibrosis, cirrhosis, and even cancer [1]. Liver injury can be caused by a combination of oxidative stress, necrosis, inflammation, and apoptosis [2]. Carbon tetrachloride (CCl4) is frequently used to induce liver injury and to study the effects of chemical compounds on the liver [3]. Existing hepatoprotective drugs are used sparingly because of their side effects [4]. Therefore, there is an urgent need to develop safer hepatoprotective drugs.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important leucine zipper-containing, redoxsensitive transcription factor that can induce the production of antioxidant enzymes via the antioxidant response element [5]. By activating heme oxygenase-1 (HO-1) expression, Nrf2 can suppress the nuclear translocation of NF-κB and subsequent inflammatory responses [6]. Thus, interventions that target the Nrf2/HO-1 signaling pathway have recently been suggested to be important therapeutic approaches for the treatment of alcoholic liver injury [7]. Antioxidants not only remove free radicals, but are also cytoprotective [8]. For example, the combination of metformin and luteolin has been reported to reduce liver injury induced by CCl4 by activating the Nrf2/HO-1 pathway [9]. The Nrf2/HO-1 pathway thus appears vital for treating liver injury.
Aloperine, a compound that can be extracted from bitter beans, plays an important role in certain inflammatory disorders, such as allergic contact dermatitis and experimental colitis [10,11]. A recent study showed that aloperine protects mice from ischemia-reperfusion-induced renal injury by reducing levels of oxidative stress [12]. Furthermore, aloperine was found to attenuate allergic airway inflammation by regulating the Nrf2/HO-1 signaling pathway [13]. Therefore, whether aloperine could alleviate liver injury remains to be investigated. The present study was undertaken to explore the influence of aloperine pretreatment on acute liver injury caused by CCl4 in mice and determine the possible involvement of the Nrf2/HO-1 pathway in this process.

EXPERIMENTAL Chemical and reagents
Aloperine and CCl4 were purchased from Abcam (Cambridge, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

Mice and treatments
The animal study was approved the Medical Ethics Committee of Qinhai Provincial People's Hospital (approval no. 20191016) and conducted according to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health [14]. Twenty-four male C57BL/6 mice (6-8 weeks of age, 16-18 g; Animal Research Center of Nanjing University (Nanjing, China) were randomly divided into four groups (six mice/group): Control, CCl4, CCl4 + 50 mg/kg aloperine, and CCl4 + 100 mg/kg aloperine. In the CCl4 group, mice were intraperitoneally (i.p.) injected with CCl4 [10 mL/kg, 0.5% (v/v), dissolved in olive oil]. In the CCl4 + 50 mg/kg aloperine and CCl4 + 100 aloperine groups, mice were orally administered aloperine (50 and 100 mg/kg, respectively, resuspended in ethanol) for 7 days. Two hours after the last dose of aloperine, mice were injected with CCl4. Mice in the control and CCl4 groups were given the same dose of solvent. Mice were sacrificed 24 h post-CCl4 injection. Blood samples were taken from the orbital vein, while liver tissues were obtained after dissection and used for the following analyses.

Hepatic histopathological examination
Liver tissues were fixed, embedded, and stained with hematoxylin and eosin (H&E). The extent of injury was scored from 0 to 4, based on the severity of the vacuolization of hepatocyte cytoplasm, sinusoidal congestion, and parenchymal necrosis [15]. Ten different views of an image were randomly chosen and scored by two pathologists independently.

Evaluation of biomarkers of liver function
Serum AST, ALP ALT, and total bilirubin levels were measured utilizing commercial assay kits (Jiancheng Biological Technology, China).

Antioxidants in hepatic tissues
Hepatic tissues were homogenized and centrifuged. The supernatants of the liver tissue homogenates were used for the determination of malondrialdeline (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels. Commercial assay kits (Jiancheng Biological Technology) were used to examine the effects of the antioxidants on hepatic tissues.

Protein extraction and western blotting analysis
Western blotting was conducted, as described previously [16,17]. Total protein was extracted, subjected to SDS-PAGE, and then transferred onto PVDF membranes. The membranes were then blocked with 5% nonfat milk and incubated with the following antibodies: anti-Nrf2 (Abcam); anti-HO-1(Cell Signaling Technology, Danvers, MA, USA), anti-iNOS (Abcam); and anti-COX-2 (Abcam). The level of β-actin was used as an internal control. Proteins were visualized and quantified.

Statistical analysis
The results are expressed as mean ± SD and analyzed by one-way ANOVA among the four groups using SPSS software. P < 0.05 was considered statistically significant.

Aloperine attenuates CCl4-induced liver injury in mice
To verify the role of aloperine in reducing hepatic damage, liver tissues were collected from mice in the different groups and examined by histological analysis and scored. Compared to control mice, CCl4 treatment resulted in severe liver damage (Figure 1). The hepatic damage was alleviated to a certain extent by treatment with 50 mg/kg aloperine, but was ameliorated to a much greater extent by treatment with 100 mg/kg aloperine. These findings demonstrate that aloperine reduces CCl4-induced liver damage in mice.

Aloperine treatment decreases CCl4-induced serum levels of hepatic functional markers in mice
The effects of aloperine on the serum levels of hepatic functional markers were determined. Compared to the control group, the serum levels of AST, ALT, ALP, and total bilirubin were significantly increased in mice by CCl4 treatment. However, aloperine treatment (50 mg/kg) markedly reduced the levels of these hepatic functional markers compared to mice with CCl4 treatment. Moreover, the serum concentrations of these markers were lower in the CCl4 + 100 mg/kg aloperine group than in the CCl4 + 50 mg/kg aloperine group (Figure 2).

Aloperine reverses the effects of CCl4 treatment on the regulation of the Nrf2/HO-1 axis in mouse liver
The results shown in Figure 4 showed that, compared to the control group, the protein levels of Nrf2, HO-1, iNOS, and COX-2 were significantly increased in the CCl4 group. On the other hand, Nrf2 and HO-1 were upregulated, whereas iNOS and COX-2 were downregulated in mouse liver in CCl4 + aloperine group compared to the CCl4 group in a concentrationdependent manner.

DISCUSSION
The liver has numerous important functions that are essential for life, including protein synthesis, glucose homeostasis, and detoxification. Although the liver has a strong regenerative capacity, it can still become damaged due to exposure environmental toxins, resulting in organ dysfunction and metabolic abnormalities. The underlying molecular mechanism of acute liver injury has been found to be associated with oxidative stress, apoptosis, and inflammation [1]. CCl4 treatment is known to stimulate lipid peroxidation, reactive oxygen species production, and centrilobular necrosis and steatosis; it has been extensively used to induce acute hepatic damage in a mouse model of liver toxicity [18]. Some natural products have been shown to relieve CCl4-induced liver damage. For example, curcumin, an extract from turmeric rhizomes, can inhibit oxidative stress and inflammation, ameliorating CCl4-induced liver injury [19]. In addition, sesamin has been suggested to regulate the JNK pathway and inhibit hepatic oxidative stress induced by CCl4 [20]. In the present study, aloperine treatment effectually relieved hepatic dysfunction and histopathologic damage by downregulating serum AST, ALT, and ALP activities, and total levels of bilirubin. The combination of two or more agents may result in synergistic effects to alleviate hepatic injury, which could potentially lessen the dose of a single drug and reduce untoward side effects. For example, the combination of metformin and luteolin has been shown to exhibit a potential synergy for the treatment of hepatic injury mediated by CCl4 [9]. Whether these medications can be used in combination with aloperine to prevent liver damage deserves further study.
Aloperine has been found to possess some beneficial medicinal functions. It protects mice against DSS-induced colitis via the suppression of the PI3K/Akt signaling pathway [21]. Aloperine pretreatment has also been shown to reduce inflammation, the apoptosis of tubular cells, and renal damage caused by ischemia-reperfusion [12]. Furthermore, aloperine was shown to inhibit the proliferation and differentiation of fibroblasts, alleviating lung fibrosis induced by bleomycin [22]. The present study demonstrated that aloperine could inhibit oxidative stress responses and elicit hepatoprotective effects. Based on these findings, aloperine may have other medicinal benefits that should be explored in the future.
Nrf2 has been shown to regulate antioxidant genes; its activation might provide effective cellular protection by regulating intracellular redox status [8]. Previous studies have suggested that natural antioxidants may activate Nrf2 and HO-1. For example, curcumin, an activator of the Nrf2 pathway, was shown to stimulate the activity of antioxidants and suppress the oxidative stress caused by CCl4 [19]. In addition, morin [23], anwulignan [24], and andrographolide [25] have been shown to upregulate Nrf2/HO-1, exert antioxidative functions, and protect against mouse liver damage. Therefore, the stimulation of the Nrf2/HO-1 pathway is a promising strategy for the clinical treatment of liver injury.

CONCLUSION
The findings of this study indicate that aloperine pretreatment suppresses mouse hepatic injury, partly via the enhancement of Nrf2/HO-1 pathway and stimulation of its antioxidant activity. These findings may thus lead to the development of novel drug candidates for the management of acute liver injury.

DECLARATIONS
terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.